University of Southern California Dissertations and Theses

Studies of polymeric immunoglobin receptor (pIgR) trafficking pathway and evaluation of rAV-LifeAct-TagRFP function in rabbit lacrimal gland acinar cells

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Studies of polymeric immunoglobin receptor (pIgR) trafficking pathway and evaluation of rAV-LifeAct-TagRFP function in rabbit lacrimal gland acinar cells

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Content STUDIES OF POLYMERIC IMMUNOGLOBULIN RECEPTOR (pIgR)
TRAFFICKING PATHWAY
AND
EV ALUATION OF rAV-LifeAct-TagRFP FUNCTION IN RABBIT LACRIMAL
GLAND ACINAR CELLS

by

Linlin Ma

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

August 2012

Copyright 2012 Linlin Ma
ii

DEDICATION
This work is dedicated to my parents, Mr. Hongjun Ma and Mrs. Meiling Chen, without
whose caring supports it would not have been possible.
iii

ACKNOWLDEGEMENTS
I sincerely thank Shi Xu for not only his brilliant ideas which have enabled this project,
but for his sagely advice, patient instruction and unconditionally support.

Special thanks to Francie Yarber and Hua Pei for their hard work on cell preparations and
virus purification as well as amplification.

I also appreciate every single advice from my other lab colleagues: Aaron Hseuh,
Eunbyul Evans, Guoyong Sun, Janette Contreras, Juhi Firdos, Lilian Chiang, Mihir Shah,
Maria Edman, Srikanth Janga and Zhen Meng. Thank you all for making my experience
full of joy and values.

Above all, I would like to give my greatest gratitude to my mentor, Dr. Sarah
Hamm-Alvarez for her valuable guidance. This two year’s lab experience became
cherishing and inspired due to her patience and encouragement.

Finally, I thank my committee members: Dr. Okamoto and Dr. Garner for their valuable
time and insightful suggestions to the project.
iv

TABLE OF CONTENTS

DEDICATION ii
ACKNOWLDEGEMENTS iii
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT x
Chapter 1. INTRODUCTION 1
Lacrimal gland physiology 1
Description of polymeric immunoglobulin receptor (pIgR); structure and
function 4
Characteristics of Rab GTPase 5
Description of myosin Vb and myosin Vc 6
rAV-LifeAct-TagRFP-a novel F-actin marker 10
Goals and experiment design 12
Chapter 2. METHODS AND MATERIALS 14
Production of recombinant adenovirus constructs 14
Preparation and the treatment conditions of primary LGACs 16
The transduction of adenovirus recombinant constructs into LGAC 16
Immunostaining 17
Secretion assay 18
Uptake of pIgR-specific binding antibody 19
Immunostaining fluorescence image by confocal microscope 19
Time-lapse live cell imaging by confocal microscope 19
Reagents 20
v

Chapter 3. RESULTS 22
Expression of human pIgR-EGFP construct in primary LGACs 22
The transcytotic pathway of pIgR in LGACs 27
Endocytosed pIgR would be sorted to early endosomes and Rab11a vesicles
during the transcytosis pathway 32
Transportation of pIgR in transcytotic pathway requires the functionality of
Rab11a 39
The secretory pathway of pIgR is associated with Rab3D-enriched vesicles 42
Myosin Vc is critical in regulating Rab3D secretory pathway 44
Myosin V motor proteins play different roles in regulating the trafficking of
pIgR in LGACs 46
The distinction between the pIgR transcytotic pathway and regulated secretory
pathway 48
Secretion assay differentiates two pIgR trafficking pathways 55
MT and MF participate in regulating pIgR trafficking in LGACs 59
Visualization of F-actin locations and dynamics with rAV-LifeAct-TagRFP in
LGACs 65
Chapter 4. DISCUSSION 71
REFERENCES 78

vi

LIST OF FIGURES
Figure 1: In culture primary LGACs schematic structures 3
Figure 2: Schematic structure of myosin Va 9
Figure 3: Distribution pattern of pIgR in non-transduced LGACs 24
Figure 4: Anti-human SC antibodies identify hpIgR-EGFP in LGACs 25
Figure 5: Colocalization of hpIgR-EGFP with Rab11a 25
Figure 6: Anti-GFP antibody identifies hpIgR-EGFP 26
Figure 7: Trafficking of hpIgR-EGFP in transcytosis pathway with dIgA uptake 30
Figure 8: Trafficking of hpIgR-EGFP in transcytosis pathway with anti-human SC
antibody 31

Figure 9: Competitive experiment to verify the specificity of sheep anti-rabbit SC
antibody 35
Figure 10: The trafficking of endocytosed pIgR in LGACs 36
Figure 11: Endocytosed pIgR entered Rab11a vesicles in transcytotic pathway 37
Figure 12: Transcytosis of pIgR in LGACs requires the functionality of Rab11a 40
Figure 13: pIgR is associated with Rab3D-enriched secretory vesicles 43
Figure 14: Colocalization of myosin Vc construct with mCherry-Rab3D in
LGACs 45
Figure 15: Myosin Vb did not participate to regulate secretory pathway 47
Figure 16: Myosin V motors regulate different pathways in pIgR trafficking 49
Figure 17: CCh accelerates the transcytotic pathway of pIgR in LGACs 53
vii

Figure 18: The release of pIgR on apical membrane was dependent on myosin Vb
motor protein through transcysotic pathway 54
Figure 19: CCh increase SC release in LGACs over-expressing hpIgR-EGFP 57
Figure 20: The inhibition of SC under CCh treatment by over-expressing
mCherry-myosin Vb tail 58
Figure 21: MT network regulates the intracellular trafficking of pIgR in LGACs 61
Figure 22: Inhibition of SC release by expressing DN-PKC-ε in LGACs 63
Figure 23: DN-PKC-ε does not change localization of Rab11a and Rab3D 64
Figure 24: Visualization of F-actin with rA V-LifeAct-TagRFP in LGACs 67
Figure 25: The expression of Lifeact-RFP and GFP-actin in LGACs 68
Figure 26: Time-course confocal fluorescence microscopy of LGACs
co-expressing rA V-LifeAct-TagRFP and GFP-actin in CCHstimulation
reveals significant actin remodeling 69
Figure 27: Working model of the trafficking of pIgR in LGACs 75

viii

ABBREVIATIONS
Ad Adenovirus
APM Apical membrane
BLM Basolateral membrane
CA Constitutively Active
CCh Carbachol
dIgA Dimeric immunoglobulin A
DN Dominant Negative
EGFP Enhanced green fluorescent protein
LGAC Lacrimal gland acinar cells
MDCK Martin-Darby canine kidney
MTOC Microtubule organizing center
PCM Peter's complete medium
pIgR Polymeric immunoglobulin receptor
RFP Red fluorescent protein
SC Secretory component
SV Secretory vesicle

ix

WT Wild type
YFP Yellow fluorescent protein

x

ABSTRACT
In lacrimal gland acinar cells, the polymeric immunoglobulin receptor is responsible for
binding and transporting dimeric IgA from the basolateral membrane to subapical region
to maintain the immune defense. Using confocal microscopy as a major research tool;
two different trafficking pathways with unique Rab GTPases and motor proteins have
been defined. Using Rab11a and myosin Vb, the transcytotic pathway of pIgR mainly
mediates dIgA transportation and release under resting conditions. However, with
stimulation by the cholinergic agonist, carbachol, the transcytotic pathway is also highly
active and secretory component release is accelerated. The regulated secretory pathway is
dependent on Rab3D and myosin Vc. In this pathway, pIgR which is directly synthesized
but not routed via the basolateral membrane serves as the major source. With acute
exposure to carbachol, the regulated secretory pathway release considerable SC. The
microtubule network serves as a track for pIgR basal-to-apical transport and the
microfilament network determines the final release step of SC. rAV-LifeAct-TagRFP is a
novel and reliable marker for F-actin visualization. With no physiological functional
effect on the F-actin, this marker, expressed using adenovirus transduction, has a wide
application in studying F-actin location and dynamics in LGACs.
1

Chapter 1. INTRODUCTION

Lacrimal gland physiology
The lacrimal gland is a small organ located above the eye which is important to produce
tear proteins as well as maintain a healthy ocular surface (Rolando & Zierhut, 2001). It
has been confirmed that roughly 80% of the lacrimal gland is composed of secretory
epithelial cells, which is in charge of the aqueous layer of the eye (Dartt, 1994). The
lacrimal gland epithelial cells are bounded by cortical actin filaments underneath the
plasma membrane, with a thicker layer beneath the apical plasma membrane which aids
in identification of their polarized domains. These cells display a polarized organization
that orients their apical membrane towards a central lumen. When reconstituted in culture,
rabbit lacrimal gland epithelial cells re-form small cluster of acini and maintain
three-dimensional formation (Figure 1). The tear factors that the lacrimal gland packages
include antibacterial factors, e.g. secretory IgA, growth factors, and lysosomal hydrolases,
et al. (Chiang et al., 2011; Y . Wang et al., 2007)

Most LGACs produce secretory fluid that flows into the lumen. Beneath the lumen
bounded by the apical plasma membranes of each cell, there is an F-actin network, which
2

is also known as the terminal web (Valentijn, Valentijn, Pastore, & Jamieson, 2000). The
secretory pathway in LGACs has been characterized: Proteins that are destined to be
secreted into the lumen are packaged in secretory vesicles. These vesicles carrying
proteins bud from the trans-Golgi network then moved all the way towards apical
membrane, possibly along with microtubules network. It was also believed that certain
Rab proteins facilitate the trafficking steps with high specificity (Jerdeva, Yarber et al.,
2005; Seabra & Coudrier, 2004).

