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A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

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A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

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Content A DYNAMIC APICAL ACTIN CYTOSKELETON FACILITATES
EXOCYTOSIS OF TEAR PROTEINS IN RABBIT LACRIMAL
ACINAR EPITHELIAL CELLS
by
Galina Vladislavovna Jerdeva
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
May 2005
Copyright 2005 Galina Vladislavovna Jerdeva
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UMI Number: 3180375
Copyright 2005 by
Jerdeva, Galina Vladislavovna
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Dedication
This dissertation is dedicated to my family, my husband Dmitri, and to our beloved
daughter Alyona.
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iii
Acknowledgements
First and foremost I am deeply thankful to my advisor, Sarah Hamm-Alvarez,
who has been an inspiration and role model for me. Over the years we have worked
together she was always there for me during good times as well as during difficult
moments. I will always remember my years spent in the lab as the best years.
I would also like to thank my committee members, Prof. Austin Mircheff,
Curtis Okamoto, Judy Garner and Wei-Chiang Shen, for their kind help and
guidance. Thank you for all you help and advice! I am also very thankful to my
friends and lab members for their friendship and scientific help. I am especially
thankful for Francie Yarber for her technical instructions, professionalism, kindness,
and good humor. I could never done it without Francie.
I would also like to thank Prof. Joel Schechter and his laboratory for the
assistance in acquiring and interpretation of electron microscopy images. I would
like to express special thanks to Prof. Melvin Trousdale and his laboratory for the
help in preparation of adenoviral vectors.
Last, but certainly not least, I would like to thank my parents, Abakumov
Vladislav Arkadievich and Prikhodko Nina Stepanovna for their years of love, care,
and support. They have inspired and helped me at all times of my life.
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iv
Table of Contents
Page
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abbreviations ix
Abstract x
Chapter I: Introduction
Lacrimal acinar cells and their function 1
Overview of regulated secretion in lacrimal acinar cell 3
Cytoskeleton, actin filaments and their regulation 9
The role of the cytoskeleton in secretion 14
Actin targeted drugs 17
Myosin and its function 19
PKCs and its function 20
Relevance to Pharmaceutical Sciences 22
Chapter II: Methods
Solutions used in cell isolation and culture 24
Cell isolation and culture 25
Reagents 26
Production and purification of recombinant Adenovirus (Ad) 28
Detection of GFP-actin in transduced acini 30
Confocal fluorescence microscopy 30
FRAP analysis 32
Electron microscopy (EM) 35
Secretion assays 35
Quantitation of Western blots 36
Detergent extraction 37
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V
Biochemical analysis of PKCs-actin filament binding 37
Chapter III: A dynamic apical actin cytoskeleton facilitates exocytosis of 41
tear proteins in rabbit lacrimal acinar epithelial cells
Actin filaments are reorganized following acute exposure to CCH 43
GFP-actin coassembles with endogenous actin in transduced lacrimal 49
acini
Time-lapse confocal fluorescence microscopy analysis reveals 52
substantial reorganization of apical actin filaments only in CCH-stimulated
acini
FRAP analysis reveals increased apical actin filament turnover 52
BDM and ML-7 suppress CCH-induced increases in apical actin 59
dynamics while inhibiting apical exocytosis.
BDM and ML-7 reduce CCH-stimulated syncollin-GFP secretion 64
LAT B decreases apical actin filaments while enhancing secretory 69
responses
Chapter IV: Discussion. A dynamic apical actin cytoskeleton facilitates 76
exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells
Chapter V: Dominant negative PKCs impairs apical actin remodeling in 82
parallel with inhibition of carbachol-stimulated secretion in rabbit
lacrimal acini
Lacrimal acinar PKCs associates with actin filaments 83
Introduction of DN-PKCs alters acinar actin organization 89
CCH-stimulated release of syncollin-GFP is inhibited by DN-PKCs 98
CCH-stimulated apical release of SC from plgR is inhibited by DN- 102
PKCs
Chapter VI: Discussion. PKCs participates in the regulation of actin 108
filament remodeling during stimulated secretion
Chapter VII: Conclusions 118
Chapter VIII: Bibliography 124
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v i
List of Tables
Page
Table I. List of Replication-defective Adenovirus constructs used in the 40
study.
Table II. Mf values for apical actin in BDM- and ML-7-treated acini 74
expressing GFP-actin without and with CCH.
Table III. Effects of Ad-syncollin-GFP on lacrimal acinar secretion of 0- 107
hexosaminidase.
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v ii
List of Figures
Page
F ig .l Schematic diagram of a lacrimal acinus reconstituted from 4
four individual cells
Fig. 2 Electron micrograph o f rabbit lacrimal acinar cells 5
reconstituted in culture
Fig. 3 Actin filament dynamics 12
Fig. 4 Actin filaments of reconstituted lacrimal acinar cells 14
Fig. 5 Confocal fluorescence microscopy reveals changes in actin 45
filaments in CCH-stimulated lacrimal acinar epithelial cells
Fig. 6 EM images of resting and CCH-stimulated lacrimal acinar 46
epithelial cells
Fig. 7 Analysis of GFP-actin expression and distribution in 50
transduced lacrimal acinar epithelial cells
Fig. 8 Time-lapse confocal fluorescence microscopy of GFP-actin in 54
lacrimal acini reveals substantial remodeling of apical and
basolateral actin in CCH-stimulated cells
Fig. 9 FRAP analysis of GFP-labeled apical actin filaments in 57
lacrimal acini
Fig. 10 BDM and ML-7 treatments alter CCH-mediated actin 60
remodeling while inhibiting protein secretion.
Fig. 11 Time-lapse confocal fluorescence microscopy of GFP-actin in 65
lacrimal acini exposed to BDM or ML-7 prior to addition of
CCH reveals stabilization of apical actin and of actin-coated
subapical structures.
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Fig. 12
Fig. 13
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 21
Fig. 22
Fig. 23
BDM and ML-7 reduce the CCH-stimulated exocytosis of
syncollin-GFP
Syncollin-GFP is enriched in actin coated structures in acini
exposed to BDM or ML-7.
LAT treatment enhances CCH-stimulated secretion in parallel
with depletion of actin filaments at the APM.
PKCs is an actin binding protein in lacrimal acini
PKCs is enriched with apical actin and actin-coated
invaginations in lacrimal acini
High efficiency transduction of lacrimal acini with Ad-DN-
PKCs results in co-localization of overexpressed DN-PKCs
with actin filaments.
Transduction of lacrimal acini with Ad-DN-PKCs is
associated with changes in acinar shape.
DN-PKCs inhibits CCH-stimulated release of syncollin-GFP
into culture medium in co-transduced lacrimal acini
DN-PKCs inhibits CCH-stimulated release of syncollin-GFP
into culture medium in co-transduced lacrimal acini.
plgR is enriched with actin and PKCs at the APM in lacrimal
acini
Transduction with Ad-DN-PKCs inhibits CCH-stimulated
release of SC from lacrimal acini.
Working model
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Abbreviations
Ad: replication-defective adenovirus serotype 5
APM: apical plasma membrane
BDM: 2-3 butanedione monoxime
CCH: carbachol
DAPI: 4’,6-diamidino-2-phenylindole, dilactate
DN: dominant negative
FRAP: fluorescence recovery after photobleaching
GFP: green fluorescent protein
MF: microfilament
Mf: mobility fraction
mSV: mature secretory vesicle
PE: phenylephrine
plgR: polymeric immunoglobulin A receptor
PKC: protein kinase C
ROI: region of interest
SC: secretory component
Tc: tetracycline transcriptional element
tTA: tetracycline transcriptional activator
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X
ABSTRACT
Lacrimal acinar cells exocytose mature secretory vesicles at their apical
membranes in response to secretagogues. Here I use time-lapse confocal
fluorescence microscopy and fluorescence recovery after photobleaching to
investigate the changes in apical actin during exocytosis evoked by the muscarinic
agonist, carbachol (100 pM). Time-lapse confocal fluorescence microscopy of
reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus
(Ad) containing GFP-actin revealed a quiescent apical actin array in resting acini.
Carbachol increased apical actin filament turnover and promoted transient actin
assembly around apparent fusion intermediates. Fluorescence recovery after
photobleaching measurements revealed significant (p<0.05) increases and decreases,
respectively, in mobile fraction (Mf) and turnover times (t'A) for apical actin
filaments in carbachol-stimulated acini relative to untreated acini. 2,3-butanedione
monoxime and ML-7 each significantly decreased carbachol-stimulated secretion of
bulk protein and the exogenous secretory vesicle marker, syncollin-GFP. This
inhibition was accompanied by accumulation of apical actin filaments, suppression
of the increased M f of apical filaments elicited by carbachol, and accumulation of
actin-coated structures enriched, in Ad-syncollin-GFP transduced acini, in syncollin-
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x i
GFP. In contrast, latrunculin B significantly increased carbachol-stimulated
secretion of bulk protein and syncollin-GFP.
I also investigated the involvement of the PKCs in apical actin remodeling
during exocytosis. Lacrimal acinar PKCs bound to actin filaments and was co-
immunoprecipitated with anti-actin antibody. Confocal fluorescence microscopy and
biochemical analysis showed increased association of PKCs with apical actin in
stimulated acini. Overexpression of dominant negative (DN) PKCs in lacrimal acini
resulted in formation of actin-coated structures at the apical surface and extension of
actin-enriched processes from the basolateral surface. Ad-DN-PKCs transduction
significantly inhibited carbachol-stimulated secretion of bulk protein and 0-
hexosaminidase. Co-transduction of acini with Ad-syncollin-GFP and Ad-DN-PKCs
significantly inhibited carbachol-stimulated release of syncollin-GFP; Ad-DN-PKCs
transduction also suppressed carbachol-stimulated release of secretory component.
My findings suggest that the increased turnover of apical actin filaments,
driven in part by non-muscle myosin II, is an essential step in stimulated exocytosis
in lacrimal acinar cells. I also propose that PKCs is essential in the apical actin
remodeling in exocytosis.
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1
Chapter I: Introduction
Lacrimal acinar cells and their function
The main function of lacrimal acinar cells is production and stimulated
release of tear fluid which protects and supports the ocular surface. In humans, the
lacrimal gland is the size and shape of an almond and located above the eyeball,
within the orbit, on the upper and outer conjunctival sac. Together with conjunctival
goblet cells and stratified ocular surface epithelium, the lacrimal gland supplies tears
to bathe and protect cornea. Tear fluid is a complex biochemical mixture of
antimicrobial proteins, growth factors, mucins and immunoglobulins. The precorneal
tear film consists of a superficial lipid layer, a middle aqueous/mucin phase, and an
anchoring layer. It is demonstrated that proteins, soluble mucus with other soluble
components, electrolytes and aqueous fluid form a hydrated gel (Nichols, 1985;
Chen, 1997). The composition of the mucus gel has been extensively studied, but yet
has not been completely characterized. This gel forms chemical bonds with
underlying membrane-bound mucins (glycocalix) to connect with the corneal
epithelium. Besides water and electrolytes, tear film contains immunoglobulins,
including dimeric IgA, antimicrobial proteins, such as lysozyme and lactoferrin, and
various growth factors including transforming growth factor-a, epidermal growth
factor, and hepatocyte growth factor (Van Setten et al., 1994; Van Setten et al.,
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2
1989). Some of the mucins detected in the ocular surface epithelium and tear fluid
include MUC-1 (Inatomi et al., 1995), SMS (sialomucin complex)/MUC4
(Pflugfelder et al., 2000a), MUC-5AC (Jumblatt et al., 1999), and MEM (mucosal
epithelial membrane mucin, Pflugfelder et al., 1997).
Maintenance of ocular fluid of appropriate composition is essential for
preservation of corneal health. Growth factors in tears aid in corneal and conjunctival
wound healing, whereas bacteriostatic factors (IgA, lactoferrin, lysosomal
hydrolases) are essential for ocular clearance of pathogens. Deficiencies in tear fluid
production increases the susceptibility of the ocular surface to environmental insults
and may result in the development of pathological conditions such as
Keratoconjunctivitis sicca (dry eye) and Sjogren’s Syndrome, a severe condition
associated with immunological destruction of the lacrimal gland. Dry eye is
classified by the National Eye Institute based on the cause of the tear film disorder:
1) dry eye caused by deficiency of produced tear film or 2) dry eye caused by
excessive evaporative loss (Lemp, 1995). In both cases tear film disorder is
accompanied by symptoms of discomfort associated with the damage to the
interpalpebral ocular surface (Pflugfelder et al., 2000b). Dry eye is one of the most
common reasons of patient visits to the ocular clinical specialist. Women are more
prone to dry eye then men, especially in physiological states accompanied by altered
levels of androgen and prolactin (Mircheff et al., 1996).
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Overview of regulated secretion in lacrimal acinar cells
Tear fluid secretion by the lacrimal glands is mostly reflexive through
stimulation of the ocular surface and nasal mucosa (Heigle and Pflugfelder, 1996).
Lacrimal gland acinar cells employ a classical mechanism of regulated secretion:
secretory products are synthesized, packaged, and stored in the form of mature
secretory vesicles (mSV) ready to release their contents into the nascent tear fluid in
response to stimuli through muscarinic and/or adrenergic pathways. Contents of
mSVs (depicted as blue spherical structures in Figure 1) are released into the
lumenal space (L) defined by the membranes of the neighboring cells forming the
acinus (Figure 1) in response to stimuli. Apical plasma membrane (APM) is
separated from mSVs by an apparent barrier formed by a filamentous network of
actin filaments (Figure 1). mSVs in the lacrimal acinar cells are heterogeneous in
their size (0.5-3 micron) and density as reveled by electron microscopy analysis
(Figure 2) suggesting coexistence of several vesicular populations in the subapical
cytoplasm beneath the APM.
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Lacrimal Gland Acinus
4
Figure 1. Schematic diagram of a lacrimal acinus reconstituted from four individual cells. L -
lumen; APM - apical plasma membrane; BLM - basolateral plasma membrane; N-nucleus. Actin
filaments are represented as dense cortical crosslinked structures (in red) beneath the APM and as a
fainter filamentous network at the basolateral side. The apical actin network poses an apparent barrier
for the mature secretory vesicles (depicted as spherical blue structures) to fuse with the apical plasma
membrane.
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Figure 2. Electron micrograph of rabbit lacrimal acinar cells reconstituted in culture. The
image shows two representative lumenal regions with adjacent secretory vesicles which are different
in their size and density . L, lumenal region; SV, secretory vesicle and bar, 500 pm.
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Most tear protein stores are released at the APM of lacrimal acini from
pre-formed mSVs that are coated with the small GTPase, rab3D. These mSVs fuse
with the APM within a few minutes of secretagogue stimulation (Wang et al., 2003).
The rab family of small GTPases belongs to the Ras oncogene superfamily of
proteins. Due to their diversity and specific compartmental locations, it is believed
that rab proteins confer specificity to endocytic and exocytotic trafficking (Novick
and Zerial, 1997) and act as molecular switches cycling between GTP- and GDP-
bound states. Rab3 proteins have been specifically implicated in regulated secretion,
with the rab3D isoform expressed in gastric chief cells and in parotid, pancreatic, and
lacrimal acini (Ohnishi et al., 1996; Valentijn et al., 1996; Raffaniello et al., 1996;
Wang et al., 2003). Rab3D has been most extensively studied in pancreas. Rab3D
associates with the cytoplasmic face of zymogen granules by lipid anchors composed
of geranylgeranyl groups. Upon stimulation with secretagogue, rab3D relocates to
the Golgi or to another unidentified cytoplasmic compartment.
In the lacrimal gland, a subpopulation of secretory vesicles is also enriched in
vesicle-associated membrane protein 2 (VAMP2). Originally identified on neuronal
secretory vesicles, VAMP2 has been identified in acinar epithelial cells from several
exocrine tissues including lacrimal gland (Fujita-Yoshigaka et al., 1996). Studies in
parotid and pancreatic acini implicate VAMP2 in secretagogue-stimulated exocytosis
(Gaisano et al., 1994; Fujita-Yoshigaka et al., 1996; Hansen et al., 1999). However,
in many systems, VAMP2 and rab3D are thought to associate with the same vesicles,
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while they seem to label distinct vesicle populations in lacrimal acini (Wang et al.,
2003).
Besides the regulated exocytotic route of the release, some components of
tear fluid can utilize transcytotic and/or constitutive routes of secretion to mediate
normal physiological functions. These routes of secretion can be accelerated by
stimuli such as the muscarinic receptor agonist, carbachol (CCH). The classical
example of the transcytotic mechanism of secretion is the release of dimeric
immunoglobulin A (IgA) into the nascent tears utilizing transcytotic traffic from the
basolateral membrane (Zeng et al., 1998). The extracellular domain of the polymeric
IgA receptor, either alone (secretory component or SC) or complexed to dimeric IgA
(secretory IgA) can be released at the APM via the transcytotic pathway. Studies on
plgA receptor trafficking in model epithelial cell systems have shown that the
receptor is synthesized in biosynthetic compartments and then delivered to the
basolateral membrane. Once at the basolateral membrane, the receptor is
internalized (either unoccupied or bound to dimeric IgA) and transcytosed to the
apical surface. At the APM, the ligand-binding domain is cleaved from the receptor
and released into external secretions. This proteolytic fragment is known as SC, and
if dlgA is bound to the released SC, the complex is termed secretory IgA.
Enrichment of SC and secretory IgA in ocular fluid is one of the highest in the body
(Gudmondsson et al., 1985) indicating the importance of this immunoglobulin in the
protection of ocular surface from pathogens and extracelullar environment. In other
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cultured cell models, hormones and secretagogues, including cholinergic agonists,
modulate the traffic of plgA receptor to the APM to accommodate the changing
needs of the mucosal surfaces . The main role of the SC is thought to be to protect
secretory IgA from proteolytic degradation; however in the light of recent studies it
is also evident that SC by itself can serve as a non-specific microbial scavenger,
especially if SC is abundantly present in mucosal surface (Dallas and Rolf, 1998).
Even though SC is abundantly present in the lacrimal gland cells and released to the
nascent tears through the transcytotic pathway, the secretagogue-dependent
mechanism of its release at the APM and identity of the membrane compartments at
this terminal step has not been characterized in the lacrimal acinar cells.
