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A study of the role of Rab27 in lacrimal gland acinar cell secretory trafficking

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A study of the role of Rab27 in lacrimal gland acinar cell secretory trafficking

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A STUDY OF THE ROLE OF RAB27 IN LACRIMAL GLAND ACINAR CELL
SECRETORY TRAFFICKING

by

Lilian Chiang

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

December 2010

Copyright 2010 Lilian Chiang

ii

Dedication

I dedicate this work to my family and friends, who have always been so supportive of my
endeavors, and especially to my parents who have always and completely believed in my
aspirations, and to Jeff, who reminds me of the reasons why I decided to pursue my
doctorate in the first place.

iii

Acknowledgements

I sincerely thank Francie Yarber for not only her hard work in everything from the
weekly cell prep to Adenovirus purifications, but for her companionship during the
longer work days and for her thorough support through the entire project. I also thank Dr.
Maria Edman for her sagely advice, patience, and support, Dr. Kaijin Wu for her valuable
input in molecular biology, Ben Xie for his hard work in preparing density gradient
fractions, and all the kind faculty who I’ve sought random bits of expertise from
throughout the years. In addition, I’d like to acknowledge our collaborators in the Seabra
lab, Dr. John Williams for his recombinant Ad-Xpress-tagged Rab27b constructs, Dr.
Chris Rhodes for recombinant Ad-syncollin-GFP constructs, Mike Pidgeon for his expert
help with the TEM, and Dr. Roberto Weigert at the NIH for his knowledge on stereology.

And above all, I would like to thank my advisor, Dr. Sarah Hamm-Alvarez, my labmates
in the Hamm-Alvarez laboratory, Thomas Ng for his advice and support for this project,
and my committee for the help and inspiration they gave for this project.

iv

Table of Contents

Dedication ii

Acknowledgements iii

List of Tables vii

List of Figures viii

Abbreviations xi

Abstract xiii

Chapter 1: Introduction
1.1 Lacrimal gland physiology 1
1.2 The regulated secretory trafficking pathway in the 3
lacrimal gland acinar cell
1.3 RabGTPase characteristics and activity 5
1.4 Rab27 is unique in its role in human diseases 6
1.5 Identification and characterization of Rab27 in hematopoietic- 9
derived cells
1.6 Studies in melanocytes reveal a relationship between Rab27 10
and its effectors
1.7 The current state of Rab27 studies in polarized secretory 11
epithelial cells
1.8 The degradative pathway performs a critical role in 13
maintaining cell health
1.9 Goals and experimental design 14

Chapter 2: Evidence of Rab27b knockout effect on organelle
morphology in lacrimal gland models
2.1 General description of classical mouse gland section as 16
observed by transmission electron microscopy
2.2 Rab27b knockout glands show strong differences relative to 17
those from C57BL/6 and Ashen
2.3 Definitions and quantifications of changed organelle 19
morphologies
2.4 Possible link between the secretory pathway and 22
degradative pathway

v

2.5 Example of possible increased mitochondrial vesiculation in 25
Mocha mice
2.6 Additional observations of knockout tissue with H&E and 26
oil red staining
2.7 Discussion and implications 28

Chapter 3: Target identification and characterization of endogenous
expression
3.1 Rab27b shows high expression in lacrimal gland tissue 30
3.2 Cultured acinar cells retain Rab27b expression 32
3.3 Biochemical confirmation of Rab27b expression and clues to 34
localization
3.4 Rab27b co-localization with other secretory pathway markers 40
and its re-localization with stimulation
3.5 Quantification of co-localizations of Rab27b 42
3.6 Discussion and implications 43

Chapter 4: Establishment of live-cell protocols using a virus entry study
4.1 Addition of Adenovirus 5 and capsid proteins induces 46
membrane ruffling
4.2 Translation of qualitative observations into quantitative data 49
4.3 Effects of Heparin on Ad5 uptake 51
4.4 Discussion and implications 52

Chapter 5: Functional activity state affects Rab27b localization
5.1 Characterization of wild-type and mutant Rab27b expression in 54
lacrimal gland acinar cells
5.2 Rab27b mutants altered secretory content localization 56
5.3 Rab27b-enriched secretory vesicles do not co-localize with the 56
lysosomal pathway
5.4 Secretory vesicle content release is positively regulated by 58
Rab27b
5.5 Quantification of changes in enriched vesicle diameter with 62
constructs
5.6 Discussion and implications 63

Chapter 6: Rab27b-enriched secretory vesicles utilize cytoskeletal
components at different stages
6.1 Rab27b-enriched secretory vesicles bud from an unidentified 65
nascent vesicle site
6.2 Co-localization study of Rab27b with a dynein motor component, 67
p150
Glued
6.3 Disruption of the microtubule network sequesters nascent vesicles 68
at the nascent vesicle site

vi

6.4 The search for the identity of the nascent vesicle site 70
6.5 Rab27b co-localizes with Myosin 5C on secretory vesicles in the 73
subapical region
6.6 Co-localization study of Rab27b with actin filaments 76
6.7 Disruption of the actin network alters the terminal apical 77
membrane fusion and release of enriched secretory vesicles
6.8 Co-localization study of Rab27b with truncated melanophilin 79
6.9 Discussion and implications 80

Chapter 7: Materials and methods
7.1 Reagents 86
7.2 Primary rabbit lacrimal gland acinar cell culture and treatments 87
7.3 Mouse lacrimal gland isolation and analysis 87
7.4 Production and amplification of recombinant adenovirus 88
7.5 Generation of recombinant proteins 89
7.5 Ad transduction of constructs into lacrimal gland acinar cells
7.6 Confocal fluorescence microscopy 90
7.7 Time-lapse live cell imaging 91
7.8 Preparation of lacrimal gland tissue and lacrimal gland acinar cell 93
lysate for western blotting
7.9 Analysis of lacrimal gland acinar cell secretion 94
7.10 Subcellular fractionation analysis 94
7.11 Transmission electron microscopy 95
7.12 Statistical analysis 95

Chapter 8: Conclusions and future perspectives
8.1 Function of Rab27b in the lacrimal gland 97
8.2 Mechanisms behind the movement of enriched vesicles in 100
trafficking
8.3 Potential therapies, analysis, and future approaches with Rab27b 102

References 108

vii

List of Tables

Table 1: Examples of Rab27a versus Rab27b expression in tissue and 8
cell lines

Table 2: Summary of morphology counts from TEM 21

Table 3: Quantification of Rab27b signal co-localized with other 42
markers in the subapical region

viii

List of Figures

Figure 1: Representation of lacrimal functional unit and the three layers 1
of the tear film

Figure 2: Model of lacrimal gland acinar cell intracellular trafficking at 4
resting state

Figure 3: C57 background mouse lacrimal gland TEM sections 16
demonstrate cell polarity and organization

Figure 4: TEM from 27bKO and DKO lacrimal glands are 18
morphologically distinguishable from C57 and Ashen glands

Figure 5: Detailed comparison and quantification of organelle morphology 20
in mouse lacrimal glands lacking Rab27b

Figure 6: Secretory vesicle distance to the center of the lumen in DKO 22
versus C57

Figure 7: DKO mouse lacrimal tissue show increased expression of 24
degradative markers

Figure 8: Mocha mouse lacrimal glands show some increased 25
mitochondrial cristolysis compared to GR/J mice

Figure 9: 27bKO and DKO lacrimal sections show increase of signal for 27
oil droplets

Figure 10: Rab27b is expressed in mouse lacrimal gland acinar cells 31

Figure 11: Rab27b expression is enriched on membranous secretory 33
vesicle structures in the subapical region of acinar cells

Figure 12: Rab27b is detectable biochemically in lacrimal gland lysate 35

Figure 13: Immunofluorescence study shows that Rab27b antibody 36
is specific for Rab27b in acinar cells

ix

Figure 14: Rab27b localization in membrane fractions shifted with 37
carbachol stimulation

Figure 15: Rab27b association with membrane fractions at resting 38
stages and after carbachol stimulation

Figure 16: Rab27b co-localization with SV markers is consistent with 41
a role in lacrimal exocytotic processes

Figure 17: Time-lapse confocal fluorescence microscopy of membrane 47
ruffling in acinar cells treated with Ad5 and capsid proteins

Figure 18: Ruffling induces some uptake of dextran 10 48

Figure 19: Quantification of membrane-ruffling events elicited by 50
Ad5 and capsid proteins in acinar cells

Figure 20: Heparin affects the macropinocytosis effect of Ad5 52

Figure 21: Rab27b activity also affects its distribution 55

Figure 22: Expression of mutant Rab27b affects the distribution of 57
secretory vesicle content

Figure 23: Rab27b-enriched secretory vesicles do not co-localize 58
with lysosomes

Figure 24: Rab27b positively regulates secretory vesicle exocytosis 60

Figure 25: Rab27b-enriched secretory vesicles bud off from a 66
nascent vesicle site

Figure 26: Rab27b co-localizes with p150 67

Figure 27: Disruption of microtubule network hinders nascent 69
Rab27b-enriched vesicle production

x

Figure 28: Rab27b may bud off from a late Golgi compartment or from 71
a separate NVS

Figure 29: Rab27b co-localizes highly with M5C in the 74
subapical-most region of the lacrimal gland acinar cell

Figure 30: Rab27b-enriched secretory vesicles lay beneath the actin 77
membrane but do not co-localize at resting state

Figure 31: Disruption of the actin network alters terminal apical membrane 78
fusion of Rab27b-enriched secretory vesicles

Figure 32: Rab27b co-localizes with Mlph 81

Figure 33: Trafficking schematic of Rab27b-enriched secretory vesicles 101

xi

Abbreviations

CA Constitutively Active
CCH Carbachol
CSA Chondroitinase ABC
DN Dominant Negative
GalNAc-T2 RFP-tagged N-acetylgalactosaminyltransferase 2
GAPs GTPase activating proteins
GEFs Guanine nucleotide exchange factors
GFP Green fluorescent protein
GFP-M5C-tail GFP-tagged dominant negative Myosin 5C
GFP-M5C GFP-tagged Myosin 5C-full length
HS-GAGs Heparin sulfate-glycosaminoglycans
LGAC Lacrimal gland acinar cells
M5A Myosin 5A
M5C Myosin 5C
Mlph Dominant-negative melanophilin, red fluorescent protein-tagged
NVS Nascent vesicle site
p150 p150
Glued
RFP Red fluorescent protein
Slp Synaptotagmin-like protein
SV Secretory vesicle

xii

Sync Syncollin, green fluorescent protein-tagged
WT Wild-type
Xp-Rab27b Xpress
TM
-tagged Rab27b
YFP-Rab27b Yellow fluorescent protein-tagged Rab27b
YFP Yellow fluorescent protein

xiii

Abstract

Tear proteins are supplied by the regulated fusion of secretory vesicles with the apical
surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet
uncharacterized. We investigated the role of Rab27b in regulating the exocytotic pathway
of these secretory vesicles. Evaluation of morphological changes by transmission
electron microscopy of lacrimal glands from Rab27b
-/-
and Rab27
ash/ash
/Rab27b
-/-
mice,
but not Ashen mice deficient in Rab27a, showed significant changes in organelle
morphology which included an approximate 50% decrease in abundance of secretory
vesicles and decreased vesicle localization in the subapical region. Along with an
apparent secretory pathway effect, knockout of Rab27b also resulted in a two-fold
increase in the number of lysosomes, four-fold increase number of damaged
mitochondria, two-fold increase in the formation of autophagosome-like organelles, and
observed increased ER swelling and vesiculation. Confocal fluorescence microscopy
analysis, also confirmed by biochemical assays, of primary cultured rabbit lacrimal gland
acinar cells revealed that Rab27b was enriched on the membrane of large subapical
vesicles that were highly colocalized with Rab3D (73.6±2.0% at rest) and Myosin 5C
(58.3±7.4% at rest). Stimulation of cultured acinar cells with the secretagogue,
carbachol, resulted in apical fusion of these secretory vesicles with the plasma membrane.
We also established live-cell protocols for examination of cultured lacrimal gland acinar
cells by measuring quantifiable markers for adenovirus entry; these protocols were
applied to the remainder of the live cell studies. Tagged-Rab27b constructs expressed in

xiv

lacrimal gland acinar cells allowed for visualization of Rab27b dynamics in real time.
Wild-type Rab27b was expressed on a large subpool of mature secretory vesicles in the
subapical region of the cell. Constitutively-active Rab27b increased the average size but
retained the subapical distribution of Rab27b-enriched secretory vesicles, while
dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm.
These secretory vesicles did not colocalize with the lysosomal pathway.
Functional studies measuring release of a co-transduced secretory protein, syncollin-GFP,
showed that constitutively-active Rab27b enhanced (up to ~1.5-fold of wild-type), while
dominant-negative Rab27b suppressed (no significant change from resting state),
stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical
membrane but did not disrupt homotypic fusion. Rab27b–enriched secretory vesicles also
required intact components of the cytoskeletal network. Rab27b colocalized highly with
the dynein motor protein subunit, p150
Glued
. In a series of stimulation-recovery
experiments, real time image series showed for the first time clear visualization of
Rab27b-enriched nascent vesicles formation on the surface of a Nascent Vesicle Site
(NVS) located in the basal region of the acinar cell and their gradual movement towards
the subapical region to join an existing pool of secretory vesicles as they matured over a
60 min time frame. Disruption of microtubule polymerization sequesters secretory
vesicles on the NVS in close proximity to the trans-Golgi network, although
colocalization with specific markers was low. These data leave two possible conclusions:
that the NVS as a contiguous membrane compartment of the trans-Golgi network, or that
the NVS is a completely separate organelle. We also examined Rab27b colocalization

xv

with another cytoskeletal component, F-actin. Rab27b also colocalized with Myosin 5C
but not directly with actin filaments. Depolymerization of actin filaments prevents mature
vesicles from fusing with the apical membrane. Finally, Rab27b shows high co-
localization with a truncated form of one of its binding effector proteins, melanophilin,
although localization was not obviously altered by overexpression of truncated
melanophilin.
Previous studies have suggested that Rab27b is a participant of the last steps of secretory
trafficking in other cell types, but have not looked at its role as one in a continuous
pathway. In this study, we demonstrate a chronological and multi-step role for Rab27b
that is critical in the secretory pathway of lacrimal gland acinar cell. In conclusion, we
have shown in this study that knockout of Rab27b results in a major secretory, as well as
degradative pathway, effect on lacrimal gland acinar cell morphology. Rab27b is highly
enriched on the membrane of secretory vesicles and is associated with components of the
exocytotic pathway. Rab27b-enriched secretory vesicles require intact microtubules for
―long distance‖ transport of nascent vesicles to replenish subapical Rab27b-enriched
vesicle pools. Upon stimulation, actin is necessary for vesicle-to-apical membrane fusion.
Overexpression of constitutively active Ra2b7b up-regulates secretion, while dominant
negative Rab27b suppresses the effect of stimulation. These last functional studies
correlate with in situ morphological observations of decreased secretory vesicle numbers
in Rab27b knockout mice.

1

Chapter 1. Introduction

1.1 Lacrimal gland physiology

The lacrimal gland is important to the production of tear proteins and fluid critical to the
maintenance of a healthy ocular surface. Constituting over 80% of the total lacrimal
mass, the gland’s polarized acinar epithelial cells (LGACs) are highly active secretory
cells which, reconstituted in culture, re-form small clusters of acini mimicking their in-
Figure 1. Representation of the lacrimal functional unit and the three layers of
the tear fluid.

2

situ three-dimensional formation. These cells are clearly polarized as delineated by the
organization of actin filaments, which are especially dense at the apical membrane. Large
pools of large, fragile secretory vesicles (SVs) are located beneath the apical membrane
of each cell and upon stimulation in vivo, empty their contents into a lumen contiguous
with the tear duct where they then flow to the ocular surface (Figure 1). The classical tear
film consists of the mucin, aqueous, and lipid layers; it is to the middle aqueous layer that
the lacrimal gland is the major supplier of its constituents (93). The lacrimal gland
packages a wide range of tear factors which contribute to the aqueous layer, including:
nutrients and growth factors, like lacritin and EGF, antibacterial and antiviral factors,
such as secretory IgA and lactorferrin, and an array of proteases and lysosomal
hydrolases (11, 97, 116, 118). The gland itself is innervated by parasympathetic,
sympathetic, and sensory nerves, which includes the activation of M
3
muscarinic
receptors in the basolateral regions of these cells (19). In response to minor stimulation,
tear film turnover in humans can increase up to 500% (56). Thus, the lacrimal gland is
characterized not only by a well-structured and highly active secretory pathway which is
capable of producing and packaging a wide range of tear factors, but is also in a highly
dynamic though highly regulated state, in order to accommodate for immediate changes
in the environment contiguous with the ocular surface.

Perhaps the most obvious illustration of the importance of the lacrimal gland’s role is in
its disease models. It is known that malfunction of lacrimal gland secretion manifests
itself as a number of dry eye conditions related to a decrease in quantity or quality of the

3

aqueous layer, which can lead to scarring and eventual blindness in advanced cases.
Documented causes of decreased tear secretion from the lacrimal gland include Sjögren’s
syndrome, senile hyposecretion, lacrimal gland excision, vitamin A deficiency, immune
gland damage in sarcoidosis or lymphoma, sensory or motor reflex loss, and scarring of
the conjunctiva due to chemical burns or contact lens wear (93). Despite its
physiologically significant role, studies in the lacrimal gland have been challenged by
technical issues concerning the fragility and heterogeneity of the large SV pool, due to a
constitution of dilute content combined with heavy carbohydrate-laden proteins which
results in fragmentation during common isolation procedures such as those used for dense
granules in the pancreas and parotid gland, as well as the preservation of the 3D polarized
acinar LGAC structure. Our solution to these technical limitations applies innovative cell
imaging techniques complemented with biochemical data, along with an established cell
culture protocol.

1.2 The regulated secretory trafficking pathway in the lacrimal gland acinar cell
Control over the specificity of each intracellular trafficking step is key to the productivity
of regulated secretory cells such as the LGAC. In response to specific signals, each step
of the pathway recruits different protein factors which may, in turn, recruit other factors,
instigating events like budding, transport, and fusion. Thus, trafficking steps are
separated both temporally and spatially by the association of trafficking vehicles with
specific markers, although the mechanisms behind these interactions are not well
understood. In the classical exocytosis pathway, such as shown in Figure 2, proteins are

4

believed to be sorted into nascent SVs in the Golgi apparatus located in the basolateral
region, possibly budding off from the trans-Golgi network which is also the membrane
source of the vesicle (36). These immature SVs may then associate with a microtubule
motor protein such as dynein, thus allowing for movement towards the minus-end of the
microtubule in the subapical region (10, 126). As they become mature SVs of
approximately 1 μm in size, they also acquire enrichment of such markers as Rab3D.
Upon stimulation to secrete such as with the muscarinic agonist, carbachol (CCH), SVs
undergo compound fusion concurrent with a loss of Rab3D and move towards the apical

Figure 2. Model of lacrimal gland acinar cell intracellular trafficking at resting state. As SVs
mature and approach the APM along the cytoskeletal network, they acquire Rab3D and other effector
proteins.

5

membrane possibly in association with actin motor proteins such as myosin 5C (69). It is
believed that the final steps include a ―catching‖ of the SV by loosely tethering proteins,
docking to the apical membrane, and then pairings with cognate soluble N-
ethylamaleimide-sensitive factor receptor proteins which then allows fusion between the
bilayers of the vesicle and the target membrane (16). Each of these trafficking steps
require a high level of specificity—a need perhaps served by RabGTPases (100).

1.3 RabGTPase characteristics and activity
In the last two decades, an increasing amount of interest has been paid to the study of Rab
proteins. With almost seventy members thus far discovered, Rab proteins form the largest
branch of the diverse Ras superfamily of GTPases and have notably been reported to be
involved in cell-specific and step-specific regulation of protein trafficking, targeting, and
fusion of membrane-bound organelles (12, 113). A single Rab protein can interact with
multiple effectors, suggesting that a given Rab may regulate several difference
biomechanisms depending on spatio-temporal recruitment. Two structural characteristics
set Rab proteins apart from other proteins. First, Rabs are formed from the Golgi as
soluble proteins which are then guided by Rab escort proteins which bring the Rabs to the
Rab geranylgeranyltransferase. Here, the carboxy-terminal domain of Rab proteins
undergo post-translational modification by addition of geranylgeranyl groups to cysteine
residues on the C-terminus of Rab proteins, allowing its association with the plasma
membrane and simultaneously providing an interdependent effector binding domain (2,
16, 18). This prenylation allows for continuous cycling of Rab proteins between being

6

membrane-bound in various compartments and localization in the cytoplasm. The other
conserved protein sequence of Rab proteins is its on/off ―switch,‖ as defined by its
binding of GTP and GDP. Classical Rab proteins cycle between GTP-bound active states
and GDP-bound inactive states, as assisted by guanine nucleotide exchange factors
(GEFs) or GTPase activating proteins (GAPs), respectively (82, 103). The ability for the
cell to actively control the on/off state of Rab proteins, in addition to the participation of
Rab associated proteins such as GEFs and GAPs, provide yet another level of
downstream regulation. While some Rab proteins have been identified as organelle-
specific or trafficking step-specific markers, such as the mature SV marker Rab3D in
relation to other secretory markers as studied previously by our group (20), much of the
mechanisms behind trafficking is not yet understood (83).

1.4 Rab27 is unique in its role in human diseases
Rab27 expression is highly conserved in mammals, although the expression of either or
both of its two isoforms (Rab27a, Rab27b) varies across different tissues and cell types.
Some of these expressions are summarized in Table 1. Rab27a is a 221 a.a. and
approximately 25 kD protein, while Rab27b is a 218 a.a. and approximately 27 kD
protein, sharing 71% a.a. sequence homology with Rab27a (6, 87). What sets Rab27 apart
from the other Rab proteins is that Rab27 is one of only two known Rab proteins thus far
to be directly associated with human disease. Griscelli syndrome is a human autosomal
recessive disorder caused by a loss-of-function in Rab27a, which causes loss of
pigmentmentation, neurological defects and, in many cases, immunological disorders (3,

7

102). Interestingly enough, studies show that the disease phenotype appears to reflect
isoform expression. Patients with Griscelli syndrome show phenotypes of
immunodeficiency and albinism because Rab27a is the only, or at least major, isoform
expressed in cytotoxic T-lymphocytes and melanosomes. In melanosomes of mouse
model, Ashen, secretory granules accumulate in the perinuclear region and do not reach
the apical plasma membrane, although transport is rescue-able by an EGFP-Rab27a
fusion protein (86, 110). Rab protein localization is dependent on the prenylation state of
the protein, and disease phenotypes related to the state of Rab27 prenylation reflect
trafficking disorders such as partial albinism due to errors in melanosomal trafficking,
tendency for bleeding due to errors in platelet production, and intracellular lysosomal
accumulation. For example, choroideremia results from a defect in Rab escort protein-1,
a protein which normally prenylates membrane-associated proteins including Rab27 (26).

8

9

Similar at the molecular level, Hermansky-Pudlak syndrome and its mouse model
gunmetal occur due to a loss-of-function in the Rab prenylation regulator Rab
geranylgeranyl transferase, which likewise prevents Rab membrane association (18, 102,
105). Together, defective Rab27 appears to manifest itself as diseases related to
intracellular trafficking malfunction and are indicative of the Rab27 protein’s active role
in transport of organelles within the secretory pathway of diverse cell types.

1.5 Identification and characterization of Rab27 in hematopoietic-derived cells
Studies associating Rab27 to trafficking pathways began in the late 1990s in
hematopoietic-derived cells, namely the characterization of Rab27a/ram in rat
megakaryocytes and Rab27b/c25KG in platelet cells (6). Platelets are released in
mammalian bone marrow megakaryocytes and store self-agonists such as ADP and
serotonin in dense core granules regulated by Rab27 (104, 108). Platelets have also been
the subject of some of the more extensive studies of Rab27 in trafficking. Studies have
shown that constitutive GDP/GTP exchange allows for the prevalence of the GTP-bound
active form of Rab27 in unstimulated platelets (61). Upon granule secretion concurrent
with increased GTP hydrolysis, GTP-bound Rab27 decreased. Platelet studies in
gunmetal mice, which exhibit abnormal platelet synthesis, macrothrombocytopenia, and
partial cutaneous albinism related to Rab27a prenylation deficiencies, have also helped to
elucidate Rab27 function in disease models (18, 108). From there, studies shifted to a
more functional focus. In competition assays, Shirakawa, et al. 2004 showed that the
addition of unprenylated Rab27a to cultured cells decreased granule trafficking in

10

melanocytes. This hypoprenylation was rescued by a novel linker protein, Munc 13-4,
which associated with Rab27a (104). In later studies, Munc 13-4 as well as many other
proteins in the Munc 13, Munc 18, and the synaptotagmin-like protein (Slp) families
were shown to be participants in linking together Rab27 with motor proteins (29, 122).

1.6 Studies in melanocytes reveal a relationship between Rab27 and its effectors
Much of our current understanding of the role of Rab27 in trafficking was also derived
from studies in the melanocyte, which is possibly the best characterized cell model thus
far because of its availability to extensive in vitro assays. Melanin is stored in membrane-
bound melanosomes which, at their mature phase and under the regulation by Rab27a,
accumulate at the cell periphery until release (45). Melanocytes require actin-dependent
movement of Rab27a-enriched melanosomes which utilize actin motor proteins like
myosin 5a (M5A) (12). However, in vitro studies showed that Rab27a and M5A do not
bind directly in melanocytes, instead utilizing a linker protein that was identified as
Slac2-a/melanophilin, a member of the Slp family (23, 128). Melanocyte studies also
unveiled structural characteristics. Melanophilin binds to Rab27a by an amino-terminal
Slp-homology domain independent of the geranylgeranylation step (25). This suggests
that effector binding and membrane association occur independently. The midsection of
melanophilin binds the globular tail of M5A, and its carboxy-terminal domain binds actin
(16, 29, 46). A second effector, Slp2-1, also appeared to regulate a later stage of
melanosome transport through an interaction with phosphatidylserine in the plasma
membrane (23). Additionally, it was found that more than one class of actin motor may

11

participate in the pathway, such as the tripartite complex between myosin 7a, Rab27, and
Slac2-c/MyRIP described in a later study (63). Collectively, these studies suggest that
control of subsequent phases of melanosomal transport involves a sequential interplay of
Rab27 with different effectors and actin motors. This tripartite complex model is believed
to be key to understanding Rab27 mechanisms in trafficking: GTP-bound and
melanosome-associated Rab27a serves a M5A ―receptor‖ that regulates trafficking
through its association with the motor protein and may also participate in SNARE pairing
during fusion through an interaction with a second effector protein (128). The validity of
this model has been strengthened by the discovery of similar tripartite associations of
other Rab proteins in other pathways, although these associations have been largely
limited to biochemical studies of individual in vitro protein-protein interactions and do
not elucidate the role of Rab27 in the trafficking pathway in completion (100).

1.7 The current state of Rab27 studies in polarized secretory epithelial cells
While studies in platelets and melanocytes have explored the biochemical interactions of
Rab27 with specific effector proteins in the trafficking pathway, Rab27 studies in
polarized secretory epithelial are still relatively new, although certainly relevant in a cell
type that depends on the tight regulation of each of its secretory steps. Unlike trafficking
in platelets, melanocytes, and neuronal cells, polarized non-neuronal endocrine and
exocrine secretory cells such as LGACs secrete towards an apical/luminal side of the
plasma membrane. Trafficking in these highly active cells are generally affected by, and
participate in, a wider range of functions than many other cell types that can include

12

secretion, absorption, transcellular transport, sensation detection, and selective
permeability. Many recent studies in Rab27 have focused on polarized secretory
epithelial cells, and thus far more extensively on Rab27a than Rab27b. GFP-Rab27a
transgenic mice show expression of the fusion protein in salivary gland cells, mucous
acinar cells, and the endocrine and exocrine pancreas (110). Several effectors discovered
in platelets, melanocytes, as well as neuronal cells have also been identified in pancreatic
β-cells, including: Slp4/granuphilin, Slp5, Slac2-c/MyRIP, and Slac2-a/melanophilin
(121, 130). These studies have largely been limited to specific interactions at specific
stages of trafficking.

Conversely, the focus on Rab27b is slightly more recent than Rab27a and is particularly
relevant to secretory pathway studies. While many of its effectors overlap with Rab27a,
Rab27b may need to be examined independently: in some cell types Rab27b has been
found to have functions distinct from Rab27a (55, 80) and as aforementioned, Rab27b
expression is appears to be predominant over Rab27a especially in some secretory cell
types such as the acinar cells of the pituitary gland (133), exocrine pancreas (7), and
parotid gland (49), although Rab27b also shows high expression in non-acinar tissues
such as the brain and spleen (133). Studies have been conducted in AtT20 cells, a
pituitary endocrine cell line, demonstrating Rab27b regulation of exocytosis of dense
core granules. In the rat exocrine pancreas, in vitro studies showed that Rab27b localized
to zymogen granule membrane and that the overexpression of functional mutants affected
amylase secretion (7, 8). An independent in vitro study has also shown in the rat exocrine

13

pancreas that Rab27b forms a complex with Slac2c, an effector previously associated
with Rab27a, which may contribute to the regulation of secretory granule exocytosis (49).
Studies conducted in parotid acinar cells showed that Rab27b also re-localized in
response to isoproterenol stimulation to secrete (48). While these studies have examined
the Rab27b from the perspective that it may play similar to roles to, and shares many
effectors with, Rab27a, few groups have been able to link these in vitro studies to in vivo
data. In 2007, two groups developed Rab27b knockout and double Rab27a/Rab27b
knockout mice, both of which were created by disruption of the Rab27b gene and then
crossing the Rab27b knockout with the naturally occurring Rab27a-mutated ashen mouse
(35, 109). Usage thus far of these knockout mice has been mainly an evaluation of
phenotype across different tissues and observations in the pituitary and platelet cells of
knockout mice that show impairment of secretory granule distribution consistent with in
vitro studies.

1.8 The degradative pathway performs a critical role in maintaining cell health
Cellular homeostasis requires a balance between the metabolic rate of the cell and the
availability of energy sources. The process of autophagy is a term for the general
catabolic ―self-eating‖ of the cell’s own components such as a region of the cytoplasm or
an organelle, often in an attempt to maintain homeostasis. In classical autophagy,
autophagosomes of approximately 0.8 to 2.0 μm form in response to stress, sequestering
components for degradation after fusion with protease-containing endosomes within a
double-walled membrane (59). Although the mechanisms are not clear, autophagy has

14

been studied extensively in yeast and Drosophila and certain steps have been generalized
(62). Autophagy is considered a nonspecific process, but there are instances where
specific organelles such as the mitochondria, termed as mitophagy, are targeted. There
are a limited number of ways to detect for autophagy, which include: protein degradation
assays, lactate dehydrogenase sequestering assays, transmission electron microscopy, and
tagged markers against autophagosomal components such as MAP1-LC3. To complicate
studies further, different forms and stages of autophagy may be separately identified (70).
These include pre-autophagosomal structures in early stages of sequestration, immature
autophagosomes which have fused lysosomal compartments but incomplete formation of
the double-walled membrane, and mature autophagosomes. Whether general or
organelle-specific autophagy occurs depends on the energy needs of the cell and may also
be related to apoptosis. Additionally, organelles targeted for degradation may have
undergone fusion with lysosomes but not form a sequestering membrane, in which case
they may be identified as autolysosomes.

1.9 Goals and experimental design
Few trafficking markers have been shown to clearly associate with SVs from formation
to release, thus limiting the focus of many studies to the resolution of specific steps of
vesicle trafficking and resulting in a rather piecemeal understanding of the overall
pathway. In this study, we not only focus on determining the functional role of Rab27 in
our polarized secretory cell model, but also piece together a more complete temporal
picture of Rab27’s association with the secretory pathway in the LGAC through

15

extensive in situ and in vitro imaging studies, which are substantiated by molecular and
biochemical data. Our study of Rab27 in the lacrimal gland begins with the observation
of major morphological changes in mouse Rab27 knockout tissue, follows Rab27
expression, movement, and associations in LGAC, and finally concludes with functional
studies of Rab27 surrounding SV content secretion and release.

16

Chapter 2: Evidence of Rab27b knockout effect on organelle morphology in
lacrimal gland models

2.1 General description of classical mouse lacrimal gland as observed by TEM
Lacrimal gland tissue sections prepared from C57BL/6 (C57), the background strain of
the knockout mice used in the following study, were used to attain TEM imagery of the
Figure 3. C57 background mouse lacrimal gland TEM sections demonstrate cell polarity and
organization. A. Lacrimal glands from C57 showed clearly polarized LGAC with large pools of SV
located just beneath the apical plasma membrane (A) delineating the lumen (*). Towards the basolateral
membrane (B) lays the nucleus (N), mitochondria (Mito), and lipid droplets (lip). Throughout the
cytoplasm, several lysosomes (Lys) and well-organized endoplasmic reticulum (ER) can be detected.
Higher magnification images show representations of the dense SV population that lays beneath the
apical membrane and well-organized endoplasmic reticulum. Bar is 2 µm in lower, 1 µm in higher
magnifications. B. High magnifications of homotype SV fusion examples in the subapical region.

17

general organization of the acinar cell. LGAC were closely situated with each other and
were generally arranged around apical membranes which were folded and often displayed
multiple invaginations into the cell which are visualized in the sections as multiple
lumens. Acinar cells of C57 mice reflect that of healthy, polarized LGAC with fully
formed basolateral and apical membranes. In the basal regions lie the nucleus,
endoplasmic reticulum, lysosomes, mitochondria with intact cristae, and some lipid
droplets (Figure 3A). As SV mature, they may undergo homotypic fusion (Figure 3B),
thus attaining larger diameters as they approach the lumen (84, 90). A large pool of
seromucous SV sits in the subapical region adjacent to the lumen. Sample organelles are
shown at higher magnifications beside the larger image.

2.2 Rab27b knockout glands show strong differences relative to those from C57BL/6
and Ashen
Ashen mice, which have a naturally occurring mutation in Rab27a, were similar in
lacrimal acinar cell ultrastructural morphology to C57 mice. However, we noted
differences in general cell organization as well as in specific organelle expression in both
Rab27b knockout (27bKO; Rab27b
-/-
) and double Rab27a Rab27b knockout (DKO;
Rab27
ash/ash
Rab27b
-/-
) mice. TEM images shown in Figure 4A demonstrate the
differences in the C57 and Ashen lacrimal sections from 27bKO and DKO. Further
general observations of the morphologies of 27bKO and DKO are summarized in Figure
4B, with the DKO sample representing morphologies seen in both 27bKO and DKO. In
contrast to the abundant array of SV adjacent to the lumen as well as tightly spaced

18

endoplasmic reticulum and intact mitochondria which are visualized in Figure 3A,
27bKO and DKO lacrimal gland sections showed an apparent decrease in SV expression
in the luminal region and such abnormalities as extremely distended endoplasmic
reticulum. Also apparent was an increase in the number of electron-dense organelles such
as lysosomes and autophagosomal-like structures in the cytoplasm.

Figure 4. TEM from 27bKO and DKO lacrimal glands are morphologically distinguishable from C57
and Ashen glands . A. While Ashen lacrimal glands were morphologically akin to those from C57, DKO
and 27bKO showed changes that included some loss of polarity and increased expression of degradative
organelles (arrows). N, nucleus; *, lumen; bar is 10 µm. B. DKO tissue, and also 27bKO to a certain extent
(not shown) exhibited decreased cellular organization and polarity varying in severity from region to
region. Individual SV were less apically concentrated while increased lysosomes and other apparent
degradative organelles were prominent. Cells also displayed swollen or vesiculated ER as shown at higher
magnification along with an example of the less dense populated subapical region beside the lumen. Bar is
2 µm in lower, 1 µm in higher magnifications.

19

2.3 Definitions and quantifications of changed organelle morphologies
Noting significant differences in 27bKO and DKO mouse lacrimal gland TEM sections
compared to the background mouse strain, C57, we quantified these morphological
differences as a measure of secretory and degradative pathway change due to loss of
Rab27b. For the purpose of illustrating some of the more extreme changes, Figure 5
show examples of high magnification micrographs of C57 and DKO lacrimal sections
which reflect quantitative data, with changes in 27bKO largely represented by the DKO
micrographs. SV were identified by distinctive, large circular membranes enclosing
electron-dense contents; contents were either mucous (less dense) or serous (denser). In
C57 mouse lacrimal glands, these SV clustered around the lumen, but in DKO and
27bKO, SV appeared more scattered throughout the cytoplasm. Quantification revealed
that DKO and 27bKO lacrimal gland sections had significantly fewer vesicles than C57.
Lysosomes, recognizable by < 0.5 µm diameter and high electron density, also appeared
in significantly greater numbers in DKO and 27bKO than C57, although fewer in 27bKO
than DKO. Lipid droplets, which are void of protein and appear unstained in the
micrographs, were localized beside the basolateral membranes. While lipid droplet size
was not measured, lipid droplet counts did not show any significant differences. A
general count for all mitochondria showed a small but significant decrease in count in
DKO compared to C57, though not in 27bKO. A subgroup of these mitochondria were
additionally classified as abnormal; these showed signs of significant cristolysis often
combined with swelling and was highly prevalent in the DKO, and sometimes 27bKO,
but not in C57. Finally, dense structures often in association with lysosomes, some with a

20

Figure 5. Detailed comparison
and quantification of organelle
morphology in mouse lacrimal
glands lacking Rab27b. TEM
images taken of acinar cells from
C57, DKO, and 27BKO mouse LG
sections were individually
quantified for specific organelles
and summarized on a per cell
basis. High resolution images are
matched with quantifications as
examples of the measured
organelle. The last two sets of
images are examples of abnormal
mitochondria and degenerated
structures, respectively. The total
mitochondrial count is inclusive of
abnormal mitochondria.
Quantification was obtained from
15 to 20 TEM images per mouse,
WT: n = 5 mice, 94 cells; DKO: n
= 5 mice, 133 cells; 27BKO: n = 4
mice, 96 cells. Error bars represent
S.E.M. (F(2, 320)= clockwise:
102.65, 82.78, 35.76, 12.40, 81.56,
0.29, *, significantly different from
C57BL/6 mouse LG at P ≤ 0.003 ;
, significantly different from
DKO at P ≤ 0.003. Bar is 1 μm.

21

single or double membrane suggestive of some stage of autophagosome formation, were
classified in a general category as degradative structures. These were significantly
increased in numbers in DKO and 27bKO compared to C57. Morphological counts are
summarized in Table 2.

In order to characterize SV localization differences between the C57 and DKO lacrimal
tissues, we also quantified SV distances from the center of the lumen as a measure of
vesicle dispersion in the cytoplasm. These results are shown in Figure 6. Most SV in the
C57 tissue appeared in a roughly 10-30% radii around the lumen, whereas in DN the SV
were significantly more distanced from the lumen, many appearing within a 40% radii.
Table 2. Organelle Count Summary from TEM. Total counts for relative abundances of the
indicated organelles in Figure 3 in C57BL/6, DKO, and 27bKO mouse LG. Errors represent S.E.M.; *,
significance over WT (P < 0.05); +, significance over DKO.

Average
Cell
Area
( μm
2
)
# of
SVs
Diameter
( μm)
# of
Lysosome
# of
Mito.
# of
Abnormal
Mito.
# of
Degen.
Structure
# of
Lipid
C57 (N=5)
Cells counted =
95
Total # of SVs =
9413
220.1±
8.9
99.6±
4.3
0.82±0.02 4.1±0.2
17.3±
0.9
2.0±0.3 1.4±0.1 4.7±0.6
DKO (N=5)
Cells counted =
134
Total # of SVs =
6379
189.3±
5.1*
47.8±
2.0*
0.82±0.02 8.9±0.3*
13.2±.
0.4*
7.9±0.4* 2.8±0.2* 5.3±0.6
27bKO (N=4)
Cells counted =
96
Total # of SVs =
5040
200.1±
4.8*
52.5±
1.8*
0.79±0.01* 5.6±0.2*
+

15.5±
0.5
+

7.2±0.4* 2.9±0.1* 4.8±0.5

22

2.4 Possible link between the secretory pathway and degradative pathway
Due to the noted increases in lysosomes and degradative structures from the TEMs, we
sought to confirm the identity of these degradative pathway components. Fresh frozen
lacrimal sections were prepared for cryoimmunohistology and incubated with antibody
either against LAMP2, and an integral glycoprotein of the lysosomal membrane (24, 30)
or against LC3B, which associates with autophagic vesicles (42, 125). While there was
some variation from region to region, some general patterns were apparent. LAMP2
staining seen in Figure 7A was seen in the more basal regions of the LGAC and,
although detectable even in C57, greatly increased in DKO and 27bKO. Similarly, LC3B
Figure 6. Secretory vesicle distance to the center of the lumen in DKO versus C57. We conducted a
quantitative evaluation of SV average distance from the center of the lumen as a percentage of the maximal
distance from the basolateral membrane to the center of the lumen. Average distance from the apical
membrane of DKO SV was significantly greater (38% ± 2%) than wild-type (32% ± 2%), and most C57 SV
were within a 30% radius compared to DKO within a 40% radius. * = significance P<0.05.

23

staining appeared to be slightly increased in DKO and 27bKO (Figure 7B). For LC3B
staining, however, some areas (not shown) of secondary antibody-only negative controls
showed a low level of nonspecific staining of dense granule-like structures in the basal
peripheries of the cell, confounding the results. Negative controls for LAMP2 did not
show nonspecific binding.

24

Figure 7. DKO mouse lacrimal tissue show increased expression of
degradative markers. Expression of LAMP2 (A) and LC3B (B) both showed
increased signal in the DKO tissue compared to C57 mice. Bar is 10 μm.

25

2.5 Example of increased mitochondrial vesiculation in Mocha mice
Our previous observation
of increased mitochondrial
hvesicularization of the
cristae seen in 27bKO and
DKO is not an isolated
case; mitochondria appear
to be very sensitive to
metabolic fluxes and a
multitude of causes can
result in mitochondria
Figure 8. Mocha mouse
lacrimal glands show increased
mitochondrial cristolysis
compared to GR/J mice. TEM
images taken of lacrimal acinar
cells from Mocha and GR/J (GR)
mouse sections. A. Mocha mouse
sample morphology was similar
to GR and to previous C57
results, but did show signs of
cristolysis that were not apparent
to the other two. B. H&E and
Oil-Red O staining of Mocha and
GR did not show any strong
differences between its lacrimal
tissues, although Mocha mouse
tissue showed increased
extracellular space between cells.
n = 2 mice; measure bar in B. is
50 μm.

26

degeneration, such as the induction of autophagy (62). We found a similar example of
mitochondrial vesicularization in glands from Mocha mice, which arise from a
spontaneous mutation in AP-3 of C57BL/6J-pi mice and have been extensively studied
in for its secretory role in platelet cells, melanocytes, and synaptic vesicles (57, 65, 107).
The adaptor-like protein complex is normally involved in the budding of coated vesicles
from the trans-Golgi network during vesicle transport. In our preliminary study of LG
morphology in Mocha mice, we examined samples by TEM which were prepared in
parallel with GR/J mice, a strain usually studied for its development of mammary
tumorigenesis. Results show in Figure 8A an increased vesicularization of the
mitochondria in Mocha, compared to GR/J samples shown here and C57 samples shown
in Section 2.4, as well as possible decrease in the size of the subapical SV pool. Figure
8B are results from H&E staining and Oil-Red-O staining and did not show striking
differences between Mocha and GR/J samples except that Mocha mouse basolateral
membrane appeared thicker than GR/J samples and had increased extracellular space
around the acinar cells.

2.6 Additional observations of knockout tissue with H&E and oil red staining
In addition to examining the secretory pathway, we were interested in whether Rab27b
regulation also affected other pathways such as the storage of lipids, for which other
proteins such as Arf-COP1 have been associated with the regulation of lipid-droplet size
and number (38). Although we did not observe significant differences in lipid droplet
numbers between C57 and Rab27b mutant mouse LG sections by TEM, we did

27

observe some slight increase in oil droplets by H&E staining (data not shown) and more
clearly by Oil-Red O lipid staining of tissue sections from the same mouse samples,
especially in DKO (Figure 9).This may be due to increased lipid droplet sizes or an
increase in droplet numbers, although there was some variation from tissue region to
region in the extent of oil staining. We suggest that if this increase in lipid expression,
whether in numbers or in droplet size, is confirmed, Rab27b regulation may be directly
involved in the production of lipid droplets, or more likely, that Rab27b regulation of the
secretory pathway has an indirect consequence on the lipid pathway. Interestingly,
Mocha lacrimal glands in Section 2.5 showed relatively little Oil-Red-O staining
Figure 9. 27bKO and DKO lacrimal sections show increase of signal for oil droplets. In comparison
to C57 sections, 27bKO and especially DKO showed increased lipid droplets (red dye) by Oil-Red O
staining, although it is not clear whether this is due to an increase in droplet numbers or size. N = 2 mice
per category, multiple random images per sample. Bar = 100 µm for all images.