Previous work in our lab revealed a lot about the pathways in LGACs. In 2005, Jerdeva’s
work suggested that the exocytosis of secretory vesicles in LGACs could be facilitated by
the interaction of actin with non-muscle myosin Ⅱas well as the increased turnover of
apical actin filaments (33). In 2008, Marchelletta further confirmed that class V motor
protein, Myosin Vc in specific, had a role in maturation and the regulated exocytosis of
secretory vesicles (30). In Chiang’s paper which investigated Rab27b in LGACs, data
show that this GTPase might participate in secretory vesicle formation and release for the
first time (3). The pathways of pIgR were also studied by Evans and Xu. Evans’s work
suggested that Rab3D might play role in regulated secretory pathway of pIgR in LGACs
due to the novel localization of and interaction of Rab3D with pIgR (9). Xu, on the other
3

hand, suggested another pathway which was regulated by Rab11a as well as myosin Vb
protein (8).

Figure 1: In culture primary LGACs schematic structures
Left: 3D structure, Right: 2D cross-section of the 3D structure. (Adapted from (Xu et al., 2011))
4

Description of polymeric immunoglobulin receptor (pIgR); structure and function
Being a member of the immunoglobulin superfamily and a functional part of the mucosal
immune system, pIgR is mostly expressed in polarized epithelial cells including barrier
epithelia as well as hepatocytes. pIgR is also present in lacrimal gland acinar cells and
other salivary gland acinar cells (Evans et al., 2008; Kaetzel, 2005).

pIgR is a membrane glycoprotein composed of an extracellular domain, a single
transmembrane region and a cytoplasmic domain. The extracellular region responsible for
extracellular ligand binding contains five homologous domains, and the cytoplasmic
region is composed of 103- amino acids residues (Rojas & Apodaca, 2002).

After synthesis in rough endoplasmic reticulum, the pIgR is sent to the trans-Golgi
network for sorting. In well characterized model systems such as transfected MDCK cells
and hepatocytes like the WIF-B model (not in cells like acinar cells) (11), pIgR is
subsequently delivered from the TGN to the basolateral membrane where it binds to IgA
and IgM. With or without its natural ligand, pIgR is endocytosed and transported through
a series of endosomal compartments to the apical region, where the proteolytic cleavage
happens (Apodaca, Katz, & Mostov, 1994; Mostov, 1994; Okamoto, Shia, Bird, Mostov,
& Roth, 1992; Su et al., 2010). The cleaved extracellular domain of pIgR, without
5

binding to the ligand, is known as secretory component (SC). In the case of sIgA, dIgA is
covalently bound to SC by a single disulphide bond to the J-chain. Both free SC and sIgA
function to maintain the normal protection physiological properties of mucosal surfaces,
saliva and tears, et al (Musil & Baenziger, 1987; Norderhaug, Johansen, Schjerven, &
Brandtzaeg, 1999; Rojas & Apodaca, 2002).

Characteristics of Rab GTPase
As mentioned before, Rab GTPases play important roles in facilitating protein
transportation within LGACs, some of which are well characterized while some are not
well understood (Jerdeva, Yarber et al., 2005; Pfeffer, 2007; Seabra & Coudrier, 2004).
What is clear is that over seventy Rabs compose the largest branch of Ras superfamily of
GTPases. They differ from other proteins by two structural characteristics: Firstly, Rab
protein contains a conserved sequence which functions as a switch to control Rab binding
to GTP and GDP. Rab cycles between the GDP-bound inactive state and the GTP-bound
active state, using GAPs (GTPase activating proteins) and GEFs (guanine nucleotide
exchange factors) as assistants (Pereira-Leal & Seabra, 2000). Secondly, Rabs are
initially formed from the Golgi as soluble proteins and then are delivered to the site of
Rab geranylgeranyltrasferase. The latter acts on Rabs by adding geranylgeranyl groups to
C’terminus. This post-translational modification enables Rab proteins to associate with
6

membranes, critical for their ability to cycle between cytoplasm and membrane
compartments (Deneka, Neeft, & van der Sluijs, 2003; Detter et al., 2000).

In the previous work in our lab, some Rab proteins were identified as step-specific
markers and associated with transporting proteins. For instance, Rab3D as a mature
secretory vesicle marker was shown to have an interaction with pIgR and thus regulate its
recruitment into the pIgR secretory pathway. Rab27b participates in secretory vesicle
formation and release through LGACs. Rab11a is a vital regulator of dIgA trafficking and
also considered to be related to pIgR basal-to-apical transportation (Chiang et al., 2011;
Evans et al., 2008; Xu et al., 2011).

Description of myosin Vb and myosin Vc
Class V myosin motor proteins provide transport of organelles, membrane cargo,
secretory vesicles and protein vesicles along tracks of actin. In humans, three myosin V
proteins are known: Va, Vb and Vc. With similar structures, they participate in membrane
trafficking but play differentiated functions. Myosin Va is mostly found in neurons and
neuroendocrine cells while Myosin Vb and Vc are primarily found in epithelial cells
(Reck-Peterson, Provance, Mooseker, & Mercer, 2000; Rodriguez & Cheney, 2002).

7

Myosin V is composed of four major domains (Figure 2). At the N’ terminus, the motor
domain contains sites for actin and nucleotide binding. It uses energy that generated by
ATP hydrolysis to perform the sequential movements. The lever arm is α-helical shaped
and works to amplify small nucleotide-dependent changes into a larger power stroke after
ATP hydrolysis. The rod region is responsible for the dimerization for the myosin V
molecules. The globular tail locates at C’ terminal and binds to targeted cargo through the
linkage of some adapter proteins (Trybus, 2008).

The movement of myosin V motor proteins along actin is processive and ATP-dependent.
At a certain stage, both heads are bound to actin firmly with ADP at the active site. The
trailing head in a post-power stoke conformation will release ADP at a rate that limits the
ATPase cycle step. Then ATP binds to the trailing head and detaches it to complete power
stroke. The trailing head becomes the new leading head in a pre-power stoke
conformation, searching for the next actin binding site. The process is continuously
repeated leading myosin V to walk along the actin (Hodges, Krementsova, & Trybus,
2007).

Observations suggested that myosin Vb motor proteins were closely associated with
plasma membrane recycling compartments (Fan, Lapierre, Goldenring, Sai, & Richmond,
8

2004; Lapierre et al., 2001; Volpicelli, Lah, Fang, Goldenring, & Levey, 2002). In
polarized cells, myosin Vb was considered to participated in regulating the transcytosis of
IgG and dIgA together with Rab11a (Tzaban S et al., 2009; Xu et al., 2011). Myosin Vb is
also required for cystic fibrosis transmembrane conductance regulator recycling in apical
recycling endosomes in epithelial cells (Swiatecka-Urban A et al., 2007). In polarized
epithelial cyst cultures, Myo5B was required for apical membrane trafficking dependent
on association with Rab GTPase (Roland JT et al., 2011). On the other hand, myosin Vc
was primarily expressed in exocrine secretory tissues and localized beneath apical
membrane of epithelial cells (Rodriguez & Cheney, 2002).

Myosin V tail is a truncated version of the myosin V motor protein: it keeps the ability of
interacting with cargo proteins with the tail region, but lacks the motor region with
ATPase activity and motor functions. Thus the mCherry-tagged myosin Vb tail construct
serves as a dominant negative mutant for myosin Vb while EGFP-myosin Vc tail
construct serves as a dominant negative mutant for myosin Vc (Rodriguez OC et al.,
2002).

A lot of work had been done in our lab to investigate the role of myosin Vb and myosin
Vc motor protein in LGACs. The mature secretory vesicle exocytosis has been shown to
be associated with myosin Vc motor, which worked as a crucial regulator of actin
9

filament remodeling (Marchelletta RR et al., 2008). Myosin Vc has also been
demonstrated to participated in regulating Rab27b-enriched secretory vesicles (Chiang et
al., 2011). In contrast, myosin Vb was revealed not to alter the regulated secretory
pathway labeled by Rab27b or Rab3D, but to affect the trafficking of Rab11a-enriched
vesicles in a pathway distinct from the regulated secretory pathway in LGACs (Xu et al.,
2011). These results indicated that myosin V motor isoforms differentiated in functions in
specialized secretory epithelial cells.

Figure 2: Schematic structure of myosin Va
At the N’ terminus, the motor domain contains the actin-binding site and nucleotide-binding site.
Followed is an around 24-nm-long lever arm that binds six calmodulins (CaM) or related light
chains. The rod region of myoVa is interrupted by two major regions of non-coiled-coil. At the C’
terminus, the globular tail binds adapter proteins that link it to cargo. (Adapted from(Trybus,
2008))

10

rAV-LifeAct-TagRFP-a novel F-actin marker
First reported in 2008, lifeact is a 17-amino acid peptide fragment of a protein derived
fom Saccharmomyces cerevisiae. It specifically binds F-actin in living cells or tissues.
The advantage of lifeact is that it does not interfere with actin dynamics in any conditions,
and this advantage is not restricted to cell types. Lifeact is also the shortest actin marker
so far described exhibiting no homologous sequences in higher eukaryotes (Riedl,
Crevenna, Kessenbrock, Yu, Neukirchen, Bista, Bradke, Jenne, Holak, Werb, Sixt, &
Wedlich-Soldner, 2008b).

Currently, fluorescent proteins fused with lifeact peptide are commercially available.
Lifeact fused with RFP and GFP are available as both plasmid vectors and adenoviral
constructs. Thereby, lifeact with fluorescent proteins provide very promising and versatile
tools for investigating cytoskeleton organization and dynamics (Riedl, Crevenna,
Kessenbrock, Yu, Neukirchen, Bista, Bradke, Jenne, Holak, Werb, Sixt, &
Wedlich-Soldner, 2008b).

In our study, human adenovirus comprising Lifeact-TagRFP fusion protein is employed to
mark F-actin. This construct has a high expression level in primary cells that are usually
hard to access, like LGACs. Therefore, being advantaged in superb biocompatibility, no
11

interference with actin dynamics and excellent signal-to-noise ratio, rAV-LifeAct-TagRFP
is an ideal marker for F-actin study in LGACs.
12

Goals and experiment design
The pIgR has long been studied for its irreplaceable function in mediating dIgA
transportation and maintaining normal mucosal immune defense. The transcytotic
pathway of pIgR was well characterized in MDCK cells. However, MDCK cells only
express pIgR with transduction and they do not express other apically targeted secretory
pathway characteristics. In other words, unlike glandular epithelial cells like LGACs,
MDCK cells lack physiological regulated secretory functions. Therefore, in its natural
environment, performing a study of the trafficking of pIgR will achieve more intuitive
and precise results.