Two signal transduction pathways in lacrimal gland are responsible for the
accelerated release of tear fluid and proteins in response to the activators such as
steroid hormones and neurotransmitters. Activation of the diacylglycerol
(DAG)/Ca2 + dependent pathway occurs through interaction with muscarinic
receptors. Binding to the receptors results in activation of phospholipase C,
production of 1,4,5-inositol triphosphate (IP3) and DAG, followed by the release of
intracellular Ca2+ . The cAMP-dependent pathway is activated through Gs coupled
receptors with activation of adenylate cyclase leading to activation of PKA (Zoukhri
et al, 1998; reviewed in Dartt, 1994). Activation of these transduction pathways leads
to the phosphorylation and activation of specific target proteins, some of which may
be the trafficking effectors listed above.
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To study the molecular mechanisms of lacrimal acinar protein secretion, in
our laboratory we utilize reconstituted primary cell culture from rabbit lacrimal gland
acini. Under defined culture conditions, the acinar epithelial cells isolated from
rabbit lacrimal gland reconstitute their polarity within two days. These reconstituted
acini are also able to form secretory lumens surrounded with mSVs and to exhibit the
ability to sustain a stimulated secretory response. The release of tear fluid in vivo
elicited by the parasympathetic nervous system can be mimicked in vitro by
exposure to the cholinergic agent, CCH (Gierow et al., 1996). This agent was the
principal secretagogue used in my studies. The effects of the stimulated secretory
response in acini can be studied biochemically and microscopically.
Cytoskeleton, actin filaments and their regulation
Eukaryotic cells have the ability to change their shape, accommodate directed
cellular movement, and sustain the intracellular transport of organelles and proteins
due to the structural and functional support of the cytoskeleton. The cytoskeleton is
composed of a complex network of filamentous systems extending throughout the
cytoplasm. Each filamentous network is composed of protein subunits: actin for
actin filaments, also known as microfilaments (MFs), tubulin for microtubules
(MTs), and intermediate filament proteins (vimentin, cytokeratin or lamins) for
intermediate filaments.
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My thesis has focused primarily on MFs and to a lesser extent on MTs. The
ability of these filaments to polymerize and depolymerize is controlled by a vast
array of cytoskeleton-interacting proteins. This functional and spatial flexibility of
filaments enables the cellular machinery to instantly react to signals from the
extracellular environment. MT- and MF-based motor proteins facilitate
cytoskeleton-related transport of proteins and/or organelles along long (MTs) and
short (MFs) tracks of their respective filamentous arrays. Since bacteria lack a
comparable cytoskeletal system, establishment of the cytoskeleton in eukaryotes is
apparently an important hallmark in the evolution of living organisms.
MTs are dynamic polymers of 25 nm diameter, formed from heterodimers of
globular polypeptides a-tubulin and (3-tubulin which are linked to each other in a
head to tail fashion. Thirteen linear protofilaments form a microtubule cylinder
(Alberts, 1994). By the addition and loss of subunits, MTs can grow or shrink,
reflecting MT dynamics. MTs have intrinsic polarity, with fast-growing ends,
referred to as plus-ends and slow-growing ends as minus-ends (Hirokawa, 1998).
MT polarity serves as a structural basis for the organization of spatially and
functionally distinct cellular organelles and as the "rails" for the motor proteins that
move cargo along the MT array. The “dynamic instability” of MTs, or the ability of
MTs to polymerize and depolymerize, is an essential feature of the cytoskeleton
important for many cellular functions including cell division and movement,
membrane trafficking, endocytosis and secretion. MT distribution in lacrimal gland
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11
acinar cells is typical of MT distribution in other epithelial cells with the minus-ends
located at the apical surface and the plus-ends at the basal-lateral membrane (da
Costa et al., 1998). MT dynamics and stability can be affected by agents interfering
with the exchange of tubulin subunits such as colchicine, by agents stabilizing MT
array such as taxol, or by MT-depolymerizing drugs such as nocodazole.
MFs are 7-9 nm wide filaments composed of globular actin that also have an
ability to polymerize and depolymerize to fulfill many cellular functions including
cytokinesis (Bi, 2001), cell motility (Krause et al., 2003; dos Remedios et al., 2003)
and endocytosis (Qualmann and Kessels, 2002; da Costa et al., 2003). MFs form a
polar structure with a slow-growing minus end (also referred to as a "pointed end"
due to the arrowheaded appearance of myosin-decorated filaments (Huxley, 1963),
and a faster-growing plus-end (Figure 3).
Actin filament assembly is regulated by a host of actin-binding proteins
(Figure 3, modified from McGrath et al., 1998). In vitro, assembly of actin
monomers can be blocked by cytochalasins (Cooper, 1987), while in vivo assembly
can be altered by specialized capping proteins and/or gelsolin (Barkalow et al.,
1996).
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Barbed end
(+)
V
Monomer flux
(-)
ooo
Pointed end
OO
a
>
ATP-actin
ADP*Pi-aefcin
A AD P-actin
Capping proteins and cytochalasins
□ Cofilin
O Thymosin
Figure 3. Actin filament dynamics. At steady state, actin filaments cycle monomers from the
pointed filament ends to the barbed ends. This process is driven by the hydrolysis of ATP in the
assembled filament. Proteins or agents that bind to the barbed end prevent assembly at this end.
Pointed end disassembly is promoted by barbed end capping and by cofilin, a small protein that binds
to ADP-actin subunits enhancing the rate of their dissociation.
Actin monomer subunits are constantly recruited to free barbed filament ends
from an unpolymerized pool maintained by the sequestering proteins, |34 thymosin
and profilin . Only a fraction of filaments have exposed barbed ends to prevent total
polymerization of cellular actin. At steady state, assembly at available barbed ends is
balanced by disassembly at pointed ends, providing flux of individual actin subunits
in the filament from the barbed to the pointed end. This process is powered by the
energy of ATP-hydrolysis, releasing free ADP-actin monomers from the pointed
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13
end. Cofilin is known to accelerate this process. ADP-actin then can be recharged to
ATP-actin with the help of profiling to replenish the pool of monomeric actin
available for subsequent rounds of polymerization.
Actin filament dynamics are exquisitely sensitive to changes in the
intracellular signaling environment, enabling rapid remodeling in response to stimuli.
Actin filament dynamics are essential for their participation in a number of functions
including cytokinesis ( Bi, 2001), cell motility (Krause et al., 2003; dos Remedios et
al., 2003) and endocytosis (Qualmann and Kessels, 2002; da Costa et al., 2003; ).
Actin filaments comprise the core of microvilli and the cortical actin
underneath the cellular plasma membrane. In polarized epithelial cells, cortical actin
forms specialized dense structures which include actin crosslinking and stabilizing
proteins and actin-based motor proteins. Together with other filamentous systems of
the cytoskeletal machinery of the cell, MFs provide cells with structural support and
serve as a basis for the directed movement of actin-related motor proteins (Alberts,
1994). Like other epithelial cells, MFs in acinar cells from lacrimal gland are
detected primarily beneath cell membranes (da Costa et al., 1998). Actin labeling in
lacrimal gland acini is detected primarily in intensely-stained regions beneath the
APM, representing the enrichment of the cortical network and actin-enriched
microvilli in this region (Figure 4). Actin filaments are also detected beneath the
basolateral surfaces of the cell (Figure 4).
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Figure 4. Actin filaments of reconstituted lacrimal acinar cells. Actin distribution delineated by
labeling with rhodamine phallodin within a typical control preparation of lacrimal acini as viewed by
confocal fluorescence microscopy (left). Schematic diagram corresponding to the fluorescence image
on the left (right). Thick lines depict the subapical actin network beneath the APM while * denote
lumenal regions. Bar, 10 pm.
The role of the cytoskeleton in secretion
It has been demonstrated that MT-based motors contribute significantly to the
directed release of secretory products during the process of regulated secretion in the
lacrimal acini. In particular, previous work has established the inhibitory effect of
MT-targeted drugs on regulated secretion (Robin et al., 1995; da Costa et al., 1998).
Recently we have addressed the role of cytoplasmic dynein in stimulated traffic to
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15
the apical plasma membrane in lacrimal acinar cells (Wang et al., 2003). In
particular, we have demonstrated that MT-dependent recruitment of cytoplasmic
dynein and dynactin is required during the process of regulated secretion in
reconstituted rabbit lacrimal acinar cells (Wang et al., 2003). However, the role of
MFs and actin binding proteins (ABP) in regulated secretion has been largely
disputed over the years in different cell systems. A precise role for actin
cytoskeleton in exocytosis in the lacrimal acinar cells has not been established.
Given the dense structure of the cortical subapical actin network beneath the APM, it
has been proposed in pancreas that apical MFs form a restrictive barrier to
spontaneous release of secretory vesicles to the apical surface. This physical barrier
imposed by the apical actin would require a transient reorganization or disassembly
of actin filaments to allow secretory vesicles to pass and fuse with the APM (Perrin
et al., 1992; Jungerman et al., 1995). Some evidence also suggests that actin-based
molecular motor-based short range movement of mSVs might be required at the
latest steps of regulated secretion (Valentijn et al., 1999). Earlier attempts to
evaluate the role of actin filaments in lacrimal acinar exocytosis using the MF-
targeted agents, cytochalasin D and jasplakinolide (da Costa et al., 1998; da Costa et
al., 2003b), did not reveal major changes in acinar secretion nor affect resting or
CCH-stimulated distributions of the mature secretory vesicle marker, rab3D.
However, it was unclear from these studies whether the actin filament array beneath
the APM was substantially affected by these treatments. Apical actin filaments in
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16
epithelial cells are more resistant to actin-targeted drugs than basolateral actin
filaments (Ammar et al., 2001). Our previous attempts to measure changes in the
filamentous (F-actin) to free (globular or G-actin) ratio elicited by CCH stimulation
using biochemical methods, while demonstrating no changes (da Costa et al., 1998),
may have been insufficiently sensitive to detect changes in particular
microenvironments. Moreover, biochemical analysis is particularly problematic if
actin reorganization involves both disassembly and reassembly within a rapid time
interval, which is possible during stimulated exocytosis.
Spurred by my recent confocal fluorescence microscopy analysis of actin
filament organization in fixed lacrimal acini, which suggested that transient changes
in apical filaments were elicited by acute exposure to CCH, I have reevaluated actin
filament participation in exocytosis in live acini, using carefully chosen actin-
targeted agents, time lapse video microscopy and FRAP (fluorescence recovery after
photobleaching) analysis. My data, which are reported in Chapter III, suggest that
dynamic reorganization of the apical actin network beneath the lumenal regions is
required to accommodate stimulated apical secretion in rabbit lacrimal acinar cells.
Additional data reported in Chapter V suggests that a novel actin-binding kinase,
PKCe, plays a major regulatory role in actin filament remodeling.
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17
Actin targeted drugs
One of the common approaches to investigate participation of actin filaments
in various cellular functions is to affect actin dynamics and stability by using actin-
targeted drugs. These agents differ in their origin, potency, and mechanism of
action. One of the widely used actin-destabilizing drugs is Cytochalasin D (CD).
Cytochalasins are membrane-permeable fungal metabolites, which bind to the barbed
end of MFs, inhibiting association and dissociation of actin subunits from that end.
This mode of action is similar to that of capping proteins which can bind the barbed
end of MF and control MF polymerization in vivo (Cooper, 1987). However, CD
when used in our acinar cell culture, is more potent in altering basolateral MF than
affecting the apical MF network. The apical MF network is considerably more
resistant to actin-targeted drugs than the basolateral array. The macrolide, latrunculin
B (LAT B), is another actin-destabilizing agent widely used in cytoskeleton research.
LAT B is a lethal toxin from sea sponge, which is known to destabilize actin
filaments through binding and sequestration of actin monomers (Spector et al.,
1983). Besides its ability to destabilize basolateral MFs, LAT B is able to strongly
affect apical MFs, an effect crucial in the evaluation of actin filament contribution to
stimulated exocytosis at the APM in lacrimal acini. One report in parietal cells
suggests that doses of LAT B required to elicit changes in apical actin relative to
basolateral actin are 50x higher (Ammar et al., 2001).
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If one wants to investigate the role of actin-based motor proteins in the
cellular processes, inhibitors of the acto-myosin system such as 2, 3-butanedione
monoxime (BDM), myosin light chain kinase inhibitor 7 (ML-7), and blebbistatin
can be used to perturb myosin function. BDM has been extensively utilized as an
uncompetitive inhibitor of myosin ATPase activity (Higuchi and Takemori, 1989;
Herrmann et al., 1992). Exposure of cells to BDM dissociates myosin from actin
filaments, impairing myosin involvement in events as varied as muscle contraction
(Herrman et al., 1992) and myosin-based vesicle transport (Bennett et al., 2001;
Neco et al., 2002; Duran et al., 2003). ML-7 is a cell permeable, potent and selective
inhibitor of myosin light chain kinase, which inhibits myosin II function at relatively
low concentrations (Ki=300 nM) (Saitoh et al., 1987). Blebbistatin was demonstrated
to have inhibitory effects on cells migration, blebbing and spreading also due to
effects on myosin II function. The inhibitory effect of blebbistatin is thought to be
due to binding to the myosin-ADP-P; complex and interfering with the phosphate
release process. Therefore blebbistatin prevents binding of myosin II to actin,
preventing actomyosin cross-linking (Kovacs et al., 2004). However, it is unclear
what cell types can be affected by each of these agents. Moreover, investigations
could be hindered by the fact that, in many cases, the exact identities of myosin
families expressed in particular cell systems not always known.
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Myosin and its function
The actin filament-based motor proteins, myosins, can be segregated into two
categories: the conventional, or muscle myosins, and the unconventional myosins
including all other myosins. The growing myosin superfamily includes 18 distinct
classes, with only one class representing conventional myosins. All myosins have an
N-terminal globular head region which contains ATP and actin binding domains.
The middle “Neck” region contains one or more “IQ-domains” that serve as sites for
interaction with light chains. Diverse C-terminal “Tail” domains are believed to
mediate association between motors and their cargoes (Reilein et al., 2001).
Non-conventional myosin II is abundantly present in non-muscle cells
including neurosecretory cells (Rose et al. 2002) and pancreatic acini (Torgerson
and McNiven, 2000), and is known to participate in many cellular functions such as
cell blebbing (Kovacs et al., 2004) and regulated secretion of insulin (Wilson et al.,
1999). Since myosin II has been implicated in secretory functions in other systems, I
have evaluated the ability of myosin II inhibitory drugs such as the broad spectrum
inhibitor, BDM, to affect regulated secretion and/or actin filament remodeling in
lacrimal acinar cells.
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PKCs and its function
The area under the APM in lacrimal acini is enriched in a dense network of
actin filaments which appear to form a barrier preventing uncontrolled SV release in
unstimulated acini. Several studies have shown that extensive remodeling of the
underlying actin network accompanies stimulated secretion in acinar epithelial cells
including pancreatic acini (Muallem et al., 1995; Valentijn et al., 2000; Nemoto et
al., 2004), parotid acini (Perrin et al., 1992) and lacrimal acini (Jerdeva et al.,
submitted and Chapter III). This remodeling includes increased turnover as well as
transient formation of actin coats beneath fusing SVs, which we term actin-coated
fusion intermediates. Normally these actin-coated intermediates are rapidly
assimilated back into the apical actin array but their stabilization is associated with
inhibition of exocytosis. Little is known about the effectors that act to regulate the
extensive actin filament remodeling in acinar exocytosis.
I have investigated the contribution of the novel protein kinase C (PKC)
isoform, PKCs, to actin filament remodeling and apical exocytosis. The PKC family
has multiple subspecies, each with distinct cellular localizations and functions. Three
subfamilies of PKC are distinguished based on their requirements for activation: 1)
the Ca2 + -dependent or conventional PKCs; 2) the Ca2 + -independent or novel PKCs;
and 3) the phorbol ester-insensitive or atypical PKCs (reviewed in Dekker and
Parker, 1994; Newton, 1993). All PKCs consist of a single polypeptide chain
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containing regulatory N-terminal and catalytic C-terminal domains. Four conserved
domains (C1-C4) interrupted by five variable regions (VI-V5) constitute the
conventional PKCs. Conventional isoforms have both the Cl domain, which binds
diacylglycerol and phosphatidylserine, and a C2 domain which is important for Ca2 +
binding. Novel PKC isoforms lack the C2 domain, rendering them Ca2 + -insensitive,
and making them maximally responsive to diacylglycerol and phorbol esters. All
isoforms have a C3 region representing the catalytic domain and containing the
ATP-binding sequence, as well as a C4 region containing the pseudo-substrate-
binding site which regulates substrate interactions (Aksoy et al., 2004).
Distinguishing it from other novel PKCs, PKCs also has a unique actin-binding site
spanning amino acids 223-228 which is located between Cl and C2 domains
(Prekeris et al., 1996; reviewed in Akita, 2002). Association of PKCs with actin
filaments is triggered by binding of diacylglycerol or phorbol esters to the Cl
domain; conversely, association of PKCs with actin filaments maintains the kinase in
the active state. It is thought that the phosphorylation of specific substrate proteins
by PKCs is regulated by its targeting to actin filaments or its other partner, Golgi
beta’-COP. The targeting brings the activated kinase into close proximity to target
molecules.
Based on all of the background information presented in this section, the
focus of my investigations was to demonstrate the role of apical actin filaments and
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actin-associated proteins in regulated secretion in lacrimal acinar cells. The
following hypotheses have been addressed in the indicated chapters:
1. Whether a dynamic apical actin cytoskeleton is required during the process of
stimulated secretion in cultured lacrimal acinar cells and whether acto-myosin
contractile force is required to accommodate exocytosis. (Chapter ID).
2. Whether the actin-binding protein, PKCs, regulates the apical actin remodeling
during stimulated secretion in the lacrimal acini. (Chapter V).
Relevance to Pharmaceutical Sciences
Lacrimal deficiency-associated diseases range from dry eye, or
keratoconjunctivitis sicca, to corneal damage and autoimmune destruction of the
gland (Sjogren's syndrome). Recent progress in the treatment and diagnosis of dry
eye reflects the increase of the knowledge about the pathogenesis and composition of
the precorneal tear film. In spite of the fact that diagnosis and therapy of dry eye is
considerably advanced over the past decade, treatment of these diseases remains
primarily symptomatic, including application of artificial tears, anti-inflammatory
therapy (Kazwan et al., 1988), application of topical corticosteroids (Marsh et al.,
1999), systemical administration of androgenic hormones (Sullivan et al.,1996) or
use of autologous human serum (Tsubota et al, 1999) Understanding of the
mechanisms of lacrimal acinar secretion at the cellular level will dramatically
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23
advance the understanding of these diseases. An increase in our knowledge of
regulated secretion and its effectors under both normal and pathophysiological
conditions will provide the groundwork for the successful drug design and treatment
of lacrimal deficiency-associated diseases. Expanding knowledge of the key
effectors of the secretory pathway and their regulation may provide necessary
information about proteins affected by the diseases. Therefore, the investigations
directed to uncover regulation of key effectors of secretory pathway in the lacrimal
gland ultimately will be important in understanding of disease pathogenesis and
design of new therapies.
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Chapter II: Methods
Solutions used in cell isolation and culture
For the isolation and culture of lacrimal acinar cells, solutions were used as
described (Gierow et al., 1996). Hank’s balanced salt solution was supplemented
with 2 mM NaEDTA and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid (HEPES) and brought to pH 7.6 with 1 N NaOH (s-Hanks’). Ham’s F-12
medium was supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM
glutamine, 0.3 pM linoleic acid, 2 mM sodium butyrate, 50 pg/ml soybean trypsin
inhibitor, and 5 mg/ml bovine serum albumin and brought to pH 7.6 with 1 M NaOH
(s-Ham’s). Just before the start of the cell isolation procedure, 266 U/ml collagenase,
53 U/ml DNase , and 667 U/ml hyaluronidase were dissolved in s-Ham’s (CDH) and
filtered through 0.45-pm filters. The culture medium (PCM) was made from a 1:1
mixture of Ham’s F-12 medium and DME supplemented with 100 U/ml penicillin,
0.1 mg/ml streptomycin, 2 mM glutamine, 0.3 pM linoleic acid, 2 mM sodium
butirate, 5 nM hydrocortisone, 5 pg/ml transferrrin, 5 pg/ml insulin, and 30 nM
sodium selenite. Im m ediately before use, 0.1 pM CCH , 5 pg/m l lam inin, and 1 nM
thyroxine were added.
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Cell isolation and culture
Isolation of lacrimal acini from female New Zealand white rabbits (1.8-2.2 kg)
obtained from Irish Farms (Norco, CA) was in accordance with the Guiding
Principles for Use of Animals in Research. Lacrimal acini were isolated as described
(da Costa et al., 1998; Yang et al., 1999; Qian et al., 2002). The glands were
collected into a beaker containing s-Ham’s that had been equilibrated at 37°C with
95% O2 -5% CO2 . The fragments were rinsed with s-Ham’s, placed in a 3-ml pool of
s-Ham’s on ultraviolet -irradiated dental wax, and cut into 1 mm3 pieces with a pair
of scalpels. The pieces were transferred to a 50-ml Erlenmeyer flask and swirled in
20 ml of s-Ham’s. The supernatant was aspirated, and the swirling procedure was
repeated with 20 ml of s-Ham’s and then with 20 ml of s-Hanks’. The pieces were
resuspended and distributed into three 50-ml Erlenmeyer flasks containing 15 ml of
s-Hank’s each. The flasks were flushed with 95% O2 -5% CO2 for 5 s, capped , and
incubated for 15 min in a 37°C shaking water bath set at 120 oscillations/min. The
supernatants were aspirated, and the fragments were washed once with 12 ml of s-
Ham’s per flask. The wash solution was replaced with 15 ml of CDH per flask, and
the fragments were incubated with shaking for 15 min. The suspensions were
transferred to three 50-ml conical centrifuge tubes, and cells and remaining tissue
fragments were sedimented by centrifugations in a swinging bucket rotor at 100 g for
5 min at room temperature. The resulting supernatants were aspirated, and each
pellet was resuspended in 20 ml of s-Hanks’. The suspention was pelleted once
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more, resuspended in 15 ml of s-Hanks’, transferred back to the Erlenmeyer flasks,
and incubated with shaking for another 15 min before a final 25-min treatment with
CDH. The cells were pelleted, washed twice with s-Ham’s, and filtered through 500-
and 25-pm nylon mesh. The filtrate was carefully layered on top of a discontinuous
gradient of 10 ml each of 4,3, and 2% Ficoll in s-Ham’s and centrifuged at 50 g for
15 min. The pellet was resuspended in s-Ham’s and washed once with s-Ham’s and
once with PCM. The final cell pellet was resuspended in PCM and distributed to
150-mm plastic tissue culture plates, to 12-well or 24-well plates with or without
coverslips, or to 35-mm round glass-bottom dishes. The cells were incubated at 37°C
in air with 5% CO2 for 40 h. Cells cultured under these conditions aggregate into
acinus-like structures; individual cells within these structures display distinct apical
and basolateral domains and a polarized cytoskeleton, and maintain a robust
secretory response (da Costa et al., 1998).
Reagents
Carbachol (CCH), phenylephrine (PE), rhodamine-phalloidin, BDM and goat anti
rabbit secondary antibody conjugated to FITC were obtained from Sigma Chemical
Co (St. Louis, MO). Rabbit polyclonal antibody to rab3D was generated against
recombinant mouse rab3D (Antibodies, Inc., Davis CA) and purified using protein
A/G Agarose. Sheep anti-rabbit antibody to SC was generated against purified SC
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from rabbit bile (Pel-Freeze, Rogers, AK) by preparative gel electrophoresis and
used to produce sheep anti-rabbit SC polyclonal antiserum (Capralogics, Hardwick,
MA). The antiserum was of sufficient titer to use diluted for Western blotting. For
immunofluorescence, antibodies were purified from antiserum using protein G-
Sepharose (Amersham-Pharmacia). Mouse monoclonal anti-syncollin antibody was
kindly provided by Dr. Michael Edwardson (Cambridge University). Rabbit
polyclonal antibody to PKCs was obtained from Santa Cruz Biotechnology, Inc
(Santa Cruz, CA) and used for Western blotting and immunofluorescence labeling.
Mouse monoclonal antibody to PKCs was purchased from BD Transduction
Laboratories (Lexington, KY) and used for immunofluorescence labeling in acini
transduced with Ad-DN-PKCs. Rabbit ProLong antifade mounting medium and
4',6-diamidino-2-phenylindole, dilactate (DAPI), goat anti-rabbit secondary antibody
conjugated to Alexa Fluor-568 or Alexa Fluor-647 and Alexa Fluor-647-phalloidin
were from Molecular Probes (Eugene, OR). Cell culture reagents were from Life-
Technologies. Rabbit polyclonal antibodies against actin and green fluorescent
protein (GFP) were purchased from NOVUS (Littleton, CO) or Santa Cruz
Biotechnology (Santa Cruz, CA), respectively. BD Adeno-X™ virus purification
and Adeno-X™ rapid titer kits were obtained from BD Biosciences (Palo Alto, CA).
Goat anti-mouse, anti-rabbit and donkey anti-sheep IRDye™800-conjugated
secondary antibodies were purchased from Rockland (Gilbertsville, PA). CCH and
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PE were used at 100 jjM unless indicated. BDM treatment was for 15 min at 10
mM.
Production and purification of recombinant Adenovirus (Ad)
QB1 cells, a derivative strain from HEK293 cells, were infected with Ad-Tc-GFP-
Actin, Ad-tTA or with Ad expressing DN-PKCs (and GFP separately;Ad-DN-
PKCs), Ad encoding GFP alone (Ad-GFP) and Ad encoding a syncollin-GFP fusion
protein (Ad-syncollin-GFP). Table I at the end of this Chapter provides information
about all Ad constructs used in this study. Cells were grown at 37°C and 5% CO2 in
DMEM (high glucose) containing 10% fetal bovine serum for 66 hours until
completely detached from the flask surface. The BD Adeno-X™ virus purification
kit was used for virus purification and Adeno-X™ rapid titer kit for viral titration or
using cesium chloride ultracentrifugation as described (Wang et al., 2003). For some
constructs viral titers were determined using the tissue culture infectious dose5 o assay
on 293 cells.
For expression of GFP-actin, cells were co-transduced with AD-Tc-GFP-
Actin and Ad-tTa. Co-transduction of Ad-Tc-GFP-actin and Ad-tTA allows
controlled expression of GFP-actin (Gossenet et al., 1995). Tet-Off and Tet-On gene
expression systems allow high-level expression of cloned genes in response to
varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline. In
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the Tet-Off system, the gene of interest is cloned into a site controlled by the Tc
operator sequence which is turned on when Tc or Dox is absent from the culture
medium. The coexpressed hybrid protein encoded by Ad-tTA, tet-controlled
transcriptional activator (tTA), binds the Tet operator sequence (tetO) to activate
transcription. When Tc or Dox is present in the culture medium, Tc binds to tTA
preventing transcription. tTA is a fusion protein of the wild-type Tet repressor
(TetR) to the VP16 activation domain of herpes simplex virus. In our system, Tc was
absent from the culture medium.
Transduction with Ad constructs involved exposure for 1-3 hrs on day 2 of
culture at a multiplicity of infection (MOI) of 5, followed by washing with
Dulbecco’s phosphate buffered saline (DPBS) and incubation in fresh culture
medium for 18-20 hrs at 37°C and 5% CO2 . Cells were analyzed on day 3 of culture.
Transduction efficiency was maintained at -70-90%, consistent with previous reports
(Wang et al., 2003). For co-transduction studies, an MOI of 5 was used for each Ad
construct. Dual transduction efficiency was difficult to quantify using flow
cytometry since the constructs of interest both expressed GFP (Ad-DN-PKCs co
expressing GFP and Ad-syncollin-GFP). However, analysis by confocal
fluorescence microscopy in fixed acini showed co-expression of cytosolic PKCs and
large syncollin-containing vesicles in -70% of lacrimal acini, consistent with
efficiencies seen for co-transduction by other constructs in lacrimal acini.
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Detection of GFP-actin in transduced acini
Lacrimal acinar cells cultured on Matrigel-coated coverslips in 12-well plates at a
density of 2 x 106 cells/well were co-transduced with Ad-Tc-GFP-Actin and Ad-tTA
at MOIs ranging from 1.5-6 at 37°C and 5% CO2 for 2 hrs. Virus was removed and
cells were rinsed with DPBS before addition of fresh culture medium and incubation
for 20 hrs. Non-transduced cells and cells transduced under identical conditions with
Ad-GFP served as controls. Cells were rinsed with DPBS and lysed in RIPA buffer
containing protease inhibitor cocktail (da Costa et al., 1998) on a rocker platform at
4°C for 1 hr. The lysates were clarified by centrifugation. Equal amounts of total
proteins from each sample were resolved by SDS-PAGE, and resolved proteins on
the gel were transferred to nitrocellulose membranes. Membranes were blocked with
Odyssey blocking buffer, followed by hybridization with appropriate primary and
IRDye™800-conjugated secondary antibodies and quantified using an Odyssey
Scanning Infrared Fluorescence Imaging System (Li-Cor, Lincoln Nebraska).
Confocal fluorescence microscopy
For analysis of actin filaments and other proteins in fixed cells, reconstituted rabbit
lacrimal acini cultured on Matrigel-coated coverslips were processed as described
(Wang et al, 2003; da Costa et al., 2003). Briefly, acini were rinsed with DPBS,
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fixed and permeabilized with ethanol at -20°C for 10 min, rehydrated in DPBS and
blocked with 1% bovine serum albumin. Acini were then incubated with appropriate
primary and fluorophore-conjugated secondary antibodies and/or rhodamine
phalloidin. In some cases, DAPI was added to label nuclei. Most confocal images
were obtained with a Zeiss LSM 510 Meta NLO imaging system equipped with
Argon, red HeNe and green HeNe lasers as well as a Ti-Sapphire tunable Coherent
Chameleon laser (720-950 nm) mounted on a vibration-free table and attached to an
incubation chamber controlling temperature, humidity and CO2 . The ability of this
system to acquire fluorescence emission signals resolved within narrow ranges in
multitrack mode, and the use of singly-labeled control samples ensured the validity
of co-localization studies. Some confocal images were acquired with a Nikon PCM
Confocal System equipped with Argon ion and green HeNe lasers attached to a
Nikon TE300 Quantum inverted microscope. All panels were compiled in Adobe
Photoshop 7.0 (Adobe Systems Inc, Mountain View, CA).
For live cell imaging of GFP-actin transduced acini or acini transduced with
syncollin-GFP, rabbit lacrimal acini seeded on Matrigel-covered glass-bottom round
35 mm dishes (MatTek, Ashland MA) at a density of 4 x 106 cells/dish for 2 days
were co-transduced with appropriate Ad constructs at an MOI of 6 for each (for Ad-
Tc-GFP-actin and Ad-tTA) or MOI of 5 for other constructs for 1-3 hrs. Cells
were then rinsed in DPBS and cultured in fresh culture medium for 18-24 hrs to
allow protein expression. On day 3, lacrimal acini were analyzed by time-lapse
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confocal fluorescence and DIC microscopy or FRAP analysis using Zeiss Multiple
Time Series V3.2. and Physiology V3.2. software modules. All live cell analyses
were performed at 37°C. For time-lapse confocal fluorescence/DIC microscopy
analysis, acini of similar size (4-6 cells arranged around a central lumen) were
chosen. DIC images and GFP fluorescence were acquired simultaneously using the
488 line of the Argon Laser.
FRAP analysis
A 30 mW Argon Laser (488 nm) set at 60% power with 100% transmission was used
to photobleach a circular region of interest (ROI) ~l-2 pm in diameter; image
acquisition post-bleach was at 0.1% of transmission with the same laser power
without frame averaging to avoid photobleaching of the ROI during imaging
acquisition. The fluorescence associated with the entire acinus was simultaneously
recorded as a control to ensure that image acquisition did not significantly reduce the
fluorescence associated with the cells under study. The loss in total cellular
fluorescence did not exceed 10-20% during 90 sec of observation. Also, since
recording of the fluorescence of the whole cell area was available together with
recording of the fluorescence of circular bleached ROI, it was obvious whether the
ROI moved out of focus. If this occurred during the experiment, the data were
discarded. A region of comparable size within the cytosol (containing G-actin) was
photobleached in parallel and shown to exhibit almost complete recovery (-95%)
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over the time period of interest, demonstrating that the parameters chosen for
photobleaching were appropriate. Additional controls were performed in each
experiment as recommended (Snapp et al., 2003, Lippincott-Schwartz et al., 2003).
Pilot experiments and published rates of actin filament turnover suggest that
complete filament exchange normally requires time scales of minutes. We limited
our observation time to 90-100 sec due to the extreme mobility of the apical actin
filaments in stimulated acini; during this shorter time scale, problems associated with
remodeling out of the plane of focus or away from the photobleached spot were
minimized. This time frame of observation was sufficient to demonstrate dramatic
differences in the mobile fraction (Mf) of apical GFP-actin under the different
conditions in our study. Mf was calculated from the equation:
Mf = Y x (F„ - F0)/( Fj - F0 ) x 100%
Where F; - initial fluorescence recorded right before the bleach, Fo -
fluorescence recorded right after the bleach, F .„ - fluorescence at the end of
observation time, Y - correction factor for the bleach during observation.
Axelrod et al. (1976) related half-times of recovery curves for FRAP to the
De ff, effective diffusion coefficient. This classical approach was chosen to evaluate
the mobility of fluorescent actin by estimating the De ff from FRAP recovery curves.
For analysis, data were converted to the fractional fluorescence (F-Fo/Fi-Fo) and
fitted to equation (12) published by Axelrod et al. (1976) by the method of least
squares using Solver function of Microsoft Excel program.
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A series solution for Fk(t) valid for all K and t describes unrestricted two-
dimensional diffusion in a circular bleaching area:
Fk(t)=(qP0 C0 /A) Z [(-K)7n!][l + n(l +2t/ti/2 )]'1
Where Po -total laser power, Co - initial fluorophore conc., A - attenuation factor
of the beam during observation of recovery, q - product of all quantum efficiencies,
K - “amount” of bleaching; n of 20 was used in all calculations.
Turnover time t/2 (ortoeff) is related to D e fr by the equation:
tD efr = w2 y/ (4De ff)
Where w - is the half-width of the intensity at e'2 height, D eff - effective diffusion
coefficient, y - is a correction factor for the amount of bleaching. More details on the
principles and applications of FRAP analysis are discussed in Reits and Neefjes
(2001). For statistical analysis paired two sample for means t-tests were utilized to
compare CCH-treated samples with their own internal dish controls. M f and ty2 were
evaluated at four intervals after CCH stimulation: 1-4 min CCH, 5-10 min CCH, 10-
12 min CCH, and 15-18 min CCH. Results are from five separate cell preparations
with 3-7 dishes used from each preparation at each time point.
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Electron microscopy (EM)
For EM, acini cultured on Matrigel-coated Biocoat Transwell Plates (Becton
Dickinson) were processed according to Schechter et al., 2002. Control and treated
acinar cells were pelleted by centrifugation and fixed in 2% paraformaldehyde/2%
glutaraldehyde in 0.1 M cacodylate buffer, 0.02% CaCl2 at pH 7.4 for 2 hrs. After
washing, samples were post-fixed in 1.0% OsC>4 in 0.1 M cacodylate buffer, 0.02%
CaCb at pH7.4. The cells were washed again, then treated with 1.0% tannic acid
before dehydration in a graded ethanol series, stained en bloc with 2% uranyl acetate
in 50% ethanol and embedding in LR White. Subsequent thin sections were stained
with 2.0% aqueous uranyl acetate and Sato’s-modified lead stain. Samples were
analyzed using a JEOL 1200 EX transmission electron microscope.
Secretion assays
Control and pretreated or Ad-transduced rabbit lacrimal acini seeded in Matrigel-
coated 24-well plates were incubated in fresh medium before removal of an aliquot
for measurement of bulk protein content or P-hexosaminidase activity. After
treatment with or without CCH (100 pM, 0-30 minutes), a second aliquot of medium
was removed for measurement of these values. In each assay, protein and 1 3 -
hexosaminidase release were calculated from 5-6 replicate wells/treatment and
normalized to total cellular protein. Basal and total (basal plus stimulated) release
were plotted. Differences in experimental groups were determined using a paired t-
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test with p<0.05. Protein was measured with the Micro-BCA Protein Assay (Pierce)
using bovine serum albumin as standard, and P-hexosaminidase activity was
assessed using methyumbelliferyl-p-D-glucosaminide as substrate. For analysis of
SC or syncollin-GFP release into culture medium, culture medium from resting and
CCH-stimulated acini was collected, concentrated on Centricon 10 filters, equal
volumes resolved by SDS-PAGE, and proteins of interest detected by Western
blotting. Signal intensity was normalized to pellet protein in each sample and
expressed as fluorescence intensity/mg protein before normalization to control and
comparison across treatments. Differences in experimental groups in all secretion
assays were determined using a paired t-test with p<0.05.
Quantitation of Western blots
For quantitation of proteins on Western blots, the majority of blots were processed
using secondary antibodies conjugated to IRDye™800 and quantified using an
Odyssey Scanning Infrared Fluorescence Imaging System (Li-Cor, Lincoln, NE).
With tubulin as standard, we have established that this system is linear over a 12-fold
range (R2 =0.98); our scanned values fall within this range. For display, fluorescent
signals were converted digitally to black and white.
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37
Detergent extraction
Sequential detergent extraction was as described by Hollenbeck (Hollenbeck, 1989).
Lacrimal acini on Matrigel-coated dishes were exposed to extraction buffer (0.1 M
PIPES, pH 7.0, 5 mM MgSCL, 10 mM EGTA, 2 mM DTT supplemented with
protease inhibitor cocktail) containing 4% polyethylene glycol, 10 pM taxol and
0.02% saponin for 12 minutes at 37°C. Extraction buffer supplemented with 4%
polyethylene glycol, 10 pM taxol and 1% Tx-100 was then added for 8 minutes at
37°C. After rinsing, the remaining material was scraped into extraction buffer
containing 1% SDS. The distribution of proteins of interest across pools was
determined by SDS-PAGE and Western blotting. Western blots were processed
utilizing appropriate primary antibodies and either goat anti-mouse, anti-rabbit or
anti-sheep secondary antibodies conjugated to IRDye™800.
Biochemical analysis of PKCs-actin filament binding
For co-immunoprecipitation experiments, lacrimal acini were collected by scraping
on day 3 and harvested by centrifugation in conical 50 ml tubes. The cell pellet was
homogenized in RIP A buffer containing 2 pM okadaic acid, 2 pM NaF, 1 mM
sodium orthovanadate, 1 mM dithiothreitol and protease inhibitor cocktail (final
concentrations of 10 pg/ml TAME, 10 pg/ml TLCK, 1 pg/ml leupeptin, and 0.2 mM
PMSF) by shearing through a 21 gauge needle 10 times on ice followed by 5 min ice
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38
incubation and then 10 passes again through the needle. The homogenate was
centrifuged in a microcentrifuge at 4°C for 10,000 rpm, 10 min, and the supernatant
processed for immunoprecipitation. Lysate was incubated with mouse monoclonal
antibody for 1 hr at 4° C. Protein A/G Agarose suspension was added to this mixture
and mixed with end-over-end agitation for 2-3 hrs at 4°C. Beads were collected by
centrifugation at 1000 x g for 5 min at 4°C, and washed 4x with PBS followed by re
centrifugation. Samples were dissolved in sample buffer, resolved by SDS-PAGE
and analyzed by Western blotting as described below.
For actin filament binding assays, the non-muscle Actin Binding Protein
Biochemistry Kit from Cytoskeleton, Inc (Denver, CO) was utilized. Cell lysates
were prepared by shearing acinar cells in A buffer [5 mM Tris-HCl pH 8.0
containing 0.2 mM CaCL and protease inhibitor cocktail as above] through a 21
gauge needle on ice and clarified by low speed centrifugation as described above.
The supernatant fraction was concentrated in Amicon 3000 filters. The concentrated
supernatant fraction was centrifuged for 1 h at 150 000 x g at 24°C before addition to
the actin polymerization reaction. PKCs and actin contents in supernatant and pellet
fractions were measured by Western blotting. 250 pg aliquots of non-muscle actin
(APHL99) were defrosted , resuspended at 1 mg/ml in General Actin Buffer (BSA 1.
5mM Tris-HCl pH 8.0, 0.2 mM CaCl2 and 0.5 mM DTT), i.e. in 250 ul of BSA01,
and incubated on ice for 30 min. Then 25 ul of actin polymerization buffer (BSA02:
10 x solution contained 500 mM KC1, 20 mM MgC12 and 10 mM ATP) was added
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39
to the non-muscle soluble actin. This buffer was kept at 24°C for the duration of the
use and was frozen in liquid nitrogen and stored at -80°C. The resultant F-non-
muscle actin stock (23 pM) was incubated at room temperature (24°C) for lh. For
the actin binding reaction 40 pi of F-non-muscle actin stock was used per reaction
and 10 pi of concentrated supernatant fraction. Alpha-actinin (final concentration of
1.0 pM) was used as a positive control and BSA (final concentration of 1.0 pM) was
used as a negative control. Negative control of concentrated supernatant fraction
without non muscle actin was also included. Actin binding reactions and all positive
and negative controls were incubated at room temperature for 30 min. Actin
filaments were sedimented by centrifugation at 150,000 x g for 1.5 h at 24°C.
Supernatants were carefully removed and placed on ice, 10 pi of 5 x sample SDS-
PAGE gel loading buffer was added to supernatants. Pellets were resuspended in 60
pi of 1 x sample SDS-PAGE gel loading buffer. Samples (pellets and supernatants)
were separated by SDS-PAGE and probed with antibodies to PKCs and actin.
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40
Table I. List of Replication-defective Adenovirus constructs used in the study
Name of the Ad Source MOI Chapter
Construct
Ad-Syncollin-GFP
(fused with GFP)
Ad-Tc-GFP-actin
(fused with GFP)
Ad-tTA
provided by Dr. Christopher J. Rhodes
(Pacific Northwest Research Institute)
provided by Dr. Daniel Kalman
(Emory University, Atlanta GA)
provided by Dr. Daniel Kalman
(Emory University, Atlanta GA))
III, V
III
III
Ad-GFP
(as a control)
Ad-DN-PKCs
( expressing GFP
separately)
Available from various sources
provided by Dr. George King (Joslin
Diabetes Center, Harvard Medical
School)
III, V
V
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41
Chapter III
A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in
rabbit lacrimal acinar epithelial cells
The ability of actin filaments to rapidly remodel in response to changes in
intracellular signaling is essential for their participation in a number of functions
including cytokinesis (Bi, 2001), cell motility (Krause et al., 2003; dos Remedios et
al., 2003), endocytosis (Qualmann and Kessels, 2002; Engqvist-Goldstein and
Drubin, 2003) and exocytosis (Eitzen, 2003). Here I explore the changes in apical
actin dynamics and organization that occur during exocytosis in the secretory
epithelial cells that are responsible for the production and release of tear proteins into
ocular fluid, the acinar cells of the lacrimal gland. Like other epithelial cells, actin
filaments in acinar cells from lacrimal gland are detected primarily beneath cell
membranes, with an abundant enrichment beneath the apical plasma membrane
(APM) (da Costa et al., 1998).
Earlier attempts to evaluate the role of actin filaments in lacrimal acinar
exocytosis using the actin-targeted agents, cytochalasin D and jasplakinolide (da
Costa et al., 1998; da Costa et al., 2003), did not reveal major changes in acinar
secretion nor affect resting or carbachol (CCH)-stimulated distributions of the
mature secretory vesicle (SV) marker, rab3D. However, it was unclear from these
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42
studies whether the actin filament array beneath the APM was substantially affected
by these treatments. Apical actin filaments in epithelial cells are more resistant to
actin-targeted drugs than are basolateral actin filaments (Ammar et al., 2001).
Spurred by recent confocal fluorescence microscopy analysis revealing evidence for
actin filament organization in acutely-stimulated lacrimal acini exposed to carbachol
(CCH), I have reevaluated actin filament participation in exocytosis in live acini.
The green fluorescent protein (GFP) epitope tag can be attached to many
proteins, making GFP-fusion proteins excellent tools for measurements of protein
dynamics in live systems (Lippincott-Schwartz and Patterson, 2003). Several studies
have utilized GFP-actin to assess cellular actin dynamics. Choidas et al. (1998)
have systematically investigated the ability of GFP-tagged actin to co-assemble with
endogenous actin into lamellipodia, filopodia and stress fibers and to dynamically
associate with the actin comet tail induced by Listeria monocytogenes, finding that it
behaved comparably to unmodified actin. GFP-actin has been utilized to measure
aspects of actin dynamics in microvilli in the kidney epithelial cell line, LLC-PK1 by
different groups (Tyska and Mooseker, 2002; Loomis et al., 2003) while GFP-actin
has also been useful in studying the actin core of stereocilia (Rzadzinska et al., 2004)
and cortical actin remodeling in presynaptic terminals (Sankaranarayanan et al.,
2003). Here I used high efficiency (80-90%) transduction with replication-defective
adenovirus (Ad) encoding GFP-actin to label the actin filament array in live lacrimal
acini and to obtain quantitative (time-lapse imaging) and quantitative (fluorescence
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43
recovery after photobleaching or FRAP analysis) measures of its dynamics. This
approach, combined with additional functional and morphological analyses of
lacrimal acini exposed to the general myosin ATPase inhibitor, 2,3-butanedione
monoxime (BDM), and the more selective myosin light chain kinase inhibitor, ML-
7, has enabled me to demonstrate that the filamentous actin array beneath the APM
of stimulated lacrimal acini is a highly dynamic system regulated in part by non
muscle myosin II.
Actin filaments are reorganized following acute exposure to CCH. Figure 5 shows
representative confocal fluorescence micrographs of actin filaments in resting
(control or CON) acini and acini exposed to CCH for 5 or 15 min. The lumenal
regions in reconstituted acinar cells were distinguished by intense actin filament
labeling detected in circular regions (*) attributable to actin filament enrichment
beneath the APM. Fainter actin filament labeling was detected beneath basolateral
membranes. The schematic diagram in Figure 5 depicts the cellular boundaries and
organization of the acini in the control image. Actin filaments at the apical
membrane exhibited two changes after CCH stimulation (100 pM) for 5 min: 1)
decreased intensity and increased irregularity in the continuity of apical actin
filaments and 2) formation of actin-coated structures beneath the APM (arrows).
Increased intensity of actin filament labeling beneath the basolateral membrane was
detected in acini exposed to CCH (arrowheads, 5 and 15 min). Formation of actin-
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44
coated structures was not as evident at 15 min of exposure to CCH, although the
apparent lumenal extension and discontinuity of labeling was still noticeable. We
and others have previously established that CCH evokes exocytosis in cultured rabbit
or rat lacrimal acinar cells with a response curve exhibiting substantial amounts of
secretory protein release by 5 min (40-50% of the total release) (Hodges et al., 1992;
Zoukhri et al., 1994; da Costa et al, 1998; Ota et al., 2003) with a subsequent slowing
of secretory product release. This release profile is consistent with the dramatic
changes in the shape of the lumen at 5 min of stimulation, the time course associated
with maximal rates of exocytosis, and also with the restoration of lumenal
dimensions to the resting state by 15 min when rates of secretion have diminished.
As shown in Figure 6A, EM analysis of the subapical cytoplasm of resting (Con)
rabbit lacrimal acini periodically revealed areas of abundant filament enrichment
(arrows) which were periodically localized between apical stores of subapical SVs
and the APM, suggesting that they might restrict access of SVs to the APM. As
shown in Figure 6A (CCH), we could frequently detect larger swaths of filaments
that assembled beneath groups of SVs in acini acutely exposed to CCH (rows 2 and
3, arrowheads); we also occasionally detected filament bundles associated closely
with fusing SVs (rows 3 and 4, arrows). Sampling of individual filament diameters in
these bundles revealed average diameters between 4-7 nm (average ± S.D. was 5 ±
0.28 nm, n=20 fields).
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45
ms s s ma
Figure 5. Confocal fluorescence microscopy reveals changes in actin filaments in CCH-
stimulated lacrimal acinar epithelial cells. Confocal fluorescence micrographs of control acini
(CON) and acini exposed to 100 pM CCH for 5 min (CCH 5) or 15 min (CCH 15). After treatments,
acini were fixed and processed as described in Chapter II to label actin filaments. The top right
panel shows a schematic outline of the actin filaments in the control preparation with thick lines
representing the apical actin filaments and the thinner lines representing basolateral actin filaments.
*, lumenal regions, arrows, apparent actin-coated fusion intermediates elicited by acute CCH,
arrowheads, increased basolateral actin; bar, 5 pm.
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46
Figure 6. EM images of resting and CCH-stimulated lacrimal acinar epithelial cells. A.
Filament distribution in resting (CON) and CCH-stimulated (5 min, 100 pM) lacrimal acini. B.
Filament distribution in BDM-treated (10 mM, 15 min) and ML-7-treated (40 pM, 15 min) lacrimal
acini exposed to CCH (5 min, 100 pM). Boxed regions in the left column are magnified in the right
column. L, lumen; SV, secretory vesicle; arrowheads, filaments beneath the APM or, in CCH-
stimulated acini, assembled beneath multiple SVs and arrows, filaments associated with individual
SVs. I would like to thank Prrof. Schechter and his laboratory for the help in EM image acquisition
and interpretation.
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47
S O O -nfg
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48
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49
GFP-actin coassembles with endogenous actin in transduced lacrimal acini. Co
transduction of lacrimal acini with Ad-Tc-GFP-actin and Ad-tTA resulted in a dual
transduction efficiency of 80-90% in each experiment, similar to previous reports for
other Ad constructs in this range in lacrimal acini (Wang et al., 2003). Western blot
analysis of lysates from co-transduced lacrimal acini confirmed that GFP-actin was
expressed at the expected MW of -66 kDa (Figure 7A). Consistent with previous
work (Choidas et al., 1998), GFP-actin represented only a fraction of endogenous
actin but this was sufficient to label actin filaments. This label was colocalized with
actin filaments labeled with rhodamine-phalloidin, indicating co-assembly with
endogenous actin (Figure 7B).
Analysis of GFP-actin fluorescence in live acini revealed intense labeling
beneath the APM surrounding the lumenal region (Figure 7C), similar to non
transduced acini. GFP-actin was also detectable at basolateral surfaces while fainter
fluorescence could be detected throughout the cytoplasm which was excluded from
large circular regions identified as SVs from the paired DIC image. Occasionally,
some SVs were ringed with apparent actin filaments (arrowheads).
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50
Figure 7. Analysis of GFP-actin expression and distribution in transduced lacrimal acinar
epithelial cells. A. Western blots of lysates (100 pg protein/lane) from non-transduced and
transduced lacrimal acinar cells developed in parallel using a polyclonal anti-actin antibody (left) and
a polyclonal anti-GFP antibody (right) and goat anti-rabbit IRDye™800-conjugated secondary
antibody. Lane 1, rabbit lacrimal acinar cells without transduction; lane 2, rabbit lacrimal acinar cells
transduced with Ad-GFP, MOI of 6; lanes 3-5, rabbit lacrimal acinar cells co-transduced with Ad-Tc-
GFP-Actin and Ad-tTA at MOIs of 1.5, 3 and 6, respectively. B. Co-transduced lacrimal acini fixed
as described in Chapter II and processed to label actin filaments with rhodamine-phalloidin (Rho-
Phall, red). Fluorescence associated with GFP-actin is shown in green while blue indicates nuclei
labeled with DAPI. *, lumenal regions; bar, 5 pm. Most soluble GFP-actin is destroyed during
fixation/permeabilization. C. Confocal fluorescence and DIC images of rabbit lacrimal acini co
transduced to express GFP-actin as described in Chapter II were acquired at 10.5 sec intervals over
16 min. The top row shows GFP-actin fluorescence, the DIC image, and an overlay of these images.
Selected images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown
in the second row. *, lumen; arrow, region of apparent actin invagination in the resting state;
arrowheads, mature secretory vesicles, some with an actin coat, bar, 5 pm. I would like to
acknowledge the assistance of Dr. Kaijin Wu in the laboratory who acquired the Western blot data
here.
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51
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52
Time-lapse confocal fluorescence microscopy reveals substantial apical actin
filament reorganization in CCH-stimulated acini. Images obtained at chosen
intervals from time-lapse confocal fluorescence microscopy sequences are shown for
Control acini (Figure 7C) and CCH-stimulated acini (Figure 8), while the entire
sequences would be available on-line once manuscript is accepted for publication
(Movies 1-3). The same resting and CCH-stimulated acinus is shown in Figures 7C
and 8A, respectively, to illustrate the remarkable increase in actin remodeling
associated with CCH while Figure 8B shows a second CCH-stimulated acinu for
comparison. Image acquisition of CCH-treated acini was initiated 30-60 sec after
CCH addition, due to the time required to refocus on the appropriate focal plane.
Therefore, the acinus in Figure 8A at 0 sec reflects the rapid appearance of GFP-
actin labeled invaginations relative to the same unstimulated acinus in Figure 7C. In
the absence of CCH, there was little global remodeling of apical or basolateral actin
filaments, although subtle changes suggestive of basal release of a few SVs at the
APM were detected after 469 sec (Figure 7C, arrow).
After CCH addition, the intensity of the apical actin filament array was
noticeably diminished in places (barbed arrows, Figure 8), suggestive of CCH-
induced disassembly. Invaginated regions underlaid with actin filaments
(arrowheads) also formed rapidly upon stimulation. These actin-coated structures
appeared to represent transient fusion intermediates, since they encircled single or
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53
multiple SVs detectable by DIC microscopy (Figure 8A, bottom row, arrowheads).
In many cases, the actin associated with these structures retracted steadily toward the
apical actin network in successively-acquired images until the underlying actin coat
could no longer be distinguished from the apical filaments. Although most of the
actin-coated intermediates formed close to the APM, some could be detected deep
within the cytoplasm.
In addition to the profound changes in actin filament organization detected at
the APM, changes in basolateral actin filaments were also seen. Within a few
minutes of CCH stimulation, GFP fluorescence associated with basolateral actin
filaments increased, followed by the appearance of small actin-coated vesicular
structures that appeared to “bubble” and extend from the surface (Figure 8, arrows).
FRAP analysis reveals increased apical actin filament turnover. The relative rates of
apical actin filament turnover in resting and CCH-stimulated acini expressing GFP-
actin were measured by FRAP. Observation time following bleaching were limited
to 90-100 sec due to the extreme mobility of the apical actin filaments in stimulated
acini; during this shorter time scale, problems associated with remodeling out of the
plane of focus or away from the photobleached spot were minimized. Representative
images clearly delineated the more complete recovery of fluorescence post-bleaching
in the ROI of CCH-stimulated samples (Figure 9A). I did not observe complete
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54
Figure 8. Time-lapse confocal fluorescence microscopy of GFP-actin in lacrimal acini reveals
substantial remodeling of apical and basolateral actin in CCH-stimulated cells. Confocal
fluorescence microscopy images of rabbit lacrimal acini transduced to express GFP-actin as described
in Chapter H were exposed to 100 pM CCH at the onset of the time-lapse sequence. Selected
images of GFP-actin fluorescence at intervals throughout the time-lapse sequence are shown. A. The
same acinus shown in Figure 7 immediately after CCH. Boxed image at 298 sec is magnified and
shown on the bottom row as GFP-actin, DIC and as an overlaid image. B. Another CCH-stimulated
acinus. Arrowheads, actin-coated structures; arrows, basolateral actin filament “bubbling”; barbed
arrows, regions of apical actin filament thinning. Bars, 5 pm.
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55
224 S 64 s
A
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56
recovery of filaments at 100 sec, possibly indicative of the presence of some capped
actin filaments and consistent with previous findings that cytochalasin D did not
significantly disassembly the apical actin array (da Costa et al., 1998). However,
CCH-stimulated acini always exhibited more recovery that control acini. Figure 9C
shows composite data from multiple experiments comparing Mf values (% of F;)
detected under each condition at defined time intervals after addition of CCH. The
M f is significantly (p<0.05) increased by CCH stimulation when FRAP is conducted
immediately (1-4 min) or up to 15-18 min after stimulation. This increase is
attributable to an overall increase in actin dynamics associated with CCH-stimulated
apical exocytosis.
Figure 9B shows two representative plots of fluorescence recovery under
each experimental condition, plotted as F/Fj. These recovery curves were biphasic,
with the second phase exhibiting a more pronounced CCH effect including an
increased slope. Figure 9D shows composite data obtained from measurement of
individual turnover times (t/2 ) calculated from individual recovery plots such as those
in Figure 9B as described in Methods. Turnover time is an overall parameter, likely
involving a variety of complex protein-protein interactions associated with actin
filament turnover, motility and diffusion. Actin filament t > /2 was significantly
decreased in CCH-treated acini by ~2-fold compared to unstimulated controls,
confirming a reduced lifetime for these apical filaments relative to those in resting
acini. Even though at 15-18 min interval observation time actin filament remodeling
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57
Figure 9. FRAP analysis of GFP-labeled apical actin filaments in lacrimal acini. A.
Representative scans of apical actin intensity before [Pre-(-ls)], during [Bleaching 0 s] and after
[Post-100 s] photobleaching in unstimulated (CON) and CCH-stimulated (100 p.M, 10 min) lacrimal
acini. The circular region is the ROI and bar, 5 pm. B. Typical plots of fluorescence over initial
fluorescence (F/FJ for resting and CCH-stimulated (100 pM, 10 min) acini. Fractional fluorescence
was calculated by F-F0 /Fi-F0. C. Mf values for apical actin filaments in resting and stimulated acini
analyzed after the indicated exposures to CCH. D. Turnover times (t/2 ) for apical actin filaments in
resting and stimulated acini analyzed after the indicated ranges of exposure to CCH as described in
Chapter IL Results from C. and D. were obtained from 3-7 dishes in each preparation from n=5
preparations; *, p<0.05.
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58
Pre- (-1 s) Bleaching (0 s) Post- ( 1 00 s recovery)
B CON CCH i 0 min
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59
was visibly lessened in the time-lapse microscopy sequence, the Un for this time
interval was not different from any of the earlier time tested (Figure 9).
Measurements of actin filament dynamics suggest that actin filament turnover is
significantelly enhanced during early points as well as during later time points of
observation up to 15-18 min.
BDM and ML-7 suppress CCH-induced increases in apical actin dynamics while
inhibiting apical exocytosis. We analyzed the effects of BDM and ML-7 on actin
filament organization and secretory functions in rabbit lacrimal acini. Preliminary
dose-response studies for effects of these agents on cell morphology and protein
secretion allowed us to identify optimal doses for effects in acini. In resting lacrimal
acini, BDM (10 mM, 15 min) and ML-7 (40 pM, 15 min) did not significantly alter
actin filament organization in resting acini; however, CCH stimulation of BDM- or
ML-7-treated acini caused the formation of large actin-coated structures at and
beneath the APM by 5 min (Figure 10A, arrows). These actin-coated structures
persisted stably up to 30 min in contrast to their rapid turnover in untreated acini.
Acini exposed to BDM or ML-7 then treated with CCH retained a more intense
labeling of apical filaments relative to basolateral filaments compared to CCH-
stimulated acini alone (Figure 5), suggestive of possible increased stability or
assembly. The apparent stabilization of apical actin as well as the trapping of
apparent actin- coated structures by BDM and ML-7 was accompanied by a
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60
Figure 10. BDM and ML-7 treatments alter CCH-mediated actin remodeling while inhibiting
protein secretion. A. Confocal fluorescence micrographs of BDM-treated (10 mM, 15 min) or ML-
7-treated (40 pM, 15 min) lacrimal acini without or with 100 pM CCH for 5 min fixed and labeled to
detect actin filaments. Arrows, actin-coated fusion intermediates; *, lumena. B. Bulk protein
secretion in acini treated with BDM and ML-7 as described above and stimulated with CCH for 5, 10
or 30 min. Basal release, total release and the stimulated component (total minus stimulated) are each
plotted. Values were normalized to cell protein before comparison across samples. Bars, s.e.m; *,
significant at p<0.05 from comparable value in control cells and n=5-6 preparations. C. Myosin II
(green) and actin (red) distributions in acini exposed without or with BDM or ML-7, then exposed to
CCH for 5 min as above. Bar, 5 pm. The Western blot shows the signals for myosin II in ~300 pg
protein loaded from clarified acinar lysate (L), the membrane pellet (Pi) and supernatant fractions (Si).
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61
A
CON CCH
B
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C CON CCH 2ryAb alone
Myosin II
BDM + CCH ML-7 + CCH
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62
significant inhibition of CCH-stimulated protein secretion that was detectable by 5
min of CCH exposure (Figure 10B). ML-7 at a dose of 10 pM elicited an apparent
stabilization of actin filaments in live acini in parallel with a trend towards inhibition
of protein secretion which was not statistically significant.
Since these agents, in particular ML-7, are commonly employed as inhibitors
of non-muscle myosin II, we investigated its distribution in lacrimal acini under
these conditions. The antibody to non-muscle myosin II recognized an intense band
at 220 kD in concentrated supernatant fractions from cultured lacrimal acinar
epithelial cells, which was also present in crude cell lysates and in membrane
fractions (Figure 10C). Myosin II immunofluorescence in resting lacrimal acini
displayed a diffuse, hazy labeling pattern, while stimulation of acini with CCH for 5
min resulted in detection of punctate immunofluorescence (arrows), some which was
colocalized with apical actin filaments (Figure 10C). Myosin II distribution in
acini exposed to BDM or ML-7 prior to stimulation with CCH exhibited increased
punctate fluorescence concentrated at the apical pole of the cells which was
associated with apical actin-coated structures increased in these acini (Figure 10C,
arrows). In particular, in the acinus treated with ML-7 + CCH, these punctate spots
of myosin II immunofluorescence were located at what appeared to be the points of
attachment of actin-coated structures with the apical actin network (see arrows).
Analysis of live lacrimal acini expressing GFP-actin and exposed to BDM or
ML-7, then CCH by time-lapse confocal fluorescence microscopy revealed
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63
remarkable changes. The intensity of the apical actin filament network in these
treated acini appeared unchanged by CCH, unlike acini in the absence of these
agents (Figure 8), suggesting a suppression of CCH-stimulated actin filament
turnover. Measurements of Mf for apical actin filaments in acini exposed to BDM
+CCH and ML-7 + CCH at all time intervals of stimulation confirmed that these
agents prevented the significant CCH-induced increases in M f elicited by CCH
(compare Table II values with those in Figure 9C).
Moreover, EM images revealed thick filament bundles beneath the APM in
acini treated with either agent prior to CCH stimulation (Figure 6B, arrowheads);
less frequently, bundles of filaments could be detected beneath SVs (Figure 6B,
arrow, top left). It was surprising, given the abundance of actin-coated structures
detected by confocal fluorescence microscopy, that we were unable to frequently
detect these coats by EM. However, these actin-coated structures stabilized by BDM
and ML-7 may be comprised of only a few filaments rather than the abundant
filament bundles detected beneath the APM. Also, filaments may not completely
envelope the SVs, making the acquisition of a filament coat within a thin EM section
more difficult than by confocal fluorescence microscopy analysis. Representative
sampling of individual filament diameters in these bundles revealed diameters
between 4-7 nm, consistent with actin filament diameter.
Also remarkable was the effect of BDM and ML-7 on the actin-coated
structures which formed sequentially adjacent to the APM after CCH stimulation
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64
(Figure 11A and B, arrowheads). Once formed, these actin-coated structures were
persistent in contrast to their transient nature in CCH-stimulated acini (Figure 8).
The actin filaments associated with actin-coated structures in ML-7-treated acini
exposed to CCH appeared to first accumulate and then to condense; actin-coated
structures in BDM-treated acini appeared, in contrast, to retain their vesicular shape.
In addition to the suppression of apical actin filament dynamics (Table II), BDM
and ML-7 permit actin filament assembly beneath fusing SVs in CCH-stimulated
acini but suppress the subsequent retraction of these filaments toward the APM seen
in CCH-stimulated acini. Similar stabilization of basolateral actin associated with
blebbing was seen in BDM-treated (Figure 11 A, arrow) and ML-7-treated acini
(data not shown). The complete time-lapse sequences for lacrimal acini exposed to
BDM or ML-7 and stimulated with CCH are available online (Movies 4-5).
BDM and ML-7 reduce CCH-stimulated svncollin-GFP secretion. Studies with a
syncollin-GFP fusion protein have shown labeling of large protein-enriched SVs in
diverse systems including zymogen granules in pancreatic acini (Hodel and
Edwardson, 2000), large dense core vesicles in AT-20 cells (Hodel and Edwardson,
2000) and insulin granules in pancreatic J3-cells (Ma et al, 2004). Syncollin-GFP
therefore appeared to be an excellent candidate for selective labeling of lacrimal
acinar SVs. . As shown in Figure 12A, syncollin-GFP in transduced, unstimulated
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65
Figure 11. Time-lapse confocal fluorescence microscopy of GFP-actin in lacrimal acini exposed
to BDM or ML-7 prior to addition of CCH reveals stabilization of apical actin and of actin-
coated subapical structures. Rabbit lacrimal acini transduced to express GFP-actin as described in
Chapter H, exposed to BDM (10 mM, 15 min, A) or ML-7 (40 pM, 15 min, B) were imaged
immediately upon CCH addition (100 pM). Selected images of GFP-actin fluorescence at intervals
throughout the time-lapse sequence are shown. Arrowheads, actin-coated fusion intermediates;
arrow, actin bubble stabilized at the basolateral membrane; bar, 5 pm.
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66
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67
Figure 12. BDM and ML-7 reduce the CCH-stimulated exocytosis of syncollin-GFP. A.
Confocal fluorescence microscopy images acquired at the indicated times after CCH addition (100
pM) without inhibitor treatments (CCH) or with ML-7 treatment (40 pM, 15 min) or BDM treatment
(10 mM, 15 min) prior to CCH addition. DIC and overlay images are also shown at 0 sec. *, lumen
and bars, 5 pm. Plots to the right of each treatment group depict 2.5 D graphical reconstructions of
the overall intensity profile of the imaged areas at 0 and 600 sec of stimulation with CCH, illustrating
individual intensities per pixel utilizing the rainbow scale. The resolution is ~10 pixels per pm. B.
Syncollin-GFP release (plotted as % of control) in resting and CCH-stimulated (100 pM, 30 min)
acini without or with BDM and ML-7 pretreatment as described above. *, significant from paired
control at p<0.05. Although the time-lapse sequences indicate rapid release of syncollin-GFP, the
signal in the culture medium was more intense after 30 min, likely due to the time taken for
exocytosed material initially trapped in the lumen to diffuse into culture medium.
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68
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69
acini was detected in a series of large vesicles, discernable by DIC microscopy, that
were enriched around a lumenal region (*). Exposure of acini to CCH resulted in
loss of this punctate fluorescence within a few minutes that increased up to 10 min,
as shown in the 2.5 D-graphical reconstruction of syncollin-GFP intensity at 0 and
600 sec. Some released syncollin-GFP appeared to be retained in the lumenal
region. This loss in vesicular syncollin-GFP fluorescence was accompanied by a
3.5-fold increase in the recovery of syncollin-GFP in the culture medium (Figure
12B). Although syncollin-GFP labeled SVs with the same intensity in acini treated
with ML-7 or BDM, stimulation with CCH appeared to discharge only a fraction of
the syncollin-GFP stores. This observation was consistent with findings that these
agents significantly decreased syncollin-GFP recovery in culture medium by ~50-
60% (Figure 12B). Confocal fluorescence microscopy revealed that actin-coated
structures trapped by BDM and ML-7 were frequently rich in syncollin-GFP (Figure
13, arrows), confirming that these actin-coated structures were transient fusion
intermediates.
LAT B decreases apical actin filaments while enhancing secretory responses. The
macrolides, LAT A and LAT B, are lethal toxins from sea sponge which destabilize
actin filaments through binding and sequestration of actin monomers (Spector et al.,
1983). LAT A and B elicit more disassembly of the resistant apical actin filament
array in epithelia, relative to other actin-targeted agents. To further investigate
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70
Dual Actin Syncollin-GFP
Figure 13. Syncollin-GFP is enriched in actin coated structures in acini exposed to BDM or
ML-7. Lacrimal acini were transduced with Ad-Syncollin-GFP on day 2 of culture as described in
Chapter II. On day 3 of culture acini were stimulated with CCH (100 pM) for 5 min after
pretreatment without (CON) or with BDM (10 mM) or ML-7 (40 pM) for 15 min. Cells were
immediately washed and fixed with 4% paraformaldehyde to preserve Syncollin-GFP fluorescence
(green) and labeled in parallel with rhodamine phalloidin to visualize actin filaments (red). Bar, 5
pm.
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71
the barrier role of apical actin filaments in exocytosis, we conducted morphological
and functional analyses of LAT-treated acini. Figure 14A shows that LAT B
decreased the intensity of labeling of apical and basolateral actin, an effect increased
by CCH stimulation. LAT B also elicited a modest but significant release of bulk
protein in the absence or presence of CCH (Figure 14B, left) and a modest and
significant enhancement of CCH-stimulated exocytosis of syncollin-GFP release in
Ad-syncollin-GFP transduced acini (Figure 14B, right).
Preliminary investigations comparing LAT B and the related agent, LAT A, in
lacrimal acini showed comparable effects on secretion and actin filaments (data not
shown). Although LAT A was not utilized extensively in biochemical assays due to
its higher cost, we utilized it in EM analysis of acinar morphology (Figure 14C and
D) The image on the left depicts a representative lumenal region enriched in SVs in
a LAT A-treated acinus, while a higher magnification image of the boxed region to
the right shows SVs poised at the APM (arrowheads) without the intervening actin
filament barrier normally restricting SVs from APM that is evident in the control
acinus in Figure 6A. The images in Figure 14D are also of LAT-A treated,
unstimulated acini, and depict secretagogue-independent fusion of an unusually large
SV at the APM (left image, arrowhead) and apparent CCH-independent compound
fusion at the APM (right image, arrows). These data are consistent with biochemical
findings that disassembly of apical actin filaments modestly enhances exocytosis.
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72
Figure 14. LAT treatment enhances CCH-stimulated secretion in parallel with depletion of
actin filaments at the APM. A. Confocal fluorescence micrographs of lacrimal acini fixed and
processed for labeling of actin filaments as described in Chapter H. Treatments included untreated
lacrimal acini (CON) and acini exposed to LAT B (10 pM, 15 min) in the absence (LAT) or presence
(LAT + CCH) of CCH (100 pM, 15 min). *, lumenal regions; bar, 5 pm B. Effects of LAT B on
bulk protein secretion (left) and syncollin-GFP release (right) without or with CCH stimulation.
Values were normalized to cell protein before comparison across samples. *, significant at p<0.05
from paired control value; n=6 for protein release and n=3 for syncollin-GFP release. C. Left, EM
image at lower magnification from rabbit lacrimal acini exposed to LAT A (1 pM, 15 min) and right,
boxed region at higher magnification. Arrowheads depict SVs at the APM that are beginning to fuse.
D. Images showing premature fusion of mSVs in LAT A-treated, unstimulated acini. L, lumenal
region; SV, secretory vesicle.
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73
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74
Table II. Mf values for apical actin in BDM- and ML-7-treated acini expressing
GFP-actin without and with CCH.
Treatment
Mr
Control (BDM series) 39 ± 3% (n=9)
BDM 40 ± 5% (n=8)
BDM + CCH (1-4 min) 39% ± 3% (n=6)
BDM + CCH (5-7 min) 41% ± 5% (n=7)
BDM + CCH (10-12 min) 42% ± 5% (n=4)
BDM + CCH (15-18 min) 29% (n=2)
Control (ML-7 series) 41% + 5% (n=4)
ML-7 45% + 3% (n=4)
ML-7 + CCH (1-4 min) 52 % + 4% (n=4)
ML-7 + CCH (5-7 min) 51% + 5% (n=4)
ML-7 + CCH (10-12 min) 45% + 5% (n=4)
ML-7 + CCH (15-18 min) 37% + 4% (n=4)
BDM treatment was 10 mM, 15 min and ML-7 treatment was 40 pM, 15 min prior to
addition of CCH (100 pM). None of the values were significantly different than that
measured in resting acini using a paired t-test with p<0.05 in comparison to the
significant increases elicited by CCH in Mf at each time point (Figure 9).
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75
Movie legend 1: Time-lapse confocal fluorescence microscopy of the resting acinus shown in Figure
3 co-transduced to express GFP-actin. Images were acquired at 10.5 sec intervals over 16 min.
Movie legend 2: Time-lapse confocal fluorescence microscopy of the acinus shown in Figure 4A co
transduced to express GFP-actin and then exposed to CCH (100 pM) at the onset of image
acquisition. Images were acquired at 10.5 sec intervals over 16 min.
Movie legend 3: Time-lapse confocal fluorescence microscopy of the acinus shown in Figure 4B co
transduced to express GFP-actin and exposed to CCH (100 pM) at the onset of image acquisition.
Images were acquired at 5 sec intervals over 17 min.
Movie legend 4: Time-lapse confocal fluorescence microscopy of the acinus shown in Figure 7A co
transduced to express GFP-actin, exposed to BDM (10 mM, 15 min), and exposed to CCH (100 pM)
at the onset of image acquisition. Images were acquired at 11 sec intervals over 17 min.
Movie legend 5: Time-lapse confocal fluorescence microscopy of the acinus shown in Figure 7B co
transduced to express GFP-actin, exposed to ML-7 (40 pM, 15 min), and exposed to CCH (100 pM)
at the onset of image acquisition. Images were acquired at 5 sec intervals over 15 min.
Full sequences of the time-lapse confocal fluorescence microscopy should be
available online once submitted manuscript is published.
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76
Chapter IV: Discussion
A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in
rabbit lacrimal acinar epithelial cells
Here I demonstrate for the first time using time-lapse confocal fluorescence
microscopy and FRAP that lacrimal acini maintain an actin filament structure
beneath the APM that is actively remodeled during apical exocytosis. GFP-labeled
apical actin filaments showed rapid CCH-induced remodeling including thinning of
the apical actin network and transient formation of actin-coated structures. These
transient actin-coated structures, when stabilized by BDM or ML-7, frequently
contained exogenous syncollin-GFP, suggesting that they were fusion intermediates.
FRAP measurements revealed increased dynamics of the apical array caused by
CCH stimulation: specifically, CCH elicited a significant increase in Mf and a
significant decrease in ty2 for apical actin filaments. The calculated ty2 for apical actin
filament turnover in lacrimal acini was comparable to published turnover times in
other cells (Amato et al., 1986; Theriot and Mitchison, 1991; McGrath et al., 1998).
Although a considerable amount of work has been conducted on mechanisms of
neuronal and neuroendocrine secretion including the role of actin filament
remodeling in models such as chromaffin cells, much less is known about the
mechanisms of acinar SV exocytosis. Lacrimal, parotid and pancreatic acini are
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77
quite different in many aspects from neuroendocrine cells including 1) the absence of
a readily-releasable, pre-docked SV pool; 2) maintenance of SVs of significantly
larger diameter; 3) slower SV release kinetics; 4) release at only one domain within
the polarized epithelial cells and 5) significantly restricted surface area available for
fusion at the APM. My studies represent one of the first such detailed analyses of
actin filament dynamics in live acinar epithelial cells.
Stabilization of the apical actin filament array by BDM and ML-7 suppressed
CCH-stimulated actin filament dynamics while increasing actin filament bundling
beneath the APM. These agents also stabilized actin-coated fusion intermediates
formed transiently in the presence of CCH. Actin stabilization was associated with
inhibition of exocytosis of bulk protein and also of the exogenous marker, syncollin-
GFP. BDM has been utilized as an uncompetitive inhibitor of myosin ATPase
activity (Higuchi and Takemori, 1989; Herrmann et al., 1992). Exposure of cells to
BDM dissociates myosin from actin filaments, impairing myosin involvement in
events as varied as muscle contraction (Herrman et al., 1992) and myosin-based
vesicle transport (Bennett et al., 2001; Duran et al., 2003). Previous work in
chromaffin cells has demonstrated that BDM suppresses chromaffin granule
dynamics in parallel with inhibition of secretion (Neco et al., 2002; Neco et al.,
2003). ML-7 and the related inhibitor, ML-9, have been utilized extensively as
selective inhibitors of myosin light chain kinase (Saitoh et al., 1987). In rat
pancreatic acinar cells, both ML-9 and BDM inhibited stimulated secretion of
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78
amylase (Torgerson and McNiven, 2000). These workers also observed a dramatic
change in cell shape and an increase in non-muscle myosin II phosphorylation
associated with stimulation of secretion.
The ability of BDM to inhibit myosins other than myosin II family members
has recently been questioned (Ostap, 2002), and other work suggests that it may
affect actin dynamics through myosin-independent mechanisms (Yarrow et al.,
2003). The similarity of the effects elicited by BDM and ML-7 suggest common
effects on non-muscle myosin II by both agents.
I propose that apical actin filament barrier remodeling by non-muscle myosin
II plays an important role in exocytosis in lacrimal acini. The stabilization of the
apical actin barrier by BDM or ML-7 resulted in accumulation of punctate myosin II
immunofluorescence with the apical actin network in parallel with inhibition of
exocytosis. The converse was also true; LAT B-induced disassembly of apical actin
filaments was associated with a modest though significant increase in protein and
syncollin-GFP release by lacrimal acini. Previous work in chromaffin cells has
investigated the role of non-muscle myosin II in modulation of cortical actin: unlike
acinar epithelial cells, these cells maintain a readily-releasable docked pool of SVs,
but like acinar epithelial cells these cells must then transport new SVs past a cortical
actin barrier. In one study, BDM did not inhibit the first phase of exocytosis from
the readily-releasable, docked pool but impaired the movement of new SVs to the
plasma membrane to sustain the response (Rose et al., 2002). However, other work
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79
by Neco et al. (2004) has shown through the use of ML-9 and by overexpression of
the non-phosphorylatable form of the myosin regulatory light chain that non-muscle
myosin II is involved in the release of the readily-releasable SV pool and in the
movement of new SVs to the plasma membrane. Work by Manneville et al. (2003)
in endothelial cells also determined that cortical actin functioned to restrict SV
mobilities at the plasma membrane, consistent with my interpretation. Findings in
related systems which engage in stimulated exocytosis are therefore consistent with
my findings that cortical actin, modulated by non-muscle myosin II, is involved in
regulating access of SVs to the APM.
The function of the actin filaments associated with the fusion intermediates in
exocytosis is still unclear. Once formed, the actin associated with these
intermediates appeared to rapidly but steadily retract toward the APM where it
apparently converged with the apical actin network. These actin-coated
intermediates were stabilized by BDM and ML-7, suggesting that filament
remodeling associated with retraction of the underlying network to the APM also
involves non-muscle myosin II. My findings are similar to those found in fixed
pancreatic acini which demonstrated trapping of actin-coated granules at the APM of
BDM-treated pancreatic acini in parallel with inhibition of exocytosis (Valentijn et
al., 2000). It is possible that myosin-mediated actin filament contraction around the
base of fusing SVs aids in the extrusion of SV contents. However, inhibition of actin
assembly with LAT B modestly enhanced exocytosis and actin-coated fusion
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80
intermediates were not detected in acini exposed to LAT B and CCH. No major
inhibitory effects on exocytosis were associated with the inability of lacrimal acini to
form actin-coated fusion intermediates in LAT B-treated, stimulated acini relative to
untreated, stimulated acini, although it is possible that the amount of material
exocytosed would have been even greater if actin-coated intermediates had formed in
the absence of an actin barrier. It is also possible that, because the lumena in the
cultured acini are more distended than are the lumena in intact gland, that ready
diffusion of secretory products into the medium can take place in the in vitro cultures
whereas force generation is required for extrusion of secretory products in the intact
gland.
Alternatively, the actin filament coating around fusing SVs may be important
for stabilization of the fusion complex formed between the APM and SV and/or in
compensatory membrane endocytosis. Previous studies in pancreatic acini have
suggested that the fusion pore formed between fusing granules and the APM is
extremely long-lived; despite this, there is no lateral exchange of components
between the granule membrane and APM (Thorn et al., 2004). It is possible that the
actin coats formed around fusing intermediates are important for preserving the
integrity of the fusing SV granule, including prevention of lateral mixing prior to
endocytosis. This model may explain the findings by Neco et al. (2004) that actin
filaments and myosin II are required to maintain normal kinetics of release from the
readily-releasable pool in chromaffin cells. Finally, actin filaments associated with
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81
fusion intermediates might also participate directly in endocytic retrieval of SV
membrane by facilitating clathrin-mediated or other mechanisms (da Costa et al.,
2003).
Several groups have reported that the apical actin network beneath the APM
of acinar epithelial cells from pancreas and parotid gland undergoes reorganization in
stimulated acini (Perrin et al., 1992; Valentijn et al., 1999), leading to the “barrier”
hypothesis proposing that apical actin filaments restrict access of SVs to the APM in
resting acini while permitting access in stimulated acini. Other studies in pancreatic
acini have shown that some actin filaments are necessary for exocytosis to proceed
(Muallem et al., 1995), possibly for force generation. My study suggests roles for
both actin filament disassembly and reassembly mediated by non-muscle myosin II
and other factors.
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Chapter V
82
Dominant negative PKCs impairs apical actin remodeling in parallel with
inhibition of carbachol-stimulated secretion in rabbit lacrimal acini.
The acinar cells of the lacrimal gland are the major source of tear proteins
released into ocular surface fluid. Most tear protein stores are released at the APM of
lacrimal acini from mSVs that are coated with the small GTPase, rab3D (Ohnishi et
al.,1996; Wang et al., 2003). These mSVs fuse with the APM rapidly upon
secretagogue stimulation (Wang et al., 2003). Additional tear proteins such as the
extracellular domain of the polymeric immunoglobulin receptor (plgR), either alone
(secretory component or SC) or complexed to dimeric IgA (secretory IgA) can be
released at the APM through the transcytotic pathway. The area under the APM in
lacrimal acini is enriched in a dense network of actin filaments which appear to form
a barrier preventing uncontrolled mSV release in unstimulated acini. Several studies
have used different techniques to suggest that extensive remodeling of the underlying
actin network accompanies stimulated secretion in acinar epithelial cells from
pancreas (Muallem et al., 1995; Valentijn et al., 2000; Nemoto et al., 2004) and
parotid gland (Perrin et al., 1992). Additionally, my work using GFP-actin to label
the lacrimal acinar actin filament array and my application of time-lapse confocal
fluorescence microscopy and FRAP definitely shows remodeling of apical actin
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83
(Chapter in). This remodeling includes increased actin filament turnover beneath
the APM as well as transient formation of actin coated invaginations thought to
represent mSV fusion intermediates. Stabilization of actin coated structures is
associated with inhibition of exocytosis in pancreas (Valentijn et al., 2000) and in
lacrimal acini (Chapter ID). Little is known about the effectors that regulate actin
filament remodeling in acinar exocytosis.
In this study, I have investigated the contribution of the novel protein kinase
C (PKC) isoform, PKCs, to actin filament remodeling and apical exocytosis. As
discussed in Chapter I, I am particularly interested in this kinase since it is known to
bind to actin filaments directly and also to be one of several kinases activated in
lacrimal acini in response to various secretagogues (Zoukhri et al., 1997).
Lacrimal acinar PK C s associates with actin filaments. To demonstrate that
lacrimal acinar PKCs associates with actin, I performed several in vitro and intact
cell assays. Actin binding proteins can be identified by their co-sedimentation with
polymerized non-muscle actin. As shown in Figure 15A, addition of polymerized
actin to lacrimal acinar cytosol resulted in sedimentation of PKCs in the pellet
(+Actin, Pell fraction) while equivalent sedimentation in the absence of polymerized
actin did not result in sedimentation of PKCs (-Actin, Pell fraction). Analysis of
PKCs in the supernatant remaining after sedimentation of actin also revealed
depletion of the cytosolic pool. As further verification, immunoprecipitation from
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84
cell lysates with anti-actin antibody resulted in co-immunoprecipitation of PKCs
with actin (Actin Ab+Lys), while beads alone (B+Lys) failed to bring down this
protein (Figure 15B).
Since PKCs is activated by secretagogue exposure in lacrimal acini (Zoukhri
et al., 1997) including agents acting through M3 muscarinic receptors (CCH) and
a 1-adrenergic receptors (phenylephrine or PE), I investigated whether the interaction
between PKCs and actin was increased in cytosolic fractions isolated from
stimulated lacrimal acini. PKCs present in cytosolic fractions from CCH-
stimulated acini was substantially depleted relative to its content in cytosolic
fractions from untreated acini (data not shown). When actin filament binding assays
were performed with stimulated extracts, less PKCs was detected in association with
actin filaments because there was very little PKCs in the starting sample. I
hypothesized that the depletion of PKCs from the cytosolic fractions from stimulated
acini might in fact reflect tighter binding to the cytoskeletal network, much of which
is removed with the cellular debris by low speed centrifugation. To test this, I
employed an alternative protocol based on isolation of subcellular protein pools with
sequential detergent extraction to analyze PKCs partitioning between soluble
(saponin-soluble), membrane (Triton X-100 soluble) and cytoskeletal (SDS-soluble)
pools isolated from unstimulated and CCH-stimulated lacrimal acini.
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85
A
Actin Binding
C
Sequential Detergent
Extraction
B)ot: PM i
Blot: Actin
< 97
<66
< 4 6
B
CCH: - + - + - +
Sol Mem Cyt
Blot: PKCe i
Blot: Actin
Actin: - + - +
Pell Sup
Co-immunoisolation
Blot: PKCe
Blot: Actin
x j * 5 > x
S ’ ^
< 9 7
<66
< 4 6
D
Summary - Extraction
O
O 6 0
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I 40-
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BCCH
Figure 15. PKCs is an actin binding protein in lacrimal acini. A. Soluble fractions from lysates
from lacrimal acini were incubated without (-) or with (+) non muscle actin and filaments were
pelleted by centrifugation. PKCs and actin contents of the resulting pellet (Pell) and supernatant
(Sup) fractions were analyzed by Western blotting with the antibodies indicated. B. Lysates (Lys)
from lacrimal acini were incubated with beads (B) or beads plus mouse monoclonal anti-actin
antibody (Actin Ab) and processed for immunoprecipitation and analysis of the indicated proteins as
described in Chapter II. Western blots of the Actin Ab and Lys are shown as controls. C. Acini
without or with CCH (100 pM, 15 min) lacrimal acini were subjected to sequential detergent
extraction to isolate soluble (Sol), membrane (Mem) and cytoskeletal (Cyt) pools as described in
Chapter n. Equal volumes of each of the fractions were resolved by SDS-PAGE and the sample
content of PKCs and actin determined by Western blotting. D. Composite values reflecting
PKCs enrichment within soluble, membrane and cytoskeletal pools from acini without or with CCH
as described in C. and expressed as a percentage of total cellular PKCs. Stimulation did not affect the
recovery of marker proteins in the three fractions (data not shown). Results are from n=3 experiments;
error bars represent sem; *, p<0.05.
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86
A representative blot of PKCs and actin contents of each pool from
unstimulated and CCH-stimulated acini (100 pM, 15 min) is shown in Figure 15C
while Figure 15D shows summary data from several preparations. It should be
noted that PKCs and actin in the cytoskeletal pool migrate more slowly, due to the
presence of high amounts of detergent in this fraction necessary to solubilize
filaments. In unstimulated acini, most PKCs (-60%) was recovered in the cytosolic
pool with only a trace amount in the membrane pool and the remainder (-30%)
enriched in the cytoskeletal pool. In CCH-stimulated acini, a significant (p<0.05)
shift in the partitioning of PKCs was noted, with only -30% recovered in the
cytosolic fraction and -55% detected in the cytoskeletal fraction containing most of
the cellular actin. This finding suggested that PKCs translocation to actin filaments
occurred in response to CCH stimulation. Similar translocation of PKCs to the
cytoskeletal pool in the sequential detergent extraction was detected in acini exposed
to PE (data not shown). This effect was not as prominent since PE is a weak
secretagogue in rabbit lacrimal acini (Qian et al., 2003).
The cellular localizations of PKCs in unstimulated and CCH-stimulated acini
were investigated in parallel. Figure 16 shows results from a representative
preparation with actin filament labeling in red and PKCs in green. Cytoskeletal
organization in reconstituted lacrimal acini has been characterized previously (da
Costa et al., 1998; da Costa et al., 2003). Briefly, the apical or lumenal regions in
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87
Figure 16. PKCs is enriched with apical actin and actin-coated invaginations in lacrimal acini.
A. Confocal fluorescence micrographs of rabbit lacrimal acini without (Con) and with CCH
stimulation (100 pM, 15 min) that were fixed and processed as described in Chapter II to label actin
filaments (red) and PKCs (green). Arrows, enrichment of PKCe at the APM; *, lumenal regions and
bar, 5 pm. B. Expanded views of boxed regions of the CCH-treated acinus in A., showing areas of
co-localization of actin filaments and PKCs in actin-coated structures, possibly fusion intermediates,
docked at the APM (arrowheads). *, lumenal regions and bars, 1 pm.
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A
CON CCH
B
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89
lacrimal acini can be distinguished by the more intense actin filament labeling
detected in roughly circular regions (marked by *) associated with actin filament
enrichment beneath the APM and within microvilli. As shown in Figure 16A,
apical actin filaments in unstimulated acini appear continuous, without multiple
invaginations. Fainter actin filament labeling can also be detected beneath
basolateral membranes. In unstimulated acini, some PKCs was co-localized with
apical actin (Figure 16A, arrows and Figure 16B, box 1 enlargement) and also in
the cytoplasm in punctuate structures. CCH stimulation increased the co-localization
of PKCs with apical actin (Figure 16A, arrows). CCH stimulation also caused
formation of transient actin-coated structures, seen as apparent invaginations of the
apical actin array from the lumenal region. PKCs was prominently enriched on these
apical actin-coated invaginations (Figure 16B, box 2 and 3 enlargements,
arrowheads). PE treatment caused a modest increase in co-localization of PKCs with
apical actin (data not shown). Biochemical and confocal fluorescence microscopy
data in Figures 15 and 16 collectively suggested a strong interaction between
lacrimal acinar PKCs and actin filaments that was increased in acini exposed to
secretagogues, particularly CCH.
Introduction o f DN-PKCs alters acinar actin organization. Point mutations in the
PKC ATP binding domain render the enzyme inactive (Soh et al., 1999). Such a
Dominant Negative (DN) mutation in PKCs has been generated (K437R) and
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90
inserted into an Ad expression vector (Kaneto et al., 2002). I transduced lacrimal
acini with Ad-DN-PKCs and examined the efficiency of transduction using the co
expressed marker protein, GFP. As shown in Figure 17A, exposure of lacrimal acini
to Ad-DN-PKCs for 1-2 hrs at a MOI of 5 followed by overnight recovery resulted
in GFP expression in -80-90% of cells. Analysis of PKCs content in lysates of
acini transduced at different MOIs revealed considerable overexpression of the DN-
PKCs at an MOI of 5, with little additional expression elicited above that dose.
Subsequent experiments utilized a MOI of 5 for transduction with Ad-DN-PKCs or
Ad-GFP (as a control). The increased PKCs immunofluorescence detected in
transduced acini was extensively co-localized with apical and basolateral actin
filaments while organization of actin at these domains was markedly different
(Figure 17C and D) At the APM, actin accumulation was evident in acini
overexpressing DN-PKCs, and lumena had a compressed appearance. Additionally,
there were abundant actin-coated invaginations (Figure 17D, arrowheads) similar to
the actin-coated structures detected transiently in CCH-stimulated acini (Figure
17B). However, the actin coats in acini expressing DN-PKCs were always detected.
Transduced acinar cells also exhibited increased attachment and spreading at the
basolateral domains, resulting in an extended or elongated appearance (Figure 17D,
arrows) relative to their normally globular shape (Figure 16).
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91
Figure 17. High efficiency transduction of lacrimal acini with Ad-DN-PKCs results in co
localization of overexpressed DN-PKCs with actin filaments. A. GFP expression in rabbit
lacrimal acini transduced with Ad-DN-PKCs co-expressing GFP at an MOI of 5 as described in
Chapter II. Bar, -30 pm. B. Western blot showing the expression of PKCs in lysates of lacrimal
acini transduced with Ad-DN-PKCs at the indicated MOI. Equal amounts of protein were loaded in
each lane. C. Confocal fluorescence micrographs of lacrimal acini transduced with Ad-DN-PKCs
and fixed and processed as described in Chapter n to label actin filaments (red) and PKCs (green).
Note, cytosolic GFP fluorescence co-expressed by the Ad-DN-PKCs construct is destroyed during
fixation. D. Confocal fluorescence micrographs showing actin filament organization in lacrimal acini
transduced with Ad-DN-PKCs. Arrowheads in C. and D. indicate accumulation of actin-coated
structures at the APM; arrows point to basolateral actin filaments associated with areas of cell
spreading and process formation; *, lumenal regions; bars, 5 pm.
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92
A Overlay DIC GFP
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93
In some cases, small processes could be detected extending from the basolateral
surface. Transduction with Ad-GFP alone elicited no change in actin cytoskeleton
(data not shown), as previously published (Wang et al, 2003).
To better visualize the morphological changes associated with overexpression
of DN-PKCs in the lacrimal acini, I performed 3-dimensional reconstruction of serial
sections acquired in lacrimal acini. Figure 18 shows the 3D reconstructed shapes of
non-transduced acini and acini overexpressing DN-PKCs (Ad-DN-PKCs) rotated at
different angles for better visualization of the features induced by DN-PKCs
(available on the supplemental CD). The straight arrows indicate elongated neuritic-
like projections extending from the basolateral surface that are commonly detected in
acini transduced with DN-PKCs but not in non-transduced acini.
The effects of DN-PKCs overexpression on basal and stimulated secretion of
bulk protein and the secretory product, (3-hexosaminidase, are shown in Figure 19.
Many lumena in reconstituted acini are open to the culture medium, so that secretion
can be measured by collecting culture medium, measuring the marker of interest, and
normalizing the signal to pellet protein as previously described (da Costa et al., 1998;
da Costa et al., 2003; Wang et al., 2003). CCH is a robust secretogogue in
reconstituted rabbit lacrimal acini, eliciting ~3.5-fold increase in both bulk protein
and (3-hexosaminidase release. GFP overexpression minimally affected this pattern
of release.
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94
Figure 18. Transduction of lacrimal acini with Ad-DN-PKCs is associated with changes in
acinar shape. 3D reconstruction of the actin filament network labeled with Alexa Fluor 647-
phalloidin in non-transduced acini (CON, left panel) or acini transduced with Ad-DN-PKCs (right
panel) as described in Chapter H Images were acquired at Z intervals of 0.5 pm and were
reconstructed into a 3D movie file which could be rotated to demonstrate the structure viewed at
different angles. Selected frames are presented at 0°, 22.5 °, 45 °, 67.5 °, and 90 °. Straight arrows
indicate projections at the basolateral side of acini; curved arrows indicate the direction of the rotation
of the acinar projection; *, lumenal regions; bar, 5 pm.
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CON Ad-DN-PKCe
0°
< *
22.5°
4 ?
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96
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Figure 19. DN-PKCs inhibits secretagogues-stimulated release of protein and p-hexosaminidase
in lacrimal acini Lacrimal acini grown on Matrigel-coated dishes were transduced with Ad-DN-
PKCe or Ad-GFP on day 2 of culture as described in Chapter II and analyzed on day 3 for secretion.
Bulk protein secretion (top) and p-hexosaminidase activity (bottom) in transduced acini exposed to
CCH (100 pM, 30 min, left) or PE (100 pM, 30 min, right) is shown. Basal (unstimulated) release is
shown in white bars; total release (basal plus stimulated) is shown in grey bars; the stimulated
component (total minus basal) is shown in black bars. Values were normalized to cell protein before
comparison across samples. N=7 separate preparations for CCH stimulation and 4 separate
preparations for PE stimulation; error bars represent sem; *, significant at p<0.05 from Ad-GFP
transduced acini; #, significant at p<0.05 from Control (non-transduced).
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97
In contrast, DN-PKCs caused a significant (p<0.05) increase in basal protein and P-
hexosaminidase release that was significant relative to Ad-GFP-treated acini, with a
concomitant significant decrease in both total and stimulated components of the
release. I tried to evaluate the effects of DN-PKCs on secretion evoked by PE in
parallel. Consistent with previous results (Qian et al., 2003), PE only weakly
increased secretory functions, promoting only a 0.5x increase in bulk protein release
and minimally affecting P-hexosaminidase release. A trend towards increased basal
release relative to Ad-GFP transduced acini and a complete inhibition of PE-
stimulated release were noted in these assays, although the magnitude of the effect
made it difficult to distinguish actual changes from the normal variation inherent in
the assay.
These results suggested that the normal pathways of CCH-stimulated
secretion were inhibited by introduction of DN-PKCs. However, because bulk
protein is a relatively non-specific marker of secretion, it was difficult to discern
whether the inhibition was exclusively due to alterations in apical targeting and
release or whether it was complicated by changes in the basolateral release profile.
Although p-hexosaminidase is a secretory protein, it is also present in basolateral
membrane compartments which may, under some conditions, contribute to
basolateral exocytosis. I therefore investigated whether DN-PKCs could modulate
the CCH-stimulated release of two apically-targeted proteins, syncollin-GFP and SC,
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98
into culture medium. PE treatment was not further pursued because of its limited
efficacy as a secretagogue.
CCH-stimulated release o f syncollin-GFP is inhibited by DN-PKCz
As shown in Chapter III, syncollin-GFP labels apparent mSVs in lacrimal
acini, and the subapical stores of this protein are depleted in response to CCH. I felt
that this would be an excellent marker to study the effects of PKCs specifically on
exocytosis of mSVs. However, I was unable to analyze lacrimal acini dually
transduced with Ad-syncollin-GFP and Ad-DN-PKCs (which co-expresses GFP) by
live cell confocal fluorescence microscopy because syncollin-GFP-enriched vesicles
could not be resolved above the strong cytosolic GFP fluorescence. As shown in
Figure 17, fixation irreversibly quenches cytosolic GFP fluorescence. Syncollin-
GFP can be visualized in fixed, transduced acini using appropriate primary and
fluorescently-labeled secondary antibodies in parallel with other markers. Figure
20A shows syncollin-GFP (green) detected by immunofluorescence in lacrimal acini
co-transduced with Ad-syncollin-GFP and Ad-DN-PKCs (red) relative to Ad-
syncollin-GFP alone. These images show that overexpression of Ad-DN-PKCs
(detected easily by the intense PKCs immunofluorescence associated with apical and
basolateral actin) does not affect the abundance of syncollin-GFP-enriched vesicles
or their apical enrichment.
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99
Figure 20. DN-PKCs inhibits CCH-stimulated release of syncollin-GFP into culture medium in
co-transduced lacrimal acini. A. High magnification view of the APM region of acini co
transduced with Ad-syncollin-GFP and Ad-DN-PKCs, or Ad-syncollin-GFP alone. Transduced acini
were fixed and labeled as described in Chapter H to detect syncollin (green), PKCs (red), and actin
filaments (purple). Overlay shows all three fluorescence labels as well as the paired DIC image.
Fixation quenches the intrinsic GFP fluorescence present in cytosol and on syncollin in these
transduced acini. *, lumenal region; arrow, co-localization of syncollin-GFP with an actin-coated
invagination; bar, 5 pm. B. Western blots showing syncollin-GFP release into culture medium in the
absence (-) and presence (+) of 100 pM CCH for 30 min in lacrimal acini transduced with Ad-
syncollin-GFP without or with Ad-GFP or Ad-DN-PKCs. Syncollin release was detected with an
anti-syncollin antibody combined with an appropriate IRDye™800 conjugated secondary antibody. C.
Syncollin-GFP release under each experimental condition was quantified as shown in A., normalized
to cell protein in the pellet, and compared across treatments. White bars show basal (CON) release
while grey bars show CCH-stimulated release. N=3 separate preparations; error bars show sem and *,
significant at p<0.05 from samples co-transduced with Ad-GFP.
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100
A Ad- Syncollin-GFP + Ad-DN-PKCs
DN PKCe Syncollin-GFP Actin Overlay
Ad- Syncollin-GFP
Syncollin-GFP Actin Overlay
B
CCH:
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-* -6 6
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Ad-Syn-GFP Ad-Syn-GFP Ad-Syn-GFP MW
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101
Interestingly, some co-localization of syncollin-GFP was detected with the actin-
coated structures induced by overexpression of DN-PKCs (arrows, green and purple
labels), consistent with our proposal that these actin-enriched structures were mSV
fusion intermediates.
Although I was unable to use live cell confocal microscopy to investigate the
effect of DN-PKCs on exocytosis of syncollin-GFP in real time, I was able to utilize
Western blotting to measure syncollin-GFP release into culture medium under
different experimental conditions. As shown in Figure 20B, CCH stimulation
caused a 3-fold increase in the recovery of syncollin-GFP in the culture medium of
Ad-syncollin-GFP transduced acini which paralleled the loss of subapical
fluorescence seen in live acini exposed to CCH (Chapter III, Figure 12). Co
transduction of acini with Ad-DN-PKCs but not Ad-GFP significantly inhibited
CCH-stimulated release of syncollin-GFP into culture medium (Figure 20B and C).
Co-transduction of acini with Ad-DN-PKCs and Ad-syncollin-GFP moderately
reduced syncollin-GFP expression relative to co-transduction with GFP and
syncollin-GFP (data not shown), an effect that was likely due to non-specific
changes associated with overexpression of three exogenous proteins in co-transduced
acini (GFP and PKCs by Ad-DN-PKCs and syncollin-GFP by Ad-syncollin-GFP).
The percentage of total cellular syncollin-GFP released in response to CCH under
each experimental condition represented -20-30% of total cellular syncollin-GFP,
suggesting that sufficient cellular stores were available for exocytosis under all
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102
conditions (data not shown). Abundant syncollin-GFP immunofluorescence could
also be detected in acini co-transduced with Ad-DN-PKCe (Figure 20A). Finally,
acini dually transduced with Ad-syncollin-GFP and Ad-DN-PKCe or Ad-GFP
exhibited the same general profiles of p-hexosaminidase release reported for acini
transduced with Ad-DN-PKCe or Ad-GFP alone in Figure 19 (Table III). These
findings indicated that the overall acinar secretory pathway was intact in acini
expressing syncollin-GFP, and that DN-PKCe had the same magnitude of inhibitory
effect on p-hexosaminidase release regardless of syncollin-GFP co-expression.
From these data, I concluded that DN-PKCs significantly and specifically inhibited
apical exocytosis of syncollin-GFP in lacrimal acini.
CCH-stimulated apical release o f SC from plgR is inhibited by DN-PKCs Free SC
and its dimeric IgA bound counterpart, secretory IgA, are enriched in ocular surface
fluid (Gundmundsson et al., 1985; Van Haeringen, 1981); they presumably derived
by proteolytic cleavage of transcytosed plgR, either free or bound to dimeric IgA,
respectively, at the APM. I evaluated the intracellular localization of plgR in
unstimulated and CCH-stimulated lacrimal acini by confocal fluorescence
microscopy using an antibody against the extracellular domain of the plgR. This
antibody therefore recognizes both intact plgR and free SC. As shown in Figure
21A, plgR immunofluorescence is recovered in punctuate spots throughout the
cytoplasm, and at basolateral (arrows) and apical (arrowheads) domains
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103
Figure 21. plgR is enriched with actin and PKCs at the APM in lacrimal acini. A. Lacrimal
acini were fixed and processed as described in Chapter II to label plgR (green) and actin filaments
(red) for detection by confocal fluorescence microscopy. Arrows, basolateral labeling; arrowheads
labeling around lumenal regions. *, lumenal regions. B. High magnification view of the APM region
of control (CON) lacrimal acini labeled as described in Chapter n to detect plgR (green), PKCs
(red), and actin filaments (purple). The triple overlay reveals extensive co-localization of all three
constituents together at the APM (arrows) and *, lumenal region. C. High magnification view of the
APM region of CCH-stimulated (100 pM, 5 min) acini fixed and processed as in B. The triple
overlay shows actin-coated invaginations co-localized with plgR and PKCs (arrowheads) and *,
lumenal region. Bars, 5 pm.
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A plgR Actin Dual
B plgR PKCe
Actin Overlay
C plgR PKCe
Actin Overlay
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105
Figure 21B shows a higher magnification of the subapical plgR
immunofluorescence in acini also labeled to detect endogenous PKCs. As shown in
the triple overlay, there was considerable co-localization of plgR, PKCs and apical
actin (arrows). Co-localization of plgR, PKCs and apical actin was also detected in
CCH-stimulated acini; furthermore, the intensity of PKCs and plgR
immunofluorescence was increased. Also, recruitment of plgR and PKCs
immunofluorescence to actin-coated structures was evident (Figure 21C,
arrowheads). These findings suggested that traffic of plgR to the actin-enriched
region beneath the APM was enhanced by CCH, and further that PKCs associated
with apical actin filaments was enriched in areas actively undergoing plgR
trafficking.
The cleaved extracellular domain of the plgR, SC, can be detected in culture
medium from rabbit lacrimal acini. Remarkably, CCH stimulated release of SC from
lacrimal acini by 3.5-fold (Figure 22). Transduction of lacrimal acini with Ad-GFP
did not affect the CCH-stimulated release of free SC, but transduction with Ad-DN-
PKCs significantly inhibited CCH-stimulated SC release. Examination of lacrimal
acini transduced with Ad-DN-PKCs revealed abundant plgR immunoreactivity
detected at and beneath the apical actin array of both unstimulated and CCH-
stimulated acini, similar to that seen in the CCH-stimulated acinus in Figure 21, and
consistent with accumulation of plgR at the APM or subapical stores (data not
shown).
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106
A
CCH: - + - +
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Figure 22. Transduction with Ad-DN-PKCe inhibits CCH-stimulated release of SC from
lacrimal acini. A. Western blots showing SC release into culture medium in the absence (-) and
presence (+) of 100 pM CCH for 30 min in non-transduced acini (no Ad), or acini transduced with
Ad-GFP or Ad-DN-PKCe. SC release was detected with an antibody to the extracellular, cleaved
domain of plgR combined with an appropriate IRDye™800 conjugated secondary antibody. B. SC
release under each condition was quantified as shown in A., normalized to cell protein in the pellet,
and compared across treatments. White bars show control (CON) release while grey bars show CCH-
stimulated release. N=3 separate preparations; error bars show sem and *, significant at p<0.05 lfom
samples co-transduced with Ad-GFP.
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107
Table III. Effects of Ad-syncollin-GFP on lacrimal acinar secretion of 1 3 -
hexosaminidase.
Cell Treatment Experimental Treatment p-hexosaminidase release
(% of non-transduced
control)
No Ad Basal 100% + 8%
Total (in presence of CCH) 174% + 34%
Stimulated (difference) 74% + 34%
Ad-syncollin-GFP Basal 97% + 2%
+
Total (in presence of CCH) 159% + 29%
Ad-GFP Stimulated (difference)
62% + 27% (84% of the
value elicited in non-
transduced)
Ad-syncollin-GFP Basal 106%+ 6%
+
Total (in presence of CCH) 149% + 25%
Ad-DN-PKCs Stimulated (difference) 43% + 21% (58% of the
value elicited in non-
transduced)
Results are averaged from n=3 separate preparations and errors indicate sem. CCH
stimulation was for 30 min at 100 pM.
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108
Chapter VI: Discussion
PKCe participates in the regulation of actin filament remodeling
during stimulated secretion
My studies using time-lapse confocal fluorescence microscopy and FRAP
clearly revealed evidence for dynamic actin remodeling during exocytosis. Inspired
by the fact that PKCs is the only PKC with an actin-binding site (Akita Y, 2002),
and by observations that it is activated both by muscarinic and a l adrenergic
receptor agonists in lacrimal gland (Zoukhri et al., 1997), I investigated whether
PKCs participated in regulation of apical actin and/or apical exocytosis. I found that
the association of PKCs with apical actin filaments and actin-coated structures was
increased after CCH stimulation in lacrimal acini. To probe its functional role in
exocytosis, I wanted to selectively inhibit its activity. I chose a strategy of using DN
mutants inhibit PKCs activity in acini, using replication-defective Ad to introduce
the DN-PKCs. Lacrimal acini were readily transduced at high efficiency with Ad
constructs, enabling me to express the DN-PKCs in essentially all cells and to then
identify individual transduced cells by immunofluorescence for examination of other
features. Overexpression of DN-PKCs resulted in profound changes in apical and
basolateral actin filament organization in parallel with inhibition of stimulated
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109
secretion. The effects on stimulated secretion included inhibition of bulk protein and
P-hexosaminidase secretion as well as inhibition of the release of two markers of
apical secretion established in this study, syncollin-GFP and SC. I propose a direct
relationship between the alterations in apical actin filaments and the inhibition of
exocytotic and transcytotic traffic.
The feature of PKCs indicative of activation that I focused on in this study was its
translocation to actin filaments. However, PKCs activation in lacrimal acini is likely
to be a complex, multistep process. Inactive PKCs exists in a closed conformation,
maintained by interactions between the catalytic and pseudosubstrate domains (Pears
et al, 1990). All unphosphorylated or hypophosphorylated PKCs requires priming
phosphorylation steps to convert enzyme from an immature into a mature form that
is responsive to second messengers (Akita, 2002). The current dogma suggests an
initial phosphorylation at a conserved threonine residue in the active site (Thr-566)
followed by consequent phosphorylation at two key C-terminal sites which allows
the enzyme to be stabilized in the catalytically active conformation (Ser-729 and Thr
710) (Rybin et al., 2003). Once primed, the mature form of PKCe can be activated
by diacylglycerol, 1,4,5-inositol triphosphate and other fatty acids (Akita, 2002).
Similar to other novel PKCs, phospholipid-dependent activation results in a
conformational change which allows enzyme translocation to the particulate fraction
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110
(in this case actin filaments and/or membranes) through interaction with anchoring
proteins termed RACKs (receptor of activated C-kinase) (Mochly-Rosen, 1995).
In preliminary studies, I did not detect a major increase in PKCs
phosphorylation associated with CCH stimulation, by Western blotting of
immunoprecipitated PKCs with anti-Phospho-PKCs (Ser 729) although some
phosphorylation could be detected in the resting state (data not shown). These
studies suggest that resting acini may maintain a relatively high steady-state level of
mature (phosphorylated) PKCs in resting acini that is primed for activation by
second messengers. Such a phenomenon has been established for other novel PKCs
in the resting state when cell cultures were grown in highly supplemented culture
medium (Parekh et al., 1999). Our lacrimal acini are cultured in a laminin-enriched
rich medium which additionally contains very low levels of carbachol (10 nM),
either of which may result in PKCs priming by phosphorylation in the resting state.
Since I clearly observed PKCs translocation to the actin cytoskeleton in response to
CCH, another hallmark of activation, I did not conduct comprehensive analyses to
resolve this issue of altered phosphorylation.
I hypothesize that release of second messengers triggered by CCH
stimulation normally results in PKCs activation followed by its translocation to
apical actin filaments. The activated PKCs acts by phosphorylating key targets
associated with actin filaments that result the remodeling of apical actin in ways that
facilitate SV exocytosis, including the transient formation of actin-coated structures.
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I l l
We and others have shown that actin filament remodeling is an integral part of apical
exocytosis in acinar epithelial cells (Muallem et al., 1995; Perrin et al., 1992;
Valentijn et al., 1999; Valentijn et al., 2000; Chapter III, this study). Further,
stabilization of actin-coated structures, thought to represent fusion intermediates, is
correlated with inhibition of acinar exocytosis as shown in Chapter III.
In acini transduced with Ad-DN-PKCs, overexpressed PKCs was detected
with actin filaments of altered organization even in unstimulated acini. The changes
caused by DN-PKCs included accumulation of actin at the APM in the underlying
filament network as well as accumulation of actin-coated structures representing
prospective fusion intermediates. I hypothesize that the inhibitory effects of DN-
PKCs on apical exocytosis are caused by stabilization of apical actin, either in the
underlying actin filament network or in actin-coated structures, either of which could
impair SV exocytosis. These effects are likely due to the absence of catalytic
activity in the DN-PKCs which, when recruited to apical actin, is unable to
appropriately phosphorylate actin-associated proteins that are normally required for
its remodeling. The inhibitory effect on DN-PKCs on SC exocytosis suggested a
comparable role for actin filament remodeling in the terminal events associated with
its release (through transcytotic or exocytotic pathways), a hypothesis supported by
the co-localization of plgR and PKCs with actin-coated structures in CCH-
stimulated acini. My studies implicating PKCs as a major effector of apical actin
remodeling in exocytosis in lacrimal acini are consistent with findings from other
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112
systems. Prekeris et al. (Prekeris et al., 1996) found that PKCs was necessary for
exocytosis of synaptic vesicles in hippocampal neurons. DN-PKCs also significantly
reduced glucose-stimulated exocytosis of insulin in insulin-secreting INS-IE cells
(Mendez et al., 2003). The latter study also demonstrated glucose-induced
association of endogenous PKCs with insulin granules, confirming a specific
translocation to vesicles involved with exocytosis. Although its function in
exocytosis was not specifically investigated, PKCs was also detected at the APM of
pancreatic acini (Bastani et al., 1995).
Our previous investigations on the mechanisms of exocytosis in lacrimal
acini have been hampered by our inability to track specific secretory products
released exclusively at the APM. Syncollin-GFP, previously established as a marker
of large dense core vesicles and zymogen granules in related systems (Hodel and
Adwardson, 2000; Ma et al., 2004), was enriched in large, 1 pM diameter, spherical
structures beneath the APM, consistent with its incorporation into mature SVs.
Additional syncollin-GFP was also present in smaller vesicles ranging from 300-500
nm in diameter; these vesicles were detected in the cytoplasm as well as beneath the
APM. This pattern suggested specific labeling of a subpopulation of mature 1 pM
SVs that might be generated from the smaller, more abundant 300-500 nm SVs
representing -60% of the syncollin-GFP population. The fluorescence associated
with these subapical vesicles was substantially diminished after CCH stimulation in
parallel with the increased recovery of syncollin-GFP in the culture medium,
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113
confirming that the syncollin-GFP-enriched vesicles were in fact SVs. The
inhibition of syncollin-GFP release in acini co-transduced with Ad-DN-PKCs but
not Ad-GFP enabled me to conclude that apical exocytosis, in particular, was
affected.
Little is known about the cellular mechanisms underlying the release of SC
and secretory IgA at the APM of lacrimal acinar cells, although common wisdom
suggests that some stores are transported via the transcytotic pathway as in other
epithelial cells. One previous study investigated the release of SC from cultured
lacrimal acini over 4-7 days, demonstrating that CCH modestly but significantly
inhibited its release (Kelleher et al., 1991). The inhibitory effect of CCH in this
previous study might have been influenced by the duration of the exposure, which
could have influenced the expression of signaling effectors or affected other acinar
functions. My study reports that CCH stimulated the acute (30 min) release of SC
from plgR at the APM of lacrimal acini. Studies on purified lacrmal acinar cells
performed in similar culture system , cells grown in Matrigel rafts, demonstrated
that CCH stimulation resulted in increased release of beta-hexosaminidase and SC
from the rafts (Schechter et al., 2002). Here I demonstrate that DN-PKCs
significantly inhibited the CCH-stimulated release of SC from lacrimal acini,
suggesting a general role for PKCs in regulation of exocytotic and transcytotic
pathways. Our laboratory is currently investigating whether these pathways function
independently in acini, or whether they converge at a common apical intermediate.
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Some of my findings obtained with DN-PKCs overexpression in acini remain
perplexing. A significant amount of overexpressed DN-PKCs was associated with
actin filaments even in the absence of stimulation. In fact, the intensity of
immunofluorescence labeling was so great in unstimulated acini that the increased
association of DN-PKCs triggered by CCH stimulation could not be distinguished by
confocal fluorescence microscopy. PKCs content in supernatant fractions from cell
lysates from CCH-stimulated acini transduced with DN-PKCs was markedly
decreased relative to its content in untreated acini (data not shown), suggestive of
increased sedimentation with actin filaments as seen in non-transduced acini and also
consistent with the existence of a pool of DN-PKCs that was still recruited to actin
by CCH stimulation. Acini may maintain a certain percentage of total PKCs in
association with the actin cytoskeleton even in the resting state in order to maintain
unique structures or morphology. The substitution of DN-PKCs for wild type PKCs
in transduced acini may underlie some of the remarkable changes in apical actin
morphology seen even in the resting state. However, it is clear that the amount of
DN-PKCs associated with actin cytoskeleton in resting transduced acini is far greater
than the amount of endogenous PKCs associated with actin cytoskeleton in resting
acini.
PKCs was not detected with basolateral actin in resting or CCH-stimulated
non-transduced acini. However, DN-PKCs was extensively co-localized with
basolateral actin in parallel with a profound change in the organization of basolateral
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115
actin filaments into neuritic like-extensions. PKCs overexpression is well
established as a contributor to cell transformation and cell metastasis (Akita, 2002),
processes which involve extensive actin filament remodeling, branching and
extension. However, recent work on the role of PKCe in neurite outgrowth has
reported that overexpression of PKCs or overexpression of the regulatory domain of
PKCs (containing the actin-binding but not catalytic domains) exerted the same
effects on outgrowth (Zeidman et al., 1999; Zeidman et al., 2002). It is possible that
association of the regulatory domain present in the DN-PKCs with the acinar actin
cytoskeleton may be able to modulate actin filament reorganization in the absence of
catalytic activity, influencing both basolateral and apical actin filament dynamics.
Another possible explanation for the drastic change in the cell shape is the
effect of PKCs on the pathway controlling cell growth and tumorigenesis. The
profound transformation of the cellular shape is reminiscent of cells experiencing
oncogenic transformations. The ability of PKCs to promote tumorigenesis has been
observed in various systems. Oncogenic activity of PKCs has been reported in
several fibroblast and colonic as well as prostatic epithelial cell lines (Mischak et al.,
1993; Perletti et al., 1998; Wu et al., 2002; Akita 2002). An increasing body of
evidence suggests a role for PKCs in tumor metastasis. Also, epidermis-specific
transgenic overexpression of wild type PKCs caused development of metastatic
carcinomas in mice (Jansen et al., 2001). These effects are mostly associated with
alterations in the ras-signaling cascade at the level of Raf-1 or cyclin D l.
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116
Cell spreading is accompanied by actin cytoskeleton reorganization, critically
important in cell survival and motility. Integrins play important role in linking the
extracellular matrix with cytoskeleton and signaling machinery of the cell. A positive
effect of PKCs on adhesion and motility is also known, and believed to be regulated
by its interaction with pi integrin through RACK1 (receptor for activated C kinase
1) and F-actin (Besson et al., 2002; Tachado et al., 2002; Akita, 2002).
Transformation of cell shape and cytoskeleton associated with altered PKCs function
has been reported in neuronal, neuroblastoma cells, HeLa cells, fibroblasts, and CHO
cells (Kim et al., 1997; Chun et al., 1996). Studies on the role of integrin pi
cytoplasmic domain demonstrated that coexpression of PKCs can restore cell
spreading inhibited by tac- pi, a DN inhibitor of integrin function in normal human
fibroblasts and CHO cells; an intact kinase domain was required for this process
(Berrier et al., 2000). Exactly why the DN form of the overexpressed kinase elicits
potentially transforming effects is so far unknown in our study, but this may have to
do with other domains of the protein apart from the catalytic site.
Although a complete consideration of the mechanisms underlying the increased
association of DN-PKCs with basolateral actin is beyond the scope of this study, it
should be noted that such changes could also influence apical exocytosis.
Interactions between lacrimal acini and extracellular matrix can regulate exocytosis,
while integrins, in concert with actin cytoskeleton are thought to modulate some of
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117
these interactions (Chen et al., 1998). Previous work has established that (3 1 integrin
interacts with PKCs and actin filaments through its receptor, RACK1, and that these
interactions regulate cell migration and adhesion (Akita, 2002; Besson et al., 2002;
Tachado et al., 2002). Recent work has also suggested that the regulatory (actin-
binding) domain of PKCs can inactivate RhoA, and that this mechanism is involved
in the switch from an adherent (stress fiber-enriched) to a motile (neuritic process)
state in neuronal cells (Ling et al., 2004). RhoA has recently been implicated as a
positive effector in the formation of actin-coated fusion intermediates in pancreatic
acini (Nemoto et al., 2004).
I have established here that PKCs is an actin-binding protein recruited
transiently to apical actin filaments and actin-coated structures, possibly representing
fusion intermediates, in CCH-stimulated lacrimal acini. I have further established
that its inhibition, through overexpression of DN-PKCs, stabilized actin-coated
structures in parallel with inhibition of stimulated exocytosis of a variety of secretory
products at the APM. Future work will focus on the identification of actin-
associated proteins that are likely to facilitate apical actin remodeling and that serve
as targets of PKCs.
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118
Chapter VII: Conclusions
Clinical significance
Most of the studies on the mechanisms of regulated secretion in the lacrimal
gland in our laboratory were done in the ex vivo cultured system of isolated rabbit
epithelial lacrimal gland cells, however some studies utilized established transgenic
mouse model.
Another model system to study regulated secretory pathway used in our
laboratory is established mouse model for Sjogren’s Syndrome, non-obese diabetic
or NOD mouse. This mouse is known to develop insulin-dependent diabetes
mellitus, as well as infiltrates in submandibular and lacrimal glands accommodated
with decrease in tear flow (Nguyen et al., 2000; Van Blokland and Versnel, 2002;
Barabino and Dana, 2004). The resulting lymphocytic infiltration is accompanied by
decreased production of lacrimal fluid. Clinical manifestations of SjS are detectable
in males by ~4 months. When glands from NOD mice compared to the control age-
matched B ALB/c mice, distribution pattern of key signaling effectors such as rab3D
and M3 muscarinic receptor of secretory pathway were affected. Also, dramatic
changes in actin cytoskeleton were notices in Sjogren’s Syndrome mouse model. As
demonstrated by the confocal fluorescence microscopy study of lacrimal glands from
1 months NOD mice, their cortical actin exhibited increased intensity of the of the
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119
basolateral actin compared to age-matched controls. By 4 months, NOD mouse
exhibited diminishing of the overall amount of actin filament labeling detected
primarily in the areas of extensive tissue destruction or lymphocytic infiltration,
when compared to BALB/c or 1 month NOD mouse glands (da Costa et al.,
manuscript submitted).
NOD mouse study demonstrated that in Sjogren’s Syndrome mouse model
altered intracellular signaling environment and early changes in SV morphology
correlate with dramatic changes in actin cytoskeleton resulting in the development of
the disease. Therefore, understanding the mechanism of secretagogue-driven process
of actin remodeling and pathological aberrations of actin remodeling can provide
valuable insights into design and development of new treatments of this malignancy.
Conclusions
My results are consistent with the following working model (Figure 22).
Secretagogue stimulation of lacrimal acini promotes rapid, transient disassembly of
actin filaments beneath the APM in sites destined for fusion of mature secretory
vesicles. Exocytosis of mature secretory vesicles is then initiated at these sites, with
actin filaments reassembling around the perimeter of the fusing mature secretory
vesicles. My data suggest that actin coats may envelope single or multiple fusing
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120
vesicles, so each scenario is depicted in the model. The actin coat surrounding the
fusing secretory vesicle appears to contract toward the APM, with the underlying
actin filaments ultimately merging with the apical actin array. The apparent sliding
or contraction of actin filaments (arrows) on the actin-coated intermediates is likely
mediated by one or more myosin motors and may be important in the extrusion of
secretory vesicle contents. At the same time CCH causes activation and translocation
of PKCe to the apical actin filaments. PKCs, while anchored to the filaments,
phosphorylates targeted ABPs. Activation of these ABPs effectors alternatively or
together with myosins results in the actin remodeling accompanied by changing in
the rates of MFs disassembly and turnover. Restoration of the APM to the resting
state (not shown) also requires the retrieval of excess membrane through clathrin-
dependent endocytosis (da Costa et al., 2003b) and possibly additional endocytotic
mechanisms.
Altogether, my data strongly suggest involvement of apical actin cytoskeleton
remodeling and actin-based motors in the process of regulated secretion in rabbit
lacrimal acinar cells. My findings support both the actin barrier hypothesis and the
force-generation hypothesis. The actin-binding protein, PKCs, appears to represent a
physiological candidate which anchors to the apical actin cytoskeleton and
phosporylates targeted ABPs at that site during the process of activation with CCH.
The consequent activation of downstream effectors associated with the actin
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121
cytoskeleton results in apical actin remodeling and increased MF turnover, which is
also accompanied by the activation of the contractile acto-myosin system.
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122
Figure 22. Working model. CCH stimulation results in increased turnover of apical actin filaments
(red), allowing access of fusing mature secretory vesicles to the APM. These vesicles acquire an actin
coat that may encompass single or multiple fusing mature secretory vesicles, as indicated. This
structure is equivalent to the actin-coated firsion intermediate. Contraction of actin filaments around
the perimeter of the fusing mature secretory vesicles (arrows), a process likely mediated by one or
more myosin motors, facilitates extrusion of mature secretory vesicle contents. PKCs is translocated
to the apical actin filaments upon stimulation with CCH, which results in the phosphorylation of the
targeted substrate - actin-binding proteins. These effectors are likely to contribute to the apical actin
dynamics associated with regulated secretion.
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123
CCH
CCH
I I A
sssSS^wsss ssR se s
I 1
rarasssa* * ' * ' 5* ' 58
Actin filaments (MFs);
Targeted actin-binding proteins (ABPs);
Phosphorilated ABPs;
PKCs;
Activated PKCs
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124
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Creator Jerdeva, Galina Vladislavovna (author)

Core Title A dynamic apical actin cytoskeleton facilitates exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

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

Tag biology, cell,biology, molecular,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Hamm-Alvarez, Sarah (committee chair), Garner, Judy A. (committee member), Mircheff, Austin K. (committee member), Okamoto, Curtis T. (committee member), Shen, Wei-Chiang (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-344427

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