28

comparable to C57 lacrimal glands, which would imply little effect of lipid formation by
dysfunction of AP3- protein.

2.7 Discussion and implications
Observations of general as well as organelle-specific changes in lacrimal gland tissue of
C57 as well as Rab27b mutant mice indicate strongly that Rab27b plays an important
functional role in the regulation of the secretory pathway. These mouse studies also
resulted to our decision to focus on Rab27b, along with preliminary biochemical data
suggesting that Rab27b expression is higher than Rab27a in LGAC, as the effect of
Rab27b knockout appeared to affect the secretory pathway of LGAC much more
extensively than Rab27a. In a cell type that is normally packed with an abundance of SV,
Rab27b appears to decrease SV biogenesis or perhaps its transport, which is part of the
SV maturation process. Perhaps even more interesting is that the dysfunction of Rab27b
appears to affect pathways beyond just the secretory or exocytotic- it affects
mitochondrial and ER morphology and increases the expression of components of the
degradative pathway. We can simplify the implications of this into two theories: that
Rab27b directly regulates more than one pathway (i.e., is a negative regulator of the
degradative pathway), or one that may be more likely based on other literature, that the
regulation of one pathway influences that of another. Borrowing a phrase applied to
scaffolding proteins by Dell’Angelica’s review paper (15), by effectively blocking one of
the main functions of the LGAC, Rab27b dysfunction creates conglomerates of otherwise

29

normally scarce proteins and pathway factors which jam the organelle traffic in other
pathways.

30

Chapter 3: Target identification and characterization of endogenous expression

3.1 Rab27b shows high expression in lacrimal gland tissue
We confirmed the expression of endogenous Rab27b by cryoimmunohistology in C57
mouse lacrimal glands, using primary antibody against Rab27b, DAPI dye to identify
nuclei lying in the basal regions, and fluorescent phalloidin to detect actin and help
delineate the cells’ basolateral and apical plasma membrane domains, the latter of which
is bound by an abundant and extensive actin filament array that is more prominent than
that of the cortical basolateral actin filament array (Figure 10). All imaging studies were
conducted in parallel with negative controls and when appropriate, positive controls, to
ensure the detection of the correct entity. Rab27b was expressed in the acinar cells of the
lacrimal gland tissues, localizing primarily in the subapical region and trailing out
sparsely into the basal regions. While expression was strong in the subapical region and
could sometimes be detected as enrichment of the membranes of visible SV, the Rab27b
antibody may have been somewhat affected by the sample preparation steps necessary for
cryoimmunohistology and were more diffuse and less distinctly membrane-enriched as in
cultured LGAC (shown in the next section). However, Rab27b localization patterns were
consistent with its role in the secretory pathway and with later expression in culture
LGAC.

31

Figure 10. Rab27b is expressed in mouse lacrimal gland acinar cells. Rab27b (green) is detected by
cryoimmunohistology in C57 mouse lacrimal glands mainly in the subapical region (arrows), but also
trailing out to the basal regions of the cell (arrowheads). The plasma membrane is delineated by actin
filaments (red) and is especially thick at the apical membrane (A) beside the lumen (L). B represents the
basolateral membrane, blue is DAPI staining of nuclei. Bar is 5 μm.

32

3.2 Cultured acinar cells retain Rab27b expression
In order to proceed to functional studies, we also established that Rab27b is
endogenously expressed in cultured, re-formed LGAC harvested from rabbit. For
orientation, Figure 11A shows a schematic of polarized cultured lacrimal acini indicating
the positioning of basolateral and apical membranes, along with numerous SV gathering
in pools around the lumen. Rab27b was detected on the membranes of these SV (Figure
11B), apparently marking a sub-pool of the total SV population as Rab27b-enriched. A
lower magnification image of Rab27b enrichment demonstrates its localization in
proximity to the plasma membranes and the nuclei (Figure 11C). Carbachol is used in
vitro to simulate the regulated exocytosis of mature SV which occurs in vivo. These
reconstituted acinar cells form lumina which are accessible to the bathing cell media, thus
making the cell accessible to treatment. Within 15 min of addition of CCH to minimal
cell media, LGAC undergo actin restructuring along with Rab27b re-localization to the
apical membrane where Rab27b-enriched SV undergo fusion (Figure 11D). After 15 min
of CCH, LGAC display considerable loss of Rab27b-enriched SV, and the remaining SV
assume a compressed appearance possibly due to actin restructuring and deformation of
the lumen. As isoform-specific controls, preabsorption of primary Rab27b and Rab27a
antibodies, respectively, effectively blocked any positive immunofluorescence signal
(data not shown).

33

Figure 11. Rab27b
expression is enriched
on membranous
secretory vesicle
structures in the
subapical region of
acinar cells. A.
Schematic of the
reconstituted lacrimal
acinar cluster; lumen
(L). SV (red) are
located in the subapical
regions, while nuclei
(N, blue) are more
basal. B. High
magnification of
Rab27b on apparent SV
membranes in the
subapical region.
Arrows point to
punctate structures. Bar
is 5 μm. C. Probing for
Rab27b (red), with a
boxed region
representing the image
magnified in B. Actin
(white) and the nuclei
(DAPI, blue) are shown
for orientation. Bar is 5
μm. D. LGAC treated
with 100 μM CCH
showed endogenous
Rab27b (green) re-
localization. Nuclei
(blue) and actin (red)
are shown for
orientation.
Arrowheads point to
Rab27b-enriched
vesicles. Bar is 10 μm.

34

3.3 Biochemical confirmation of Rab27b expression and clues to localization
Biochemical data was also consistent with imaging results. Endogenous expression of
Rab27b in LGAC was confirmed, in parallel with virally transduced overexpression of
wild-type (WT) and mutant (CA; DN) Rab27b used in later studies. For general purposes,
these samples were run in parallel with Rab27a as well. A band at ~29kD representing
endogenous Rab27b expression was detected in lysates from both untreated as well as
virally transduced LGAC expressing a YFP-tagged variant of Rab27b (~50kD) (Figure
12A). On the other hand, little or no endogenous protein was detected by Rab27a,
although there was some low level of cross reactivity between isoforms at levels higher
than endogenous in the overexpressed YFP-tagged Rab27b proteins. There was a low
level of cross reactivity between the isoforms at above-endogenous protein expression
levels with recognition of overexpressed YFP-tagged Rab27b protein by anti-Rab27a
antibody ~10% and lower nonspecific recognition by the anti-Rab27b antibody. Serial
dilutions of purified His-tagged Rab27a and Rab27b were detected with anti-Rab27a and
Rab27b antibody, respectively (Figure 12B). While endogenous Rab27b represented
roughly less than 0.001% of total protein in lysate, although this is not unreasonable
when compared to more abundant structural proteins such as tubulin (~0.6%, chicken
erythrocytes) (78). Negative controls for Rab27b demonstrating antibody specificity are
shown in Figure 13. Pre-absorption of Rab2b antibody with purified Rab27b protein
shows saturation of antibody binding sites, resulting in little or no detection of signal for
Rab27b. Negative control with using only the fluorescently-conjugated secondary
antibody also shows very little or no background staining.

35

Figure 12. Rab27b is detectable biochemicaly in lacrimal gland lysate. A. Fresh mouse LG lysate
was prepared in RIPA buffer with protease inhibitors. 10 μg of lysate protein from non-transduced (c)
or mutant Rab27b-transduced (WT, CA, DN) LGAC were resolved by SDS-PAGE and analyzed by
Western blotting. Endogenous Rab27a and Rab27b is detected at its predicted molecular weight (~27-
29kD) and in transduced cells the tagged protein at 52kD. γ-Adaptin was used as control for equal
loading. B. Serial dilution of purified recombinant His-tagged Rab27a/b protein in μg. Exposure to
only secondary antibody showed no bands in this region (data not shown). *, lumen.

36

In order to confirm microscopic observations of membrane localization, we conducted
two different fractionation studies. In the first, we conducted a general survey of the
Figure 13. Immunofluorescence study shows that the Rab27b antibody is specific for Rab27b in
acinar cells. In order to test for antibody specificity for Rab27b, mouse Rab27b primary antibody was
pre-absorbed with His-tagged recombinant Rab27b protein overnight (~10 μg protein per 1 μg
antibody), prior to immunostaining procedures in cultured LGAC as described in Experimental
Procedures. In the positive control, non-absorbed Rab27b (green) labels the subapical region beneath
lumina delineated by thick actin (red) labeling. This Rab27b signal is quenched with a pre-absorption
step. A secondary only negative control is shown to indicate that the secondary for Rab27b does not
show nonspecific background in LGAC. Bars are 10 μm.

37

density distribution of the pool of endogenous Rab27b-enriched SV, as evaluated based
on previous analysis in LGAC (33, 40, 75, 118). Isolated LGAC were collected at rest or
after CCH stimulation for the gradient analysis (Figure 14). Roughly 13% of total protein
from non-treated LGAC at resting phase was detected in the fraction 1, which according
to previous analysis of LGAC gradients, consists of basolateral membrane and fragments
of specialized microdomains from mature SVs. This was also comparable to published
results, as well as results run in parallel, for Rab3D which also showed strong peaks in

fraction 1 at resting stage, suggesting association of Rab27b with mature SV
compartments. The remainder of the Rab27b signal was much more broadly spread
throughout fraction 4-11, which previous studies have shown contain additional SV
compartments enriched in secretory proteins such beta-hexosaminidase as well as Golgi
apparatus, trans-Golgi network, and the two compartments for the endoplasmic
reticulum, perhaps reflecting different states of Rab27b enrichment on nascent, immature
and mature SV. With CCH stimulation, we saw a shift of protein peaks from fraction 1
to fraction 3-7, reflecting a proportional shift of mature SV to SV in biosynthetic
Figure 14. Rab27b localization in membrane fractions shifted with carbachol treatment. Rab27b
were detected in membrane fractions which included SV membrane fragments. After the addition of
CCH, Rab27b re-localized to fractions containing biogenesis compartments such as the Golgi. *,
significance (P<0.05)

38

compartments, as mature, subapical SV were released at the apical plasma membrane. A
slight decrease in peaks 8-11 may be reflective also of an efflux of SV from earlier
biosynthetic compartments, such as the Golgi apparatus and trans-Golgi network.

In the second study, we used LGAC which expressed the Xpress
TM
(Xp)-tagged
constructs for Rab27b WT and DN forms in order to attain comparable experimental
groups while determining whether CCH had a stimulatory effect on the localization of
Rab27b on SV membranes. Examining soluble and membrane-bound fractions of LGAC
for Xp-Rab27b obtained from direct centrifugations of LGAC in lyses buffer, we found
that approximately 25% of total detected Rab27b using this methodology of preparation
was membrane-bound (in the pellet fraction) (Figure 15).

With CCH stimulation, a significant loss of membrane-bound Rab27b occurs,
presumably due to release of Rab27b from the membrane of SV upon fusion with the
apical membrane. In comparison, the abundance of DN Rab27b on pellet fractions did
Figure 15. Rab27b association with
membrane fractions at resting stages
and after carbachol stimulation.
Lysate from LGAC transduced with
Xp-tagged WT or DN Rab27b
constructs was used to measure Rab27b
expression in the pellet versus
supernatant fractions. Rab27b
expression is in the pellet fraction
shown here is analyzed by western blot
by detecting with an antibody against
Xpress
TM
, and total expression in the
fraction is taken as WT or DN at resting
stage. WT, n=4; DN, n=3; *, significant
from resting (P < 0.05).

39

not significantly change with CCH stimulation, suggesting a loss of the CCH effect on
Rab27b-localization. We note here that the percentage of total Rab27b detected in the
pellet fraction was surprisingly small compared to that found in the cytoplasmic fraction
(~25% for WT was in pellet). Visually, we observed almost all signal of Rab27b came
from that enriched in SV membrane. DN Rab27b expression in the pellet fraction
approximately the same at resting state to WT. There are several implications of these
results. First, we measured expression of Rab27b in LGAC overexpressing Rab27b WT
or mutant proteins which may result in a saturation of binding of effector proteins which
recruit Rab27b to the membranes. This would suggest that the overexpression of Rab27b
results in a large quantity of the protein which is cytoplasmic, although these do not show
a noticeably high detectable signal by imaging. Alternatively, these results may have to
do with a technicality. Mature Rab27b associates with membrane after prenylation of
their carboxy-terminus (34, 108). Utilizing the same protocol that has generated
fractionation data for M5C (69) and other lacrimal gland studies, it is possible that sheer
force from centrifugation or mild detergents from the cell lysis buffer is sufficient to
separate Rab27b association from the membrane. We have tested several different lysis
methodology which resulted in similar data (data not shown) but these yielded similar
results to that shown. A third complication is that Rab27b is recruited early in vesicle
biogenesis, as we will discuss in Chapter 6; therefore, a proportion of Rab27b is
expressed on the tiny nascent vesicles located in the basal regions of the LGAC. These
nascent vesicles may be small enough to be separated into the supernatant fraction rather
than the pellet fraction, and extensive fractionation studies, such as one in which the

40

fractions were embedded in a solid phase material and scrutinized by TEM for vesicle
size, would be necessary to analyze the fractions.

3.4 Rab27b co-localization with other secretory pathway markers and its re-
localization with stimulation
Dual immunofluorescence labeling of cultured LGAC for endogenous Rab27b and other
known lacrimal SV markers strengthened ties between Rab27b and the secretory
pathway. Figure 16 shows high co-localization between Rab27b and Rab3D in the
subapical region. Rab3D is an established marker of mature, resting SV that is known to
be released into the cytosol concomitant with stimulation and vesicle fusion with the
apical plasma membrane (119). While co-localization in the subapical region was high,
labeling patterns between the two Rabs were also observably different and not all vesicles
enriched in one were enriched in the other. Consequently, these data reinforced previous
findings that the LGAC vesicle pool is constituted of multiple sub-pools of SV
heterogeneous in markers and likely also content, as has been suggested by TEM analysis
(69). Rab27b also showed a high degree of co-localization with the actin-based motor
protein, Myosin 5C (M5C), which has been shown in LGAC to enrich SV and negatively
affects CCH-stimulated exocytosis when inhibited (69). Co-localization between Rab27b
and M5C was especially high in the subapical-most region of the LGAC, which perhaps
reflects association of Rab27b-enriched SV with actin filaments via the M5C tether in
this region.

41

Figure 16. Rab27b co-localization with SV markers is consistent with a role in lacrimal exocytotic
processes. LGAC were processed for detection of endogenous Rab27b (green) in parallel with the
mature SV marker, Rab3D. Arrows depict regions within the cell exhibiting high colocalization,
although Rab3D appeared to label a larger pool of vesicles. Dual staining of LGAC also showed a high
degree of colocalization (arrows) between Rab27b and M5C, a highly expressed actin motor protein in
acinar cells. Rab27b distribution is more widespread than M5C. Nuclei are shown in the overlay images
and were detected with DAPI (blue). Actin is shown in white. Bar is 10 μm. *, lumen.

42

3.5 Quantification of co-localizations of Rab27b
Rab27b co-localizations with Rab3D, M5C, and actin, were quantified and shown in
Table 3. While Rab27b co-localized highly with both Rab3D and M5C at resting stages,
stimulation with CCH for 15 min resulted a significant decrease in co-localization with
Rab3D but not in M5C, suggesting that these two effectors may be involved with Rab27b
at different steps of the secretory pathway. Additionally, as co-localization values
between Rab27b and the two markers both showed >50% co-localization, we sought a
marker which visually shows low co-localization with Rab27b. For comparison, F-actin,
which is a cytoskeletal protein enriched in the cell cortex but detected throughout the cell
and is not specific to the secretory pathway only (76), showed minimal co-localization
values with Rab27b.

Table 3. Rab27b subapical colocalization with proteins involved in SV trafficking. Fluorescent
pixel co-localization of endogenous Rab27b with different markers in fixed reconstituted LGAC
was measured with the Enhanced Colocalization tool of the Zeiss LSM Meta 510 in a region of
interest (ROI) defined as the area within a circular radius around the lumen equal to half of the
maximal distance from the apical to basal membrane. At least 4 random fields of view per
preparation (n) were taken, in 4-6 preparations per set of data. Co-localization values reflect the
percentage of total fluorescent pixels associated with each marker in the ROI that was co-localized
with Rab27b-Xpress that was expressed in the LGAC. Errors represent S.E.M.
Rab3D at rest 73.6% ± 2.0% (n=4)

+ CCH 39.0% ± 7.4% (n=4)
Myosin 5C at rest 58.3% ± 8.1% (n=6)

+ CCH 53.4% ± 6.1% (n=4)
F - Actin at rest 41.4% ± 6.2% (n=4)

+ CCH 22.0% ± 2.0% (n=4)

43

3.6 Discussion and implications
At the beginning of this study, we observed that the knockout of Rab27b in mouse
lacrimal glands has an effect on acinar cell organelle morphology and general cell
organization. In this chapter, we confirmed endogenous expression of Rab27b in both
mouse lacrimal tissue and rabbit cultured acinar cells and began to characterize its
localization and possible pathway associations. Confirmation of Rab27b expression in
cultured LGAC was particularly valuable. In order to study these functions more
extensively, we transitioned our studies from mouse tissue to purified cultures of rabbit
LGAC due to several reasons. A recent review by Schechter, et al., discusses several key
benefits and limitations in studying the lacrimal gland in different models (98), including
the fact that in some physiological and protein marker-related aspects, rabbit glands are
actually more similar to human than rat or mouse. Rabbit glands are 0.5 grams, compared
to a mouse which yields approximately 50 mg of tissue. Also, the culture of rabbit
LGAC, while maintaining similar polarity to in vivo studies, makes cells accessible for
viral transduction and drug treatments and increases ease for imaging studies as well. The
availability of both mouse as well as rabbit glands for study further strengthens our
studies of endogenous Rab27b expression.

We also briefly examined the possibility that both Rab27 isoforms, and not just Rab27b,
was expressed, which would imply that Rab27a may also have a significant role in the
LGAC. Although the knockout mice studies indicated that Rab27b played a more
functionally important role in LGAC secretion, as Ashen mouse lacrimal glands did not

44

show a significant phenotype compared to our observations in 27bKO and DKO mouse
lacrimal glands, we were interested in obtaining an approximate comparison of protein
expression in the isoforms given the resources available. Biochemically, we detected
endogenous Rab27b but not endogenous Rab27a (Figure 12A), although there was some
nonspecific binding of overexpressed YFP-tagged Rab27b by Rab27a. As positive
controls, we demonstrated the potency of Rab27a and Rab27b antibodies against their
respective purified proteins. Although not shown in this chapter, we have also noticed
that while detection levels for Rab27b is consistently high in immunofluorescent
imaging, Rab27a detection levels are very low and sometimes not detectable. These data
collectively indicated that Rab27b is the prevalent isoform both in function and in
expression in the lacrimal gland. A more thorough exploration into isoform expression
would require the sequencing of rabbit Rab27a and Rab27b for the generation and
development of siRNA for knockdown functional studies. For the purposes of this study,
we focused on identifying a major regulatory protein of the secretory pathway rather than
conducting a detailed analysis of Rab27a versus Rab27b functional roles. For the
development of drug targets against Rab27b however, because of their high isoform
homology (amino acid homology is 71%) and because it is not yet clear whether the
isoforms play similar or different roles in different tissues (3, 80), a closer examination of
Rab27a expression and function in the lacrimal gland may be necessary.

Immunofluorescence imaging data also strengthened Rab27b ties to the secretory
pathway. Rab27b co-localized highly with two secretory markers, Rab3D and M5C,

45

which have previously shown to play significant roles in exocytosis in LGAC.
Furthermore, we showed that Rab27b localization correlated with an SV exocytosis
response to CCH. Upon stimulation to secrete, Rab27b co-localization with Rab3D
decreased, although co-localization with M5C did not change significantly. The
decreased association with Rab3D may correlate with the loss of Rab3D immediately
upon fusion with the apical membrane, while co-localization measurements with M5C
suggests that Rab27b association with M5C lingers even after stimulation and may be
independent of Rab27b’s association with the SV.

46

Chapter 4: Establishment of live-cell protocols using a virus entry study

4.1 Addition of Adenovirus 5 and cased proteins induces membrane ruffling
In order to pursue more extensive imaging studies of Rab27b activity in living LGAC, we
needed to first optimize conditions for live-cell imaging which would yield distinct cell
states (resting, stimulated) and then convert our observations into quantitative data.
Qualitative studies of live-cell imaging, which can reveal more about cell processes in
real time than biochemical and molecular studies, can be converted into quantitative data
for further analysis, as was done as part of the study in our publication (129).
Macropinocytosis is form of bulk uptake of fluids and solid cargo which, in normal cell
function, participates in the regulation of both endocytic and exocytic membrane
trafficking (21). It has been proposed that viruses in mammalian cells may hijack natural
uptake pathways such as macropinocytosis to gain entry into the intracellular trafficking
pathway (72, 73). In this example, we transduced LGAC with a GFP-tagged F-actin
(GFP-actin), which coassembles with endogenous actin into filaments which allowed for
the visualization of the plasma membrane in live cells. In Figure 17A, LGAC at resting
stages show little movement at the actin-rich basolateral membranes. However, addition
of Ad5 caused actin rearrangement that immediately latrunculin B, which sequesters
monomeric F-actin and thus promotes actin depolymerization, appeared to prevent
ruffling events. Similarly, the addition of Ad5 capsid proteins fiber (Figure 17C) and
knob (data not shown) also resulted in membrane ruffling. Again, pre-treatment of LGAC

47

with latrunculin B also appeared to prevent ruffling caused by the addition of fiber and
Figure 17. Time-lapse
microscopy of
membrane ruffling in
LGAC treated with
Ad5 and capsid
proteins. Rabbit LGAC
are transduced to
express GFP-actin.
These LGAC are then
treated with DMSO or
LatB prior to being
incubated with Ad-LacZ
(MOI 100 PFU/cell) or
recombinant fiber (20
µg/ml), and then
warmed to room
temperature for 5 min
prior to the onset of the
time-lapse sequence.
Select GFP-actin
fluorescence and DIC
images at regular
intervals are shown.
Arrows indicate
basolateral actin
remodeling. Boxed
images were magnified
and shown in the insets.
Bars are 10 µm. From
JOURNAL OF
VIROLOGY, Dec.
2006, p. 11833–11851
Vol. 80, No. 23
Copyright © 2006,
American Society for
Microbiology. All
Rights Reserved.

48

knob. However, penton base (data not shown), as well as control proteins purified from
the same background as fiber (GST protein from baculovirus in Sf9 cells) and the knob
(rab3DQL from E. coli) did not cause any detectable change in actin arrangement. These
data indicated that Ad5 and its capsid proteins fiber and knob, but not nonspecific protein
introduction, are sufficient for membrane ruffling and may enable Ad5 entry into LGAC.

Further live cell imaging appeared to confirm that stimulation of LGAC with Ad5 and
some of its subunits instigated macropinocytotic events. Figure 18 shows that with the
addition of a fluorescent marker, texas-red tagged dextran 10, shown in green, a glucose
polysaccharide added to the cell media, we visually captured one area which depicted
uptake of dextran 10 apparently through the basolateral membrane into LGAC. This also
contributed to evidence that LGAC membranes are exposed to the cell media and thus is
able to uptake labeled particles, such as dextran, from the media into the cell.

Figure 18. Ruffling induces some uptake of dextran 10. Following binding of AdlacZ on ice for
one hour to the surface of LGAC, dextran 10- texas red was added to solution for a 1 mg/ml final
concentration. After 15 min. of CCH stimulation, actin-GFP transduced cells (red) are seen uptaking
some dextran (green) . It is not clear whether the thick intracellular actin towards which the dextran
are transported to is part of a lumen.

49

4.2 Translation of qualitative observations into quantitative data
Ruffling events such as those described in Figure 17 were defined as the rearrangement
of the actin-rich plasma membrane which causes a distortion, or a ―bleb‖. These blebs
were measured according to their maximal diameters (Figure 19A). Bleb events occurring
over a 10-min interval per preparation examined was recorded in the graph showing the
average number of events of a specific diameter per field under different experimental
conditions (Figure 19B). Acini that did not have virus or viral capsid protein additions
showed very few bleb events, and those that did occurred were small in diameter. With
addition of Ad5, fiber, and knob treatments, but not penton base or control proteins (GST,
Rab3DQL) (Figure 19C), we saw significant increases in the number of bleb events.
These reaffirmed the qualitative observations made previously.

50

Figure 19. Quantification of membrane-ruffling events elicited by Ad5 and capsid proteins in
acinar cells. A. A representative image of a transduced LGAC exposed to Ad-LacZ (MOI = 100
PFU/cell) from one frame within a 600-s analysis period is shown, with the diameters of several
membrane extensions marked. B. Treatment of LGAC with Ad5 (n=6), fiber (n=5), and knob (n=7)
cause significant increases in membrane-ruffling events per sequence under experimental
conditions, with ruffling events grouped by largest diameter using the LSM Image Browser Overlay
tool to measure diameter. Penton base was also tested (n=5). C. Comparable values obtained from
control (n=5), GST (n=4), and Rab3DQL (n=2) are show. *, significant at P<0.05 based on results
for the control; error bars represent S.E.M. From JOURNAL OF VIROLOGY, Dec. 2006, p.
11833–11851 Vol. 80, No. 23. Copyright © 2006, American Society for Microbiology. All Rights
Reserved.

51

4.3 Effects of Heparin on Ad5 uptake
Previous studies have suggested that heparan sulfate-glycosaminoglycans (HS-GAGs)
participate in the uptake of such viruses as Ad2 and Ad5 (13, 14, 31, 88). We tested
whether Ad5 binds to the HS-GAGs using live cell imaging studies to track differences in
the numbers of macropinocytosis events resulting from the addition of an HS-GAG
analog, heparin. A decrease in membrane ruffling may demonstrate a functional role of
HS-GAGs in viral uptake in the LGAC. As a control, we also pre-incubated LGAC with
chondroitinase ABC (CSA), an enzyme which depolymerizes a variety of GAG
substrates but not heparin sulfate or heparin. In Figure 20, changes in ruffling events were
most apparent in smaller blebs. Consistent to the previous results, while control (no virus
added) and heparin only groups showed low numbers of events, Ad5 addition resulted in
increased number of events in the lower diameter ranges 0 to 7.5 nm. Ad treatments were
also affected by the addition of 5mg/ml and 1mg/ml treatments of Heparin for lower
diameter blebs and were significantly decreased from Ad5 alone, though still greater than
control and Heparin only. However, treatment with CSA did not appear to affect Ad5
stimulation of macropinocytosis.

52

4.4 Discussion and implications
By clearly defining certain events that occur during live cell imaging, we were able to
quantify these observed events and use the data for comparison between treatment
groups. Ad5, as well as its capsid proteins fiber and knob, activated membrane ruffling
indicative of macropinocytosis and suggested a mechanism for viral entry into LGAC.
These events may utilize HS-GAGs as a binding target, since the addition of heparin
effectively decreased ruffling events. However, inhibition by heparin was not complete,
perhaps suggesting also that alternative binding sites may be targeted by Ad5.

Figure 20. Heparin affects the macropinocytosis effect of Ad5. Preincubation of LGAC
with Heparin (Hep) and CSA affected the number of macropinocytosis events in each size
category. N = 4-7 per treatment; Δ, significance from Ad5; *, significance from Control;
P<0.05.

53

The analysis of macropinocytotic events conducted in this study demonstrated the usage
of a valuable tool in imaging. We established a live cell imaging protocol with designated
temporal parameters and environmental conditions necessary to promote LGAC
expression at resting state. The ability to obtain clear imaging with distinct cell states,
resting versus stimulated, was especially important since LGAC are very sensitive to any
changes in environment. Extracellular factors such as temperature fluctuations, presence
of certain metal ions, and even the air flow rate within the incubation chamber can cause
cell stress or a false positive signal such as ruffling during the macropinocytosis study.
We also successfully converted our observations into quantifiable data by providing strict
definitions for the events or objects we measured or counted. We transferred these skills
to the imaging studies of Rab27 in the next chapters, including the acquisition of the live
cell images of Rab27 mutant expression at rest compared to with CCH stimulation
(Figure 24A-C) and to follow live cell events over time (Figure 25, Figure 27, Figure 31),
and to calculate SV size measurements to compare the effects of mutant Rab27b
expression (Figure 24D).

54

Chapter 5: Functional activity state affects Rab27b localization

5.1 Characterization of wild-type and mutant Rab27b expression in lacrimal
gland acinar cells
After establishing a protocol for live cell conditions and for quantification studies, we
proceeded with our Rab27b study. We tested Rab27b’s functional role in exocytosis by
transducing Xp- or YFP-tagged WT or mutant Rab27b into LGAC, which provide labels
for Rab27b optimized for biochemical or immunofluorescent detection, respectively. Xp-
and YFP- epitome tags were attached to the same WT and mutant Rab27b sequences
constructs (7). WT Rab27b expresses a fully functional protein, while CA proteins are
locked into a GTP-bound state and DN proteins have low affinity for both GTP and GDP.
Figure 21 shows Xp-tagged Rab27b expression patterns as representatives of both tags.
By fixed cell imaging, WT Rab27b expression was similar to endogenous expression,
appearing on a large subpool of SV in the subapical region. With CCH stimulation,
approximately 50% of this signal is lost presumably due to fusion and release of SV
content into the lumen, while the remainder of the vesicle population is concentrated at
the apical-most region of the LGAC. CA Rab27b expression appeared similar in pattern
to WT Rab27b but enriched a larger number of vesicles, and retained a similar response
to CCH stimulation. On the other hand, DN Rab27b expression appeared much more
punctuate and dispersed even at resting state and did not obviously appear to be recruited
to the membranes of rounded SV as had been the case with WT and CA Rab27b. LGAC
expressing DN Rab27b appeared somewhat depolarized with difficult-to-identify lumina,

55

and DN Rab27b was not re-localized in response to CCH. Testing of cell viability for
both CA and DN Rab27b indicated that the constructs were not cytotoxic to the LGAC;
thus, the results of DN Rab27b expression suggested a regulatory effect during SV
formation.

Figure 21. Rab27b activity also affects its distribution. Reconstituted LGAC were transduced with
Rab27b-Xpress
TM
-tagged Adenoviral constructs (MOI = 3-5) encoding full-length wild-type Rab27b
(WT), a constitutively active Q78L form of Rab27b which is GTP-locked (CA), or a dominant
negative GDP-locked form (DN). After fixation of resting and CCH-treated (100 m, 15 min) cells,
the samples were labeled to detect Rab27b (green) and actin filaments (white). Transduced WT
labeling patterns were similar to endogenous Rab27b in acini at rest and after CCH stimulation,
showing CCH-induced redistribution to the APM and some loss of subapical labeling likely due to the
release of SV (arrows). CA was also enriched subapically in the resting state and redistributed with
CCH-stimulation (arrows). DN exhibited a more cytoplasmic and dispersed labeling in the resting
state; arrowheads point to discrete regions of labeling of cytoplasmic DN Rab27b away from the
luminal region. Furthermore, fixed DN-transduced acini showed fewer and smaller lumina. *, lumen.
Bar is 10 μm.

56

5.2 Rab27b mutants altered secretory content localization
The expression of Rab27b mutants in LGAC also appeared to change the distribution of
the SV content (Figure 22). LGAC were co-transduced to express both YFP-tagged
Rab27b and GFP-tagged syncollin (Sync), an exogenous content marker that has been
used extensively in exocytosis studies (44, 53, 54, 69). Some of these co-transduced
LGAC expressed either or both tagged proteins, and because of the close spectral
wavelengths of the two protein tags, these were detected using fingerprinting imaging
analysis (Figure 22A, B top row). In cells expressing both WT Rab27b and Sync, Sync
appeared encapsulated within a subpopulation of Rab27b-enriched SV localized in the
subapical region. An increased abundance of Sync appeared to be localized in the CA
Rab27b-enriched SV, while in DN Rab27b expressing cells, signal was low and Sync
appeared diffused throughout the cell cytoplasm (Figure 22B bottom two rows).

5.3 Rab27b-enriched secretory vesicles do not co-localize with the lysosomal
pathway
Lysosomes play an integral role in the degradative pathway, engulfing particles and
organelles which are then enzymatically digested and broken down (24). These
organelles carry out a digestive role in multiple pathways, including phagocytosis,
endocytosis, and autophagy (96). Using the LysoTracker
TM
(Invitrogen), a fluorescent
live-cell probe which detects acidic compartments, we identified lysosomal/late

57

Figure 22. Expression of mutant Rab27b affects the distribution of secretory vesicle content. A.
LGAC transduced with constructs for YFP-tagged WT Adenoviral construct (red) (MOI 3-5) and a GFP-
tagged syncollin construct (Sync; green) (MOI 5) show that in cells expressing both tagged proteins,
Rab27b is enriched on the membranes of SV carrying Sync as cargo protein. B. Vesicle content, as
represented by Sync (green) remained localized primarily in subapical vesicles in LGAC transduced with
WT and CA Rab27b constructs (red) showed subapical localization. DN and Sync also showed
depolarization, and exhibited few clearly detectable lumens and few, if any, Rab27b-enriched SV.
Instead, Rab27b appeared diffused. Very little Sync appeared as enclosed content within membrane-
bounded organelles and when they were so encased, these organelles were not clearly subapical
(arrowheads). *, lumen; arrowheads, basal Sync labeling. Bars are 5 μm.

58

endosomal compartments in LGAC expressing YFP-tagged Rab27b. The three-
dimensional projections of live cell z-stack series images we took are summarized in
Figure 23 and show little or no co-localization between Rab27b and the more basally
localized lysosomal marker. These results indicate that the Rab27b-enriched SV remain
separate from the lysosomal degradative pathway.

5.4 SV content release is positively regulated by Rab27b
After establishing that the expression of mutant Rab27b alters Rab27b-enriched SV
distribution, we tested the functional effect of these mutants on SV content secretion.
Cultured LGAC reconstitute into acinus-like structures which have lumina contiguous
with the culture media (10). Using Sync as a marker of vesicle content release (54), we
co-transduced LGAC to express the Xp-tagged Rab27b WT, CA, or DN protein with
Sync, stimulated the cells with CCH, and then measured Sync release in the culture
media western blot analysis. Samples of culture media were collected from cells treated
Figure 23. Rab27b-enriched secretory vesicles do not co-localize with lysosomes. LGAC expression
the YFP-tagged Rab27b (green) were imaged by z-stacks in live cells after treatment with Lysotracker
(red), along with DAPI (blue). A flattened projection is shown here. *, lumen; Bar is 5 µm.

59

in parallel for each experimental group over a 30 min period and then the release was
calculated as a percentage of maximal release in stimulated cells to obtain comparative
data, rather than an absolute measure per treatment. Figure 24 shows results from these
functional secretion assays, alongside live cell image examples of YFP-tagged Rab27b
which correspond to intracellular events occurring at the same time. At basal levels in
WT Rab27b-enriched LGAC (Figure 24A), secretion occurs at low levels, corresponding
with minimal movement in live cell imaging. With CCH stimulation, these LGAC
undergo an immediate and significant increase and sustained release of Sync over the 30
min period. This release occurs as Rab27b-enriched SV become compressed (arrows) and
undergo sequential compound fusion and fusion with the apical plasma membrane. In
comparison to WT, YFP-tagged CA expression resulted in a significant increase of basal
release in Sync and an even greater magnitude of release with CCH stimulation (Figure
24B). In live cell imaging, we also noticed an increase in the periodic ―bursts‖ of
vesicles, and even more so with CCH stimulation, which signify fusion events between
vesicles or between vesicles and the apical membrane (arrows). In contrast to WT and
CA, however, acini expressing YFP-tagged DN did not exhibit a secretory response to
CCH and by 30 min after CCH, showed significantly less secretion levels than the same
conditions in WT-expressing LGAC (Figure 24C). In corresponding images, YFP-tagged
DN Rab27b appeared dispersed and not clearly recruited to SV, and no SV movement
was detected upon CCH stimulation.

60

Figure 24. Rab27b positively regulates secretory vesicle exocytosis. Reconstituted
LGAC expressing Sync and WT, CA, or DN Xp were plotted in wells and stimulated
with 100 μM CCH before collection at set time points. Results are given as a
percentage of maximal Sync release in WT. A. Live cell images compare resting with
CCH-stimulated cells, the latter of which show Rab27b-enriched SV closely localized
with the subapical region and undergoing compound fusion concurrent with SV
fusion with the APM (arrows). In correlation, biochemical data shows that
stimulation of WT-expressing cells with CCH resulted in an increase of Sync release.
B. SV appear to increase participation in fusion events in CA-expressing cells at
resting and even after CCH stimulation. C. Rab27b did not to enrich subapical SV in
LGAC expressing the DN form and also showed loss of response to CCH stimulation
and secretion levels. WT, CA N=7; DN N=6; *, significant change compared to
basal; Δ, significant change compared to WT; (P < 0.05); *, lumen; arrows, SV
fusion events with the APM; bar is 10 μm. D. The diameters of Rab27b (green)
enriched SV membranes were individually measured in resting acini and at 10 min
after CCH stimulation. Results reveal that in acini expressing WT, a shift towards
larger vesicles occurred with CCH addition. CA exhibited a broader range of and
larger vesicle diameters than did LGAC expressing WT. DN was not quantified
because Rab27b signal was diffused. F. SV diameter measurements are summarized
in a box-and-whisker plot demonstrating the range of data.

61

Figure 24, Continued

62

5.5 Quantification of changes in enriched vesicle diameter with constructs
An earlier study in secretory trafficking has indicated that SV size can be affected by the
absence of the Rab secretory effector, Rab3D (90). Riedel, et al. found that in the
exocrine pancreas and the parotid gland of Rab3D knockout mice, exocytosis release
kinetics upon stimulation did not appear to change. What was affected by the knockout of
Rab3D was secretory granule size, suggesting the Rab3D regulated granule maturation
rather than final fusion steps. We examined Rab27b’s functional effects on SV diameters
by measuring YFP-tagged WT and CA Rab27b-enriched SV diameters from time-lapse
videos at resting state and 15 min after CCH stimulation (Figure 24D). Consistent with
data suggesting that SV sizes increase after undergoing compound fusion prior to the
expulsion of SV content (53), WT-enriched SV significantly increased to approximately
0.87 µm after CCH stimulation, compared to 0.72 µm at resting state. CA-enriched SV
were approximately 30% larger than WT and retained a CCH response by increasing
further in size (arrows). YFP-tagged DN Rab27b was not measured because of its
dispersed signal and failure to label discrete SV structures. This decrease in DN Rab27b
expression that is detectable on membrane correlates with results from our TEM
morphology quantifications summarized in Table 2 which demonstrated that although the
DKO mouse lacrimal glands did not show any significant difference in SV diameter
compared to C57, there was a significant decrease in the number of SV. This would
imply that Rab27b helps to regulate SV formation, which affects SV number, but that its
dysfunction does not affect homotypic fusion during the maturation stage which
determines SV size. However, overexpression of CA Rab27b can influence SV size.

63

5.6 Discussion and implications
In our previous chapters, we have shown that the loss of Rab27b affects lacrimal gland
organelle morphology and that Rab27b is involved in the secretory pathway. We have
shown in this chapter, using a combination of imaging techniques that include optimized
live-cell conditions and guidelines for image quantifications, that Rab27b plays a positive
role in the regulation of SV exocytosis. Evaluation of these imaging and biochemical
studies of Sync content release indicate that the increased release of Sync and movement
and fusion events of SV at the apical plasma membrane are collectively tied to the apical
exocytosis which occurs upon stimulation of these acinar cells (Figure 22, Figure 24).
The expression of CA Rab27b appears to increase both basal and stimulated secretion,
while DN Rab27b results in a loss of CCH responsiveness for Sync release in LGAC.
Furthermore, Rab27b appears to enrich a vesicle population that is related to the secretory
pathway and Rab27b-enriched organelles do not appear to overlap with the late
endosomal/lysosomal pathway (Figure 23). These results establish Rab27b’s role in the
positive regulation of exocytosis.

However, our collective data present an interesting and complex scenario for Rab27b.
The overexpression of CA Rab27b results in a significant increase in Rab27b-enriched
SV diameters compared to WT Rab27b (Figure 24D), which would suggest that the
maturation of SV is regulated by Rab27b activity. Yet, DN Rab27b appear to affect the
formation of Rab27b-enriched SV, and a complete loss of Rab27b in 27bKO mice does
not affect SV diameters but does decrease vesicle numbers. These data suggest that

64

Rab27b regulates a step upstream of SV maturation, such as SV formation and budding.
A possible interpretation of this, which we reinforce in the next chapter, is suggested by
the fact that Rab27b is recruited early in SV biogenesis and remains with the SV until its
fusion with the apical membrane upon stimulation. Rab27b is recruited to SV membrane
much earlier than many other known SV markers such as Rab3D, the latter of which
recruited to mature SV in the subapical region and has been established as a late stage
exocytosis effector (90, 99, 114). It is possible that Rab27b plays multiple roles in the
same secretory pathway and thus explaining its ability to associate with numerous
effectors such as M5C and Rab3D, as we discussed in Chapter 3, and also with effectors
in as studied in other cell types (51). This would indicate that Rab27b regulates an early
biogenesis step of SV (formation or fission) and also can promote homotypic fusion
during maturation of SV, although this latter function is not necessary for maturation to
occur. We sought further clarification of the mechanisms and associations behind
Rab27b’s role in exocytosis in the next chapter.

65

Chapter 6: Rab27b-enriched secretory vesicles utilize cytoskeletal components at
different stages

6.1 Rab27b-enriched secretory vesicles bud from an unidentified nascent vesicle
site
Once LGAC are stimulated and regulated exocytosis occurs, only a small proportion of
the original Rab27b-enriched SV pool (<50%) remained (Figure 25A). We observed that
over time, a large SV pool was regenerated beneath the apical membrane of the cells,
suggesting that nascent vesicles were being produced and transported to the subapical
region after stimulated release. We explored the origins of these nascent vesicles by
designing a live cell study of LGAC expressing YFP-tagged Rab27b. LGAC were
stimulated for 15 min, after which CCH was washed out and the cells were allowed to
rest and replenish the subapical SV pool. Live cell images of the recovery over a 55 min
time frame are shown in Figure 25B. Within 15 min of recovery, nascent SV enriched in
YFP-tagged Rab27b could be seen forming on unidentified organelles in the basal region
of the cells. These organelles, which we designated the ―NVS,‖ tended to be faintly
labeled with YFP-Rab27b, were spherical, and were roughly 3-4 times the size of a SV
(Figure 25B, see schematic in time-series). Within the given time frame, the nascent SV
were transported to the subapical region and joined the remainder of the SV pool.

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Figure 25. Rab27b-enriched secretory vesicles bud off from a nascent vesicle site. A. YFP-tagged
Rab27b is expressed in reconstituted LGAC and imaged in live cells which retain both basolateral (B)
and apical (A) plasma membranes, the latter of which delineates the lumen (L). At resting stage, a large
pool of SV lie beneath the apical membrane, but after 15 min CCH stimulation, undergo compound
fusion concurrent with actin contraction and SV fusion with the apical membrane. B. Live cell imaging
of LGAC recovering after CCH stimulation showed that YFP-tagged Rab27b enriched SV associated
with a faintly Rab27b-labeled structure and over the course of 55 min, moved to the subapical region
besides the apical membrane of the cell where it replenished the resting vesicle pool. A schematic at 15
min identifies the NVS from which SV appear to bud off from. Arrowheads represent the site of nascent
vesicle emergence; arrows follow the movement of these vesicles to the apical membrane. Bar is 10μm.

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6.2 Co-localization study of Rab27b with a dynein motor component, p150
Glued
Trafficking studies in secretory cells, including in the lacrimal gland, have suggested that
SV movement is restricted by its tethering to motor proteins, and this tethering is aided
by Rab proteins (1, 37). Previous studies in the lacrimal gland indicate involvement of
cytoplasmic dynein with the transport of apically-targeted SV labeled with VAMP2 to the
apical plasma membrane, although the extent and purpose of this association is not
entirely clear (39, 58, 119). In LGAC, microtubules are polarized with minus-ends
organized towards the apical membrane, allowing for unidirectional cytoskeletal-based
transport to occur (10). Two classes of mechanochemical ATPase motor proteins can

transport cargo along this microtubule highway: kinesins, which classically transport
cargo towards the plus-ends of microtubules, and cytoplasmic dynein, which transports
cargo towards the minus-ends. Cytoplasmic dynein itself is a large multi-subunit protein
complex which associates with an accessory complex known as dynactin, of which
p150
Glued
(p150) is a member (4). We initially explored Rab27b association with p150 as
Figure 26. Rab27b co-localizes with p150. Co-immunofluorescence analysis of LGAC transfected with
YFP-tagged Rab27b (green) and labeled for (red) showed Rab27b co-localization with p150 (arrows).
Actin (pink); DAPI (blue); Bar is 10 μm.

68

a possible motor linking the SV to microtubules (64, 85). Co-localization imaging
analysis shown in Figure 26 indicated that YFP-tagged Rab27b-enriched SV was co-
localized with endogenous p150 in the subcortical actin region.

6.3 Disruption of the microtubule network sequesters nascent vesicles at the
nascent vesicle site
Rab27b co-localization with p150, as well as previous indications of SV transport
associated with microtubules in the lacrimal gland (5, 10, 126), indicated a likely
association between Rab27b-enriched SV and the microtubule cytoskeleton. We
examined the association between Rab27b-enriched SV and the microtubule network
more closely, using extensive live-cell imaging studies with an additional treatment with
nocodazole, which prevents microtubule repolymerization after enhancement with a cold
treatment and also caused fragmentation of the Golgi apparatus (91). This results in
extensive microtubule depolymerization in LGAC, effectively nullifying microtubule
function (10). Three-dimensional z-stack reconstructions of live LGAC cultures were
taken at rest prior to any stimulation, after a pulse of CCH to stimulate exocytosis for 15
min, and following washouts of CCH, after a 60 min recovery phase, to determine the
extent of subapical SV pool recovery. For the depolymerization of microtubules,
nocodazole was re-perfused into the cell media after CCH addition. Results are shown in
Figure 27. At resting stages, nocodazole-treated LGAC (Figure 27A) were very similar
to non-treated cells (Figure 27B) and expressed large, subapical Rab27b-enriched SV
pools. Similarly, nocodazole treatment did not affect the process of CCH stimulation and

69

release of SV, as Rab27b-enriched SV retained fusion and release activities (arrows in
zoom images) very similar to non-treated cells.
However, we observed a striking difference between treatment groups during the nascent
SV recovery phase after CCH washout. While non-treated LGAC replenished a
significant portion of the subapical mature SV pool within 60 min of recovery,
nocodazole-treated cells exhibited reduced subapical vesicle recovery and exhibited a
Figure 27. Disruption of the microtubule network hinders nascent Rab27b-enriched vesicle
production. A. LGAC transduced with YFP-tagged Rab27b were treated with or without 33 µM
nocodazole (Noc) for 60 min, and for CCH-stimulated cells, with the addition of 100 µM CCH in the last
15 min. After incubation, CCH was washed out and cells were allowed to recover for 60 min in the
presence of Noc. Z-stack images were acquired of cells in the resting stage or 15 min after CCH
stimulation. The outline of the acinus, which corresponds to the flattened z-stack image of YFP-Rab27b
(green) beside it, can be seen in the DIC image. In non- treated cells, resting stage Rab27b-enriched SVs
are grouped in the subapical region of the cells (top row). With CCH stimulation, these SVs fuse with the
apical membrane (middle row). Arrows point to subapical regions with noticeable loss of the vesicle pool.
Over the 60 min recovery after CCH washout, the cell regenerates a new pool of Rab27b-enriched SVs
which are then again recruited to the subapical region. B. Noc-treated cells appear similar to non-treated
cells in the resting stage and also undergo apparent fusion of Rab27b-enriched SVs upon stimulation with
CCH (top and middle rows). However, recovery of a new subapical pool of vesicles is severely limited;
instead, many Rab27b-enriched membranes or small SV gather at nascent vesicle sites (NVS) located
towards the basal region of the cell. Insets represent magnifications of the boxed regions; * represents
lumen; arrows point to NVS. Bar represents 5 µm.

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large number of apparent early-phase SV which appeared to be sequestered on the NVS
membrane (Figure 27B, bottom row). These Rab27b-enriched small or fragmented SV
appeared in continuity with the NVS, almost reminiscent of the NVS structure detected in
Section 6.1 except that the associated Rab27b-enriched nascent vesicles were much
smaller, in greater numbers, and did not dissociate and move towards the subapical
region during the recovery period. These results suggest that microtubules plays a critical
role in the formation at and/or transport of nascent Rab27b-enriched SV from the NVS,
although it is possible that the previously noted effect of nocodazole on Golgi
fragmentation may also be tied to these results. However, intact microtubules are not
critical for SV fusion and release at the apical plasma membrane.

6.4 The search for the identity of the nascent vesicle site
Although we consistently detected budding Rab27b-enriched SV sequestered on NVS
during recovery after CCH in LGAC treated with nocodazole, the identity of the NVS
compartment was not clear. In the classic scheme, secretory proteins are synthesized in
the ER and then modified in the Golgi stacks before exiting the trans-Golgi compartment,
although many mechanisms are not yet well understood (36, 83). We tested whether the
NVS could be identified with different Golgi markers in co-localization analysis with
Rab27b-enriched SV. These Golgi markers, golgin97, a large peripheral membrane
protein with a C-terminal GRIP domain localized in the trans-Golgi network (132), and γ-
adaptin, a subunit of the adaptor protein complex involved in late-Golgi or trans-Golgi
clathrin-mediated trafficking (124), showed some differences in co-localization patterns

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with Rab27b under these conditions. Interestingly enough, in non-treated LGAC during
recovery from stimulation, Rab27b was co-localized in some regions with golgin97

Figure 28. Rab27b may bud off from a late Golgi compartment or from a separate NVS. YFP-
tagged Rab27b cultured acinar cells were fixed and immunostained for different trans-Golgi markers,
A. Golgin 97 (Golgin) and B. γ-Adaptin. Overlay images showing co-expression of Rab27b with the
trans-Golgi markers are paired with actin, which identifies the lumina and some plasma membrane.
Arrowheads point to regions of co-localization. C. A schematic of two possible origins of the budding
vesicles suggesting either direct budding from a compartment of the Golgi or from a physically separate
NVS compartment close to the Golgi stacks. D. Live cell z-stack reconstruction of LGAC doubly
transduced with YFP-tagged Rab27b and RFP-tagged GalNAc-T2 show close localization of Rab27b-
enriched SV to the late-Golgi/TGN. * represents lumens, A represents apical membrane, BLM
represents basolateral membrane, bars represent 5 μm.

72

(Figure 28A, arrowheads) but not with γ-adaptin (Figure 28B), whereas the reverse was
confirmed with nocodazole treatment during otherwise similar conditions. Taken
altogether, these data indicate that Rab27b is recruited early in SV biogenesis in the
proximity of the Golgi, but also that distinctive sorting pathways or sub-domains in the
trans-Golgi network may be in play, as has been suggested by others (17). Based on this
study, we derived two simple interpretations of the NVS (Figure 28C): that the NVS is an
attached compartment of the trans-Golgi network, but that budding off occurs in such a
brief moment that few examples are detected in the moment of image capture thus
resulting in reduced co-localization. Alternatively, it is possible that the NVS is actually a
distinct structure which is largely free of trans-Golgi markers, and what we see as co-
localizations are mis-expressed proteins. We did not find any literature supporting the
latter scenario, although the process of SV biogenesis in acinar cells is poorly defined.

Due to the fragility and loss of structure of the LGAC during the dehydration process
required for cell fixation, we sought to obtain live cell images showing more precise
localization of Rab27b-enriched SV relative to the trans-Golgi network. The limitations
of live cell imaging, however, include the lack of a variety of highly specific markers to
test for the expression of multiple sub-compartments of the trans-Golgi network. Live
cell imaging strengthened the first scenario aforementioned: that the enriched SV bud off
directly from a compartment of the trans-Golgi network. We observed this in LGAC
expressing YFP-tagged Rab27b and RFP-tagged N-acetylgalactosaminyltransferase 2
(GalNAc-T2), which is a late Golgi/TGN marker (94, 123). These nocodazole-treated

73

cells, as in the z-stack image shown in Figure 28D, exhibited the fragmented Golgi
pattern as well as a close association, but not direct co-localization, of Rab27b with
GalNAc-T2. An intensity profile is shown corresponding to two Rab27b-enriched SV
adjacent to a GalNAc-T2 structure (white arrow and graph).

6.5 Rab27b co-localizes with M5C on secretory vesicles in the subapical region
Once SV cargo reach the subapical cytoplasm, studies in secreting cells indicate that that
an intact actin cytoskeleton is critical for successful exocytosis (77). Although its
mechanistic role is not yet clear, it has been suggested that actin may act as the receiving
end of a ―hand off‖ of the SV from a microtubule- to actin-based transport via an
exchange of effectors (60, 77, 95, 126). From a physiological standpoint this is logical, as
apical plasma membrane is underlaid with an abundant array of intertwined actin
filaments which are believed to participate in SV fusion with apical membrane, extrusion
of SV contents, stabilization of the fusion intermediate and/or retrieval of excesses of SV
membrane (22, 53, 60, 112, 115). SV fusion necessitates SV access to the apical plasma
membrane so that SNARE proteins may form fusion pores, a process which may be
mediated by the myosin superfamily, which facilitate tethering of SV to the actin
filaments (71, 76). In vertebrates, class 5 myosins (M5A, Myosin 5B, and M5C) have
been affiliated with the tethering and transport mechanisms of vesicles in a variety of cell
(66, 67, 79, 127). Of these class 5 myosins, M5C is expressed highly in

74

exocrine secretory tissues and has been shown in the lacrimal gland to be localized to the
apical-most SV in resting acini and to facilitate apical exocytosis (52, 69). Due to these
previous studies and also the general physiology of the cell, we speculated that M5C was
a candidate motor protein which enabled the ―switchover‖ of SV from earlier
Figure 29. Rab27b co-localizes highly with M5C in the subapical-most region of the lacrimal gland
acinar cell. We studied the effects of Rab27b function on the localization of M5C, an actin motor protein
highly expressed in LGAC. LGAC were co-transduced with WT, CA, or DN Rab27b-YFP constructs
(green) and the full-length functional transcript for M5C-GFP (red). Acinar cells expressing both
constructs were live imaged using Zeiss Online Fingerprinting to distinguish between YFP and GFP
spectra. In WT-Rab27b enriched SVs, M5C appeared to colocalize on the membrane of SVs in the
subapical-most region. In fact, in multiple instances M5C labeling seemed much heavier on the apical
side of the SVs, such as shown by the arrowhead. CA-Rab27b enriched SVs also appeared colocalized
with in the subapical region with M5C (arrows). However, DN-Rab27b expression was diffuse and was
generally not enriched on SV membrane. Furthermore, M5C-enriched SVs localization was not apically
polarized. *, lumen. Bar is 10 μm. B. In the reverse, a nonfunctional form of M5C-GFP (M5C tail) which
competitively binds SVs but not actin was co-transduced with WT-Rab27b-YFP. In cells doubly
transduced, larger accumulations of Rab27b-enriched (green, arrows) SVs appeared in some subapical
regions of the acini (inset), although in general localization of these SVs did not appear significantly
altered. M5C tail (red, arrowheads) is expressed as punctate labels on the membrane of SVs. *, lumen.
Bar is 10 μm.

75

microtubule-mediated transport to the later actin-mediated transport. In LGAC co-
expressing both GFP-tagged M5C as well as YFP-tagged Rab27b imaged in real time
(Figure 29A), we noted that co-localization not only occurred to the greatest extent on
the most apically-localized SV, but that co-localization was most apparent on the apical-
side of the vesicles (large arrowhead). This is perhaps representative of the recruitment
of M5C to the surface of SV in preparation for the final fusion with the apical membrane.
In LGAC co-expressing GFP-tagged M5C with the YFP-tagged CA Rab27b mutant, we
observed a similar if not increased high degree of co-localization. However, co-
localization with the YFP-tagged DN Rab27b mutant was very low. Instead, M5C
appeared recruited to basally-oriented large SV-like structures which were not Rab27b-
enriched, suggesting a shunt Rab27b-independent pathway which enables association of
M5C with SV in the event that active Rab27b is not available.

In the reverse situation, we examined the effects of the expression of a GFP-tagged
dominant-negative M5C (GFP-M5C-tail) on full length Rab27b localization (Figure
29B). This GFP-M5C-tail retains a putative cargo interaction site but not the actin-
binding domain, and in LGAC it has been shown to negatively affect the secretion of SV
content (52, 69). In cells co-expressing these proteins, GFP-M5C-tail appeared to
aggregate in regions localized to the surface of subapical SV. However, Rab27b
localization did not appear to be obviously altered, although SV appeared even more
abundant than typically seen. This study indicates that while Rab27b and M5C are
associated to the same SV pool, at least on a portion of the vesicles, they can each be

76

recruited independent of the dominant negative form of the other. These observations
also suggest that the uncoupling of the M5C motor protein does not affect Rab27b-
enriched SV transport to the subapical region, although it may affect their maturation
through homotypic fusion, thus resulting in an increase of total SV. This could mean that
contrary to studies in melanocytes indicating the formation of ternary complexes are
necessary for SV transport along the actin cytoskeleton (46), Rab27b association with an
effector which has an actin-binding domain, such as Slac-2c, is sufficient for Rab27b-
enriched SV association with actin (49). However, it would be necessary to first test the
possibility that endogenous M5C or other myosin motors are enabling Rab27b-enriched
vesicles with the actin filament.

6.6 Co-localization study of Rab27b with actin filaments
Live cell imaging of LGAC expressing GFP-tagged actin and YFP-tagged Rab27b
confirmed earlier observations that Rab27b-enriched SV lie beneath the actin-rich apical
plasma membrane (Figure 30). We noted that while the subapical-most SV were in close
proximity to the actin filaments, sometimes appearing to nest within the filaments, there
was no indication of co-localization at resting states.

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6.7 Disruption of the actin network alters the terminal apical membrane fusion
and release of enriched secretory vesicles
The role of actin in the terminal exocytotic trafficking of Rab27b-enriched SV was tested
by treating LGAC with latrunculin B, which disrupts F-actin polymerization (53). Unlike
nocodazole-treated cells, latrunculin treatments did not affect the replenishment of
mature SV at the subapical region following CCH stimulation and recovery (data not
shown). However, the addition of latrunculin caused noticeable changes in the final
stages of exocytosis. These are shown in Figure 31, which compares live cell images
from LGAC immediately after CCH stimulation, with and without latrunculin treatment.
In untreated cells (Figure 31A), SV are localized within the subapical region at the
moment of stimulation and begin to undergo multiple instances of compound fusion
Figure 30. Rab27b-enriched
secretory vesicles lay beneath the
actin membrane but do not co-
localize at resting state. Rab27b
(red) and actin filaments (green)
were both expressed by Ad-
constructs. Arrows point to Rab27b
localization, *, lumen; bar is 5 µm.

78

(white arrowheads) as well as fusion with the apical plasma membrane (red arrowheads)
over a 10 min stimulation period. With latrunculin treatment (Figure 31B), LGAC
retained a large pool of Rab27b-enriched SV at the beginning of stimulation and

Figure 31. Disruption of the actin network alters terminal apical membrane fusion of Rab27b-
enriched secretory vesicles. A. An untreated acinus expressing YFP-tagged Rab27b was imaged in time-
series. Transduced cells expressing Rab27b (green) are outlined with white to delineate the cell borders.
The remaining images in the series are higher magnifications of the boxed region around the lumen (L).
Within 100 s after CCH addition, the region around the lumen begins to constrict and SV move towards
the lumen, fusing with each other (white arrowheads) and with the APM (red arrowheads). Within 600 s,
approximately 50% of the SV were in apparent contiguity with the lumen, suggesting discharge of
contents. B. An acinus treated with 10 µM latrunculin B for 60 min at 37°C was similarly imaged. With
CCH addition, SV failed to move toward the lumen. Although many SV underwent homotypic fusion
(white arrowheads), membrane of internal fused SV failed to fuse with the APM. *, lumen. Bar is 10 µm.

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underwent multiple instances of homotypic fusion (arrowheads), but showed few, if any,
fusion events with the apical plasma membrane. Over the stimulation period, compound
fusion led to the production of large SV or possibly vacuoles with only a weak and
dispersed Rab27b labeling on the surface. These results suggest that actin is required
during the docking process to the apical plasma membrane.

6.8 Co-localization study of Rab27b with truncated melanophilin
Thus far, our studies have indicated that Rab27b-enriched SV required microtubules for
vesicle maturation and transport to the subapical regions and also intact actin filaments to
undergo fusion with the apical membrane upon stimulation to secrete. As
aforementioned, studies in other secretory cell types have proposed that Rab27b functions
through a tripartite complex by associating with specific effectors which then bind to
cytoskeletal motor proteins such as myosin 5 (28, 29, 45, 51, 122). Although in Section
6.5 it was not clear whether active Rab27 was necessary for M5C function in the
secretory pathway, or vice-versa, based on the lack of effect of either Rab27 or M5C
dominant-negative expression on the other’s localization, it is possible that multiple
alternate effectors provided compensatory function which minimized these effects. We
then questioned whether saturation of the Rab27b binding site with a dominant negative
effector protein could prevent Rab27b association with myosin 5 (or any other motor
protein) and effectively change Rab27b localization. To accomplish this, we utilized
melanophilin, an effector protein of Rab27a/b expressed in melanocytes which regulates
polarized melanosomal trafficking by binding Rab27 and M5A at its C-terminus and to

80

actin filaments at its N-terminus, providing a bridge between Rab27-enriched
melanosomal membrane with the cytoskeletal network (46, 122). In a preliminary
examination, we exogenously expressed V5-tagged dominant-negative melanophilin
(Mlph), which has a truncated c-terminus at a.a. 266 that removes the binding site for
M5A but retains the binding site for Rab27a/b. Co-localization of endogenous Rab27b
with Mlph, as expressed by Ad5, was very high in LGAC (Figure 32). Expression
occurred largely at the subapical region but also appeared more sparsely in parts of the
basal regions, consistent with endogenous Rab27b localization shown in Figure 10 and
11. However, we did not detect any obvious differences in these Mlph-expressing LGAC
from untransduced cells. These results are not completely surprising: if Rab27b-enriched
SV require intact actin filaments only for the final step of fusion with the apical plasma
membrane as suggested in our live cell data in Section 6.7, then we would only expect to
see differences in localization after stimulated release.

6.9 Discussion and implications
We have already shown that Rab27b plays an important regulatory role in exocytosis, and
in this chapter we examined potential associations which enable Rab27b-enriched SV to
mature and transport to the subapical region. For the first time, we provide clear imaging
evidence that Rab27b is recruited to budding SV early in biogenesis in the basal regions
of the acinar cells (Figure 25) and remains associated through vesicle fusion and release
in contrast to Rab3D, which is largely recruited to mature SV in the subapical region. We

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also determined that Rab27b maturation and transport to the subapical region requires an
intact microtubule network; disruption of the microtubule filaments sequesters apparent
budding SV on the surface of a compartment we named the NVS (Figure 27).
Conversely, while Rab27b does not appear to co-localize with actin filaments directly
(Figure 30), an intact actin network is critical for the final fusion step of Rab27b-enriched
SV (Figure 31).

Figure 32. Rab27b co-localizes with Mlph. Mlph (red), a dominant-negative effector of Rab27b, shows
high co-localization with endogenous Rab27b (green) in the subapical regions and, at a lower level of
intensity, in the basal regions of LGAC. Also shown is actin (white). *, nucleus; bar represent 10 µm.

82

Our imaging studies raised an unexpected question: are Rab27b-enriched SV really
budding from the trans-Golgi network, as according to the classical SV biogenesis
scheme? From our images, we identified the NVS (labeled in Figure 25, Figure 27,
Figure 28), a circular, basolateral compartment faintly coated with Rab27b which was
approximately 2-3 µm in diameter. We attempted to positively identify these NVS with
trans-Golgi network markers (Section 6.4) by detecting for co-localization of Rab27b
with the trans-Golgi markers after disrupting the microtubule network during the
recovery period after CCH stimulation. Interestingly, the two trans-Golgi markers co-
localized with Rab27b differently. We examined the specific functional roles of golgin97
and γ-adaptin to explain the difference localization. In Figure 28A and 28B, with the
depolymerization of microtubules, nascent Rab27b-enriched SV may be sequestered at
the final fission step with γ-adaptin, which is normally recruited for clathrin-mediated
vesicle fission. Conversely, golgin97 enriches a difference sub-compartment of the trans-
Golgi network different from where the SV were sequestered. This resulted in the
detection of increased co-localization with Rab27b with γ-adaptin but decreased co-
localization with golgin97. In live cell imaging using the GalNAc-T2 marker, we
identified a possible compartment labeled with GalNAc-T2 which was close to, but not
co-localized with, Rab27b-enriched SV. Therefore, while we are certain that Rab27b-
enriched SV bud from a compartment that is in close proximity to the trans-Golgi
network, we cannot yet determine whether the NVS is a contiguous sub-compartment of,
or a separate organelle in close proximity to, the trans-Golgi network (Figure 28C).
Notably, we also observed increased fragmentation of the Golgi membranes after

83

addition of nocodazole especially clearly in live cell images (Figure 28D), a phenomenon
consistent with previous studies (91). While the mechanisms behind this Golgi
fragmentation are not well understood, it is conceivable that this may also have a
negative impact on SV formation.

Results from Section 6.5 showed that overexpression of WT and CA Rab27b proteins did
not affect M5C recruitment to the subapical-most vesicles in LGAC. Moreover, the
overexpression of the dominant-negative form of Rab27b, which in Section 5.1 showed
loss of ability to recruit to SV membrane, did not prevent M5C recruitment to SV-like
structures, although these structures were basal and not subapical. These results were
surprising because the ability of M5C to recruit to SV-like structures without Rab27b
activity indicates that a M5C-Rab27b association, direct or indirect, is not required.
However, localization of these M5C enriched SV in the basal regions also indicates that
while active Rab27b is not critical to the recruitment of M5C to SV, active Rab27b is
critical for SV localization to the subapical membrane. Vice-versa, the dominant-negative
form of M5C did not appear to prevent Rab27b-enrichment of subapical SV. These data
suggest that Rab27b and M5C do not bind and may function independently, contrary to
the theory that Rab27 functions through a tripartite complex (29). Extensive further
studies would be necessary, however, to more thoroughly examine this conclusion. First,
association of Rab27b with the other members of the myosin 5 family need to be tested in
order to determine that it is indeed M5C, and not another myosin, which specifically
interacts with Rab27b. Secondly, Rab27b co-localization of M5C-tail should also be

84

analyzed upon CCH addition, to test whether co-localization occurs after stimulation.
These tests would contribute to our understanding of the relationship between Rab27b
and the myosin 5 family in the LGAC.

The co-expression of Rab27b and Mlph also prompted new questions concerning the
function of Rab27 effectors. In theory, Mlph binds Rab27b at its effector binding site,
thus blocking other effectors which bind the same domain. Since Mlph retains its Rab27
binding domain but not its M5A binding domain, this effectively prevents Rab27b-
enriched SV from being transported on actin filaments via M5A. What was interesting
was that Rab27b and Mlph co-localized almost completely. This co-localization can be
discounted by the fact that Mlph was an overexpressed exogenous protein. However, if
the extent of Mlph co-localization is not the result of overexpression and is consistent
with endogenous effector binding patterns, this would suggest that Mlph can be recruited
to the Rab27b-enriched SV prior to transport of the SV to the subapical region where
melanophilin in theory would link Rab27b-enriched SV to the actin cytoskeletal network
during stimulated exocytosis, as published in melanosomal studies (46). If Rab27b
function is determined by its specific association with different effectors (27), then how is
the binding of effectors regulated? An answer suggested by Fukuda, et al. based on in
vitro studies is that perhaps a hierarchy exists for effector binding affinities to the Rab27
protein (23), although it is not clear how this hierarchy would be put into practice in vivo.
Another question concerns the specificity of the effector protein itself. Melanophilin, as
endogenously expressed in the melanocyte, binds to M5A. However, studies have shown

85

that in vitro, melanophilin can be used in competitive binding studies for binding to
Rab27b (122). What does Mlph bind when exogenously expressed in LGAC? We know
that LGAC highly express M5C, but studies in this direction would require biochemical
assays studying the binding affinities of effector proteins to motor proteins in the LGAC.
Further studies into Rab27b effectors will be critical to our understanding of Rab27b
function but will clearly unveil a complex pathway.

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Chapter 7: Materials and methods

7.1 Reagents
Matrigel
TM
was obtained from Collaborative Biochemicals (Bedford, MA); carbachol,
Nocodazole (Methyl-(5-[2-Thienylcarbonyl]-1H-Benzimidazol-2-YL) Carbamate),
heparin, chondroitinase ABC, protease inhibitors pepstatin A, N-tosyl-
L
-phenylalanine
chloromethyl ketone, leupeptin, N -p-tosyl-
L
-arginine methyl ester, and
phenylmethylsulfonyl fluoride were from Sigma-Aldrich (St. Louis, MO), and latrunculin
B from EMD Biosciences (San Diego, CA). Commercial antibody for Rab27a was from
Santa Cruz Biotechnology (goat polyclonal N20, Santa Cruz, CA) or Novus Biologicals
(mouse MAb M02, Littleton, CO) while Rab27b antibodies were from Santa Cruz
Biotechnology (goat polyclonal C20), IBL America (rabbit polyclonal, Minneapolis,
MN), or Novus Biologicals (mouse polyclonal B01P, Littleton, CO). Rabbit polyclonal
antibody to recombinant Rab3D was generated by Antibodies, Inc. (Davis, CA) (20).
Other commercial primary antibodies utilized included: mouse MAb to GFP (Santa Cruz
Biotechnology, Santa Cruz, CA), mouse MAb to p150 and mouse MAb to γ-adaptin (BD
Transduction Laboratories, San Jose, CA), mouse MAb CDF4 to Golgin 97 (Molecular
Probes, Invitrogen, Eugene, OR), mouse MAb to V5 epitope tag (Invitrogen, Eugene,
OR) and mouse MAb to Xpress
TM
tag (Invitrogen, Carlsbad, CA). Rabbit polyclonal
antibody to Myosin 5C was a gift from Dr. Richard Cheney (University of North
Carolina). Secondary antibodies and fluorescent affinity probes for microscopy were
purchased from Molecular Probes/Invitrogen (Carlsbad, CA). Purified recombinant his-

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tagged Rab27a and Rab27b protein were prepared as described (101). All other chemicals
were reagent grade and obtained from standard suppliers.

7.2 Primary rabbit lacrimal gland acinar cell culture and treatments
All animal procedures were in accordance with the Guide for the Care and Use of
Laboratory Animals, published by the National Institutes of Health (NIH Publication No.
85-23, Revised 1996) (10, 32, 33) and were approved by the University of Southern
California IACUC. LGAC isolated from rabbit LG from New Zealand White rabbits
(1.8-2.2 kg) (Irish Farms, Norco, CA) were sequentially incubated in Mg2
+
- and Ca
2+
-
free HBSS supplemented with EDTA and Ham’s medium supplemented with
collagenase, hyaluronidase, and DNAse, and cultured for 2 days in Peter’s serum-free
culture medium (33). These cultured cells re-form acinus-like structures displaying
distinct apical and basolateral domains (10, 32, 53). For imaging of fixed cells, LGAC
were seeded on coverslips at 2 x 10
6
cells per well in 12-well plates coated with
Matrigel
TM
. For live cell imaging, LGAC were seeded on 35 mm dishes with glass
coverslip bottoms at 6 x 10
6
cells per dish (MatTek, Ashland, MA). For nocodazole
treatments, cultured cells were iced for 5 min to induce depolymerization of microtubule
filaments prior to the addition of 33μM nocodazole for 60 min at 37°C.

7.3 Mouse lacrimal gland isolation and analysis
LG from 3-4 month old male mice were surgically removed and processed (9). For
immunocytochemical and immunofluorescence labeling and analysis, tissue was

88

immediately immersed in 4% paraformaldehyde for 2 hours at room temperature,
transferred to 30% sucrose overnight, and then frozen in Tissue-Tek
®
O.C.T. (Sakura
Finetek, Torrance, CA). The embedded tissue was sectioned to 5-8 μm thickness and
thaw-mounted onto warm glass slides. For TEM, fresh tissue was carefully minced into 1
mm
3
pieces and fixed with 3% glutaraldehyde in 0.1M cacodylate buffer overnight.
Samples were postfixed with 1% osmium/0.8% potassium ferricyanide in 0.1M
cacodylate buffer, dehydrated, infiltrated in 100% Spurrs resin: by weight 23.6%
ERL4221, 14.2% DER736, 61.5% NSA, 0.7% DMAE (EMS, Hatfield, PA). Thin
sections were prepared with an RMC MTX ultra microtome (Boeckeler Instruments Inc.,
Tucson, AZ) and counterstained with Sato’s lead stain and 2% uranyl acetate.

7.4 Production and amplification of recombinant adenovirus
Adenovirus (Ad) constructs were amplified in QBI cells at 37 C and 5% CO2 in DMEM
(4.5 g/mL glucose, GIBCO/Invitrogen, Carlsbad, CA) containing 10% FBS until cells
showed the characteristic cytopathological effect. Cells were then harvested and purified
using CsCl gradient gradient ultracentrifugation (119), and viral titers were measured by
the formation of viral plaques in sequential dilutions. The following replication-deficient
Ad constructs were used: Ad-syncollin-GFP (kindly provided by Dr. Christopher Rhodes,
University of Chicago) (68), Ad5 containing the β-galactosidase reporter gene, Ad-LacZ
(120), and Ad-GFP (129). Mouse Rab27 sequences, fused to epitope tags on their N-
termini, were expressed using the following constructs: Ad-Rab27bQ78L-Xpress™
(constitutively active; CA Xp), Ad-Rab27bN133I-Xpress™ (dominant negative; DN Xp)

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and Ad-Rab27b-Xpress™ (wild-type; WT Xp), which were kind gifts of Dr. John
Williams, University of Michigan (7, 133); and Ad-Rab27b-YFP (WT YFP),
Rab27bQ78L-YFP (CA YFP), Rab27bN133I-YFP (DN YFP) as described (106). For
Myosin 5C studies, GFP-tagged full-length human myosin 5C (WT M5C) and a
dominant-negative tail of myosin 5C (GFP-M5C-tail) were prepared as described (69,
92). Truncated Melanophilin (Mlph) 1-266 a.a. was prepared as described (46).

7.5 Generation of recombinant protein
Recombinant protein used in the technical study in Chapter 4: recombinant knob, GFP-
knob, penton base, and rab3DQL, were produced in Escherichia coli as His6-tagged
fusion proteins, and recombinant fiber protein and control protein (glutathione-S-
transferase [GST]) produced in a baculovirus expression system, as described previously
(89, 129).

7.6 Ad transduction of constructs into lacrimal gland acinar cells
Initial studies showed that the Xpress
TM
tagged protein expression yielded better quality
images for fixed cell analysis; while YFP tagged protein expression enabled visualization
of intact SV in living cells. For imaging of exogenous proteins, cultured LGAC were
transduced with Ad constructs at MOI 4-6 and incubated for an additional 18-24 hours to
optimize expression levels (53). Previous studies have consistently shown a 70-80%
transduction efficiency using this low viral titer (129). Transduction efficiency with
Rab27b constructs was >80%. For assays analyzing the release of syncollin-GFP, LGAC

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were doubly-transduced with Ad-syncollin-GFP (MOI 2-3) and WT/DN/CA Xp (MOI 2-
5) or with an Ad-GFP control. Ad-syncollin-GFP transduction was 60-70%, but because
of the higher transduction efficiency of all other constructs, in dual transduction
experiments most LGAC expressing Sync also expressed the transduced form of the
Rab27b construct. Expression levels of epitope-tagged Rab27 constructs were ~20- to 50-
fold that of endogenous protein as determined by western blot analysis of transduced
lysates. Expression of constructs was validated by confocal fluorescence microscopy.
Cell viability of the acini expressing the DN Rab27b constructs, which showed loss of
epithelial cell polarity, was tested using the LIVE/DEAD® Cell Viability Assay Kit for
mammalian cells (Invitrogen, Carlsbad, CA). Expression of RFP-tagged late
Golgi/trans-Golgi network marker, GalNAc-T2, was conducted by the use of a
commercial kit, Bacmam 2.0 Cell Light Golgi (Invitrogen, Carlsbad, CA), as per
manufacturer’s instructions.

7.7 Confocal fluorescence microscopy
For immunofluorescence, LGAC were fixed with ethanol, blocked with 1% BSA and
incubated with primary antibody, followed by the appropriate secondary antibody, and
mounted on glass slides with Prolong anti-fade mounting medium (Molecular Probes,
Eugene, OR). For frozen sections from LG tissue, fresh-cut sections were fixed with 4%
paraformaldehyde and permeabilized with 0.1% Tx-100 before blocking with 1% BSA,
followed by labeling with the primary and secondary antibodies. Slides were imaged
using a Zeiss LSM 510 Meta NLO (Thornwood, NY) confocal imaging system equipped

91

with Argon, red HeNe, and green HeNe lasers and a Coherent Chameleon Ti-Sapphire
laser mounted on a vibration-free table. LSM images were converted into .tiff by Adobe
Photoshop 8.0 (Adobe Systems Inc, Mountain View, CA). Further analysis of
colocalization coefficients associated with distinct chromophores linked to proteins of
interest was conducted using the Zeiss Enhanced Colocalization Tool software in parallel
with non-colocalizing specimens. To distinguish between the close spectral fluorescence
emission values of GFP and YFP, the Zeiss META Emission Fingerprinting program was
used to acquire a complete lambda stack, followed by linear unmixing.

7.8 Time-lapse live cell imaging
Live cell imaging was conducted in cultured LGAC expressing a tagged protein in a
controlled incubation chamber as described (53, 129). For live cell time series studies,
reconstituted acini of similar size were analyzed at the resting stage for 10 min and upon
stimulation after addition of 100 µM CCH (15 min). To analyze the reliance of Rab27b
YFP enriched vesicle movements on actin-dependent transport, 10 µM latrunculin B was
incubated with the cells at 37°C for 60 min prior to CCH addition. Negative controls for
the latrunculin B studies included pretreatment with equivalent amounts of
dimethylsulfoxide. DIC images collected in parallel with fluorescence allowed the
identification of the apical/luminal and basolateral membranes. Z-stacks were taken to
identify relative position of signals within the cell and reconstructed with the Zeiss LSM
Projection Tool and with the NIH ImageJ 1.41a.

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To visualize membrane surface activity, LGAC were co-transduced with Ad-Tc-GFP-
actin and Ad-tTA at an MOI of 8pfu/cell. (Dr. Dan Kalman, Emory University). Cells
were then placed in 1 mL binding buffer (HBSS+.01% BSA+0.02% HEPES) and treated
on ice for one hour with appropriate amounts of 1. dPBS (neg control) 2. Heparin (neg
control, Sigma) 3. non-treated AdLacZ (MOI=100) 4. AdLacZ (MOI=100) pretreated 1
hr@RT with 1mg/mL Hep 5. AdLacZ (MOI=100 PFU/cell) pretreated 1 hour at RT with
5mg/mL Hep 6. AdLacZ (MOI=100) pretreated 1 hr@ RT with 1mg/ml CSA (a GAG,
positive control) 7. AdLacZ (MOI=100) pretreated 1hr@RT with 5mg/ml CSA. Cells
were washed and warmed 37°C for 5 min prior to analysis by time-lapse confocal
fluorescence. Quantification of membrane ruffling events was completed by comparing
acinar clusters of similar cell numbers and transduction efficiencies within a 600s period
representing the highest membrane ruffling activity in each sequence. Each membrane
protrusion was measured and recorded at its largest diameter using the LSM Image
Browser overlay tool and classified into diameter ranges. The number of ruffling events
in each range was divided by treatment and standardized by dividing by the number of
sequences used to obtain an average value per sequence for each treatment. The exact
numbers of sequences used are: ctl (n=8), ctl w/hep (n=7), Ad (n=8), 5mg/1ml Hep
(n=8), 1mg/1ml Hep (n=8), 5mg/ml CSA (n=8), 1mg/ml CSA (n=4). Error bars represent
s.e.m.
For analysis of vesicle diameter, acini from cell treatments were analyzed at specific time
points: 5 min prior to CCH addition and 10 min after CCH addition. Vesicles clearly
enriched in Rab27b were measured using the Image J measurement analysis tool (NIH,

93

Bethesda, MD). Samples are from 15-20 random regions taken from 12 preparations:
WT (25 cells, 412 SV), WT+CCH (22 cells, 297 SV), CA (23 cells, 431 SV), CA+CCH
(22 cells, 324 SV); error bars represent S.E.M.; P < 0.05. E. Schematic of SV diameter
and distance measurements. Variations in vesicle density prevented usage of high-
throughput quantitative analysis programs.

7.9 Preparation of lacrimal gland tissue and lacrimal gland acinar cell lysate for
western blotting
Cultured rabbit LGAC were lysed in RIPA buffer containing protease inhibitors as
previously described (117). LGAC lysate were passed through a 20½G syringe needle 20
times, followed by 20 passes through a cell press (H & Y Enterprises, Redwood City,
CA, USA). LG tissue from mice was collected and homogenized with three 30s pulses on
ice in 2 mL RIPA buffer with protease inhibitors using a PT-MR-2100 Polytron tissue
homogenizer. Crude tissue homogenate was pre-cleared at 7,700 x g for 10 min at 4°C
(Hermle Labnet Z216MK, Woodbridge, NJ), and the supernatant was collected. Lysate
protein concentrations were determined with the Pierce BCA Assay using albumin as
standard protein (Thermo Fisher Scientific Inc., Rockford, IL) and proteins of interest
were resolved by SDS-PAGE. Western blot analysis was conducted on nitrocellulose
membranes using appropriate primary and IRDye
TM
-conjugated secondary antibodies
(Rockland, Gilbertsville, PA), the Odyssey infrared imaging system, and the Odyssey
imaging software version 2.1 for quantification of immunoreactive band intensities (Li-

94

Cor, Lincoln, NE). Semiquantitative analysis of the Rab27a and Rab27b antibody
reactivities to its prospective purified recombinant protein was carried out.

7.10 Analysis of lacrimal gland acinar cell secretion
As a measure of regulated secretion, syncollin release was measured in LGAC co-
transduced with Ad-syncollin-GFP and the WT, CA or DN Xp constructs as described
previously (44, 53, 69). Bathing media from LGAC, which is continuous with the apical
lumina of the cultured cells, was collected at time points after CCH addition. The
bathing medium was concentrated on Centricon YM-10 filters (Millipore, Billerica, MA)
and resolved by SDS-PAGE. A GFP signal was detected by western blotting and its band
intensity was measured using the Odyssey imaging software. Secretions were normalized
to total cell protein in each well. In data plots, values were expressed as a percentage of
total release from stimulated acini of cells expressing WT in the presence of CCH at the
maximal time of release (30 min) in order to conduct a comparative study to WT release,
rather than total protein release per treatment. Lysate samples were also resolved by SDS-
PAGE to ensure equal loading by detection for actin or Xpress™.

7.11 Subcellular fractionation analysis
Non-treated and carbachol treated cultured acinar cells (100 μM, 15 min), with or without
a nocodazole treatment, were collected after the appropriate treatments and lysed by
Balch press in culture and Ham’s media with added protease inhibitor cocktail and
DNAse. Fractions were then obtained by sorbitol gradient centrifugations as described

95

previously (40, 74, 119). The final fraction, the pellet, was designated Fraction 13.
Sedimented fractions were snap frozen in liquid nitrogen and stored at -80°C until
analysis by Western blot with the appropriate primary and secondary antibodies for
endogenous protein expression. Blot signal intensities were measured by Odyssey
imaging software (Li-Cor, Lincoln, NE). Fractions were quantified as a percentage
recovery of total signal from all thirteen fractions. Significant changes between treatment
groups was determined by Student’s t-test (P<0.05).

7.12 Transmission electron microscopy
Mouse LG tissue was prepared as described under LG isolation. Samples were analyzed
with a digital 11 megapixel JEOL 1011 TEM (JEOL USA, Inc., Portland, OR). A large
systematic random sampling (131) of mouse sections was used to examine the detailed
morphology of the knockout mouse LG tissue compared to that from WT. SV numbers,
diameters, and distances from the lumen, as well as cell size and organelle counts, were
measured with the Image J measurement and counting tools (NIH, Bethesda, MD). Cell
area was also measured and showed no significant differences between mice, although a
per cell count was used to evaluate the data in order to avoid the reference trap in the
fluid-filled LGAC.

7.13 Statistical analysis
Data analysis was conducted to compare between data sets using either Student’s two-
sample t-test (for assays of Ad-syncollin-GFP secretion) or a one-way ANOVA followed

96

by posthoc analysis using the Games-Howell test (PHAST TM, by Phillip R. Stanwood,
Copyright 2007) (for comparison of organelle abundance in different mouse models) as
appropriate. The criterion for significance was at least P < 0.05.

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Chapter 8: Conclusions and future perspectives

8.1 Function of Rab27b in the lacrimal gland
The scope of this study encompasses the characteristics and functions of a protein which,
newly identified in the lacrimal gland acinar cell, provides strong and unique perspectives
into the mechanisms behind the secretory pathway. We have taken the current knowledge
of intracellular trafficking in the LGAC, including the understanding that CCH
stimulation increases apical actin filament turnover and promotes the transient assembly
of actin coats around SV fusion and fluid content which leads to fusion and release of the
SV (54, 81), and identified a new marker which is a direct participant in this pathway.
Drawing from biochemical studies alongside electron microscopy and extensive confocal
fluorescent microscopy imaging analysis of fixed tissue and cultured cell, as well as live
cell data, we established the expression and localization of Rab27b in LGAC and also
considered the expression of Rab27a. While Rab27b expression was strong and
underwent re-localization in response to CCH stimulation, at least in preliminary
biochemical and imaging studies, Rab27a expression appeared relatively weak. We also
found that 27bKO and DKO, but not Ashen mice, showed considerable secretory disease
phenotype. These early data resulted in our decision to focus on Rab27b in the lacrimal
gland, especially since knockout of Rab27b alone is sufficient to create a significant
effect. However, we do not exclude the possibility that even low expression of Rab27a
may affect or compensate for Rab27b dysfunction.

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Functionally, we have shown that Rab27b is a critical positive regulator of exocytosis in
LGAC. Rab27b-enriched SV respond to CCH stimulation and relocalize to the subapical
region where they fuse with the apical membrane and are released. GTP-locked Rab27b
expressed in LGAC affect both biochemically the Sync secretion levels and qualitatively
increase the number of fusion events as well as the size of the mature vesicle pool,
whereas in LGAC expressing the dominant-negative form, Rab27b appears diffuse and
although Sync secretion levels still occur at a basal level, they LGAC show a loss of
response to CCH stimulation. This latter observation also suggests that basal release of
SV may still occur in spite of the dominant-negative form either because of the
expression of endogenous Rab27b in DN Rab27b-transduced cells, or because there is an
alternative and independent basal secretion pathway that does not require Rab27b
activity. In 27bKO and DKO mice, loss of functional Rab27b does not only affect the
secretory pathway but also affects global aspects of cellular membrane organization and
function.

Rab27b is recruited on the membranes of SV during biogenesis and can be detected as
early in SV maturation as during budding in the basal regions of the cell. There, Rab27b-
enriched SV emerge from lightly-Rab27b coated NVS. The identity of the NVS is not yet
clear, although classical biogenesis suggests that SV content is gathered in the ER and
then sorted through the Golgi apparatus to the trans-Golgi network. We surmised that the
NVS should be either a sub-compartment of the trans-Golgi network or an affiliated but
separate organelle altogether. Fixed cell co-localization with trans-Golgi network markers

99

and live cell imaging with a more general late-Golg/trans-Golgi network marker
collectively indicate at least a close proximity of nascent SV with the trans-Golgi,
although further analysis and better markers are necessary to yield a definite answer. The
early recruitment of Rab27b on to the nascent SV suggests that it has a functional role
either in budding and fission, although we saw no indication of this in our live cell study,
or during transport to the apical membrane during SV maturation.

We also determined that SV maturation requires intact microtubules. Not only does
Rab27 co-localize with p150, a marker of the dynein motor protein complex, but the
depolymerization of microtubules leaves nascent SV stranded at the surface of the NVS
during recovery phase. It was also notable that depolymerization of microtubules did not
inhibit initial stimulated release of the mature SV pool beneath the apical membrane.
This indicated that microtubules were critical to Rab27b-enriched SV maturation to the
subapical region, but not during SV docking and apical fusion. We suggest that Rab27b-
enriched SV utilize the microtubule network for transport between the NVS and the
subapical region. While it is not yet clear why Rab27b is recruited so early in biogenesis
and what its function might be, we do know that Rab27b has the ability to interact with
different effectors with different functions (25, 27, 41, 47, 50, 51, 121). We propose that
the recruitment of Rab27b to SV just as they bud from the NVS in turns enables the
recruitment of effectors which, down a chain of command, allow recruitment of motor
proteins such as dynein which associate with microtubules.

100

Once the LGAC is stimulated, Rab27b-enriched SV then require the actin cytoskeleton
during the final docking and fusion steps of vesicle release. In the subapical region, we
have shown that Rab27b co-localizes with M5C especially strongly on the face of the SV
closest to the subapical region. This bias towards the side of the SV which will fuse with
the apical membrane may indicate that Rab27b-M5C interactions, perhaps similar to
Rab27a/b-M5A interactions described in other cell types (46, 49, 122), are required for
fusion and provides a mechanism which explains how Rab27b-enriched vesicle interacts
with the high concentration of actin in the subapical region. Depolymerization of actin
appears to suppress this final docking and fusion step of Rab27b-enriched SV during
stimulation.

8.2 Mechanisms behind the movement of enriched vesicles in trafficking
We summarize our working model for Rab27b-enriched membrane trafficking supported
by our data in the schematics shown in Figure 33. (A). In resting acini, cytoplasmic
Rab27b associates with the NVS in the basal region of the cell. As new SV form at this
domain, nascent Rab27b-enriched SVs recruit an effector which allows their association
with a possible dynein motor protein, which in turns allows for their association with
microtubules, budding from the NVS, and directed transport towards the subapical
region. They are captured by association with M5C to actin filaments in the subapical
region remain tethered in this region. (B) Upon stimulation to secrete, these mature
vesicles now in the subapical region are compressed by the transient actin coats that form
around fusion intermediates, using associated and activated M5C to move on additional

101

filaments to fusion sites at the apical plasma membrane. Actin serves as the substratum
which allowed directed transport to the apical plasma membrane as well as the
compressing force which squeezes together SVs to close enough proximity for vesicle-
vesicle fusion and for fusion of these late-stage vesicles with the apical membrane. It is
unclear whether another effector is required for the final docking and fusion of the
Figure 33. Trafficking schematic of Rab27b-enriched secretory vesicles. LGAC representations are
shown at resting state (A), with CCH stimulation (B), with treatment of nocodazole at resting state (C),
and with latrunculin B treatment upon stimulation (D).

102

vesicles for exocytosis, or whether actin compression is sufficient for fusion and release.
(C) Depolymerization of microtubules still allows for exocytosis of subapical SV, but
inhibits the subsequent accumulation of nascent vesicles and sequesters vesicle buds on
the NVS. (D) Depolymerization of actin leaves the trafficking from the NVS to the
subapical region intact, but inhibits final vesicle fusion with the apical membrane in
CCH-stimulated acini. SVs continue to undergo compound fusion, possibly as a result of
compression force.

8.3 Potential therapies, analysis, and future approaches with Rab27b
The LGAC is a prime model of study for the regulation of exocytosis both as a highly
active and physiologically important secretory epithelial cell and as a therapeutic target
for the many patients suffering from dry eye diseases. Our studies provide exciting new
insight into the characteristics and functions of a Rab protein in secretory cells. We meet
our goal to identify a protein which has potential as a target for manipulating the
upregulation of SV-specific exocytosis in exocrine secretory disorders such as dry eye
diseases, as evidenced by the ability of CA Rab27b to enhance stimulated exocytosis.
Increased stimulated exocytosis would enable an increase of tear proteins in patients with
low protein to fluid ratio. This study also validates the use of Rab27b as a marker of the
exocytosis pathway. While Rab3D has previously been used as a mature vesicle marker
(20, 115), Rab27b expression on SV begins earlier at biogenesis, thus allowing for its
usage as a general SV marker. Furthermore, Rab3D regulates homotypic fusion but does

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not appear to be essential for secretion (90), thus suggesting that Rab27b may play a
more significant role in trafficking than Rab3D.

As interesting as our data is in showing that Rab27b can be used to upregulate protein
secretion in vitro, there are many more issues which must be considered before we can
begin design drug delivery therapies which target Rab27b. We made an assumption that
the upregulation of protein secretion from the lacrimal gland would aid patients with dry
eye syndromes. One main clinical issue that must be addressed is how much
improvement patients with dry eye would experience with an increase of tear proteins in
tear fluid and whether it is possible to design a means to target Rab27b upregulation
without having the side effects and risks of the treatment outweigh the benefits. Dry eye
diseases can arise from different causes. For instance, dry eye due to Sjögren’s Syndrome
is caused by a systemic autoimmune attack on several exocrine glands including the
lacrimal gland, while evaporative dry eyes is a term describing rapid evaporation of tears
due to changes in tear constituency. While the source of the aqueous portion of the tear
fluid is usually attributed to the ductal cells in the lacrimal gland, acinar cells contribute a
watery serous secretion inclusive of the critical protein components (19). To understand
the benefits of Rab27b upregulation, we would need to examine the contents of the
Rab27b-enriched SV population and what these contents contribute to the ocular surface.
Increased protein secretion from the lacrimal gland, especially in dry eye patients who
experience lowered protein concentrations in their tear fluid could increase protection of
the eye from external pathogens, as well as provide constituents such as lacritin and EGF

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which are necessary for the maintenance of ocular health. In examining these effects, we
may also increase our understanding of the function of specific tear components, much of
which are not yet known.

The usage of largely imaging-based studies also has its limitations. The first is a technical
limitation- many of our studies depend on the availability of specific antibodies, which
means that not only do the antibodies have to be commercially available or designed and
produced by ourselves, but that sufficient information must be available of markers for
specific organelles or pathway steps. While imaging studies yield data with high temporal
and spatial value not attainable in many other assays, these studies are limited to what can
be detected. We experienced this issue during our search for the identity of the NVS in
Section 6.4. The ambiguity of the NVS was due in part to the limited availability of
specific trans-Golgi markers, and more specific markers which were conducive to live
cell fluorescent imaging would have been helpful to this study. Secondly, our
investigation was complicated by the expression of multiple isoforms with potential for
compensatory function, such as in the co-localization study concerning the functional
mutants of Rab27b and M5C in Section 6.5. Results suggested that Rab27b and M5C did
not associate, since M5C was able to recruit to SV membrane without functional Rab27b,
and Rab27b was able to localize in the subapical region without functional M5C.
However, it was possible that low level expression of Rab27a and M5A/B respectively
were able to rescue some of the effects of the dominant-negative Rab27b and M5C.
Moreover, we could not discount the possibility that the same transduced LGAC

105

expressing the mutant forms could also express a proportionally lower level of the
endogenous protein which may be sufficient for function. While we were able to establish
that Rab27b and M5C were both highly expressed isoforms compared to their respective
isoforms, even a low level of expression of another isoform could potentially bias our
results. Knockdown perhaps by the design of siRNA, or the development of assays which
test highly specific interactions between certain isoforms, would be necessary to
eliminate the possibility of compensatory functions in other isoforms. Lastly, the
secretion studies such as those conducted in Section 5.4 provide relative but not absolute
secretion information. This means that the percentage of Sync recovered, for example, in
the DN Rab27b is only comparable to the recovery value of WT Rab27b. In order to
obtain absolute values, we would have to complete a similar assay in vivo.

There are many directions to take Rab27b studies in the lacrimal gland, but all are
important. First, the advancement of Rab27b studies as a potential target for the
upregulation of tear protein secretion requires understanding of the mechanisms behind
Rab27b interactions with its effector proteins. It is the belief of this author that Rab27b
plays multiple temporally and spatially separated roles which eventually result in
regulated secretion- similar to a role as a gatekeeper of many gates- and that the key to
this is in Rab27b’s interactions with specific effectors at each gate. These effectors will
need to be identified by extensive assays testing for biochemical interactions of LGAC
proteins. Secondly, we have identified a specific pool of SV which are Rab27b-enriched
but with unknown content. An important goal is to establish SV content markers

106

endogenous to LGAC. This will require extensive biochemical analysis of protein in
Rab27b-enriched SV, but perhaps even more challenging, the development a protocol
which will enable the isolation of Rab27b-enriched SV pool. This is particularly difficult
given that LGAC SV are prone to fragmentation using most isolation protocols. Thirdly,
in clinic, tear studies are already underway and efforts are being made to utilize tear
markers to understand eye diseases, although little is known yet of many tear proteins
(11, 43, 111). Collection of tears from DKO and 27bKO mice could yield clues to SV
content changes resulting from loss of Rab27b function. Additionally, analysis of the
DKO and 27bKO mouse ocular surface would yield useful information regarding
whether Rab27 function affects the disease state. Finally, an observation was made in this
study that suggested that the loss of functional Rab27b in 27bKO and DKO mice affects
not only SV numbers, but influences general cell health and increases degradative
pathway activity. Future studies exploring the relationship between Rab27b and the
degradative pathway would provide interesting data and potentially demonstrate a
connection between the secretory pathway and cell homeostasis. Specific experiments
relating to this include the confirmation of autophagic organelle expression, which can be
completed with the use of a grid on TEM samples in parallel to immunofluorescent
studies to detect for autophagic markers, or usage of immunogold labeling of autophagic
markers.

This study establishes Rab27b as a positive regulator of exocytosis, as well as a SV
marker in LGAC, through detailed imaging systems supported by biochemical and

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functional studies. This is also the first demonstration of a phenotype associated with loss
of Rab27b in any exocrine tissue, substantiating its role in exocrine secretory trafficking.

108

References

1. Abu-Hamdah, R., W. J. Cho, J. K. Horber, and B. P. Jena. 2006. Secretory
vesicles in live cells are not free-floating but tethered to filamentous structures: a
study using photonic force microscopy. Ultramicroscopy 106:670-3.

2. Anant, J. S., L. Desnoyers, M. Machius, B. Demeler, J. C. Hansen, K. D.
Westover, J. Deisenhofer, and M. C. Seabra. 1998. Mechanism of Rab
geranylgeranylation: formation of the catalytic ternary complex. Biochemistry
37:12559-68.

3. Barral, D. C., J. S. Ramalho, R. Anders, A. N. Hume, H. J. Knapton, T.
Tolmachova, L. M. Collinson, D. Goulding, K. S. Authi, and M. C. Seabra.
2002. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli
syndrome. J Clin Invest 110:247-57.

4. Burkhardt, J. K., C. J. Echeverri, T. Nilsson, and R. B. Vallee. 1997.
Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts
dynein-dependent maintenance of membrane organelle distribution. J Cell Biol
139:469-84.

5. Busson-Mabillot, S., A. M. Chambaut-Guerin, L. Ovtracht, P. Muller, and B.
Rossignol. 1982. Microtubules and protein secretion in rat lacrimal glands:
localization of short-term effects of colchicine on the secretory process. J Cell
Biol 95:105-17.

6. Chen, D., J. Guo, T. Miki, M. Tachibana, and W. A. Gahl. 1997. Molecular
cloning and characterization of rab27a and rab27b, novel human rab proteins
shared by melanocytes and platelets. Biochem Mol Med 60:27-37.

7. Chen, X., C. Li, T. Izumi, S. A. Ernst, P. C. Andrews, and J. A. Williams.
2004. Rab27b localizes to zymogen granules and regulates pancreatic acinar
exocytosis. Biochem Biophys Res Commun 323:1157-62.

8. Chen, X., A. K. Walker, J. R. Strahler, E. S. Simon, S. L. Tomanicek-Volk,
B. B. Nelson, M. C. Hurley, S. A. Ernst, J. A. Williams, and P. C. Andrews.
2006. Organellar proteomics: analysis of pancreatic zymogen granule membranes.
Mol Cell Proteomics 5:306-12.

9. da Costa, S. R., K. Wu, M. M. Veigh, M. Pidgeon, C. Ding, J. E. Schechter,
and S. F. Hamm-Alvarez. 2006. Male NOD mouse external lacrimal glands
exhibit profound changes in the exocytotic pathway early in postnatal
development. Exp Eye Res 82:33-45.

109

10. da Costa, S. R., F. A. Yarber, L. Zhang, M. Sonee, and S. F. Hamm-Alvarez.
1998. Microtubules facilitate the stimulated secretion of beta-hexosaminidase in
lacrimal acinar cells. J Cell Sci 111 ( Pt 9):1267-76.

11. de Souza, G. A., L. M. Godoy, and M. Mann. 2006. Identification of 491
proteins in the tear fluid proteome reveals a large number of proteases and
protease inhibitors. Genome Biol 7:R72.

12. Deacon, S. W., and V. I. Gelfand. 2001. Of yeast, mice, and men. Rab proteins
and organelle transport. J Cell Biol 152:F21-4.

13. Dechecchi, M. C., P. Melotti, A. Bonizzato, M. Santacatterina, M. Chilosi,
and G. Cabrini. 2001. Heparan sulfate glycosaminoglycans are receptors
sufficient to mediate the initial binding of adenovirus types 2 and 5. J Virol
75:8772-80.

14. Dechecchi, M. C., A. Tamanini, A. Bonizzato, and G. Cabrini. 2000. Heparan
sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell
interactions. Virology 268:382-90.

15. Dell'Angelica, E. C. 2007. Bad signals jam organelle traffic. Nat Med 13:31-2.

16. Deneka, M., M. Neeft, and P. van der Sluijs. 2003. Regulation of membrane
transport by rab GTPases. Crit Rev Biochem Mol Biol 38:121-42.

17. Derby, M. C., C. van Vliet, D. Brown, M. R. Luke, L. Lu, W. Hong, J. L.
Stow, and P. A. Gleeson. 2004. Mammalian GRIP domain proteins differ in their
membrane binding properties and are recruited to distinct domains of the TGN. J
Cell Sci 117:5865-74.

18. Detter, J. C., Q. Zhang, E. H. Mules, E. K. Novak, V. S. Mishra, W. Li, E. B.
McMurtrie, V. T. Tchernev, M. R. Wallace, M. C. Seabra, R. T. Swank, and
S. F. Kingsmore. 2000. Rab geranylgeranyl transferase alpha mutation in the
gunmetal mouse reduces Rab prenylation and platelet synthesis. Proc Natl Acad
Sci U S A 97:4144-9.

19. Edman, M. C., R. R. Marchelletta, and S. F. Hamm-Alvarez. 2010. Ocular
Surface: Lacrimal Gland Overview. In J. Besharse, R. Dana, and D. A. Dartt (ed.),
Encyclopedia of the Eye, 1 ed. El Sevier.

20. Evans, E., W. Zhang, G. Jerdeva, C. Y. Chen, X. Chen, S. F. Hamm-Alvarez,
and C. T. Okamoto. 2008. Direct interaction between Rab3D and the polymeric
immunoglobulin receptor and trafficking through regulated secretory vesicles in
lacrimal gland acinar cells. Am J Physiol Cell Physiol 294:C662-74.

110

21. Falcone, S., E. Cocucci, P. Podini, T. Kirchhausen, E. Clementi, and J.
Meldolesi. 2006. Macropinocytosis: regulated coordination of endocytic and
exocytic membrane traffic events. J Cell Sci 119:4758-69.

22. Felmy, F. 2007. Modulation of cargo release from dense core granules by size
and actin network. Traffic 8:983-97.

23. Fukuda, M. 2006. Distinct Rab27A binding affinities of Slp2-a and Slac2-
a/melanophilin: Hierarchy of Rab27A effectors. Biochem Biophys Res Commun
343:666-74.

24. Fukuda, M. 1991. Lysosomal membrane glycoproteins. Structure, biosynthesis,
and intracellular trafficking. J Biol Chem 266:21327-30.

25. Fukuda, M. 2006. Rab27 and its effectors in secretory granule exocytosis: a
novel docking machinery composed of a Rab27.effector complex. Biochem Soc
Trans 34:691-5.

26. Fukuda, M. 2002. Synaptotagmin-like protein (Slp) homology domain 1 of
Slac2-a/melanophilin is a critical determinant of GTP-dependent specific binding
to Rab27A. J Biol Chem 277:40118-24.

27. Fukuda, M. 2005. Versatile role of Rab27 in membrane trafficking: focus on the
Rab27 effector families. J Biochem 137:9-16.

28. Fukuda, M., and T. S. Kuroda. 2002. Slac2-c (synaptotagmin-like protein
homologue lacking C2 domains-c), a novel linker protein that interacts with
Rab27, myosin Va/VIIa, and actin. J Biol Chem 277:43096-103.

29. Fukuda, M., T. S. Kuroda, and K. Mikoshiba. 2002. Slac2-a/melanophilin, the
missing link between Rab27 and myosin Va: implications of a tripartite protein
complex for melanosome transport. J Biol Chem 277:12432-6.

30. Furuno, K., S. Yano, K. Akasaki, Y. Tanaka, Y. Yamaguchi, H. Tsuji, M.
Himeno, and K. Kato. 1989. Biochemical analysis of the movement of a major
lysosomal membrane glycoprotein in the endocytic membrane system. J Biochem
106:717-22.

31. Gaden, F., L. Franqueville, S. S. Hong, V. Legrand, C. Figarella, and P.
Boulanger. 2002. Mechanism of restriction of normal and cystic fibrosis
transmembrane conductance regulator-deficient human tracheal gland cells to
adenovirus infection and ad-mediated gene transfer. Am J Respir Cell Mol Biol
27:628-40.

111

32. Gierow, J. P., R. W. Lambert, and A. K. Mircheff. 1995. Fluid phase
endocytosis by isolated rabbit lacrimal gland acinar cells. Exp Eye Res 60:511-
25.

33. Gierow, J. P., T. Yang, A. Bekmezian, N. Liu, J. M. Norian, S. A. Kim, S.
Rafisolyman, H. Zeng, C. T. Okamoto, R. L. Wood, and A. K. Mircheff.
1996. Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a
case of mistaken identity. Am J Physiol 271:C1685-98.

34. Gomes, A. Q., B. R. Ali, J. S. Ramalho, R. F. Godfrey, D. C. Barral, A. N.
Hume, and M. C. Seabra. 2003. Membrane targeting of Rab GTPases is
influenced by the prenylation motif. Mol Biol Cell 14:1882-99.

35. Gomi, H., K. Mori, S. Itohara, and T. Izumi. 2007. Rab27b is expressed in a
wide range of exocytic cells and involved in the delivery of secretory granules
near the plasma membrane. Mol Biol Cell 18:4377-86.

36. Goud, B., and P. A. Gleeson. 2010. TGN golgins, Rabs and cytoskeleton:
regulating the Golgi trafficking highways. Trends Cell Biol 20:329-36.

37. Grosshans, B. L., D. Ortiz, and P. Novick. 2006. Rabs and their effectors:
achieving specificity in membrane traffic. Proc Natl Acad Sci U S A 103:11821-
7.

38. Guo, Y., T. C. Walther, M. Rao, N. Stuurman, G. Goshima, K. Terayama, J.
S. Wong, R. D. Vale, P. Walter, and R. V. Farese. 2008. Functional genomic
screen reveals genes involved in lipid-droplet formation and utilization. Nature
453:657-61.

39. Haghnia, M., V. Cavalli, S. B. Shah, K. Schimmelpfeng, R. Brusch, G. Yang,
C. Herrera, A. Pilling, and L. S. Goldstein. 2007. Dynactin is required for
coordinated bidirectional motility, but not for dynein membrane attachment. Mol
Biol Cell 18:2081-9.

40. Hamm-Alvarez, S. F., S. Da Costa, T. Yang, X. Wei, J. P. Gierow, and A. K.
Mircheff. 1997. Cholinergic stimulation of lacrimal acinar cells promotes
redistribution of membrane-associated kinesin and the secretory protein, beta-
hexosaminidase, and increases kinesin motor activity. Exp Eye Res 64:141-56.

41. Handley, M. T., and R. D. Burgoyne. 2008. The Rab27 effector Rabphilin,
unlike Granuphilin and Noc2, rapidly exchanges between secretory granules and
cytosol in PC12 cells. Biochem Biophys Res Commun 373:275-81.

112

42. He, H., Y. Dang, F. Dai, Z. Guo, J. Wu, X. She, Y. Pei, Y. Chen, W. Ling, C.
Wu, S. Zhao, J. O. Liu, and L. Yu. 2003. Post-translational modifications of
three members of the human MAP1LC3 family and detection of a novel type of
modification for MAP1LC3B. J Biol Chem 278:29278-87.

43. Herber, S., F. H. Grus, P. Sabuncuo, and A. J. Augustin. 2001. Two-
dimensional analysis of tear protein patterns of diabetic patients. Electrophoresis
22:1838-44.

44. Hodel, A., and J. M. Edwardson. 2000. Targeting of the zymogen-granule
protein syncollin in AR42J and AtT-20 cells. Biochem J 350 Pt 3:637-43.

45. Hume, A. N., L. M. Collinson, A. Rapak, A. Q. Gomes, C. R. Hopkins, and
M. C. Seabra. 2001. Rab27a regulates the peripheral distribution of melanosomes
in melanocytes. J Cell Biol 152:795-808.

46. Hume, A. N., A. K. Tarafder, J. S. Ramalho, E. V. Sviderskaya, and M. C.
Seabra. 2006. A coiled-coil domain of melanophilin is essential for Myosin Va
recruitment and melanosome transport in melanocytes. Mol Biol Cell 17:4720-35.
47. Imai, A., M. Fukuda, S. Yoshie, T. Nashida, and H. Shimomura. 2009.
Redistribution of Rab27-specific effector Slac2-c, but not Slp4-a, after
isoproterenol-stimulation in rat parotid acinar cells. Arch Oral Biol 54:361-8.

48. Imai, A., S. Yoshie, T. Nashida, M. Fukuda, and H. Shimomura. 2009.
Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells
after stimulation with isoproterenol. Eur J Oral Sci 117:224-30.

49. Imai, A., S. Yoshie, T. Nashida, H. Shimomura, and M. Fukuda. 2004. The
small GTPase Rab27B regulates amylase release from rat parotid acinar cells. J
Cell Sci 117:1945-53.

50. Izumi, T. 2007. Physiological roles of Rab27 effectors in regulated exocytosis.
Endocr J 54:649-57.

51. Izumi, T., H. Gomi, K. Kasai, S. Mizutani, and S. Torii. 2003. The roles of
Rab27 and its effectors in the regulated secretory pathways. Cell Struct Funct
28:465-74.

52. Jacobs, D. T., R. Weigert, K. D. Grode, J. G. Donaldson, and R. E. Cheney.
2009. Myosin Vc is a molecular motor that functions in secretory granule
trafficking. Mol Biol Cell 20:4471-88.

113

53. Jerdeva, G. V., K. Wu, F. A. Yarber, C. J. Rhodes, D. Kalman, J. E.
Schechter, and S. F. Hamm-Alvarez. 2005a. Actin and non-muscle myosin II
facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial
cells. J Cell Sci 118:4797-812.

54. Jerdeva, G. V., F. A. Yarber, M. D. Trousdale, C. J. Rhodes, C. T. Okamoto,
D. A. Dartt, and S. F. Hamm-Alvarez. 2005b. Dominant-negative PKC-epsilon
impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated
secretion in rabbit lacrimal acini. Am J Physiol Cell Physiol 289:C1052-68.

55. Johnson, J. L., A. A. Brzezinska, T. Tolmachova, D. B. Munafo, B. A. Ellis,
M. C. Seabra, H. Hong, and S. D. Catz. 2009. Rab27a and Rab27b Regulate
Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by
Independent Mechanisms. Traffic.

56. Jordan, A., and J. Baum. 1980. Basic tear flow. Does it exist? Ophthalmology
87:920-30.

57. Kantheti, P., X. Qiao, M. E. Diaz, A. A. Peden, G. E. Meyer, S. L. Carskadon,
D. Kapfhamer, D. Sufalko, M. S. Robinson, J. L. Noebels, and M.
Burmeister. 1998. Mutation in AP-3 delta in the mocha mouse links endosomal
transport to storage deficiency in platelets, melanosomes, and synaptic vesicles.
Neuron 21:111-22.

58. Kim, H., S. C. Ling, G. C. Rogers, C. Kural, P. R. Selvin, S. L. Rogers, and V.
I. Gelfand. 2007. Microtubule binding by dynactin is required for microtubule
organization but not cargo transport. J Cell Biol 176:641-51.
59. Kochl, R., X. W. Hu, E. Y. Chan, and S. A. Tooze. 2006. Microtubules
facilitate autophagosome formation and fusion of autophagosomes with
endosomes. Traffic 7:129-45.

60. Kogel, T., and H. H. Gerdes. 2010. Maturation of secretory granules. Results
Probl Cell Differ 50:1-20.

61. Kondo, H., R. Shirakawa, T. Higashi, M. Kawato, M. Fukuda, T. Kita, and
H. Horiuchi. 2006. Constitutive GDP/GTP exchange and secretion-dependent
GTP hydrolysis activity for Rab27 in platelets. J Biol Chem 281:28657-65.

62. Kundu, M., and C. B. Thompson. 2005. Macroautophagy versus mitochondrial
autophagy: a question of fate? Cell Death Differ 12 Suppl 2:1484-9.

63. Kuroda, T. S., and M. Fukuda. 2005. Identification and biochemical analysis of
Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.
Methods Enzymol 403:431-44.

114

64. Laird, F. M., M. H. Farah, S. Ackerley, A. Hoke, N. Maragakis, J. D.
Rothstein, J. Griffin, D. L. Price, L. J. Martin, and P. C. Wong. 2008. Motor
neuron disease occurring in a mutant dynactin mouse model is characterized by
defects in vesicular trafficking. J Neurosci 28:1997-2005.

65. Lane, P. W., and M. S. Deol. 1974. Mocha, a new coat color and behavior
mutation on chromosome 10 of the mouse. J Hered 65:362-4.

66. Lapierre, L. A., and J. R. Goldenring. 2005. Interactions of myosin vb with
rab11 family members and cargoes traversing the plasma membrane recycling
system. Methods Enzymol 403:715-23.

67. Lise, M. F., T. P. Wong, A. Trinh, R. M. Hines, L. Liu, R. Kang, D. J. Hines,
J. Lu, J. R. Goldenring, Y. T. Wang, and A. El-Husseini. 2006. Involvement of
myosin Vb in glutamate receptor trafficking. J Biol Chem 281:3669-78.

68. Ma, L., V. P. Bindokas, A. Kuznetsov, C. Rhodes, L. Hays, J. M. Edwardson,
K. Ueda, D. F. Steiner, and L. H. Philipson. 2004. Direct imaging shows that
insulin granule exocytosis occurs by complete vesicle fusion. Proc Natl Acad Sci
U S A 101:9266-71.

69. Marchelletta, R. R., D. T. Jacobs, J. E. Schechter, R. E. Cheney, and S. F.
Hamm-Alvarez. 2008. The Class V myosin motor, Myosin 5c, localizes to
mature secretory vesicles and facilitates exocytosis in lacrimal acini. Am J
Physiol Cell Physiol.

70. Marsh, B. J., C. Soden, C. Alarcon, B. L. Wicksteed, K. Yaekura, A. J.
Costin, G. P. Morgan, and C. J. Rhodes. 2007. Regulated autophagy controls
hormone content in secretory-deficient pancreatic endocrine beta-cells. Mol
Endocrinol 21:2255-69.

71. Mehta, A. D., R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E.
Cheney. 1999. Myosin-V is a processive actin-based motor. Nature 400:590-3.

72. Meier, O., K. Boucke, S. V. Hammer, S. Keller, R. P. Stidwill, S. Hemmi, and
U. F. Greber. 2002. Adenovirus triggers macropinocytosis and endosomal
leakage together with its clathrin-mediated uptake. J Cell Biol 158:1119-31.

73. Mercer, J., and A. Helenius. 2009. Virus entry by macropinocytosis. Nat Cell
Biol 11:510-20.

115

74. Mircheff, A. K. 1989. Isolation of plasma membranes from polar cells and
tissues: apical/basolateral separation, purity, function. Methods Enzymol 172:18-
34.

75. Mircheff, A. K., J. Xie, L. Qian, and S. F. Hamm-Alvarez. 2002. Diverse
perturbations may alter the lacrimal acinar cell autoantigenic spectra. DNA Cell
Biol 21:435-42.

76. Mooseker, M. S., and L. G. Tilney. 1975. Organization of an actin filament-
membrane complex. Filament polarity and membrane attachment in the microvilli
of intestinal epithelial cells. J Cell Biol 67:725-43.

77. Muallem, S., K. Kwiatkowska, X. Xu, and H. L. Yin. 1995. Actin filament
disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell
Biol 128:589-98.

78. Murphy, D. B., and K. T. Wallis. 1983. Isolation of microtubule protein from
chicken erythrocytes and determination of the critical concentration for tubulin
polymerization in vitro and in vivo. J Biol Chem 258:8357-64.

79. Nedvetsky, P. I., E. Stefan, S. Frische, K. Santamaria, B. Wiesner, G. Valenti,
J. A. Hammer, 3rd, S. Nielsen, J. R. Goldenring, W. Rosenthal, and E.
Klussmann. 2007. A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2
shuttle. Traffic 8:110-23.

80. Ostrowski, M., N. B. Carmo, S. Krumeich, I. Fanget, G. Raposo, A. Savina,
C. F. Moita, K. Schauer, A. N. Hume, R. P. Freitas, B. Goud, P. Benaroch, N.
Hacohen, M. Fukuda, C. Desnos, M. C. Seabra, F. Darchen, S. Amigorena, L.
F. Moita, and C. Thery. 2010. Rab27a and Rab27b control different steps of the
exosome secretion pathway. Nat Cell Biol 12:19-30; sup pp 1-13.

81. Ota, I., D. Zoukhri, R. R. Hodges, J. D. Rios, V. Tepavcevic, I. Raddassi, L.
L. Chen, and D. A. Dartt. 2003. Alpha 1-adrenergic and cholinergic agonists
activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland. Am
J Physiol Cell Physiol 284:C168-78.

82. Pereira-Leal, J. B., and M. C. Seabra. 2000. The mammalian Rab family of
small GTPases: definition of family and subfamily sequence motifs suggests a
mechanism for functional specificity in the Ras superfamily. J Mol Biol
301:1077-87.

83. Pfeffer, S. R. 2007. Unsolved mysteries in membrane traffic. Annu Rev Biochem
76:629-45.

116

84. Pickett, J. A., and J. M. Edwardson. 2006. Compound exocytosis: mechanisms
and functional significance. Traffic 7:109-16.

85. Qian, L., J. Xie, C. M. Rose, E. Sou, H. Zeng, S. F. Hamm-Alvarez, and A. K.
Mircheff. 2004. Altered traffic to the lysosome in an ex vivo lacrimal acinar cell
model for chronic muscarinic receptor stimulation. Exp Eye Res 79:665-75.

86. Ramalho, J. S., R. Anders, G. B. Jaissle, M. W. Seeliger, C. Huxley, and M.
C. Seabra. 2002. Rapid degradation of dominant-negative Rab27 proteins in vivo
precludes their use in transgenic mouse models. BMC Cell Biol 3:26.

87. Ramalho, J. S., T. Tolmachova, A. N. Hume, A. McGuigan, C. Y. Gregory-
Evans, C. Huxley, and M. C. Seabra. 2001. Chromosomal mapping, gene
structure and characterization of the human and murine RAB27B gene. BMC
Genet 2:2.

88. Rentsendorj, A., H. Agadjanian, X. Chen, M. Cirivello, M. Macveigh, L.
Kedes, S. Hamm-Alvarez, and L. K. Medina-Kauwe. 2005. The Ad5 fiber
mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel
cell entry pathway. Gene Ther 12:225-37.

89. Rentsendorj, A., J. Xie, M. MacVeigh, H. Agadjanian, S. Bass, D. H. Kim, J.
Rossi, S. F. Hamm-Alvarez, and L. K. Medina-Kauwe. 2006. Typical and
atypical trafficking pathways of Ad5 penton base recombinant protein:
implications for gene transfer. Gene Ther 13:821-36.

90. Riedel, D., W. Antonin, R. Fernandez-Chacon, G. Alvarez de Toledo, T. Jo,
M. Geppert, J. A. Valentijn, K. Valentijn, J. D. Jamieson, T. C. Sudhof, and
R. Jahn. 2002. Rab3D is not required for exocrine exocytosis but for
maintenance of normally sized secretory granules. Mol Cell Biol 22:6487-97.

91. Robin, P., B. Rossignol, and M. N. Raymond. 1995. Effect of microtubule
network disturbance by nocodazole and docetaxel (Taxotere) on protein secretion
in rat extraorbital lacrimal and parotid glands. Eur J Cell Biol 67:227-37.

92. Rodriguez, O. C., and R. E. Cheney. 2002. Human myosin-Vc is a novel class
V myosin expressed in epithelial cells. J Cell Sci 115:991-1004.

93. Rolando, M., and M. Zierhut. 2001. The ocular surface and tear film and their
dysfunction in dry eye disease. Surv Ophthalmol 45 Suppl 2:S203-10.

117

94. Rottger, S., J. White, H. H. Wandall, J. C. Olivo, A. Stark, E. P. Bennett, C.
Whitehouse, E. G. Berger, H. Clausen, and T. Nilsson. 1998. Localization of
three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of
O-linked glycosylation throughout the Golgi apparatus. J Cell Sci 111 ( Pt 1):45-
60.

95. Rudolf, R., T. Salm, A. Rustom, and H. H. Gerdes. 2001. Dynamics of
immature secretory granules: role of cytoskeletal elements during transport,
cortical restriction, and F-actin-dependent tethering. Mol Biol Cell 12:1353-65.

96. Saftig, P., and J. Klumperman. 2009. Lysosome biogenesis and lysosomal
membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 10:623-35.

97. Sanghi, S., R. Kumar, A. Lumsden, D. Dickinson, V. Klepeis, V. Trinkaus-
Randall, H. F. Frierson, Jr., and G. W. Laurie. 2001. cDNA and genomic
cloning of lacritin, a novel secretion enhancing factor from the human lacrimal
gland. J Mol Biol 310:127-39.

98. Schechter, J. E., D. W. Warren, and A. K. Mircheff. 2010. A lacrimal gland is
a lacrimal gland, but rodent's and rabbit's are not human. Ocul Surf 8:111-34.

99. Schluter, O. M., M. Khvotchev, R. Jahn, and T. C. Sudhof. 2002. Localization
versus function of Rab3 proteins. Evidence for a common regulatory role in
controlling fusion. J Biol Chem 277:40919-29.

100. Seabra, M. C., and E. Coudrier. 2004. Rab GTPases and myosin motors in
organelle motility. Traffic 5:393-9.

101. Seabra, M. C., Y. K. Ho, and J. S. Anant. 1995. Deficient geranylgeranylation
of Ram/Rab27 in choroideremia. J Biol Chem 270:24420-7.

102. Seabra, M. C., E. H. Mules, and A. N. Hume. 2002. Rab GTPases, intracellular
traffic and disease. Trends Mol Med 8:23-30.

103. Seabra, M. C., and C. Wasmeier. 2004. Controlling the location and activation
of Rab GTPases. Curr Opin Cell Biol 16:451-7.

104. Shirakawa, R., T. Higashi, A. Tabuchi, A. Yoshioka, H. Nishioka, M.
Fukuda, T. Kita, and H. Horiuchi. 2004. Munc13-4 is a GTP-Rab27-binding
protein regulating dense core granule secretion in platelets. J Biol Chem
279:10730-7.

105. Stinchcombe, J., G. Bossi, and G. M. Griffiths. 2004. Linking albinism and
immunity: the secrets of secretory lysosomes. Science 305:55-9.

118

106. Suda, J., Zhu, L., Okamoto, C., Karvar, S. 2010 (in press). Cellular distribution
and functional importance of Rab27b in gastric parietal cells. Gastroenterology.

107. Swank, R. T., M. Reddington, O. Howlett, and E. K. Novak. 1991. Platelet
storage pool deficiency associated with inherited abnormalities of the inner ear in
the mouse pigment mutants muted and mocha. Blood 78:2036-44.

108. Tiwari, S., J. E. Italiano, Jr., D. C. Barral, E. H. Mules, E. K. Novak, R. T.
Swank, M. C. Seabra, and R. A. Shivdasani. 2003. A role for Rab27b in NF-
E2-dependent pathways of platelet formation. Blood 102:3970-9.

109. Tolmachova, T., M. Abrink, C. E. Futter, K. S. Authi, and M. C. Seabra.
2007. Rab27b regulates number and secretion of platelet dense granules. Proc
Natl Acad Sci U S A 104:5872-7.

110. Tolmachova, T., R. Anders, J. Stinchcombe, G. Bossi, G. M. Griffiths, C.
Huxley, and M. C. Seabra. 2004. A general role for Rab27a in secretory cells.
Mol Biol Cell 15:332-44.

111. Tomosugi, N., K. Kitagawa, N. Takahashi, S. Sugai, and I. Ishikawa. 2005.
Diagnostic potential of tear proteomic patterns in Sjogren's syndrome. J Proteome
Res 4:820-5.

112. Turvey, M. R., and P. Thorn. 2004. Lysine-fixable dye tracing of exocytosis
shows F-actin coating is a step that follows granule fusion in pancreatic acinar
cells. Pflugers Arch 448:552-5.

113. Valencia, A., P. Chardin, A. Wittinghofer, and C. Sander. 1991. The ras
protein family: evolutionary tree and role of conserved amino acids. Biochemistry
30:4637-48.

114. Valentijn, J. A., K. Valentijn, L. M. Pastore, and J. D. Jamieson. 2000. Actin
coating of secretory granules during regulated exocytosis correlates with the
release of rab3D. Proc Natl Acad Sci U S A 97:1091-5.

115. Valentijn, K., J. A. Valentijn, and J. D. Jamieson. 1999. Role of actin in
regulated exocytosis and compensatory membrane retrieval: insights from an old
acquaintance. Biochem Biophys Res Commun 266:652-61.

116. Van Setten, G. B., L. Viinikka, T. Tervo, K. Pesonen, A. Tarkkanen, and J.
Perheentupa. 1989. Epidermal growth factor is a constant component of normal
human tear fluid. Graefes Arch Clin Exp Ophthalmol 227:184-7.

119

117. Vilalta, P. M., L. Zhang, and S. F. Hamm-Alvarez. 1998. A novel taxol-
induced vimentin phosphorylation and stabilization revealed by studies on stable
microtubules and vimentin intermediate filaments. J Cell Sci 111 ( Pt 13):1841-
52.

118. Wang, Y., C. T. Chiu, T. Nakamura, A. M. Walker, B. Petridou, M. D.
Trousdale, S. F. Hamm-Alvarez, J. E. Schechter, and A. K. Mircheff. 2007.
Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells.
Am J Physiol Endocrinol Metab 292:E1122-34.

119. Wang, Y., G. Jerdeva, F. A. Yarber, S. R. da Costa, J. Xie, L. Qian, C. M.
Rose, C. Mazurek, N. Kasahara, A. K. Mircheff, and S. F. Hamm-Alvarez.
2003. Cytoplasmic dynein participates in apically targeted stimulated secretory
traffic in primary rabbit lacrimal acinar epithelial cells. J Cell Sci 116:2051-65.

120. Wang, Y., J. Xie, F. A. Yarber, C. Mazurek, M. D. Trousdale, L. K. Medina-
Kauwe, N. Kasahara, and S. F. Hamm-Alvarez. 2004. Adenoviral capsid
modulates secretory compartment organization and function in acinar epithelial
cells from rabbit lacrimal gland. Gene Ther 11:970-81.

121. Waselle, L., T. Coppola, M. Fukuda, M. Iezzi, A. El-Amraoui, C. Petit, and
R. Regazzi. 2003. Involvement of the Rab27 binding protein Slac2c/MyRIP in
insulin exocytosis. Mol Biol Cell 14:4103-13.

122. Westbroek, W., J. Lambert, S. De Schepper, R. Kleta, K. Van Den Bossche,
M. C. Seabra, M. Huizing, M. Mommaas, and J. M. Naeyaert. 2004. Rab27b
is up-regulated in human Griscelli syndrome type II melanocytes and linked to the
actin cytoskeleton via exon F-Myosin Va transcripts. Pigment Cell Res 17:498-
505.

123. White, T., E. P. Bennett, K. Takio, T. Sorensen, N. Bonding, and H. Clausen.
1995. Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-
galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem
270:24156-65.

124. Wong, D. H., and F. M. Brodsky. 1992. 100-kD proteins of Golgi- and trans-
Golgi network-associated coated vesicles have related but distinct membrane
binding properties. J Cell Biol 117:1171-9.

125. Wu, J., Y. Dang, W. Su, C. Liu, H. Ma, Y. Shan, Y. Pei, B. Wan, J. Guo, and
L. Yu. 2006. Molecular cloning and characterization of rat LC3A and LC3B--two
novel markers of autophagosome. Biochem Biophys Res Commun 339:437-42.

120

126. Wu, K., G. V. Jerdeva, S. R. da Costa, E. Sou, J. E. Schechter, and S. F.
Hamm-Alvarez. 2006. Molecular mechanisms of lacrimal acinar secretory
vesicle exocytosis. Exp Eye Res 83:84-96.

127. Wu, X., B. Bowers, Q. Wei, B. Kocher, and J. A. Hammer, 3rd. 1997. Myosin
V associates with melanosomes in mouse melanocytes: evidence that myosin V is
an organelle motor. J Cell Sci 110 ( Pt 7):847-59.

128. Wu, X., K. Rao, M. B. Bowers, N. G. Copeland, N. A. Jenkins, and J. A.
Hammer, 3rd. 2001. Rab27a enables myosin Va-dependent melanosome capture
by recruiting the myosin to the organelle. J Cell Sci 114:1091-100.

129. Xie, J., L. Chiang, J. Contreras, K. Wu, J. A. Garner, L. Medina-Kauwe, and
S. F. Hamm-Alvarez. 2006. Novel fiber-dependent entry mechanism for
adenovirus serotype 5 in lacrimal acini. J Virol 80:11833-51.

130. Yi, Z., H. Yokota, S. Torii, T. Aoki, M. Hosaka, S. Zhao, K. Takata, T.
Takeuchi, and T. Izumi. 2002. The Rab27a/granuphilin complex regulates the
exocytosis of insulin-containing dense-core granules. Mol Cell Biol 22:1858-67.

131. Yla-Anttila, P., H. Vihinen, E. Jokitalo, and E. L. Eskelinen. 2009. Monitoring
autophagy by electron microscopy in Mammalian cells. Methods Enzymol
452:143-64.

132. Yoshino, A., B. M. Bieler, D. C. Harper, D. A. Cowan, S. Sutterwala, D. M.
Gay, N. B. Cole, J. M. McCaffery, and M. S. Marks. 2003. A role for GRIP
domain proteins and/or their ligands in structure and function of the trans Golgi
network. J Cell Sci 116:4441-54.

133. Zhao, S., S. Torii, H. Yokota-Hashimoto, T. Takeuchi, and T. Izumi. 2002.
Involvement of Rab27b in the regulated secretion of pituitary hormones.
Endocrinology 143:1817-24.

Abstract (if available)

Abstract Tear proteins are supplied by the regulated fusion of secretory vesicles with the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in regulating the exocytotic pathway of these secretory vesicles. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b-/- and Rab27ash/ash/Rab27b-/- mice, but not Ashen mice deficient in Rab27a, showed significant changes in organelle morphology which included an approximate 50% decrease in abundance of secretory vesicles and decreased vesicle localization in the subapical region. Along with an apparent secretory pathway effect, knockout of Rab27b also resulted in a two-fold increase in the number of lysosomes, four-fold increase number of damaged mitochondria, two-fold increase in the formation of autophagosome-like organelles, and observed increased ER swelling and vesiculation. Confocal fluorescence microscopy analysis, also confirmed by biochemical assays, of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were highly colocalized with Rab3D (73.6±2.0% at rest) and Myosin 5C (58.3±7.4% at rest). Stimulation of cultured acinar cells with the secretagogue, carbachol, resulted in apical fusion of these secretory vesicles with the plasma membrane.

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Asset Metadata

Creator Chiang, Lilian (author)

Core Title A study of the role of Rab27 in lacrimal gland acinar cell secretory trafficking

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 11/01/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag exocytosis,intracellular trafficking,lacrimal gland acinar cells,membrane,OAI-PMH Harvest,primary cell culture,secretory vesicles

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah F. (committee chair), McDonough, Alicia A. (committee member), Okamoto, Curtis Toshio (committee member), Shen, Wei-Chiang (committee member), Yu, Alan S. L. (committee member)

Creator Email lilian.chiang@gmail.com,lilianc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3516

Unique identifier UC1135128

Identifier etd-Chiang-4135 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-408978 (legacy record id),usctheses-m3516 (legacy record id)

Legacy Identifier etd-Chiang-4135.pdf

Dmrecord 408978

Document Type Dissertation

Rights Chiang, Lilian

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu