Experimental Eye Research 83 (2006) 84e96
www.elsevier.com/locate/yexer
Review
Molecular mechanisms of lacrimal acinar
secretory vesicle exocytosis
Kaijin Wu a, Galina V. Jerdeva a, Silvia R. da Costa a, Eunbyul Sou a,
Joel E. Schechter b, Sarah F. Hamm-Alvarez a,c,d,*
a
Department of Pharmaceutical Sciences, School of Pharmacy, 1985 Zonal Avenue,
University of Southern California, Los Angeles, CA 90033, USA
b
Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA 90033, USA
c
Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90033, USA
d
Department of Ophthalmology, University of Southern California, Los Angeles, CA 90033, USA
Received 19 July 2005; accepted in revised form 1 November 2005
Available online 10 March 2006
Abstract
The acinar epithelial cells of the lacrimal gland are responsible for the production, packaging and regulated exocytosis of tear proteins into
ocular surface fluid. This review summarizes new findings on the mechanisms of exocytosis in these cells. Participating proteins are discussed
within the context of different categories of trafficking effectors including targeting and specificity factors (rabs, SNAREs) and transport factors
(microtubules, actin filaments and motor proteins). Recent information describing fundamental changes in basic exocytotic mechanisms in the
NOD mouse, an animal model of Sjögren’s syndrome, is presented.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: exocytosis; rab3D; VAMP2; actin filaments; microtubules; cytoplasmic dynein; non-muscle myosin II; NOD mouse; Sjögren’s syndrome
1. Introduction
The acinar epithelial cells are the major cell type within the
lacrimal gland, responsible for production and regulated exocytosis of proteins and fluid onto the ocular surface. These secreted proteins are responsible for maintaining the integrity of
the ocular surface by nurturing the corneal epithelium and protecting the ocular surface from pathogens. Numerous proteins
are detected in tears including such major constituents as lysosomal hydrolases (van Haeringen and Glasius, 1980; van
Abbreviations: SjS, Sjögren’s syndrome; SV, secretory vesicle; APM, apical plasma membrane; EM, electron microscopy; SNARE, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors; VAMP,
vesicle-associated membrane protein; rSV, recruitable secretory vesicles;
Arp1, actin-related protein 1; ICA69, islet cell autoantigen (69 kDa); PKC,
protein kinase C; DAG, diacylglycerol.
* Corresponding author. Tel.: þ1 323 442 1445; fax: þ1 323 442 1390.
E-mail address: [email protected] (S.F. Hamm-Alvarez).
0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2005.11.009
Haeringen et al., 1978), secretory IgA (Fullard and Snyder,
1990; Pandit et al., 1999), lactoferrin (Fullard and Snyder, 1990;
Yoshino et al., 1996), transferrin (Salvatore et al., 1999),
lipocalins (Gasymov et al., 1999) and growth factors (Sack
et al., 2005; van Setten et al., 1996). Dry eye disorders, which
affect millions of people each year are, in many cases, associated with decreased protein and fluid secretion by the lacrimal
gland (Pflugfelder et al., 2000). One of the most severe forms
of dry eye is the autoimmune disease, Sjögren’s Syndrome
(SjS), which affects a significant subset of dry eye patients.
Diagnosis of SjS is based on presenting symptoms of dry
eye and dry mouth accompanied by a characteristic focal
lymphocytic infiltration of the lacrimal and salivary glands
(Daniels and Fox, 1992; Haga, 2002).
Recent work has provided an up-to-date description of the
complex signaling mechanisms utilized in the lacrimal gland
by cholinergic and adrenergic agonists and other regulatory
factors to transmit signals associated with receptor binding
into the cytoplasm to evoke release of secretory products
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
into tear fluid (Dartt, 2001, 2004). However, the identity of the
trafficking proteins that serve as targets of these signaling
pathways and that physically promote the rapid transport
and fusion of secretory vesicles (SVs) with the apical plasma
membrane (APM) has remained unclear. Recent work has finally begun to address the fundamental mechanisms of secretory protein exocytosis in lacrimal acini, revealing intriguing
and unique features. The advent of several in vitro culture systems for maintenance of primary lacrimal acinar cells under
conditions that allow either maintenance or reconstitution of
normal acinar polarity and elaboration of SVs has greatly enhanced these efforts (Gierow et al., 1995; Hodges et al., 2004;
Laurie et al., 1996; Rismondo et al., 1994).
All events in membrane trafficking including those involved in exocytosis are governed by effector proteins which
can be grouped into different categories: targeting and specificity factors such as rab and SNARE proteins, and transport
factors such as cytoskeletal filaments and molecular motors.
The new knowledge obtained from cellular and molecular investigations into the mechanisms of tear protein exocytosis in
lacrimal acini is reviewed within the context of these different
categories of effectors, with established participants then integrated into a working model for lacrimal acinar exocytosis.
2. Results
2.1. Targeting and specificity: rabs
Rab proteins are a family of small Ras-like GTPases that regulate membrane trafficking along the biosynthetic, exocytotic
and endocytotic pathways in all eukaryotic cells (Pfeffer,
2001). Some 60 members have been identified to date in mammalian cells (Pereira-Leal and Seabra, 2001), many of which
display distinctive distributions on different subcellular membrane organelles and regulate the directional membrane traffic
between them. As with all other members of the small Raslike GTPase family, the rabs’ activities, and therefore their
effector functions, are regulated by their cycling between an
activated, GTP-bound state and an inactive, GDP-bound state.
This cycle is regulated by intrinsic rab GTPase activity, which
is modulated by interactions with multiple proteins possessing
the following activities, each specific for a particular rab:
GTPase-activating proteins which stimulate GTP hydrolysis;
guanine nucleotide exchange factors which are involved in the
exchange of GDP for GTP; and GDP dissociation inhibitors
which stabilize the GDP bound rab. The regulation of the
GTP-bound state in turn regulates rab effector functions in
such membrane trafficking events as recruitment of coat proteins, recruitment of effector machinery for membrane fusion,
recruitment of cytoskeletal machinery for vesicle motility and
trafficking and direct binding to cargo proteins to regulate their
association with vesicle coat proteins.
The most abundant protein thus far characterized in the lacrimal acinar secretory pathway is rab3D. Rab3D is a member
of the rab3 family, each of which (rabs3AeD) are implicated
in regulated exocytosis (Darchen and Goud, 2000). Rab3D is
primarily localized to mature SVs in pancreatic, parotid and
85
lacrimal acinar cells (Ohnishi et al., 1996; Raffaniello et al.,
1999; Wang et al., 2003) although reports are still conflicting
regarding its role in acinar exocytosis. Overexpression of dominant negative rab3D, which is unable to bind GTP, impairs
pancreatic secretory granule exocytosis in functional assays
(Chen et al., 2002, 2003). Another study has suggested, in contrast, that rab3D participates specifically in the assembly of actin filaments on fusing SVs in pancreatic acini (Valentijn et al.,
2000). A recent report analyzing exocrine tissue in the rab3D
knockout mouse has revealed the presence of abnormally large
SVs in pancreatic and parotid acini lacking rab3D (the lacrimal gland was not examined in this study) (Riedel et al.,
2002). Despite the abnormal morphology of SVs, the release
kinetics of protein secretion in these glands were not affected
(Riedel et al., 2002). This work by Riedel and coworkers suggests that rab3D may play a regulatory role in modulating the
size of intracellular SVs, perhaps by controlling homotypic
fusion of smaller SVs.
Using indirect immunofluorescence, rab3D has been detected in reconstituted rabbit lacrimal acini at the periphery
of a large diameter SV population enriched beneath the APM
(Wang et al., 2003). Subsequent studies have also revealed its
association with apparent SVs in acini within mouse lacrimal
glands (da Costa et al., 2006). Estimates of the average diameter of the circular rab3D immunofluorescence pattern detected
beneath the APM in both rabbit and mouse lacrimal acini range
from 1 to 2 mm. These values are consistent with recent electron
microscopy (EM) analyses of SV diameter in reconstituted rabbit lacrimal acini (Jerdeva et al., 2005a), and with the representative EM micrographs from rabbit lacrimal gland shown in
Fig. 1A depicting large 1e2 mm SVs clustered beneath the
APM. Rab3D therefore appears to label some or all of these
large subapical mature SVs. Consistent with this model, stimulation of reconstituted rabbit lacrimal acini with the muscarinic
agonist, carbachol, consistently shows a rapid loss of subapical
rab3D immunofluorescence within the time course associated
with fusion of SVs at the APM (Wang et al., 2003). Studies
comparing the effects of dominant negative (e.g., GDP bound)
and constitutively active (e.g., GTP bound) rab3D overexpression in reconstituted rabbit lacrimal acini have thus far failed to
reveal any effects on basal or stimulated release of bulk protein
and b-hexosaminidase (unpublished data).
Other recent work suggests, circumstantially, that the
model of Riedel and coworkers (Riedel et al., 2002) based
on analysis of SV morphology in pancreas and parotid gland
in the rab3D knockout mouse may represent the most accurate description of its function in lacrimal glandde.g., rab3D
may regulate SV size through regulation of homotypic fusion. In reconstituted rabbit lacrimal acini, carbachol stimulation causes an immediate and significant increase in the
average vesicle diameter of subapical SVs prior to their fusion at the APM, suggestive of homotypic fusion of individual SVs into larger SVs prior to fusion at the APM (Jerdeva
et al., 2005a). As mentioned above, the immediate effect of
carbachol stimulation in lacrimal acini is also the disassociation of rab3D from SVs, a loss which may initiate the compound fusion that occurs prior to SV discharge at the APM.
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
86
A
mv
Lumen
sg
RER
jc
ct
N
B
C
mv
Lumen
c
d
ij
sg
jc
tj
fil
mv
D
E
sg
ij
d
tj
mv
Fig. 1. Ultrastructure of rabbit lacrimal gland acini. (A) This low-magnification electron micrograph shows key features of the organization of acini in the rabbit
lacrimal gland. The acinar cells are highly polarized, i.e., nuclei (N) and the bulk of the rough endoplasmic reticulum (RER) are located basally. At the apical
surface numerous secretory vesicles (sv) are collected prior to release by exocytosis. Some secretory vesicles are confluent while others are individual, and the
contents of vesicles are variable in electron density. The luminal surface is marked by small microvilli (mv). Junctional complexes are found just beneath the
luminal surface. The acini are enveloped by a delicate loose connective tissue (ct). Dark triangle at lower right is part of the copper support grid. (B) Flocculent
material (secretory product) is seen within the lumen just after release from the acinar cells. Microvilli project from the apical surfaces of acinar cells and junctional complexes connect adjacent acinar cells just beneath the luminal surface. (C) Part of a centriole (c) is evident in the apical cytoplasm just beneath the luminal
surface. Individual components of a junctional complex are evident connecting adjacent acinar cells: tight junction zone (tj), intermediate junction (ij) and desmosome (d). The cytoplasm beneath the apical surface is rich in actin microfilaments (fil). (D,E) Both of these images have been stained by an immunogold method
to demonstrate actin. The small round particles (overlying dense filamentous zones) represent the sites of actin protein and were found in abundance in the apical
cytoplasm just beneath the luminal surface. They were found in association with intermediate junctions (ij), which are associated with actin, but not desmosomes,
which are not associated with actin. In addition, gold particles are evident within the microvilli, i.e., the actin-rich core.
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
Although rab3D is the best marker of SVs so far identified
in the lacrimal gland, it is unlikely that this particular rab labels all lacrimal acinar SVs. SVs in lacrimal gland are clearly
a heterogeneous population, varying with age and gender in
size and in content (e.g., serous versus mucous) (da Costa
et al., 2006; Draper et al., 1998, 1999). Lacrimal acini are
also responsive to multiple secretagogues acting through cholinergic, adrenergic and growth factor pathways (Dartt, 2004).
Conceivably, different secretory products may be released in
response to different stimuli, to adjust the ocular surface fluid
to accommodate to the needs of the cornea. Consistent with
this, some studies have suggested that the release of secretory
products in response to different effectors varies in parotid acini (Castle et al., 2002; Huang et al., 2001). As well, recent
work in parotid acini has demonstrated a differential effect
of cytoskeletal-targeted agents on protein secretion evoked
by isoproterenol and carbachol, suggesting the existence of
at least two distinct SV populations (Nashida et al., 2004).
In contrast, Dartt and coworkers compared the spectrum of
secretory products released from rabbit lacrimal acini in
response to acetylcholine and VIP (Dartt et al., 1988), but
found no changes. A number of new effectors are now known
to evoke secretion in lacrimal acini including EGF; moreover,
the influence of diseases and corneal surgery on the spectrum
of tear proteins released into tears is well established [for
examples see (Glasson et al., 2003; Herber et al., 2001;
Yamada et al., 2000; Zhou et al., 2004)]. Given the advent
of new proteomics tools which may also have better resolution
for low-abundance tear proteins, this issue may deserve
re-examination in the lacrimal gland.
Possible additional candidates for secretory rab proteins include rab27a and rab27b, which are expressed in a broad range
of secretory cells (Chen et al., 2004; Imai et al., 2004b; Izumi
et al., 2003; Tolmachova et al., 2004; Yi et al., 2002). In particular, rab27b has been implicated in the stimulated release
of amylase from parotid gland (Imai et al., 2004b), and in
the exocytosis of zymogen granules in pancreas (Chen et al.,
2004). Our recent results based on Western blotting of lacrimal
gland lysates as well as immunofluorescence microscopy analysis of lacrimal acini with antibodies to rab27a and rab27b
show that both rab27 isoforms are expressed in lacrimal acini in association with large vesicles enriched adjacent to
the APM (unpublished data). By analogy with pancreas and
parotid gland, we propose that one or both of these rab27
isoforms may participate in aspects of lacrimal gland SV
maturation and/or exocytosis.
2.2. Targeting and specificity: SNAREs
SNAREs [soluble N-ethylmaleimide(NEM)-sensitive factor
(NSF) attachment protein (SNAP) receptors] are membraneassociated proteins essential for membrane fusion, a process
in which two lipid bilayers of previously separate intracellular
membrane compartments are physically merged to promote
content mixing or exocytosis (Sollner, 2003). Mammalian
SNAREs are comprised of three conserved families: synaptobrevin/vesicle-associated membrane proteins (VAMPs),
87
syntaxins, and SNAP-25 homologues. These three family
members were originally identified in neurons as key participants in synaptic vesicle exocytosis, and have been categorized as v-SNAREs (VAMPs) or t-SNAREs (syntaxins and
SNAP-25 homologues), depending on their vesicle or target
membrane localization, respectively. More recently, these proteins have been reclassified into R-SNAREs or Q-SNAREs to
reflect the presence of arginine or glutamine residues in their
core binding domains, respectively. All SNARE proteins share
at least one a-helical domain that contributes to the formation
of a highly stable coiled-coiled structure, which is believed to
pull the donor and acceptor membranes together in a complex
that may progress to form a fusion pore if primed by other
factors. Other factors including synaptotagmin and Ca2þ are
required for completion of the fusion pore. It is now known
that every cell expresses a wide variety of SNARE proteins
that are distributed to distinct intracellular membrane compartments (Chen and Scheller, 2001; Lin and Scheller, 2000). The
distinct distributions of SNARE proteins are believed to contribute to the specificity of membrane fusion (Scales et al.,
2000; Sollner, 2003).
Reconstituted rabbit lacrimal gland acini express several
SNARE proteins as determined by Western blotting and immunofluorescence microscopy. A population of apparent SVs
associated with the v-SNARE, VAMP2, is detected in the interior of resting (unstimulated) lacrimal acini. These vesicles
have been termed ‘‘recruitable’’ SVs, since they are dispersed
throughout the cytosol in resting acini and recruited to the
APM upon secretagogue stimulation (Wang et al., 2003). Interestingly, there is very little colocalization of rab3D and
VAMP2 on membrane vesicles in resting or stimulated lacrimal acini (Wang et al., 2003). The relationship between the
VAMP2-associated rSVs with rab3D-enriched SVs is still
not completely clear, but most experimental evidence suggests
that both populations are fusion competent. The cognate
t-SNARE for the VAMP2-enriched rSVs that confers the
ability to fuse at the APM is likely to be syntaxin 3, which
colocalizes with VAMP2 at the APM in stimulated rabbit
lacrimal acini (Sou et al., 2005). Syntaxin-3 and VAMP2 are
known to form an apical SNARE complex in another secretory
epithelial cell, the gastric parietal cell (Ammar et al., 2002).
The likely involvement of VAMP2 and syntaxin 3 in apical
exocytosis in lacrimal acini is consistent with other work in secretory epithelia. Acinar cells from pancreas and parotid gland
express VAMP2 on secretory granule membranes (Hansen
et al., 1999; Takuma et al., 2000). SNAP-23 has been shown
to be present on either or both the APM and basolateral membrane. Syntaxin 2 and/or 3 are mainly localized to the APM in
polarized epithelial cells, such as Caco-2, MDCK and acinar
cells (Delgrossi et al., 1997; Fujita et al., 1998; Hansen
et al., 1999; Low et al., 1998).
The finding that VAMP2 and rab3D appear to be associated
with discrete SVs in lacrimal acini (Wang et al., 2003) is in
contrast to the detection of these components in a complex
on the same zymogen granules in pancreatic acini (Jahn and
Sudhof, 1999). This finding illustrates an apparent major difference in the organization of the pancreatic acinar secretory
88
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
pathway relative to the lacrimal acinar secretory pathway. This
observation also reveals the major gaps in our knowledge of
the secretory targeting and fusion machinery in lacrimal acini. Vesicles targeted for fusion at a particular acceptor membrane must contain a unique rab for targeting and a unique
v-SNARE, which is able to recognize the acceptor membrane
t-SNARE, and to facilitate membrane fusion. If rab3D- and
VAMP2-enriched vesicles are distinct, the identification of at
least one t-SNARE/v-SNARE complex for the rab3D-enriched
vesicles and one unique rab protein for the VAMP2-enriched
rSV population remain to be established in lacrimal acini.
Likely v-SNARE candidates for participation in lacrimal
acinar exocytosis include VAMP8 and VAMP7. VAMP8, also
known as endobrevin, was originally identified on early and
late endosomes and shown to mediate homotypic fusion as a
v-SNARE (Antonin et al., 2000); recently, work in pancreatic
acinar cells has revealed that VAMP8 plays a role in regulated
exocytosis (Wang et al., 2004). In pancreatic acini, VAMP8 is
present on membranes of zymogen granules and forms a complex with syntaxin 4 and SNAP-23. Pancreatic acini isolated
from VAMP8 null mice have an excess number of zymogen
granules, while secretagogue-stimulated amylase secretion is
inhibited, indicating that VAMP8 plays a role in regulated exocytosis as a v-SNARE on zymogen granules (Wang et al.,
2004). VAMP8 is also localized on a subset of secretory granules in RBL-2H3 mast cells, again forming a complex with
syntaxin 4 and SNAP-23 and participating in regulated secretion (Paumet et al., 2000).
There is also evidence that the tetanus toxin-insensitive
VAMP (TI-VAMP, also known as VAMP7), is involved in
stimulated exocytosis. TI-VAMP, reported to be involved in
trafficking to late endosomes and lysosomes in cultured cells
(Advani et al., 1999; Martinez-Arca et al., 2003), interacts
with t-SNAREs at the plasma membrane, playing a role in
trafficking of influenza hemagglutinin to the APM of MDCK
cells (Galli et al., 1998) and in lysosomal exocytosis in
NRK cells (Rao et al., 2004). Lysosomal exocytosis is particularly interesting in the context of lacrimal acini because of
the large number of lysosomal enzymes found in tear fluid.
Expression of VAMP7 and VAMP8 has been confirmed in rabbit lacrimal acini by Western blotting (unpublished data).
Another aspect of SNARE complex assembly is its regulation to prevent spontaneous fusion events. One aspect of this
regulation is facilitated by Munc 18 proteins, which bind
tightly to syntaxins to block their interactions with cognate
SNAREs (Dulubova et al., 1999; Fisher et al., 2001). Three
isoforms of Munc18 have been identified to date (Munc18-1/
Munc18a, Munc18-2/Munc18b, Munc18-3/Munc18c). In rat
parotid acinar cells, Munc18-3/Munc18c interacts with syntaxin 4 at the APM and acts as a regulator of amylase secretion
(Imai et al., 2004a). Munc18c also interacts with syntaxin 4 at
the plasma membrane of 3T3-L1 adipocytes and is suggested
to be involved in the insulin-dependent trafficking of GLUT4
from intracellular storage compartments to the plasma membrane(Tamori et al., 1998). Western blotting and confocal fluorescence microscopy have revealed the presence of Munc 18b
and 18c in lacrimal acini (unpublished data) at the APM and in
association with SVs although additional investigations are
necessary to provide conclusive evidence for their role in lacrimal acinar secretory traffic.
2.3. Transport factors: microtubules and
microtubule-based motors
A major factor in determining cell polarity and establishing
distinct APM and basolateral membrane domains is the placement of cytoskeletal filaments. Three major filament systems
comprise the eukaryotic cytoskeleton: microtubules (MTs), intermediate filaments, and actin filaments. Of these polymers,
both MTs and actin filaments play active roles in exocytosis;
their organization is shown schematically in Fig. 2. MTs,
formed from individual ab tubulin dimers which assemble
head-to-tail first into protofilaments and then into the intact
MT, are the largest of these filaments with a diameter of
25 nm. The intrinsic polarity of the MT, conferred by the
head to tail orientation of ab tubulin subunits in the dimer,
provides the MT with biochemically distinct ends referred to
as ‘‘plus’’ and ‘‘minus’’ ends which exhibit different dynamics
in vitro and in vivo. In vivo, the MT minus-ends are usually
organized and stabilized at the MT-organizing center, which
consists of a centriole pair, while the plus-ends extend away
from the organizing center into the cytoplasm. The placement
of MT minus- and plus-ends is a defining feature in establishing cell polarity in all cells (Musch, 2004), and in facilitating
distinct sorting events to each domain.
In epithelial cells, MT-organizing centers are known to be
positioned beneath the APM (Meads and Schroer, 1995).
This orientation is clearly seen for lacrimal acinar epithelial
cells in lacrimal glands by EM (Fig. 1C). This orientation
Fig. 2. Cytoskeletal organization in lacrimal acini. This schematic model depicts the organization of microtubules (MT, green) and the orientation of their
plus and minus ends, actin filaments (red) and the junctional complexes which
separate the apical plasma membrane (APM) from the basolateral membrane
(BLM) in lacrimal acini.
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
has also been independently verified using confocal fluorescence microscopy to demonstrate the apical concentration of
g-tubulin, a major constituent of the MT-organizing center associated with MT minus-ends (da Costa et al., 1998). These
findings together confirm that MT minus-ends are organized
beneath the APM of lacrimal acini, while their plus-ends extend to the basolateral membrane. The integrity of the apical
and basolateral domains is further maintained by the presence
of junctional complexes localized in the lateral regions just
beneath the apical membranes (Fig. 1B).
Several studies have investigated the role of MTs in stimulated secretory traffic in the lacrimal gland, using specific
inhibitors of the MT array. Consistently, drugs that modulate
the MT array have the ability to blunt stimulated secretory
responses in the lacrimal gland (Busson-Mabillot et al.,
1982; da Costa et al., 1998; Herman et al., 1989; Robin
et al., 1995). Pulse-chase and other biochemical studies which
investigate the specific steps influenced by modulation of the
MT array have suggested that this array plays an important
role in production and/or maturation of SVs, but does not
directly influence their discharge (Busson-Mabillot et al., 1982;
Robin et al., 1995). A similar role for MTs in stimulated protein secretion has been demonstrated in pancreas (Nevalainen,
1975; Pavelka and Ellinger, 1983) although their participation
has not been confirmed systematically in parotid gland (Robin
et al., 1995). This may be due to the presence of multiple SV
types in parotid acini, only some of which are sensitive to MT
inhibitors (Huang et al., 2001).
The orientation of MT minus- and plus-ends provides the
directional cue for the polarized transport of membrane vesicles by MT-based motor proteins. Two different classes of
MT-based motor proteins have been identified: cytoplasmic
dyneins and kinesins. Cytoplasmic dynein was first identified
as the motor protein responsible for MT- based retrograde
transport in axons and is the major MT minus-end directed
motor in interphase cells. Subsequently, its function in many
different steps in membrane trafficking has been reported [reviewed in (Vallee et al., 2004)]. Cytoplasmic dynein consists
of two w440 kDa heavy chains containing the sites for MT
binding and ATP binding and hydrolysis, as well as multiple
intermediate chains (w70 kDa), light intermediate chains
(w50 kDa) and light chains (w8kDa). For appropriate attachment to membrane cargo, cytoplasmic dynein must interact
with the w1500 kDa, multi-protein effector complex called
the dynactin complex (Schroer, 2004). The dynactin complex
has two major structural motifs, a core filament which interacts with the membrane cargo and a protruding side arm which
interacts with cytoplasmic dynein and the MT. Major protein
constituents of this complex include actin-related protein 1
(Arp1) which assembles to form the core filament in conjunction with conventional actin and actin capping protein; the
p150Glued protein, the major component of the side arm which
facilitates tethering of the complex to MTs and also interacts
with the dynein intermediate chains; and dynamitin which
links the Arp1 core filament and the p150Glued side arm. Heterogeneity of the many subunits within the cytoplasmic dynein
motor complex has been reported, leading to the possibility
89
that different combinations of subunit isoforms provides the
ability to interact with diverse cargo.
Secretory traffic moves toward the APM and thus, towards
MT minus-ends. It therefore came as no surprise that the minus-end directed motor protein, cytoplasmic dynein, seems to
play a major role in this traffic. Analysis of the distribution of
cytoplasmic dynein and dynactin complex subunits in reconstituted rabbit lacrimal acini by confocal fluorescence microscopy revealed that they were localized in a dispersed pattern in
resting acini but exhibited a remarkable recruitment to a region
immediately beneath the APM in response to carbachol (Wang
et al., 2003). Further analysis of their association with subcellular membranes by confocal fluorescence microscopy and
subcellular fractionation revealed substantial colocalization
with VAMP2-enriched rSVs in stimulated acini. As well,
traces of colocalization were also seen with the rab3Denriched SVs.
Overexpression of the dynamitin subunit linking the core
filament and side arm of the dynactin complex is known to inhibit intracellular cytoplasmic dynein activity (Burkhardt et al.,
1997; Echeverri et al., 1996), likely through uncoupling of the
two structural motifs (filament and side arm) of the dynactin
complex and impairing dynein’s ability to attach to membranes. Dynamitin overexpression was utilized as a tool in lacrimal acini to probe the role of cytoplasmic dynein in SV
exocytosis in lacrimal acini (Wang et al., 2003). Even in resting
acini, dynamitin overexpression abolished the rab3D-enriched
SV population, suggesting that either their formation or maintenance requires cytoplasmic dynein activity. Moreover, dynamitin overexpression inhibited the carbachol-stimulated
recruitment of VAMP2-enriched rSVs into the subapical cytoplasm, showing that cytoplasmic dynein was actively involved
in the stimulated transport of rSV to the APM. Finally, dynamitin overexpression significantly inhibited carbachol-stimulated
release of protein and b-hexosaminidase, showing functionally
that cytoplasmic dynein activity in the maintenance and movement of these two vesicle populations, rSV and SV, was an essential component of exocytosis at the APM. Consistent with
this work, other studies in pancreas (Kraemer et al., 1999)
and parotid gland (Nashida et al., 2004), have shown association of cytoplasmic dynein with SVs.
The other MT-based motor family consists of the kinesins,
which share the conserved head domain but have a variable
number of tails, presumably for association with diverse cargo
(reviewed in Hirokawa and Takemura, 2004). Most of the cytoplasmic kinesins within this superfamily are MT plus-end
directed motors and, in lacrimal acini, would participate in
trafficking from the APM towards the cell interior and the basolateral membrane. Although several kinesins function in membrane trafficking in lacrimal acini including conventional
kinesin (Hamm-Alvarez et al., 1998), they have not been implicated to date in the production, maturation and release of SVs.
2.4. Transport factors: actin filaments and myosins
The smallest in diameter of any of the cytoskeletal
filaments (7e9 nm), actin filaments are formed from actin
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K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
monomers that associate in a head to tail fashion into protofilaments; two parallel protofilaments then twist around each
other to form an actin filament. Actin filaments are extensively
regulated by a number of proteins which modulate their assembly status and extent of crosslinking or bundling (Revenu
et al., 2004). 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 endocytosis and exocytosis (Eitzen, 2003; Engqvist-Goldstein and
Drubin, 2003; Qualmann and Kessels, 2002). In lacrimal acini,
actin filaments are organized into a cortical network beneath
the plasma membranes, which is particularly dense beneath
the APM (Fig. 1D,E). Actin filaments are also incorporated
into microvilli, which are detected at the APM (da Costa
et al., 2003) (Fig. 1A,B).
The role of actin filaments in stimulated exocytosis in acinar epithelial cells has been controversial for many years. In
pancreas and parotid acini, it has long been known that the apical actin network undergoes reorganization following acute
stimulation (Muallem et al., 1995; O’Konski and Pandol,
1990; Perrin et al., 1992). These and other findings led to
the development of the ‘‘barrier’’ hypothesis which postulated
that the dense apical actin filament layer beneath the APM
normally prevented access of SVs to the APM, and that a critical event in exocytosis was the disassembly of this actin barrier. Other work has, however, shown that total disassembly of
actin filaments is detrimental to exocytosis, and that some actin filaments are essential for exocytosis to proceed (Muallem
et al., 1995), possibly to participate in force generation.
Our initial attempts to assess actin filament participation in
SV exocytosis in reconstituted rabbit lacrimal acini utilized the
actin-targeted agents, cytochalasin D and jasplakinolide, but
failed to detect any significant effects of these agents on basal
or carbachol-stimulated protein or b-hexosaminidase secretion
(da Costa et al., 1998). In these studies it was unclear to what
extent the apical actin array in lacrimal acini was actually perturbed by these agents. In secretory epithelial cells in general,
many such studies have been complicated by the refractory nature of the dense apical actin array to actin-targeted agents that
normally elicit rapid actin disassembly in other cells (Ammar
et al., 2001). Subsequent re-evaluation of the role of actin
filaments in exocytosis, using high resolution confocal microscopy analysis of actin filaments in acutely-stimulated
reconstituted rabbit lacrimal acini revealed immediate effects
of carbachol on the apical actin array that were characterized
by an apparent thinning in conjunction with the formation of
novel actin-coated structures beneath the APM region (Jerdeva
et al., 2005a).
Expression of a GFP-actin fusion protein in reconstituted rabbit
lacrimal acini revealed much more information about ‘‘barrier’’ actin remodeling during apical exocytosis. Fluorescence recovery after photobleaching analysis of the GFP-actin-labeled apical array
revealed that carbachol stimulation elicited a significant increase
in the mobile fraction and a significant decrease in the actin filament turnover time (Jerdeva et al., 2005a). These changes were detected as early as 1e4 min after addition of secretagogue and
persisted for up to about 15 min, confirming unequivocally that
the apical actin array exhibits enhanced turnover when acini are actively secreting at the APM. Time-lapse confocal fluorescence microscopy of filaments containing GFP-actin also enabled a detailed
analysis of the novel actin-coated structures that formed within the
cytosol of acutely stimulated acini. These structures formed adjacent to the APM and were then retracted rapidly and steadily toward the APM until the underlying actin coat could no longer be
distinguished from the dense apical actin filament network.
Myosin motors are actin-based motors that participate in
many different aspects of motility including muscle contraction, cytokinesis, phagocytosis, and organelle trafficking (reviewed in De La Cruz and Ostap, 2004). Analogous to the
MT-based motors which move along MTs, myosin motors
move cargo (membranes or other filaments) relative to actin
filaments, also by utilizing the energy of ATP hydrolysis.
Like the kinesins, myosin motors are grouped within a large
superfamily, which includes both conventional (filament forming) and unconventional (non-filament forming) myosins. Currently, 18 subfamilies of myosin motors are known (Berg
et al., 2001). The apparent retraction of actin-coated structures
towards the apical actin array seen in stimulated lacrimal acini
suggested a role for a myosin motor in actin filament sliding;
such a role would typically be fulfilled by a filament-forming
myosin that could generate contractile force. In non-muscle
cells, this function would most likely be fulfilled by nonmuscle myosin II isoforms.
Treatment of lacrimal acini with selective inhibitors of nonmuscle myosin II revealed a stabilization of actin-coated structures in carbachol-stimulated acini, an effect accompanied by
a functional inhibition of exocytosis (Jerdeva et al., 2005a).
Confocal fluorescence microscopy analysis revealed non-muscle myosin II immunofluorescence enriched on the stabilized
actin-coated structures in the inhibitor-treated, carbachol-stimulated acini. Further verification that these actin-coated structures constituted fusion intermediates was provided by the
trapping of the exogenous fluorescent secretory protein, syncollin-GFP, in actin-coated structures stabilized by non-muscle
myosin II inhibitors in stimulated acini expressing syncollin-GFP.
Finally, measurement of the diameters of SVs and actin-coated
structures suggested that these actin-coated structures enveloped multiple SVs, and that actin filament contraction was
required for the compound fusion of these individual SVs prior
to the discharge of their contents at the APM (Jerdeva et al.,
2005a).
These studies support a dual role for actin filaments in exocytosis, one role which involves force generation through interaction with non-muscle myosin II and one role which is
independent of this motor. To achieve exocytosis of SVs, actin
filament contraction driven by non-muscle myosin II occurs
around clusters of individual SVs, promoting their subsequent
compound fusion, compression and retraction toward the
APM. At the same time, accelerated turnover of the apical actin filament network also occurs to increase the accessibility of
actin-coated fusion intermediates to the APM. These findings
in lacrimal acini explain the historically contradictory findings
that actin filament disassembly (O’Konski and Pandol, 1990;
Perrin et al., 1992) and actin filament assembly (Muallem
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
et al., 1995) are both required to facilitate discharge of SVs in
acinar epithelial cells.
2.5. Regulation of the exocytotic machinery
As recently reviewed, innervation of the lacrimal gland by
sympathetic and parasympathetic nervous systems provides
neurotransmitters which interact with basolateral receptors
through muscarinic cholinergic-, a1-adrenergic-, b-adrenergic
and VIPergic signaling mechanisms to stimulate release of secretory proteins contained in apical SVs (Dartt, 2001). Perhaps
the best characterized signaling pathway in lacrimal acini is
that triggered by cholinergic stimulation. Cholinergic agents
are linked via Gaq/11 to activation of phospholipase Cb (Meneray et al., 1997), generating two second messengers, diacylglycerol (DAG), and Ca2þ. Cholinergic agents also activate
phospholipase D (Zoukhri and Dartt, 1995). The second messenger, DAG, activates members of the protein kinase C
(PKC) family in lacrimal acini. This family has at least 11 isoforms divided into three groups: conventional (-a, -bI, -bII, -g),
novel (-d, -3, -h, -q, -m) and atypical (-l/i and -z) (Dekker and
Parker, 1994; Dekker et al., 1995). Lacrimal acini express at
least 5 PKC isoforms, including PKCa, -d, -3, -m, and l/i
(Zoukhri et al., 1997). Here we review our current understanding of the regulation by signaling proteins of specific processes
involved in apical exocytosis. Much of this discussion is speculative as it pertains to the lacrimal gland, but well supported by
studies in other systems that engage in stimulated exocytosis.
A key effector in the remodeling of apical actin filaments
associated with exocytosis is the novel protein kinase C isoform, PKC3. This particular PKC isoform is unique in that it
contains an actin-binding domain (Prekeris et al., 1998).
This isoform, along with PKCa, is responsible for the increase
in protein secretion evoked by stimulation with muscarinic agonists in rat acinar cell models (Zoukhri et al., 1997). Evaluation of PKC3 distribution in reconstituted rabbit lacrimal acini
revealed a pronounced accumulation with apical actin in acini
acutely stimulated with carbachol (Jerdeva et al., 2005b).
Moreover, inhibition of PKC3 activity in rabbit lacrimal acini
using either chemical inhibitors or by overexpression of a dominant negative mutant of the enzyme resulted in stabilization
of apical actin filaments, accumulation of actin-coated structures and inhibition of stimulated secretion (Jerdeva et al.,
2005b). These findings suggest that under normal conditions,
the secretagogue-stimulated recruitment of PKC3 to actin
may trigger key changes in phosphorylation of effectors involved in apical actin organization. Although not demonstrated specifically in lacrimal acini, data from other systems
that utilize stimulated exocytosis including parotid acini
(Imai et al., 2004a) and neurons (Barclay et al., 2003) suggest
a role for PKC isoforms in the regulated release of Munc 18
isoforms from plasma membrane syntaxins as a key factor
in formation of SNARE complexes in systems engaging in
stimulated exocytosis.
As outlined above, a major intracellular second messenger
released in response to cholinergic stimulation is Ca2þ. A vesicle-associated protein called synaptotagmin plays a key role
91
in sensing of elevations in intracellular Ca2þ in many secretory
cells that have been studied. Once intracellular Ca2þ levels
have reached sufficient levels, primed synaptotagmin is able
to aid in the completion of fusion pore formation between
t-SNAREs, v-SNARES and SNAPs (Sollner, 2003; Stojilkovic,
2005). Although not yet investigated in lacrimal acinar epithelial cells, it is likely that a comparable role for Ca2þ in priming
of fusion pore formation through synaptotagmin or a related
protein will be indicated. Studies in mast cells, which also
maintain rab3D-enriched SVs, have demonstrated the presence
of a specific rab3D-associated kinase, termed Rak3D which
is activated in response to elevations in intracellular Ca2þ
(Pombo et al., 2001). This kinase has the ability to phosphorylate syntaxin 4, thus inhibiting its ability to bind to SNAP-23.
A complex model has been proposed for the concerted function of this kinase in conjunction with a protein phosphatase
to couple increased intracellular Ca2þ to stimulated exocytosis
in mast cells, which, unlike lacrimal acini, maintain a predocked vesicle population (Blank et al., 2002). The role of
Rak3D has not yet been investigated in secretory epithelial
cells from any exocrine glands, although the prominent association of rab3D with acinar SVs suggests that regulation of
this interaction is likely to be important.
Phospholipase C activation has been well documented
in response to cholinergic stimulation (Meneray et al., 1997).
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a precursor
for second messengers generated by phospholipase C isoforms
(Oude Weernink et al., 2004). Very recent work has suggested
that PIP2-enriched microdomains in the plasma membrane aid
in clustering syntaxins and large dense core vesicles at sites of
stimulated exocytosis in PC12 cells (Aoyagi et al., 2005).
While again unexplored in acinar epithelial cells from exocrine glands, it is intriguing to speculate not only a role for
PIP2 microdomains in organizing fusion sites, but a role for
spatially segregated PIP2 hydrolysis by activated phospholipase C in the activation/unmasking of prospective fusion sites.
Little is known about the convergence of b-adrenergic or
VIPergic pathways on the exocytotic trafficking machinery.
Both of these pathways work through elevation of intracellular
cAMP and activation of cAMP-dependent signaling pathways
(Dartt, 2001). One recent study in parotid acinar epithelial
cells has demonstrated that protein kinase A is responsible
for promoting dissociation of an inhibitory factor from
VAMP2, associated with SVs, in parotid gland (Fujita-Yoshigaki et al., 1999). With the identification of specific lacrimal
acinar exocytotic effectors reviewed here, the next few years
promise to provide many advances in the understanding of
their regulation by intracellular signaling pathways.
2.6. Working model
Fig. 3 shows the working model for stimulated exocytosis
in lacrimal gland. Two populations of SVs have been confirmed: apically located, rab3D-enriched SVs which are larger
in diameter (also termed mature SVs or SVs); and VAMP2enriched rSVs which are located in the cell interior and recruited to the APM following stimulation. Cytoplasmic dynein
92
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
participates in the maturation and maintenance of the rab3Denriched SV population in resting acini. Secretagogue stimulation results in priming of rab3D-enriched SVs (an event that
includes loss of rab3D) and concomitant assembly of actin
and non-muscle myosin II filaments in a basketlike network
around clusters of adjacent primed vesicles. The contractile
force generated by interaction of actin filaments with nonmuscle myosin II aids in cytoplasmic fusion of the aggregated
SVs, and also promotes directed migration of the cluster toward the APM. Increased turnover in the apical actin network
results in increased accessibility of APM sites for the cluster
of fused SVs, resulting in exocytosis. Aspects of actin remodeling are regulated by activation and recruitment of PKC3 to
the apical actin network. At this point, the cognate SNAREs
involved in fusion of rab3D-enriched SVs are not yet determined. Also in stimulated acini, rSVs are recruited to the
APM along MT tracks driven by cytoplasmic dynein. The
v-SNARE, VAMP2, interacts with the t-SNARE, syntaxin-3, to
facilitate fusion of this smaller SV population and release of
its contents at the APM.
2.7. Changes in exocytotic trafficking in the NOD mouse,
a Sjögren’s syndrome model
Fig. 3. Working model for organization and function of well-characterized
effectors of the lacrimal acinar secretory pathway. (A) Resting lacrimal acini
maintain mature SVs (mSVs) enriched in Rab3D beneath the dense apical
actin filament network. These mSVs utilize cytoplasmic dynein for their maturation and to maintain their subapical localization. Other recruitable SVs (rSVs)
enriched in VAMP2 are present in the cytosol. (B) Upon secretagogue stimulation, there is an immediate thinning of apical actin filaments concomitant
with the loss of Rab3D from primed individual SVs in clusters accompanying
assembly of actin and non-muscle myosin II filaments around these clusters.
Contractile force generated by the acto-myosin system results in compound
fusion of individual SVs and movement of these fusion intermediates toward
the APM. Ultimately, they reach the APM where they are able to gain access
to appropriate SNARE proteins and complete the fusion step. At the same
time, rSVs in the cytosol move along MTs with the aid of cytoplasmic dynein
to the APM, where they fuse and release their contents through formation of
a SNARE pair with the rSV VAMP2 and the APM syntaxin 3.
An animal disease model is of great help in understanding
the mechanisms underlying normal physiological function and
in explaining how changes in these processes contribute to the
development of disease. Key hallmarks of SjS are a loss of lacrimal and salivary gland functions associated with development of autoantibodies and lymphocytic infiltration of these
glands. The male NOD mouse recapitulates these features (reviewed in Barabino and Dana, 2004). Recently, the changes in
morphology of SVs and in the distribution of key effectors of
exocytosis was investigated in male NOD mice at early times
prior to reported lymphocytic infiltration and at later times
after infiltration is underway (da Costa et al., 2006). Interestingly, the formation of abnormal SV aggregates was detected
by 4 weeks of age, prior to reported lymphocytic infiltration.
At this time, profound changes in the distribution of the M3
muscarinic receptor and apical and basolateral actin filaments
were also noted. This study suggests, in this particular disease
model for SjS, that some changes in membrane trafficking
may proceed independently of inflammatory responses or
may even contribute to such responses. Previous work in
NOD/scid mice, which lack the capacity to mount an autoimmune response, has shown that parotid acini exhibit fundamental changes in the spectrum of secretory products
released (Robinson et al., 1996). These studies therefore raise
the possibility that NOD mice exhibit primary changes in the
organization and function of the acinar secretory pathway that
can occur independently of inflammatory responses and that
may even trigger subsequent autoimmune responses.
One potential effector directly implicated in the trafficking
changes associated with development of SjS is a protein called
the islet cell antigen 1 (69 kDa) or ICA69. ICA69 is a common
autoantigen initially identified by screening a human islet
cDNA expression library with islet cell antibody positive
sera from relatives from patients with insulin-dependent diabetes (Pietropaolo et al., 1993). Researchers interested in exploring the role of this protein in development of diabetes in the
NOD mouse found, when they made an ICA69 knockout
mouse in an NOD background, that this knockout prevented
lacrimal gland disease and reduced salivary gland disease
(Winer et al., 2002). Although little is known thus far about
the cellular function of ICA69, it appears to be involved in
membrane trafficking. In islet cells, it is associated with the
Golgi complex and, to a lesser extent, with immature
K. Wu et al. / Experimental Eye Research 83 (2006) 84e96
insulin-containing secretory granules (Spitzenberger et al.,
2003). Involvement of ICA69 in neuronal secretion has been
suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene, ric19, in Caenorhabditis elegans (Pilon et al., 2000). Its location
is also consistent with a role for formation and/or maturation
of SVs in diverse systems (Pilon et al., 2000; Spitzenberger
et al., 2003). Characterization of its precise role in lacrimal
acinar SV biogenesis promises to shed new insights into the
development of SjS.
3. Conclusion
This review has provided an overview of the different types
of trafficking proteins that have been characterized as participants in SV exocytosis at the APM in lacrimal acinar epithelial
cells. It has also integrated their activity into a working model
for apical exocytosis in the lacrimal gland. While considerable
progress has been made, a number of effector proteins clearly
remain to be identified, while additional work will then be
necessary to clarify their role in these events and to expand
and complete the working model. The utility of completing
these subsequent characterizations in order to more precisely
define the exocytotic pathways is supported by new animal
studies suggesting a fundamental role for changes in secretory
pathways in the initiation and development of SjS.
Acknowledgements
This work was supported by NIH grants EY-11386 and EY13949 to SHA and EY-10550 to J.S. Additional salary support
to S.H.A. was from NIH grants EY-05081, NS-38246,
DK-56040 and GM-59297.
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