In 2008 and 2011, two studies successively revealed that pIgR trafficking in LGACs
might have two pathways: regulated secretory pathway and transcytotic pathway, as
suggested (Evans et al., 2008; Xu et al., 2011). The former confirmed that pIgR could be
intracellularly cleaved generating a pool of SC which responded rapidly to agonist
stimulation. This pathway is related to Rab3D-enriched secretory vesicles. The latter
depicted a Rab11a labeled transcytotic pathway responsible for the basal-to-apical
transport of pIgR-dIgA complex, similar to the model of pIgR trafficking in MDCK cells.
13

Our goal is to understand the two pathways and their interrelationships, and to identify
their regulatory proteins in depth. Not only will we elucidate the paths but also interpret
the differences between them. This is expected to give a clearer view of pIgR trafficking
in LGACs and provides an explanation for the novel secreted pool of pIgR and SC. In
addition, we also evaluated the expression and function of a novel F-actin marker
rAV-LifeAct-TagRFP in LGACs, which proved to be a promising tool to investigate
cytoskeleton dynamics.

Adenovirus encoding human pIgR fused with fluorescent protein was designed and
purified to focus on pIgR trafficking in primary LGACs. Other class V myosin protein
mutants were obtained to exclusively inhibit one of the trafficking pathways. Followed by
biochemical verification assay, confocal microscopy was used as a major tool to obtain
direct or indirect fluorescent images of targeted proteins.
14

Chapter 2. METHODS AND MATERIALS

Production of recombinant adenovirus constructs
The construction of Ad-hpIgR-EGFP involved a series of steps: PCR was employed to
amplify the human pIgR precursor cDNA sequence from the cloning vector (Genbank
Entry: BC110494.2) with primer pairs: sense primer, 5’-
TACTGCTAGCTCAACGGGAGAGAAGGAAGTGG-3’, anti-sense primer,
5’-TAGACTCGAGATAGGCTTCCTGGGGGCCGTC-3’; the product was inserted into
PCR® II-TOPO vector (Invitrogen, Carlsbad, CA). After digesting with NheI and XhoI
restriction enzymes, the human pIgR encoding fragment (2381bp) was inserted into
pEGFP-N1 vector, which was then digested with NheI and NotI restriction enzymes. The
3172bp fragment was subcloned into the pTRE-shuttle2 vector from the AdenoX
Tet-On® expression system kit (Clontech, Moutain View, CA) and finally subcloned into
Adeno X System 1 viral DNA vector. After linearization with PacI restriction enzyme, the
adenovirus construct was amplified in QBI cells at 37C and 5% CO2 in DMEM (4.5
g/mL glucose, GIBCO/Invitrogen, Carlsbad, CA)
15

The S20V constitutively active (CA) mutant of human Rab11a was a gift from Dr. James
Goldenring (Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN).
The pEGFP-C2 Rab11a S20V was digested with Nhe1 and Sal1 restriction enzymes. The
1432bp fragment encoding EGFP-Rab11a S20V was subcloned into the pTRE-shuttle 2
vector. The following procedures were same as producing Ad-hpIgR-EGFP.

Ad-mCherry-Rab3D was a construct that fused rabbit Rab3D at the N’-terminal with
mCherry. However, a 12 amino acids linker-GGSGGGSGGGSG- was inserted between
the fluorescent protein and Rab3D N’-terminal. The cloning vectors containing
mCherry-Rab3D sequences were sent to Vector Biolabs (Philadelphia, PA) for
outsourcing customized construction of Adenovirus.

The expression of Ad-hpIgR-EGFP, Ad-EGFP-Rab11a CA and Ad-mCherry-Rab3D in
LGACs requires co-transduction with Adeno-X Tet-On® regulatory virus. In addition,
0.1μg/mL doxycycline was applied for inducing protein expression.

rAV
CMV
LifeAct–TagRFP was purchased from Ibidi GmbH (Martinsried, Germany). Ad
YFP-Rab27b was a gift from Dr. Serhan Karvar (Division of Gastrointestinal & Liver,
University of Southern California). Ad EGFP-Rab11a DN, Ad mCherry-myosin Vb tail,
16

Ad EGFP-myosin Vc tail, DN-PKC-ε were obtained from previous lab work (Jerdeva,
Yarber et al., 2005; Marchelletta, Jacobs, Schechter, Cheney, & Hamm-Alvarez, 2008; Xu
et al., 2011). All constructions were amplified in QBI cells then harvested and purified
using CsCl gradient gradient ultracentrifugation (Y . Wang et al., 2003).

Preparation and the treatment conditions of primary LGACs
LGAC isolated from rabbit LG from female New Zealand White rabbits (1.8-2.2 kg)
(Irish Farms, Norco, CA) were incubated in accordance with previous protocol(Rismondo
et al., 1994). These cells were cultured for 2-3 days in PCM and reconstitute acinus-like
structures. Distinct apical and basolateral domains were observed. For live cell imaging,
LGACs were seeded onto 35mm dishes. For imaging of fixed cells stained by fluorescent
antibody, LGACs were seeded onto 12-well plates with coverslips coated with
Matrigel™.

The transduction of adenovirus recombinant constructs into LGAC
On the 2
nd
of culture, LGACs were ready for the transduction. Ad constructs at MOI 5
(multiplicity of infection) and Adeno-X Tet-On® regulatory virus were co-incubated at
37degree for 2 hours. Adeno-X Tet-On® regulatory virus is necessary due to they encode
17

a regulatory protein that recognizes the“reverse” Tet repressor upstream of the DNA
sequences encoding fluorescent protein-fused pIgR or Rab3D in our Ad constructs.
Therefore the regulatory virus induces the expression of fluorescent pIgR or Rab3D in
LGACs. After the virus was removed after 2 hours, 0.1μg/mL doxycycline was added
when replace culture medium. Ad mCherry- myosin Vb tail, Ad EGFP-myosin Vc tail,
Ad EGFP-myosin Vc full length, DN-PKC-ε, Ad YFP-Rab27b and rAV-LifeAct-TagRFP
were used at an MOI 5 for 2 hours 37degree incubation without Adeno-X Tet-On®
regulatory virus. Before analysis, LGACs were cultured for another 18-24 hours after the
virus transduction.

Immunostaining
After the transduction and incubation, LGACs cultured on glass coverslips were fixed
with 4% paraformaldehyde in PBS for 15 minutes. Then LGACs were incubated with
50mM NH
4
Cl for 5 minutes, followed by 10-minute permeabilization with 0.1% Triton
X-100. Thoroughly PBS washing should be taken in between of every step until the BSA
was applied. LGACs were blocked with 1% BSA at room temperature (25degree) for 1
hour and then incubated with appropriate fluorophore- conjugated primary and secondary
antibodies. Finally the LGACs would be mounted on glass slide and preserved by
prolong anti-fade mounting medium. (Molecular Probes, Eugene, OR)
18

Secretion assay
This assay aims to quantify the SC released by LGACs. LGACs were cultured on 12-well
plate and transfected with Ad constructs on the 2
nd
day. On the 3
rd
day, 500 μl fresh PCM
was added in to each well after the depletion of old medium to adjust the cell condition.
After 30 minutes release, the amount of SC in the supernatant during this period was
quantified as background SC release. Then the cells were treated with or without 100μM
CCh for another 30 minutes. After that, we extracted culture supernatant and dissolved
cell pellets with 0.5 M NaOH. BCA protein assay was adopted to measure the SC
secretion to protein in the cell pellets. Culture supernatant was concentrated for 10 times
using Vivaspin-500 concentrator (Littleton, MA, USA). In order to quantify SC, goat
anti-human SC primary antibody and IRDye®800-conjugated donkey anti-goat
secondary antibody were chosen in western blotting method. The image was scanned by
Odyssey® Imaging System from LI-COR and quantification was obtained with
Odyssey® 3 software. Net release of SC during the 30 minutes treatment was calculated
by using amount of SC in each treatment minus the background SC release. The
advantage of this calculation is to eliminate confounders at the most degree during
operation, for instance the SC release induced by mechanical stress.

19

Uptake of pIgR-specific binding antibody
Binding medium composed of PCM, 2mM CaCl
2,
2mM Mg Cl
2,
20mM HEPES and 3%
BSA was applied to LGACs cultured on glass coverslips. Sheep anti-SC serum or goat
anti-human SC antibody was diluted in binding medium and incubated with LGACs on
ice for 1 hour. After thoroughly washing by PCM, LGACs were transferred to 37degree
incubator and let the uptake and subsequent trafficking of antibody to process. LGACs
were fixed for the further immunostaining and observation at 0-min, 15-min, 30-min and
60-min time points, respectively.

Immunostaining fluorescence image by confocal microscope
Slides were examined by Zeiss LSM 510 Meta inverted confocal microscope equipped
with Argon, Red HeNe, and free HeNe lasers. A 63 x 0il immersion objective len NA 1.4
was adopted to take the images. Z-stacks were taken to construct 3D models of cell with
consecutive confocal planes at 0.4 μm intervals. This reconstruction was performed by
Zeiss LSM Projection Tool and Zeiss LSM image examiner.

Time-lapse live cell imaging by confocal microscope
LGACs expressing tagged proteins were imaged on the 3
rd
day of culture, in a controlled
20

incubation chamber as described (Jerdeva et al., 2005). Under the function of time series
studies, images from a single confocal plane were taken sequentially at a time interval of
2.5 seconds. Acini were analyzed at resting stage for approximately 8 minutes. After that,
another 30 minutes long video was taken upon 100 μM CCH stimulation.

Reagents
Nocodazole (Methyl-(5-[2-Thienylcarbonyl]-1H-Benzimidazol-2-YL) Carbamate) and
carbachol were purchased from Sigma-Aldrich (St.Louis, MO). Doxycycline was
obtained from Clontech (Mountain View, CA). Protease inhibitors pepstatin A, leupetin,
tosyl lysyl chloromethyl ketone, soybean trypsin inhibitor, tosyl phenylalanyl
chloromethyl ketone and phenylmethane sulphanyl fluoride were also purchased from
Sigma-Aldrich and were used as ingredients of protease inhibitor cocktail for cellular
homogenates as described in 1998 (Vilalta, Zhang, & Hamm-Alvarez, 1998). Peter’s
Complete Media (PCM) was prepared in the method described in previous study (Xu et
al., 2011). Matrigel™ was obtained from Collaborative Biochemicals (Bedford, MA).
Latrunculin B was obtained from EMD Biosciences (San Diego, CA). Commercial
antibodies Alexa Fluro®488-conjugated goat anti-mouse, Alexa Fluro®488-conjugated
donkey-anti-rabbit, Alexa Fluro®568-conjugated donkey-anti-sheep and Alexa
Fluro®568 conjugated goat-anti -mouse, Alexa Fluro®647 conjugated phallodin as well
21

as Prolong® anti fade Kit were all purchased from Molecular Probes/ Invitrogen
(Carlsbad, CA). Other antibodies included: goat-anti-human pIgR extracellular domain
(hSC) polycloncal Ab (R&D system, Minneapolis, MN), mouse-anti-GFP monoclonal Ab,
pre-immune sheep serum and mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
IRDye®800-conjugated and IRDye®700-conjugated donkey-anti-goat and
goat-anti-mouse (Rockland, Gilbertsville, PA). Some antibodies were raised and purified
as previously described, included: Rabbit anti Rab3D serum (Evans et al., 2008); Sheep
anti-serum raised against SC purified from rabbit gall bladder bile (Caproilogics,
Hardwick, MA); Rabbit dIgA (Xu et al., 2011).
22

Chapter 3. RESULTS
Expression of human pIgR-EGFP construct in primary LGACs
Several studies confirm that adenovirus can be effectively used to transduce primary
LGACs in vitro. Based on this expertise, we constructed an adenovirus encoding human
pIgR fused with EGFP at the cytoplasmic terminal. This construct (hpIgR-EGFP) is the
first known fluorescent protein-tagged pIgR construct so far (Chiang et al., 2011; Evans
et al., 2008; Xu et al., 2011).

In order to verify the expression pattern of hpIgR-EGFP, we analyzed direct and indirect
fluorescent proteins expressed in LGACs. In non-transduced cells, endogenous pIgR was
mostly located to the subapical region while a subset of pIgR was found at the basolateral
membrane (Figure 3). In LGACs expressing hpIgR-EGFP, confocal microscopy showed
that the distribution pattern of hpIgR-EGFP highly resembled that of pIgR in
non-transduced cells. Using goat-anti-human SC and mouse-anti-human SC antibody to
hpIgR-EGFP transduced LGACs, we observed colocalization of direct fluorescence from
EGFP and indirect fluorescence from antibodies. This indicated that hpIgR-EGFP could
be successfully recognized by anti-human SC antibody (Figure 4A, Figure 4B).
Furthermore, we found hpIgR-EGFP was extensively colocalized with Rab11a (Figure 5).
23

In the study of Rab11a and pIgR, Xu presented that endogenous pIgR was substantially
colocalized with exogenous EGFP-Rab11a (Xu et al., 2011). All these images support the
conclusion that hpIgR-EGFP could be expressed in rabbit LGACs with regular function
and distribution.

Western blotting also suggested that the expression of post-translational modified
hpIgR-EGFP was highly similar to the endogenous pIgR. Goat-anti-human SC antibody
recognized a SC band around 80 kD in the lysate with hpIgR-EGFP expressed, but not
the non-transduced LGACs. On contrast, sheep-anti-rabbit SC antibody identified 80kD
bands in both hpIgR-EGFP expressed and non-transduced LGACs (Data not shown).
These results suggested that hpIgR-EGFP had functional SC expressed in LGACs and
had been successfully recognized by anti-rabbit SC primary antibody. Human SC would
also be specifically identified by anti-human SC primary antibody, which distinguishes it
from endogenous rabbit SC.

Besides anti SC antibodies, we use two different anti-GFP primary antibodies to display
the hpIgR-EGFP contains GFP epitope. On one hand, Figure 6 showed that hpIgR-EGFP
had an apparent molecular weight ~150 kD, on the other, results proved that Adeno-X Tet
24

On® regulatory virus and doxycycline were necessary for inducing the expression of
hpIgR-EGFP in LGACs.

Figure 3: Distribution pattern of pIgR in non-transduced LGACs
Non-transduced LGACs were incubated for 18 hour and then fixed, permeabilized, and stained
with primary sheep anti-SC as well as secondary FITC-conjugated donkey-anti-sheep antibody.
Scale bar=5 μm, *=lumena. Results shown are representative of 6 independent experiments.
(Adapted from (9))

25

Figure 4: Anti-human SC antibodies identify hpIgR-EGFP in LGACs
LGACs were expressing hpIgR-EGFP were fixed, permeabilized for immunostaining. In A)
Goat-anti- human SC and in B) mouse-anti-human SC antibody was used as primary antibody. In
A) Alexa-Fluor-568-conjugated donkey-anti-goat and in B) Alexa-Fluor-568-conjugated
goat-anti- mouse was used as secondary antibody. Alexa-Fluor-647-conjugated phalloidin was
employed to stain actin. Scale bar=5 μm, *=lumena; white arrows indicate colocalization.

Figure 5: Colocalization of hpIgR-EGFP with Rab11a
LGACs expressing hpIgR-EGFP were fixed, permeabilized for immunostaining. Goat-anti-Rab11
antibody was used as primary antibody and Alexa-Fluor-568-conjugated donkey-anti-sheep was
used as secondary antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin.
Scale bar=5 μm, *=lumena; white arrows indicate colocalization.

26

Figure 6: Anti-GFP antibody identifies hpIgR-EGFP
LGACs transduced with or without hpIgR-EGFP, with and without the Adeno-X Tet On®
regulatory virus as well as doxycycline were incubated for 18 hours. Lysates were analyzed by
western blotting. Sample sequences in both panels are as follow: Lane 1, non-transduced LGACs
free from Adeno-X Tet On® regulatory virus and doxycycline. Lane 2, hpIgR-EGFP and
Adeno-X Tet On® regulatory virus, free from doxycycline. Lane 3, hpIgR-EGFP and Adeno-X
Tet On® regulatory virus, 0.1 μM/mL doxycycline applied. (Zhechu Peng made great
contribution to this western blotting experiment)
27

The transcytotic pathway of pIgR in LGACs
The transcytosis of pIgR in MDCK cells has been extensively studied (Apodaca et al.,
1994; Mostov et al., 1995); the trafficking of transcytotic pathway of pIgR in LGACs was
also studied and its tight relationship with Rab11a vesicles was revealed (Rojas &
Apodaca, 2002; Xu et al., 2011). This study further investigated the transcytosis of pIgR
in LGACs. To mimic the natural physiological process, we used dIgA, which is the
natural ligand to label hpIgR-EGFP at the basolateral membrane. The reason why we did
not use the the endogenous rabbit pIgR pool was that the single cohort of fluorescent
rabbit dIgA-pIgR could not yield a strong enough signal which was resolvable in live cell
fluorescent imaging experiment. Therefore, LGACs over-expressing hpIgR-EGFP were
adopted to generate a decent amount of fluorescent intensity to elevate the endocytosis of
dIgA, enabling sufficient fluorescence intensity of Rhodamine-conjugated dIgA to be
detected.

Figure 7 showed that at the 0-min time point, the internalization did not occur and all
Rhodamine-conjugated dIgA were localized at basolateral membrane due to the cold
pretreatment on ice which impairs endocytosis. After warming up to 37degree, the
transcytosis process started and clusters of Rhdamine-conjugated dIgA slowly moved
towards lumen. 15-min, 30-min and 60-min time point images clearly presented the
28

migrating trace of dIgA. Rhodamine-conjugated dIgA was endocytosed from basolateral
membrane and transported to the subapical region in the form of dIgA-pIgR complex.

Actually substantial subsets of hpIgR-EGFP were observed not to co-localize with dIgA
(Figure 7). This phenomenon indicated that a pool of hpIgR-EGFP was free from the
transcytotic pathway and might participate in other trafficking process.

After magnifying the indirect fluorescent signal by over expressing hpIgR-EGFP became
accessible, we used mouse anti human SC antibody to label pIgR, generating a
distinguishable signal for transcytosis trafficking. A similar trace was observed through
the uptake of the complex of anti-SC antibody and pIgR (Figure 8). This result was
comparable to the dIgA-pIgR complex moving pattern (Figure 7). The
mouse-anti-human SC-pIgR complex was exclusively at the basolateral membrane at
0-min time point. By the end of 60 minutes warm up and subsequent trafficking, the
complex was clustered at the subapical region as hpIgR-EGFP did at the steady-state.

The dIgA-uptake and anti-SC-pIgR-uptake experiments both suggested that hpIgR-EGFP
normally functioned and underwent transcytosis from the basolateral membrane to
subapical region with time. Worth pointing out, due to the similar result with dIgA-uptake
29

experiment, the anti-SC-pIgR-uptake method was thus considering to be a reliable tool
distinguishing basolaterally endocytosed pIgR from the total original pIgR pool.

30

Figure 7: Trafficking of hpIgR-EGFP in transcytosis pathway with dIgA uptake
LGACs expressing hpIgR-EGFP were treated with 150μl Rhodamine-conjugated-dIgA, Then
incubated cells with 450μl fresh PCM on ice for 1hour. Z-stack images were taken during
warming up to 37degree. Scale bar=5 μm, *=lumena; white arrows indicate the movement of
dIgA.
31

Figure 8: Trafficking of hpIgR-EGFP in transcytosis pathway with anti-human SC antibody
LGACs expressing hpIgR-EGFP were pre-treated on ice for an hour with mouse-anti-human SC
antibody. Then the cells were warmed to 37degree and fixed at 60-min time point for
immunostaining assay. Alexa-Fluor-568-conjugated goat-anti-mouse was used as secondary
antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm,
*=lumena; white arrows indicate colocalization, white square indicates an area of colocalization.
32

Endocytosed pIgR would be sorted to early endosomes and Rab11a vesicles during
the transcytosis pathway
We also developed a method to label endogenous pIgR using antiserum against rabbit SC.
This was necessary because hpIgR-EGFP occupied the “green filter channel”, if this
channel was free from hpIgR-EGFP, more fluorescently labeled probes could be
employed and detected.

The anti-rabbit SC serum used to label endogenous pIgR was raised in sheep. In order to
verify its specificity, a competitive SC binding experiment was conducted. Rabbit SC was
purified and adopted to compete with pIgR binding to anti-SC antibody. Non-transduced
LGACs were incubated with sheep anti-rabbit SC antibody for 1 hour on ice, and then the
cells were fixed at 0 minute time point before the transcytosis began. Indirect
fluorescence revealed that by competing with endogenous pIgR, purified rabbit SC
reduced the detectable signal largely (Figure 9). It was obvious that basolateral
membrane endocytosed pIgR fluorescence signaling decreased with the increase of rabbit
SC concentration. We could safely came to the conclusion that sheep anti-rabbit SC
antibody had high specificity binding ability to pIgR and thus providing a credible
method for labeling as well as tracking endocytosed endogenous pIgR.

33

Using the method above, we identified some critical steps during the pIgR transcytosis. In
what organelle and by what order the pIgR would be sorted into? By labeling
endocytosed pIgR from basolateral membrane with sheep anti-rabbit SC serum and
marking early endosome with EEA1, we found that the basolateral pIgR eventually
accumulated in early endosomes time by time. At the 15-min point, pIgR colocalized with
EEA1 and merged towards lumen. Substantial amount of pIgR remained in subapical
region and even at 60-min time point, partial pIgR kept staying within early endosomes
(Figure 10).

Rab11a was also labeled to explore the relationship with endocytosed pIgR from
basolateral membrane. As expected, pIgR was sorted to EGFP-Rab11a-enriched vesicle
after 30 minutes transcytosis (Figure 11B). In LGACs, pIgR were observed to colocalize
with both endogenous Rab11a and transduced EGFP-Rab11a. In the 60-min time point,
pIgR accumulated around lumen and continued remaining in Rab11a vesicles (Figure
11A, Figure 11 B).

Anti-SC uptake experiments lead us to identified two critical steps in pIgR transcytosis
through LGACs: sorting into early endosomes at roughtly 15-min time point and sorting
into Rab11a vesicles around 30-min time point. We believed that after pIgR was
34

endocytosed basolaterally, it went to early endosomes and then sorted into Rab11a
vesicles. A subset of pIgR would still remain in these organelles when substantial amount
of pIgR approached to subapical region.
35

Figure 9: Competitive experiment to verify the specificity of sheep anti-rabbit SC antibody
Non-transduced LGAGs were incubated with 0μg/ml, 20μg/ml, 50μg/ml, 100μg/ml purified
rabbit SC on ice for an hour, respectively, Sheep-anti-rabbit pIgR serum was added in the ratio of
1:20 in the medium as primary antibody. Alexa-Fluor-488-conjugated donkey-anti-sheep was
used as secondary antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin.
Scale bar=5 μm, *=lumena.
36

Figure 10: The trafficking of endocytosed pIgR in LGACs
Non-transduced LGACs were incubated with sheep anti-rabbit SC serum for an hour on ice. After
warmed up to 37degree, cells were fixed at 0-min, 15-min, 30-min and 60-min time points for
immunostaining, respectively. Mouse-anti-EEA1 was used as primary antibody and
Alexa-Fluor-488-conjugated donkey-anti -sheep as well as Alexa-Fluor-568-conjugated
goat-anti-mouse was used as secondary antibody. Alexa-Fluor-647-conjugated phalloidin was
employed to stain actin. Scale bar=5 μm, *=lumena; white arrows indicate colocalization.
37

Figure 11: Endocytosed pIgR entered Rab11a vesicles in transcytotic pathway
A) LGACs expressing EGFP-Rab11a and B) Non-transduced LGACs were incubated with sheep
anti-rabbit SC serum on ice for 1 hour. After warmed up to 37degree, cells were fixed at 0-min,
30-min. 60-min time points for immunostaining, respectively. For A),
Alexa-Fluor-568-conjugated donkey-anti-sheep was used as secondary antibody. For B), mouse-
anti-Rab11 was used as primary antibody; Alexa-Fluor-488-conjugated donkey-anti-sheep as well
as Alexa-Fluor-568-conjugated goat-anti-mouse were used as secondary antibodies. In both cases,
Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm, *=lumena;
white arrows indicate colocalization; while square indicates an area of colocalization.
38

Figure 11: Continued

39

Transportation of pIgR in transcytotic pathway requires the functionality of Rab11a
In the previous studies, the participation of Rab11a in pIgR and dIgA transcytosis was
confirmed (Mostov et al., 1995; Xu et al., 2011). We also corroborated the statement once
again with the anti-SC uptake experiment in LGACs expressing endogenous Rab11 and
EGFP-Rab11a WT (Figure 11). To further investigate the regulatory function of Rab11a,
we utilized two mutant forms of Rab11a which had been established in Wang’s study in
2000: S25N GDP-locked Rab11a, known as dominant negative (DN) Rab11a; S20V
GTP-locked Rab11a, known as constitutively active (CA) Rab11a. These plasmids were
modified to fuse with EGFP. Adenovirus constructs were adopted to generate
Ad-EGFP-Rab11a mutants (X. Wang, Kumar, Navarre, Casanova, & Goldenring, 2000;
Xu et al., 2011).

EGFP-Rab11a DN did not follow the subapical distribution pattern as EGFP -Rab11a CA
or EGFP-Rab11a WT did in the LGACs. Interpreting the result with pIgR trafficking
trace, EGFP-Rab11a DN inhibited the transcytosis of pIgR whereas (Figure 12). In
LGACs expressing EGFP-Rab11a WT, pIgR was colocalized EGFP-Rab11a CA
successfully transported endocytosed pIgR from basolateral membrane into subapical
compartment with large subset with a subset of EGFP-Rab11a WT through transcytosis.
EGFP-Rab11a CA also colocalized with pIgR in the basal-to-apical transport of pIgR
40

(Figure 12A, Figure 11A). However, EGFP-Rab11a DN seemed not to colocalize with
pIgR at all (Figure 12 B).

These observations suggested that the failure of Rab11a function inhibited the transport
of pIgR from basolateral membrane to apical surface. In other words, the transcytosis of
pIgR in LGACs requires the functionality of Rab11a.

Figure 12: Transcytosis of pIgR in LGACs requires the functionality of Rab11a
A) LGACs expressing EGFP-Rab11a CA and B) EGFP-Rab11a DN were incubated with sheep
anti-rabbit SC serum on ice for 1 hour. After warming up to 37degree, cells were fixed at 0-min,
30-min. 60-min time points for immunostaining, respectively. For A) and B),
Alexa-Fluor-488-conjugated donkey-anti-sheep was used as secondary antibodies.
Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm, *=lumena;
white arrows indicate colocalization; while square indicates an area of colocalization.
41

Figure 12: Continued

42

The secretory pathway of pIgR is associated with Rab3D-enriched vesicles
Our lab has verified that two of the Rab GTPase: Rab3D and Rab27b, are well-known
markers of secretory vesicles in LGACs. However, their relationship to pIgR trafficking
in LGACs is not extensively studied (Chiang et al., 2011; Evans et al., 2008).

We co-expressed mCherry-tagged Rab3D together with YFP-tagged Rab27b adenovirus
constructs in LGACs. Confocal fluorescence microscopy clearly showed that these two
markers identified different secretory vesicle pools (Figure 13A).

When hpIgR-EGFP was coexpressed with either mCherry-Rab3D or YFP-Rab27b, we
found that pIgR extensively colocalized with the former but not the latter (Figure 13B,
Figure 13C).

These direct fluorescent images (Figure 13) lead to the conclusion that Rab3D and
Rab27b labeled different pools of secretory vesicles and pIgR was only associated with
Rab3D-regulated secretory pathway.
43

Figure 13: pIgR is associated with Rab3D-enriched secretory vesicles
LGACS expressing A) YFP-Rab27b and mCherry-Rab3D ; B) hpIgR-EGFP and YFP-Rab 27b ;
C) hpIgR-EGFP and mCherry-Rab3D were observed with confocal microscopy to show location
of these markers. Cellular outlines were obtained by DIC image. Scale bar=5 μm, *=lumena; in B)
white arrows indicate hpIgR-EGFP and YFP-Rab27 do not exclusively colocalize; in C) white
arrows indicate colocalization.
44

Myosin Vc is critical in regulating Rab3D secretory pathway
Previous work in our lab revealed a close association of Rab3D-enriched secretory
vesicles with myosin Vc protein in LGACs. Colocalization of Rab3D and myosin Vc was
observed in cells expressing EGFP-myosin Vc tail and endogenous Rab3D by direct or
indirect fluorescence. The study furthermore suggested that myosin Vc might play a role
in pIgR trafficking pathway, since colocalization of endogenous pIgR and EGFP-myosin
Vc tail was also observed (Marchelletta et al., 2008).

The EGFP-myosin Vc tail construct was developed in 2002 from a human class V myosin
study. EGFP- myosin Vc tail lacked a motor domain, but with the tail it functionally
binds vesicles. Therefore, this fluorescent protein fused to a dominant negative construct
can compete with endogenous myosin Vc for vesicle binding sites. In our case, it can be
employed to be a powerful tool investigating the regulation of related secretory vesicles
as well as the trafficking pathway of pIgR (Rodriguez & Cheney, 2002).

To obtain more evidences proving the association of myosin Vc with Rab3D enriched
vesicles as well as pIgR, we transduced LGACs with mCherry-Rab3D to yield a high
intensity fluorescence signal for confocal microscopy experiment. Figure 14 showed that
mCherry-Rab3D not only colocalized with EGFP-myosin Vc tail but full-length-myosin
45

Vc. In addition, live cell movies displayed that in LGAC treated with CCh, full-length
myosin Vc together with Rab3D-enriched secretory vesicles underwent fusion and
turnover. In contrast, myosin Vc tail inhibited the movement of Rab3D-enriched vesicles
upon CCh stimulation. (data not shown)

Above all, our study provides more evidence to claim that myosin Vc motor protein
regulates Rab3D-enriched secretory vesicles, as well as the trafficking of pIgR in
secretory pathway.

Figure 14: Colocalization of myosin Vc construct with mCherry-Rab3D in LGACs
LGACs expressing A) EGFP-myosin Vc tail and mCherry-Rab3D, B) EGFP-full-length-myosin
Vc and mCherry-Rab3D were observed by confocal microscopy to show location of these
markers. Cellular outlines were obtained by DIC imaging. Scale bar=5 μm, *=lumena; white
arrows indicate colocalization
46

Myosin V motor proteins play different roles in regulating the trafficking of pIgR in
LGACs
Our data showed that myosin Vc motor protein facilitated the motility of Rab3D-enriched
vesicles in the pIgR regulated secretory pathway. Another study put forward the idea that
myosin Vb regulated the Rab11a-enriched vesicles with evidence (Xu et al., 2011).
Because we came to the conclusion that the pIgR transcytotic pathway requires the
functionality of Rab11a, we can therefore make an assumption that myosin Vb might be
critical in regulating the transcytosis of in LGACs.

To obtain supportive data, we used confocal fluorescence microscopy to get fluorescent
images of LGACs expressing hpIgR-EGFP, mCherry-myosin Vb tail as well as
EGFP-myosin Vc tail. mCherry-myosin Vb tail was mentioned in Xu’s study in 2011 and
works as a dominant negative mutant of motor activity(Xu et al., 2011). In Figure 15A,
hpIgR-EGFP showed partial colocalization with mCherry-myosin Vb tail. This is
reasonable because another subset of hpIgR-EGFP might be involved in secretory
pathway. Figure 15B and Figure 15C suggested that myosin Vb tail did not follow the
distribution pattern of myosin Vc tail, nor did it colocalize with the Rab3D-enriched
vesicles. Data from secretion assay also revealed the different functions of myosin Vb
and myosin Vc in pIgR pathways: each is separate but each can knock down some of the
47

basal-to-apical and stimulated secretion.(data not shown) We then concluded that myosin
V motor proteins played different roles in pIgR trafficking. Briefly, myosin Vb regulates
transcytotic pathway of pIgR with Rab11a-enriched vesicles, while myosin Vc regulates
secretory pathway of pIgR with Rab3D.

Figure 15: Myosin Vb did not participate to regulate secretory pathway
LGACs expressing A) hpIgR-EGFP, mCherry-myosin Vb tail; B) EGFP-myosin Vc tail and
mCherry-myosin Vb tail were observed by confocal microscopy. Cellular outlines were obtained
by DIC image. C) LGACs expressing mCherry-myosin Vb tail were fixed and permeabilized for
immunostaining. Rabbit-anti-Rab3D serum was used as primary antibody and
Alexa-Fluor-488-conjugated donkey-anti-rabbit antibody was used as secondary antibody.
Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Cellular outlines were
obtained by DIC image. Scale bar=5 μm, *=lumena.
48

The distinction between the pIgR transcytotic pathway and regulated secretory
pathway
After investigating the two pathways of pIgR transport in LGACs, we next focused on
finding out the connection between them. A hypothesis was put forward: Since these two
pathways were independent from each other, the over-expression of any of the myosin V
motor protein would only inhibit the pathway it was associated with but did not interfere
the other.

Data supporting the hypothesis were obtained by anti-SC uptake experiment in which
basolaterally endocytosed pIgR were labeled and tracked. In both cases that LGACs
over-expressing myosin Vb tail and myosin Vc tail, the endocytosed process of pIgR
seemed not to change compared to anti-SC uptake only (Figure 16). However, after pIgR
was endocytosed from basolateral membrane, it was trapped in myosin Vb tail labeled
compartment and did not accumulate in the subapical region (Figure 16B). On the other
hand, the basal-to-apical transport of pIgR after endocytosis was not interfered with by
over-expression of myosin Vc tail in LGACs. Figure 16A pointed out that the pIgR
accumulated around the lumen and did not extensively colocalize with myosin Vc tail. In
summary, the results seemed to support the hypothesis that transcytotic and secretory
pathways were regulated by different myosin V motor proteins. In addition, pIgR
49

endocytosed from basolateral membrane would not incline to enter the regulated
secretory pathway to a prominent extent; instead, they were sorted to the transcytotic
pathway.

Figure 16: Myosin V motors regulate different pathways in pIgR trafficking
LGACs expressing A) EGFP-myosin Vc tail; B) mCherry-myosin Vb tail were fixed and
permeabilized for immunostaining. Sheep-anti-SC serum was used as primary antibody. In A)
Alexa-Fluor-568-conjugated donkey-anti-sheep antibody and in B) Alexa-Fluor-488-conjugated
donkey-anti-sheep antibody was used as secondary antibody. Alexa-Fluor-647-conjugated
phalloidin was employed to stain actin. Scale bar=5 μm, *=lumena.

50

Figure 16: Continued

51

CCh (carbachol) is a cholinergic agonist employed to stimulate LGACs releasing more
SC. We used CCh to mimic the natural response of lacrimal gland so as to acquire more
SC in culture supernatant. This agonist enabled secretory pathway release SC in acute
phase and thus distinguishes it from the transcytotic pathway.

More confocal microscopy fluorescence analyses were performed with anti-SC uptake
experiments. We once again incubated non-transduced LGACs with sheep-anti-SC serum
on ice for 1 hour, and then let it warm up in 37degree for 30 minute. During this period of
time, endocytosed pIgR from the basolateral membrane would have sufficient time to
conduct basal-to-apical transport. The group with 30 minutes recovery in 37degree was
set up as background. A 30 minute plus 30 minute warm up group in resting stage was the
control group. CCh was added to the third group and let it be treated continuously for 30
minutes. Figure 17 showed that at the 30-min time point, anti-SC serum labeled pIgR
accumulating in subapical region, colocacalized with Rab11a. In the control group, pIgR
continued to accumulate beneath lumen. In the experimental group, 30 minutes treatment
of CCh almost depleted all the pIgR that accumulated subapically. These results indicated
that CCh boosted the transcytosis of pIgR, possibly by accelerating the very last step in
transport within the subapical region.

52

Figure 18 revealed that over-expressing mCherry-myosin Vb tail in LGACs trapped pIgR
as well as could not deplete pIgR. In contrast, LGACs over-expressing EGFP-myosin Vc
tail did not inhibit the depletion of pIgR. This observation proved that in transcytotic
pathway, the release of pIgR beneath lumen was dependent on myosin Vb motor protein,
but not myosin Vc. This was also consistent with the conclusion that it was myosin Vb
instead of myosin Vc responsible for pIgR transcytosis pathway in LGACs.
53

Figure 17: CCh accelerates the transcytotic pathway of pIgR in LGACs
Non-transduced LGAGs were incubated on ice for an hour. Sheep -anti-rabbit pIgR was also
added in the ratio of 1:20 in the medium. Then warm up the cells in 37degree for 30 minutes.
CCh was added or not in cells and incubated for another 30 minute. Cells were fixed at different
time point for immunostaining. Mouse-anti-Rab11 antibody was used as primary antibody.
Alexa-Fluor-488-conjugated donkey-anti-sheep and Alexa-Fluor-568-conjugated goat-anti-mouse
was used as secondary antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain
actin. Scale bar=5 μm, *=lumena; white arrows indicate colocalization.

54

Figure 18: The release of pIgR on apical membrane was dependent on myosin Vb motor
protein through transcysotic pathway
LGACs expressing A) mCherry-myosin Vb tail and B) EGFP-myosin Vc tail were incubated on
ice for an hour. Sheep-anti-SC serum was add in the ratio of 1:20. Then warmed up cell in 37
degree for 30 minutes. The cells were treated with or without 100 μM CCh for another 30 minutes.
Cells were fixed and ready for immunostaining. In A) Alexa-Fluor-488-conjugated
donkey-anti-sheep; B) Alexa-Fluor-568-conjugated donkey-anti-sheep antibody was used as
secondary antibodies. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale
bar=5 μm, *=lumena.
55

Secretion assay differentiates two pIgR trafficking pathways
Although direct and indirect confocal microscopy fluorescent images provided evidences
to distinguish pIgR transcytotic and regulated secretory pathways by over-expressing
myosin Vb tail and myosin Vc tail separately, more biochemical assays other than cell
imaging technology were required to solidify the conclusions. We therefore investigated
the two pathways by quantifying the SC release of pIgR into the culture supernatant.

Worth mentioning, the basis hypothesis of the secretion assay was that pIgR was able to
release much more SC from the apical domain under CCh stimulation than that in resting
stage. Besides the fluorescent images displayed in previous figures, we also did western
blotting to confirm the burst of SC release with CCh treatment. In Figure 19, the control
group with Ad-EGFP expressing showed no big difference with or without CCh
stimulation. However, in the experiment group which over-expressing hpIgR-EGFP, lane
2 and lane 4 revealed an acute increase of SC amount after CCh treated for 30 minutes
(Figure 19). Anti-human SC specific antibody was employed to identify SC in case of
confusing or cross-reacting with endogenous rabbit SC. In addition, mCherry-myosin Vb
tail and EGFP-myosin Vc tail were controlled to express at comparable levels (Data not
shown).

56

With the above conditions, we co-transduced LGACs with hpIgR-EGFP and Ad
mCherry-myosin Vb tail, EGFP-myosin Vc tail, or rAV-LifeAct-TagRFP (used as control
due to its non-interference towards LGACs), respectively.

All the data were cross-analyzed and summarized into 3 major points. Representative
figures are displayed in the graph below. First, at resting stage, compared to the control
group, LGACs over-expressing myosin Vb tail released significantly less SC while
LGACs over-expressing myosin Vc tail released just slightly less SC. This result
suggested that transcytotic pathway, regulated by myosin Vb, was primarily responsible
for transporting pIgR and releasing SC into culture supernatant. Secondly, after 30
minutes treatment, LGACs over-expressing myosin Vb tail did not significantly reduce
SC release (not as significant reduction as that in resting stage: 100% to 30% in resting,
200% to 150% in CCh stimulation) (Figure 20B ). This indicated that transcytosis was
not the only pathway that participated in SC release; the regulated secretory pathway was
likely to contribute to total SC release. In addition, the regulated secretory pathway was
not likely to be inhibited by myosin Vb tail. Thirdly, under the condition that secretory
pathway was inhibited, SC release still increased to a significant level. A possible
explanation was that the transcytotic pathway also participated in releasing SC into lumen
and myosin Vc tail was not likely to prohibit transcytotic pathway, neither.
57

In summary, upon the stimulation with a cholinergic agonist, both transcytotic and
regulated secretory pathways are likely to accelerate pIgR trafficking. In all
circumstances, these two pathways are regulated by different motor proteins and do not
cross talk to each other.

Figure 19: CCh increase SC release in LGACs over-expressing hpIgR-EGFP
LGACs expressing Ad-EGFP (lane 1, lane3) and hpIgR-EGFP (lane2, lane4). The cells were
rinsed and treated with (lane3, lane4) or without (lane1, lane2) CCh for 30minutes. Lysates were
analyzed by western blotting. Goat-anti-human SC was used as primary antibody and
IRDye800® donkey-anti-goat was used as secondary antibody. (Zhechu Peng made great
contribution to this work)

58

Figure 20: The inhibition of SC under CCh treatment by over-expressing mCherry-myosin
Vb tail
A) LGACs expressing hpIgR-EGFP, rAV-LifeAct-TagRFP (lane 1, lane3) and hpIgR-EGFP,
mCherry-myosin Vb tail (lane2, lane4) were rinsed and treated with (lane3, lane4) or without
(lane1, lane2) CCh for 30minute. Lysates were analyzed by western blotting. Goat-anti-human SC
was used as primary antibody and IRDye800® donkey-anti-goat was used as secondary antibody.
B) Relative intensity quantification of SC expression. (Zhechu Peng made great contribution to
this work)
59

MT and MF participate in regulating pIgR trafficking in LGACs
It had been suggested that the MT network was associated with the trafficking and
localization of Rab11a-enriched vesicles. Data showed that the basal-to-apical transport
of endocytosed pIgR in LGACs was extensively inhibited by nocodazole, relative to the
non-treated cells (Xu et al., 2011). Nocodazole is a well-known anti-neoplastic agent used
as a microtubule inhibitor in our case. By treating cell with or without nocodazole, we
investigated how the transcytosis of pIgR was associated with MT network.

From Figure 21A, no obvious inhibition of accumulation of endocytosed pIgR in early
endosomes was found during the 60 minutes time course, suggesting that MT network
might not be responsible for sorting basolateral pIgR into early endosomes. With both
nocodazole and CCh treatment, Rab11a and pIgR showed colocalization, indicating that
the sorting process of pIgR from early endosomes to Rab11a-enriched vesicles was
independent on the MT network (Figure 21B). Figure 21 further revealed that the
transport of pIgR was closely associated with MT, because the disassembling of MT
largely inhibited the basal-to-apical transport of pIgR, which was supposed to use MTs as
tracks.
60

Besides the MT network, MF might also be associated with certain stage of pIgR
transcytotic or regulated secretory trafficking. The rationale behind this hypothesis was
that plasma membranesmembranes, especially the subapical region, are rich in F-actin
(Chiang et al., 2011), thereby having large chances to participate in regulating the
terminal release of pIgR. What is more, myosin Vb and myosin Vc which were identified
to play vital roles in pIgR trafficking were both actin-dependent motor proteins.

DN-PKC-ε was confirmed to alter lacrimal acinar apical actin remodeling and resulting in
the inhibition of stimulated transcytosis in a study in 2005 (Jerdeva, Yarber et al., 2005).
This adenovirus constructed effector could be employed to effectively regulate the
F-actin network, for the convenience of investigating the function of MF in pIgR
trafficking. Images taken by confocal microscopy displayed that DN-PKC-ε had no
inhibition effect on endocytosed pIgR accumulated beneath lumen (Figure 22). In
LGACs expressing DN-PKC-ε, pIgR did not undergo depletion as they did in
non-transduced cells under CCh stimulation, but remained trapped in the subapical region
(Figure 22). This suggested that DN-PKC-ε significantly interfere with the release of
pIgR via the transcytotic pathway.
61

In order to eliminate the possibility that DN-PKC-ε inhibited SC release by disrupting the
functionality of Rab11a or Rab3D, we looked into the distribution patterns of Rab in
DN-PKC-ε expressing cells. Figure 23 confirmed that in LGACs expressing DN-PKC-ε,
the localization of Rab3D and Rab11a demonstrates no obvious difference than that in
non-transduced cells. The results led to the conclusion that DN-PKC-ε was most likely to
inhibit the terminal release of pIgR through the transcytotic pathway by regulating
F-actin network.

Figure 21: MT network regulates the intracellular trafficking of pIgR in LGACs
Non-transduced LGACs were incubated on ice for an hour and sheep-anti-SC serum was added in
the ratio of 1:20, then warmed up cells to 37degree. Cells were fixed at different time points for
immunostaining. In A) mouse- ant-EEA1 and In B), mouse-anti-Rab11 antibody was used as
primary antibody. In both cases, Alexa-Fluor-488-conjugated donkey-anti-sheep and
Alexa-Fluor-568-conjugated goat-anti-mouse were used as secondary antibodies.
Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm, *=lumena.
62

Figure 21: Continued

63

Figure 22: Inhibition of SC release by expressing DN-PKC-ε in LGACs
LGACs expressing DN-PKC-ε were rinsed and then incubated on ice for an hour. Sheep-anti-SC
serum was added in the ratio of 1:20. Then warmed up cells to 37 degree for up to 60 minutes.
With or without the treatment of CCh, cells were fixed at different time points and ready for
immunostaining. Alexa-Fluor-568-conjugated donkey-anti-sheep antibody was used as secondary
antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm,
*=lumena.

64

Figure 23: DN-PKC- ε does not change localization of Rab11a and Rab3D
LGACs expressing DN-PKC-ε were rinsed and then fixed for immunostaining. A)
Mouse-anti-Rab11a was used as primary antibody and Alexa-Fluor-568-conjugated
goat-anti-mouse was used as secondary antibody. B) Rabbit-anti-Rab3D serum was used as
primary antibody and Alexa-Fluor-568-conjugated goat-anti-rabbit was used as secondary
antibody. Alexa-Fluor-647-conjugated phalloidin was employed to stain actin. Scale bar=5 μm,
*=lumena.
65

Visualization of F-actin locations and dynamics with rAV-LifeAct-TagRFP in
LGACs
In this study we presented the visualization of F-actin localization in LGACs with a novel
actin marker, rAV-LifeAct-TagRFP, for the first time (Deibler, Spatz, & Kemkemer, 2011;
Riedl, Crevenna, Kessenbrock, Yu, Neukirchen, Bista, Bradke, Jenne, Holak, Werb, Sixt,
& Wedlich-Soldner, 2008a). The result is consistent with previous descriptions that
F-actin is enriched in basolateral and apical membrane, especially at the lumenal cortex
(Figure 24). The expression of lifeact was not likely to intervene the physiology of
neither actin nor cell itself. White arrows in Figure 24 indicate some actin-coated
transient structures, which is very common even in the resting stage of cells. Therefore,
lifeact is considered to be a reliable and useful tool to visualize lumen region as well as
F-actin dynamics.

Ad-Tc-GFP-actin (GFP-actin) is a well-characterized actin marker employed in many
studies to monitor actin, especially actin-coated structure dynamics (Angrisano et al.,
2012; Engelke, Heinrich, & Radler, 2010; Jerdeva et al., 2005; Washington & Knecht,
2008; Xie et al., 2006). We co-transduced GFP-actin with rAV-LifeAct-TagRFP in
LGACs to evaluate and compare their behavior. Western blotting was performed to
confirm to expression of the protein. The apparent molecular weight of GFP-actin was 75
66

kD. This is consistent with predicted because GFP has apparent molecular weight of ~28
kD while actin has that of ~45 kD. Lifeact-RFP composed of a 17-amino acid long
peptide and RFP. Since 1-amino acid is around 110 Daltons, 17-amino acid has apparent
molecular weight of ~1.7kD. Therefore, lifeact-RFP showed similar molecular weight to
RFP of around 28 kD. They did not seem to compete with each other extensively, though
the amount of protein expressed was not quantified (Figure 25).

It was analyzed in previous sections that CCh accelerated secretory vesicles carrying SC
release into the lumen. We also identified that the actin network beneath lumen might be
responsible for the terminal release step. Therefore, it is worth investigating the dynamics
of actin marked by lifeact.

Time-lapse cofocal microscopy fluorescent images and videos were obtained under a
successive treatment of CCh. Figure 26 showed that actin filament intensity beneath
lumen was considerably reduced, due to the fast turnover under stimulation. Some
actin-coated structures rapidly underwent fusion and reform in the acute CCh treated
phase. The result is consistent with Jerdeva’s work in 2005, which presented a similar
process labeled by GFP-actin (Jerdeva et al., 2005). In our case, lifeact seems not to label
those transient fusion intermediates initially, but the newly formed ‘bubble structures’
67

during the successive process. This is different from the GFP-actin labeling pattern which
clearly depicted the outline of SVs even at the resting stage (Figure 26). A complete
video showed all the steps and changes is also provided (data not shown).

Figure 24: Visualization of F-actin with rAV-LifeAct-TagRFP in LGACs
LGACs exressing rAV-LifeAct-TagRFP were analyzed by cofocal microscopy. Direct fluorescent
images were taken. Scale bar=5 μm, *=lumena. White arrows indicate actin-coated vesicles.

68

Figure 25: The expression of Lifeact-RFP and GFP-actin in LGACs
A) mouse-anti-actin was used as primary antibody and IRDye®700-conjugated anti- mouse was
used as secondary antibody. lane1, non-transduced LGACs; lane 2, LGACs expressing Ad-EGFP;
lane 3, LGACs expressing GFP-actin transduced; lane 4, LGACs expressing lifeact-RFP; lane 5,
LGACs co-expressing lifeact-RFP and GFP-actin. Bands indicated around 45 kD are actin; bands
indicated around 75 kD are GFP-actin. B) rabbit-anti-GFP was used as primary antibody and
IRDye™800 conjugated goat-anti-rabbit was used as secondary antibody. lane1, non-transduced
LGACs; lane 2, LGACS expressing Ad-EGFP; lane 3, LGACs expressing GFP-actin; lane 4,
LGACs expressing lifeact-RFP ; lane 5, LGACs co-expressing lifeact-RFP and GFP-actin. Band
indicated around 30 kD is EGFP; bands indicated around 75 kD are GFP-actin. C)
rabbit-anti-mCherry was used as primary antibody and IRDye®800-conjugated goat-anti-rabbit
was used as secondary antibody. lane1, non-transduced LGACs; lane 2, LGACs expressing
GFP-actin; LGACs expressing lane 3, lifeact-RFP; lane 4, LGACs expressing lifeact-RFP and
GFP-actin. Band indicated around 30 kD is lifeact-RFP. Lysates were prepared in parallel for
western blotting.

69

Figure 26: Time-course confocal fluorescence microscopy of LGACs co-expressing
rAV-LifeAct-TagRFP and GFP-actin in CCH stimulation reveals significant actin
remodeling
LGACs expressing lifeact-RFP and GFP-actin were analyzed by confocal microscopy. Cells were
exposed to 100μM CCh at the onset of the time-lapse live cell imaging. Selected images of
lifeact-RFP and GFP-actin fluorescence at intervals throughout the time-lapse sequence are
shown. Scale bar=5 μm, *=lumena. White arrows indicate the actin remodeling.
70

Figure 26: Continued

71

Chapter 4. DISCUSSION
One of the most important tools employed in our study is hpIgR-EGFP expressed using
adenovirus transduction. This fluorescent protein fused construct is a valuable tool for
investigating pIgR trafficking mechanism, especially in primary cells. The novelty lies in
the synchronization and observability of pIgR instant tracking, by the means of confocal
fluorescence microscopy. It also suggested the possibility of constructing an N’ terminal
tagged fluorescent protein, which can be used to do the real-time tracking of cleaved SC
in its exocrine functions. We designed a SmCherry-pIgR Ad constructs which comprising
mCherry fluorescent protein fused with pIgR N’ terminal. We also added a piece of DNA
sequence ahead of mCherry protein which supposed to play role in pIgR sorting.
However, this construct had functional defects. In LGACs expressing SmCherry-pIgR,
pIgR was observed to evenly distribute throughout the cells, whereas the endogenous
pIgR was found mostly located in subapical region. Since the structure that is responsible
for ligand binding is located on an extracellular site, it would be more complicated to
design a tagged fluorescent protein link to pIgR N’ terminal without disturbing its binding
capacity with ligand and post-expression intracellular sorting.

72

Another important method we adopted to track pIgR was through SC-uptake using a time
course. By labeling the SC domain with primary antibody, we tracked the movement of
this single cohort of receptor by confocal microscopy. The reason why we developed this
kind of method is that LGACs are primary polarized cells that do not form a monolayer
in culture. It is difficult to separate the apical and basolateral plasma membrane by simply
growing the cells in transwell dishes like MDCK cells do. Therefore, our study proposed
reliable and practical method for investigating pIgR trafficking mechanism, for future
reference.

The transcytotic pathway of pIgR in MDCK cells was well illustrated (Rojas & Apodaca,
2002). A regulated secretory pathway and a transcytotic pathway of pIgR in LGACs were
also proposed in 2008 and 2011(Evans et al., 2008; Xu et al., 2011). Our study confirmed
and further provides insight into these two pathways. Rab11a is identified to be
associated with pIgR transcytosis together with myosin Vb motor protein, whereas
Rab3D is thought to regulate the secretory pathway of pIgR with myosin Vc protein.
There are two points making our study noteworthy. Firstly, myosin Vb and myosin Vc
were compared in the same model in parallel. For the first time we intuitively saw that
they did not colocalize with each other and had different functions. Secondly, we clearly
presented the difference between the two pathways under resting and stimulated stages.
73

At resting stage, pIgR is endocytosed from basolateral surface and then sorted into
transcytotic pathway, the cleaved SC serve as the main source to provide protection
function. After treatment with CCh, the release of SC from transcytotic pathway is
accelerated. In the meanwhile, SC stored in secretory vesicle undergoes a burst of
emancipation and thus the regulated secretory pathway plays the major role.

We then looked into the tracks of pIgR transporting in LGACs. By inhibiting MTs, pIgR
was trapped in Rab11a-enriched vesicles and cease moving from basolateral surface to
subapical region. This lined up with our expectations that the process is dependent on MT
networks. In a study about Rab27b regulating exocytosis of secretory vesicles, the
authors proposed that the Rab27b-enriched SV’s final release process near apical
membrane was dependent on the actin network (Chiang et al., 2011). We performed a
similar experiment by inhibiting the reorganization of F-actin beneath lumen with
DN-PKC-ε. Results are consistent with the previous study, revealing that the terminal
release of SC is directly or indirectly impaired by F-actin depolymerization.

After making a thorough inquiry of regulator effectors as well as the cytoskeleton, we
proposed a trafficking model of pIgR in LGACs. pIgR is synthesized and recycled to
basolateral membrane, loaded or unloaded with its ligand dIgA, pIgR is sorted to the
74

early endosomes where it undergoes processing and then is sorted into Rab11a-enriched
vesicles. Together with myosinVb and other dynein complexes, Rab11a-enriched vesicles
use MTs as tracks to transport pIgR-dIgA from basolateral membrane to apical surface.
Approaching and docking to APM F-actin network, SC or sIgA is released. However, not
all the newly synthesized pIgR goes through transcytosis: a subset directly goes into
Rab3D-enriched vesicles and then move towards APM with the help of myosin Vc. The
terminal release step is also dependent on F-actin reorganization. This process is known
as the regulated secretory pathway. CCh stimulation accelerated both pathways release
SC from secretory vesicles (Figure 27).
75

Figure 27: Working model of the trafficking of pIgR in LGACs
A schematic working model of trafficking of pIgR in LGACs, red arrow indicates transcytotic
pathway and blue arrow indicates regulated secretory pathway.
76

Why do LGACs recruit two pathways for secreting SC? A plausible explanation is that
they perform their duties in different circumstance. The dIgA is transported from serum to
tears by the transcytotic pathway, playing a major protective role in the mucosal system.
When the ocular surface undergoes a dramatic change in the external environment,
secretory pathway is activated and triggers SC release in large amount.

rAV-LifeAct-TagRFP is confirmed to be an effective F-actin marker in LGACs in our
study. We presented in the results section that lifeact-RFP clearly marks the
actin-enriched cortical region in LGACs (Figure 24) as well as actin-coated structures
under CCh stimulation (Figure 26). Interestingly, when co-expressed with another actin
marker, GFP-actin, lifeact-RFP seems not to label as many vesicles like structures as
GFP-actin does in the resting stage. After CCh treatment, both Ad-Tc- GFP-Actin and
rAV-LifeAct-TagRFP labeled secretory vesicles begin to emerge and fuse with each other.
Here comes the question, how do we define the vesicle-like structure at the resting stage
that was labeled by Ad-Tc-GFP-actin but not rAV-LifeAct-TagRFP? Is it because the
rAV-LifeAct-TagRFP is not effective enough to stain the structure, or is it because
Ad-Tc-GFP-actin alters actin polymerization so as to create more vesicles than
non-transduced cells? The preferred answer is the latter one. rAV-LifeAct-TagRFP is
proved to have high transduction efficiency in LGACs. There is no reason for
77

rAV-LifeAct-TagRFP to sufficiently stain f-actin beneath the lumen but not the other
actin-coated structure in cells. Another possible explanation is that Ad-Tc-GFP-actin
actually labels the vesicles not on the membrane but in the interspace. In contrast,
rAV-LifeAct-TagRFP which only stains F-actin would not be able to yield strong enough
signal in those vesicles. More investigations would be performed to give a better
understanding of this problem.
78

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Abstract (if available)

Abstract In lacrimal gland acinar cells, the polymeric immunoglobulin receptor is responsible for binding and transporting dimeric IgA from the basolateral membrane to subapical region to maintain the immune defense. Using confocal microscopy as a major research tool

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Asset Metadata

Creator Ma, Linlin (author)

Core Title Studies of polymeric immunoglobin receptor (pIgR) trafficking pathway and evaluation of rAV-LifeAct-TagRFP function in rabbit lacrimal gland acinar cells

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/26/2012

Defense Date 06/07/2012

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

Tag actin,lacrimal gland,lifeact,microtubules,OAI-PMH Harvest,PKC-ε,polymeric immunoglobulin receptor,Rab11a,Rab3D,secretory pathway,transcytosis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah F. (committee chair), Garner, Judy A. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email linlinma@usc.edu,victoria_malin@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-67412

Unique identifier UC11288402

Identifier usctheses-c3-67412 (legacy record id)

Legacy Identifier etd-MaLinlin-1014.pdf

Dmrecord 67412

Document Type Thesis

Rights Ma, Linlin

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA