1 Department of Pharmaceutical Sciences, University of Southern California, 1985
Zonal Avenue, Los Angeles, CA 90033, USA
2 Department of Physiology and Biophysics, University of Southern California,
1985 Zonal Avenue, Los Angeles, CA 90033, USA
3 Department of Ophthalmology, University of Southern California, 1985 Zonal
Avenue, Los Angeles, CA 90033, USA
4 Department of Pathology, University of Southern California, 1985 Zonal Avenue,
Los Angeles, CA 90033, USA
5 Institute for Genetic Medicine, University of Southern California, 1985 Zonal
Avenue, Los Angeles, CA 90033, USA
* Author for correspondence (e-mail: shalvar{at}hsc.usc.edu)
Accepted 28 January 2003
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Summary |
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Key words: Microtubule, Exocytosis, rab3D, VAMP2, Dynein, Acinar secretion
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Introduction |
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Previous work has established that treatment with microtubule (MT)-targeted
drugs reduces stimulated lacrimal acinar secretion
(Robin et al., 1995;
da Costa et al., 1998
).
However, these studies did not identify the MT-based motor protein responsible
nor define the step(s) involved. Two families of MT-based motor proteins are
known, the kinesins and the cytoplasmic dyneins
(Hirokawa, 1998
). Lacrimal
acinar MTs are organized with their minus-ends beneath the apical membrane
(da Costa et al., 1998
),
suggesting that MT-dependent traffic into this region would utilize a
minus-end directed motor-like cytoplasmic dynein.
Conventional cytoplasmic dynein is a large multisubunit complex of
1400 kDa consisting of two heavy chains (500-530 kDa) and several
intermediate (DIC, 74 kDa) light/intermediate (51-61 kDa) and light (8, 14, 22
kDa) chains (Holzbaur and Vallee,
1994
). Dynein heavy chain contains sites for MT binding and ATP
hydrolysis, and is responsible for generation of mechanochemical force. The
remaining dynein subunits may specify cargo interactions and/or regulate heavy
chain function. Directed transport of vesicles by conventional cytoplasmic
dynein requires a multiprotein complex called the dynactin complex
(Gill et al., 1991
). This
complex functions as an adapter, mediating dynein binding to different cargo
structures (Allan, 1996
;
Schroer, 1996
;
Holleran et al., 1998
). The
dynactin complex exhibits two major structural features including a 37 nm
actin-like filament largely comprised of the actin-related protein, Arp1,
attached to a 24 nm sidearm largely comprised of a dimer of
p150Glued (Schafer et al.,
1994
). The p150Glued sidearm interacts with DIC
(Vaughan and Vallee, 1995
) and
MTs (Waterman-Storer et al.,
1995
). p50/dynamitin, a dynactin complex protein, may mediate the
interaction of p150Glued and the Arp1 filament
(Echiverri et al., 1996
). When
overexpressed in mammalian cells, it is thought to prevent complex formation
between the p150Glued sidearm and the membrane-associated Arp1
filament (Echiverri et al.,
1996
; Burkhardt et al.,
1997
). We refer to conventional cytoplasmic dynein and
p50/dynamitin as dynein and dynamitin.
Here we focus on CCH-induced changes in dynein and dynactin complex
association with lacrimal acinar membranes enriched in one of two secretory
vesicle markers, vesicle-associated membrane protein 2 (VAMP2) or rab3D.
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
). Proteins of the rab3 subfamily of small GTP-binding
proteins are likewise implicated in exocytosis
(Fischer von Mollard et al.,
1994
), with rab3D constituting the principal isoform associated
with secretory vesicles in pancreas, parotid gland and lacrimal gland
(Ohnishi et al., 1996
;
Valentijn et al., 1996
).
In rabbit lacrimal acini, we show by confocal fluorescence microscopy that CCH promotes accumulation of dynein and the dynactin complex in the subapical region. Colocalization studies and biochemical analysis of isolated subcellular membranes suggest that at least one vesicle population transported towards the apical membrane by dynein is enriched in VAMP2. Adenovirus-mediated overexpression of dynamitin prevents the recruitment and colocalization of dynein, the dynactin complex and VAMP2 beneath the apical membrane. Nocodazole treatment or dynamitin overexpression also depletes subapical stores of rab3D in resting acini, suggesting that dynein may also maintain this mature secretory vesicle population at the apical membrane.
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Materials and Methods |
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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
) and cultured for 2-3 days. Cells prepared in this way
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
).
Confocal fluorescence microscopy
For analysis of DIC, p150Glued or Arp1 distribution with VAMP2,
rab3D or actin filaments, acini cultured on Matrigel-coated coverslips were
rinsed with Dulbecco's PBS (DPBS), fixed and permeabilized with ethanol at
20°C for 10 minutes, rehydrated in DPBS
(Mendell and Whitaker, 1978)
and blocked with 1% bovine serum albumin. Acini were then incubated with
appropriate primary and fluorophore-conjugated secondary antibodies and/or
rhodamine phalloidin. For dynamitin detection, acini were fixed in 4%
paraformaldehyde, permeabilized in 0.1% Triton X-100 (Tx-100) and processed as
above. Samples were imaged using a Nikon PCM Confocal System equipped with
Argon ion and green HeNe lasers attached to a Nikon TE300 Quantum inverted
microscope. Images were compiled in Adobe Photoshop 7.0 (Adobe Systems,
Mountain View, CA).
Quantitation of western blots
For quantitation of proteins on western blots, the majority of blots were
processed using secondary antibodies conjugated to IRDyeTM800 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. Some experiments included in summary analyses
used secondary antibodies conjugated to horseradish peroxidase for ECL
detection and quantitation by densitometry.
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 MgSO4, 10 mM EGTA, 2 mM DTT supplemented with
protease inhibitor cocktail) containing 4% polyethylene glycol, 10 µM taxol
and 0.02% saponin for 12 minutes at 37°C. Extraction buffer supplemented
with 4% polyethylene glycol, 10 µM 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.
Subcellular fractionation analysis
Resting and CCH-stimulated acini were resuspended, lysed and analyzed by
differential sedimentation and isopycnic centrifugation on hyperbolic sorbitol
gradients as described (Hamm-Alvarez et
al., 1997; Yang et al.,
1999
; Qian et al.,
2002
). Sedimented membrane fractions were resuspended in sorbitol
cell lysis buffer and snap frozen in liquid nitrogen before storage at
80°C. Phase partitioning analyses of membranes from selected,
pooled density gradient fractions were performed as described
(Mircheff, 1989
), concentrated
by differential centrifugation and resuspended and frozen as described above.
Proteins of interest were analyzed and quantified either on western blots or
using activity measurements (ß-hexosaminidase, alkaline phosphatase) as
previously described (Mircheff,
1989
). Density gradient distributions are expressed as the
percentage of the total recovered in the 13 fractions, whereas phase
partitioning distributions are expressed as cumulative percent recovery
(Yang et al., 1999
). CCH
stimulation had no significant effect on the total amounts of each marker
recovered in the membranes. Differences between the amount of each of the
proteins in corresponding fractions from parallel CCH-stimulated and control
gradients were evaluated with Student's t-test with
P
0.05.
MT-affinity isolation of membranes
Isolation of membranes by MT-affinity was as previously described
(Goltz et al., 1992). Briefly,
supernatant containing microsomal membranes from resting and CCH-stimulated
(100 µM, 15 minutes) acini were incubated with taxol-polymerized porcine
brain MTs. Bound membranes were cosedimented with the MT pellet under
conditions that pelleted MTs but not microsomal membranes, and attached
membranes were released with excess Mg-ATP. DIC and VAMP2 contents in
ATP-releasable membrane fractions from resting and stimulated acini were
assessed by western blotting of equivalent amounts of total protein.
Generation of adenoviral vectors
Recombinant adenoviral (Ad) vectors were constructed using the AdEasy
Vector System (Quantum Biotechnologies). Human dynamitin cDNA (provided by
Janis Burkhardt, University of Chicago, with permission from Richard Vallee,
University of Massachusetts) was cloned into the shuttle plasmid, pAdTrack,
containing a CMV-driven green fluorescent protein (GFP) marker gene and arms
of homology to the left and right ends of the Ad5 genome flanking a plasmid
backbone containing the kanamycin resistance gene. Shuttle plasmids were
linearized and co-electroporated into the recombinogenic E. coli
BJ5183 strain with the 30 kb supercoiled plasmid, pAdEasy, containing the Ad
genome in an ampicillin-resistant plasmid. Transformants were selected on
kanamycin plates, mini-prep DNA from resistant colonies was screened by
restriction digest, and clones showing the correct restriction pattern were
re-transformed into E. coli DH10 to prevent recombination. Virus
stocks were produced by transfection of recombinant Ad genomes into 293 cells,
amplification and purification of harvested virus by cesium chloride
ultracentrifugation. Viral titers were determined using the tissue culture
infectious dose50 assay on 293 cells. For these studies, 2-day
acinar cultures were transduced at a MOI of 5 PFU/ml for 4 hours, followed by
rinsing and recovery for 16-18 hours at 37°C.
Secretion assays
Transduced acini seeded in Matrigel-coated 24-well plates were incubated in
fresh medium before removal of an aliquot for measurement of protein content
or ß-hexosaminidase activity. After treatment with or without CCH (100
µM, 30 minutes), a second aliquot of medium was removed for measurement of
these values. In each assay, protein and ß-hexosaminidase release were
calculated from 5-6 replicate wells/treatment and normalized to total cellular
protein. Differences between post- and pre-incubation values from unstimulated
acini represent basal release. Differences between post- and pre-incubation
values from stimulated acini represent total release (basal plus stimulated).
The stimulated value was calculated by subtracting basal release from total
release. Differences in experimental groups were determined using a paired
t-test with P0.05. Protein was measured with the
Micro-BCA Protein Assay (Pierce) using bovine serum albumin as standard, and
ß-hexosaminidase activity was assessed using
methyumbelliferyl-ß-D-glucosaminide as substrate.
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Results |
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Fig. 2 shows the distribution of p150Glued and Arp1 in resting and CCH-stimulated acini. Like dynein, p150Glued (Fig. 2A) and Arp1 (Fig. 2C) were detected in a diffuse pattern in resting acini. CCH stimulation promoted the same remarkable recruitment of p150Glued (Fig. 2B) and Arp1 (Fig. 2D) into the subapical region detected for dynein. Analysis of the dynamitin distribution also revealed comparable diffuse and subapical (data not shown) accumulation patterns in resting and CCH-stimulated acini, respectively.
Our previous investigations defined conditions for nocodazole exposure that
promoted disassembly of lacrimal acinar MTs
(da Costa et al., 1998).
Nocodazole comparably altered the resting distributions of dynein
(Fig. 1G,I) and
p150Glued (data not shown), increasing punctate labeling
(arrowhead) while decreasing diffuse labeling. Nocodazole also reduced
CCH-induced accumulation of dynein (Fig.
1J,L) and p150Glued (data not shown) beneath the apical
membrane.
CCH significantly increases dynactin recovery in a protein fraction
enriched in cytoskeletal and membrane proteins
To determine whether the CCH-induced subapical accumulations of dynein and
the dynactin complex were associated with changes in their association with
subcellular fractions, we subjected resting and CCH-stimulated acini to
sequential detergent extraction into buffers containing saponin, Tx-100 and
SDS. Analysis of the abundance (actin, tubulin, polymeric immunoglobulin A
receptor or pIgAR, VAMP2, rab3D) or activity [lactate dehydrogenase (LDH)] of
several proteins was used to define the composition of each pool
(Table 1). The pool
distributions of these proteins were not markedly affected by CCH. The
presence of most of the actin and tubulin in the Tx-100 insoluble, SDS soluble
pool was consistent with the enrichment of cytoskeletal filaments in this
pool. Most cellular pIgAR (65%), rab3D (
75%) and considerable stores
of VAMP2 (
45%) were recovered in Tx-100 soluble pools, consistent with
enrichment of membrane proteins in this pool. However,
20% of total pIgAR
and rab3D as well as
35% of total VAMP2 were recovered in Tx-100
insoluble, SDS soluble fractions, representing proteins associated with Tx-100
insoluble membranes or cytoskeleton. Approximately 15-20% of pIgAR and VAMP2
stores were recovered in saponin soluble pools, possibly reflecting release of
some membranes with saponin. Lacrimal acinar LDH has previously been
identified in cytosol and within secretory membranes
(Thurig et al., 1984
).
Consistent with this, most LDH activity (
70%) was recovered in saponin
soluble fractions with approximately 25% recovered in Tx-100 soluble
fractions. We conclude that the proteins recovered in saponin soluble
fractions are primarily cytosolic proteins, those recovered in Tx-100 soluble
fractions are primarily membrane proteins, and those recovered in SDS soluble
fractions include a mixture of cytoskeletal and Tx-100 insoluble membrane
proteins.
|
Fig. 3 shows that
approximately 60% of total p150Glued and Arp1 are recovered in the
Tx-100 insoluble, SDS soluble pool from resting acini, with the remainder
split between saponin soluble and Tx-100 soluble pools. CCH stimulation
elicited a small but significant (P0.05) increase in the recovery
of both dynactin components with the Tx-100 insoluble, SDS soluble pool, with
a concomitant depletion in their stores from the other two pools. DIC
distribution across these pools was assessed; approximately 40% of cellular
DIC was recovered in the Tx-100 insoluble, SDS soluble pool from resting
acini, with the remainder distributed equally between the other two pools. No
significant change in DIC association across pools was caused by CCH
treatment. These findings suggest that CCH promotes redistribution of dynactin
to a Tx-100 insoluble, SDS soluble pool enriched in cytoskeleton and membrane
proteins.
|
CCH increases the colocalization of VAMP2 with dynein and dynactin in
the subapical region
The CCH-sensitive recruitment of dynein and dynactin into the subapical
region under conditions associated with apical exocytosis suggested that
dynein might facilitate movement of secretory membranes. The distribution of
two effectors of the secretory pathway, rab3D and VAMP2, were investigated in
parallel with dynein and the dynactin complex. Rab3D immunoreactivity was
concentrated primarily in the subapical region in resting acini
(Fig. 4K). This large subapical
pool was rapidly depleted by CCH stimulation, resulting in dispersal of rab3D
labeling throughout the cell with retention of traces of residual rab3D
beneath the apical membrane (Fig.
4K'). VAMP2 immunoreactivity was dispersed throughout the
cytoplasm of resting acini, with traces associated with the apical membrane
(Fig. 4B,E,H). CCH stimulation
increased VAMP2 immunoreactivity in the subapical region and at the apical
plasma membrane (Fig.
4B',E',H'). The changes in these proteins in response to CCH
suggested that rab3D-enriched membranes represent mature secretory vesicles
located beneath apical membrane; release of their stores constitutes the
initial response to secretagogue. VAMP2-enriched membranes may represent a
recruitable vesicle population transported into the subapical region to
sustain the secretory response.
|
Dual labeling of VAMP2 with dynein and dynactin complex constituents in resting acini revealed traces of colocalization of DIC, Arp1 and p150Glued (arrows, Fig. 4C,F,I). However, CCH stimulation resulted in substantial increases in colocalization of DIC (Fig. 4C'), Arp1 (Fig. 4F') and p150Glued (Fig. 4I') with the VAMP2 stores specifically in the subapical region (arrows). Dual labeling experiments with p150Glued or DIC (data not shown) and rab3D revealed traces of colocalization in the subapical region of resting acini (Fig. 4L, arrowheads), which were not increased by CCH (Fig. 4L').
Increased recovery of dynein and VAMP2 in membranes isolated by
MT-affinity from CCH-treated acini
To obtain additional evidence in support of dynein-driven transport of
VAMP2-enriched membranes into the subapical region, we used MT-affinity
membrane isolation. This technique isolates membranes containing active motors
based on their ability to cosediment with taxol-stabilized MTs and to be
released in the presence of excess Mg-ATP. As shown in
Fig. 5, western blotting of
equivalent amounts of protein obtained from microsomal membranes isolated by
MT-affinity, ATP release revealed a significant (P0.05) increase
in the recovery of dynein and VAMP2 in membranes from CCH-stimulated acini
relative to resting acini. Rab3D was undetectable by western blotting of these
samples (data not shown).
|
Association of dynein and dynactin with isolated membrane
compartments
Previous studies have used sorbitol density gradient centrifugation to
isolate subcellular membranes and to map the trafficking between them
(Gierow et al., 1996;
Hamm-Alvarez et al., 1997
;
Yang et al., 1999
;
Qian et al., 2002
). We
therefore applied this technique to membranes from resting and CCH-stimulated
acini to further understand the contributions of dynein to apically targeted
secretory traffic.
p150Glued and Arp1 immunoreactivities (Fig. 6A) associated with membranes from resting acini exhibited similar distributions, with major concentrations in fractions 7-10. DIC was present in the same fractions (Fig. 6A), but it was relatively more abundant in fractions 11, 12 and P than dynactin. ß-Hexosaminidase (Fig. 6A), which is both a lysosomal and a secretory protein in lacrimal acini, resembled p150Glued and Arp1 but compared to these markers, it was slightly more abundant in fractions 11-P. Total membrane protein (Fig. 6A) resembled ß-hexosaminidase.
|
In resting acini, rab3D exhibited a pronounced concentration in fraction 1
(Fig. 6B), probably reflecting
the presence of fragments derived from a specialized microdomain of mature
secretory vesicle membranes. In addition, rab3D was broadly distributed across
the gradient. The major component resembled p150Glued and Arp1,
but, compared to p150Glued and Arp1, rab3D was relatively more
abundant in fractions 4-6. Acid phosphatase
(Fig. 6B) was broadly
distributed, but, somewhat like rab3D, it was also abundant in fraction 1. The
resting distributions of VAMP2 and -adaptin
(Fig. 6B) resembled that of
rab3D in the higher density regions of the gradient. Unlike rab3D, they were
not particularly abundant in fraction 1, but, like rab3D, they were relatively
more abundant than p150Glued and Arp1 in fractions 5-6. Consistent
with its primary localization in the trans-Golgi network (TGN), a complex
structure organized into multiple functionally distinct microdomains, rab6
appeared associated with three compartments, centered in fractions 4-5, 7-8
and 10-12 (Fig. 6B).
|
CCH stimulation promoted the following parallel changes in DIC,
p150Glued and Arp1: relative depletions centered at fraction 8 and
concomitant increases in fraction 5 and fraction P. These changes were
statistically significant (P0.05) for at least one of the three
proteins, with the same trend reflected for the others (see change plots in
Fig. 6A). CCH stimulation was
also associated with several additional apparent redistributions:
ß-hexosaminidase from fractions 7-8 to P, protein from 7-10 to P, rab6
from 7-10 to 2-3, rab3D from 1 to 4-5, and acid phosphatase from 1 to 4-6. CCH
stimulation had no significant effects on VAMP2 and
-adaptin, but the
data suggest possible redistributions of these markers to pooled fractions
11-P.
These findings as well as our data from previous studies probing the
identity of membrane compartments in acini using this method
(Gierow et al., 1996;
Hamm-Alvarez et al., 1997
;
Yang et al., 1999
;
Qian et al., 2002
) suggested
that in resting acini dynein, dynactin and VAMP2 are associated with
biosynthetic and sorting compartments, and that dynein is additionally
associated with pre-lysosomal and lysosomal compartments. The CCH-associated
redistributions of dynein and dynactin complex, as well as
ß-hexosaminidase, suggest movement away from biosynthetic compartments to
higher density compartments. In contrast, the redistributions of rab3D and
acid phosphatase suggest movement from mature secretory vesicles to
biosynthetic compartments. The redistribution of rab6 suggests pronounced
changes or translocations within the TGN.
Given the extensive overlap of these compartments on the sorbitol density gradients, we introduced a second separation dimension by conducting phase partitioning in an aqueous, dextran-polyethyleneglycol two-phase system. Phase partitioning is a liquid chromatography procedure that separates membranes according to their distribution between stationary (dextran-rich) and mobile (polyethyleneglycol-rich) phases. Fraction numbering begins at the origin that contains membranes preferring the stationary phase.
Fig. 7 depicts the
distributions of DIC, p150Glued and other markers among the
isolated compartments after phase partitioning analysis of density gradient
fractions from CCH-stimulated acini. Partitioning of fractions 11-P revealed
comigration of VAMP2, p150Glued and ß-hexosaminidase with a
dynein-rich compartment (peak j in Fig.
7). Similarly, partitioning of fraction P revealed comigration of
dynein, p150Glued VAMP2 and traces of ß-hexosaminidase in
another population (peak m in Fig.
7). We propose that these membrane populations contain
VAMP2-enriched secretory transport vesicles driven into the subapical region
by dynein. In addition, considerable amounts of dynein, the dynactin complex
and VAMP2 are associated with a series of Golgi- and TGN-related compartments
that contain varying amounts of rab6, -adaptin and rab3D (labeled b, c,
d, f, g, h and i in Fig.
7).
|
Overexpression of dynamitin in lacrimal acini using
replication-deficient Ad
Dynamitin overexpression offered the possibility to directly test whether
(1) dynein activity was required for movement of VAMP2-enriched membranes into
the subapical region and (2) dynein activity was important in basal and/or
CCH-stimulated secretion. Conditions of exposure to replication-incompetent Ad
vectors were identified that resulted in reproducible, high-efficiency
(70-90%) transduction of lacrimal acini with ß-galactosidase
(Fig. 8A,B) or GFP
(Fig. 8C,D). Likewise,
untransduced acini exhibited a diffuse cytoplasmic dynamitin labeling pattern
because of endogenous protein (Fig.
8E), whereas acini transduced with Ad-Dynt and imaged at
comparable settings exhibited an intense cytoplasmic fluorescence in almost
all cells (Fig. 8F). Dynamitin
overexpression relative to levels in untransduced acini was confirmed by
35S-labeling of cellular proteins and analysis by SDS-PAGE and
autoradiography (Fig. 8G), and
by western blotting of extracts from Ad-Dynt- and Ad-Dynt-GFP-transduced acini
(Fig. 8H). Dynamitin
overexpression in acini transduced with Ad-Dynt and Ad-Dynt-GFP was
395±70% and 363±63% of untransduced acini, respectively
(n=7).
|
Dynamitin overexpression inhibits CCH-stimulated recruitment of
p150Glued, DIC and VAMP2 to the subapical region
Resting acini transduced with Ad-LacZ exhibited diffuse labeling patterns
for p150Glued and DIC (Fig.
9A,B, respectively), whereas CCH stimulation of these acini
promoted the accumulation of p150Glued and DIC into the subapical
region characteristic of untreated acini
(Fig. 9A',B', respectively).
Similarly, Ad-LacZ transduction did not affect either the CCH-dependent
recruitment of VAMP2 into the subapical region nor its colocalization (arrows)
with p150Glued (compare Fig.
9E and Fig. 9E')
or DIC (compare Fig. 9F and
Fig. 9F').
P150Glued and DIC in Ad-Dynt transduced resting acini exhibited a
punctate labeling pattern (arrowheads,
Fig. 9C,D, similar to
nocodazole-treated acini, see also Fig.
2). CCH stimulation of Ad-Dynt-transduced acini exhibited no
accumulation of p150Glued or DIC in the subapical region
(Fig. 9C',D', respectively).
Ad-Dynt also prevented the CCH-dependent enrichment of VAMP2 and its
colocalization with p150Glued (compare
Fig. 9G and 9G') and DIC
(compare Fig. 9H and 9H').
|
To confirm that dynamitin overexpression was specifically inhibiting dynein-based movement of membranes in CCH-stimulated acini, we investigated whether CCH-induced Arp1 recruitment to membranes was affected. Dynamitin overexpression is thought to prevent the interaction of the two structural motifs of dynactin, the Arp1 filament and the p150Glued sidearm; therefore, its overexpression should not alter CCH-stimulated recruitment of Arp1 to membranes. Fig. 10A and 10A' show the localization of Arp1 in Ad-LacZ-transduced resting and CCH-stimulated acini, respectively. As seen in these images and the overlaid images in Fig. 10E and Fig. 10E', Arp1 is clearly recruited into the subapical region following CCH stimulation where it is colocalized with VAMP2, comparable to the response in untransduced acini (Fig. 4). Fig. 10B and Fig. 10B' show the localization of Arp1 in Ad-Dynt-transduced resting and CCH-stimulated acini, respectively. CCH stimulation of Ad-Dynt-transduced acini is associated with increased punctate Arp1 fluorescence distributed throughout the cytoplasm relative to untransduced acini, suggesting increased association with subcellular membranes. Fig. 10F' also shows that there is increased colocalization of Arp1 and VAMP2 (arrowheads) throughout the cytoplasm of CCH-stimulated Ad-Dynt-transduced acini, although these components are not recruited into the subapical region.
|
We analyzed the effects of dynamitin overexpression on CCH-stimulated
recruitment of Arp1 to the Tx-100 insoluble, SDS soluble pool using sequential
detergent extraction. Table 2
shows the enrichment of Arp1 in saponin soluble, Tx-100 soluble and SDS
soluble pools from resting and CCH-stimulated untransduced acini and acini
transduced with Ad-LacZ or Ad-Dynt. The effects under all conditions were
comparable, revealing a small but statistically significant
(P0.05) increase in Arp1 recovery with Tx-100 insoluble, SDS
soluble fractions comparable in magnitude to that shown previously
(Fig. 3). Moreover, the
percentage recovery of Arp1 within each pool in resting and CCH-stimulated
acini under each condition was not significantly different.
|
Dynamitin overexpression and nocodazole treatment deplete rab3D
beneath the apical membrane
Although only traces of overlap between p150Glued and rab3D were
detected by confocal fluorescence microscopy
(Fig. 4), a contribution of
dynein and MTs to maintenance of this vesicle population was suggested by the
finding that nocodazole treatment abolished subapical stores of rab3D in
unstimulated acini (compare Fig.
11E and Fig. 11F).
We examined the effects of dynamitin overexpression on rab3D-enriched membrane
vesicles. Transduction with Ad-Dynt markedly reduced subapical stores of rab3D
in resting acini relative to untransduced acini (compare
Fig. 11C versus
Fig. 11A). However, some
dispersal of the subapical pool of rab3D was also elicited by Ad-LacZ
(Fig. 11B) and Ad-GFP (data
not shown) in resting acini. In order to distinguish between the non-specific
effects of Ad vector and the specific effects of dynamitin overexpression, we
quantified the rab3D labeling pattern in untransduced (control) and transduced
acini. The labeling pattern was categorized into one of three classes:
dispersed, half apical (concentrated in the apical half of the cell) and
mostly apical. As shown in Fig.
11D, only 19% of the lumenal regions in control acini showed
dispersed rab3D labeling, with
44% of lumenal regions exhibiting compact
apical labeling and
37% exhibiting half apical labeling. In contrast,
78% of the lumenal regions in Ad-Dynt-transduced acini showed dispersed
rab3D labeling, with
19% showing half apical labeling and only
3%
showing apical labeling. Rab3D distribution in Ad-LacZ-transduced acini was
intermediate between these values, exhibiting
45% of labeling in a
dispersed pattern and retaining
39% as half apical and
17% as
apical. As shown in Fig. 11G
and Fig. 11H, respectively,
transduction with Ad-LacZ or Ad-Dynt did not alter the organization or
abundance of apical MTs. Although the non-specific effects elicited by Ad-LacZ
necessitate interpretation of these data with some caution, the comparable
effects elicited by nocodazole suggest that dynein-driven vesicle transport
may be important for maintenance of rab3D-enriched vesicles.
|
Dynamitin overexpression modifies CCH-independent and CCH-dependent
secretion of protein and ß-hexosaminidase
Basal, total and stimulated release of bulk protein and the secretory
protein, ß-hexosaminidase, were measured in lacrimal acini transduced
with Ad-GFP or Ad-Dynt-GFP (Fig.
12). Total protein and ß-hexosaminidase release in
CCH-stimulated acini were not significantly altered by dynamitin
overexpression. When we resolved the contributions of basal and CCH-stimulated
release to total release, we found that Ad-Dynt-GFP elicited a significant
(P0.05) increase in basal (CCH-independent) release of protein
with a similar but not statistically significant trend noted for
ß-hexosaminidase. Dynamitin overexpression also caused a significant
(P
0.05) decrease in the component of total protein and
ß-hexosaminidase release triggered by CCH, by 58% and 69%, respectively.
These changes in the proportion of total secretory products released in a
CCH-independent versus CCH-dependent manner were not directly attributable to
decreased protein synthesis, because labeling experiments using 35S-Translabel
revealed that bulk protein synthesis in acini transduced with Ad-GFP or
Ad-Dynt-GFP was actually slightly elevated relative to bulk protein synthesis
in untransduced acini (data not shown). We suggest that dynein inhibition
impairs the ability of acini to sequester secretory proteins in the
regulatable arm of the secretory pathway, allowing their release through a
constitutive pathway. This effect may be related to the depletion of
rab3D-enriched mature secretory vesicles in Ad-Dynt-transduced acini
(Fig. 11).
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Discussion |
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Dynein activity may also be important for maintenance of the mature rab3D-enriched secretory vesicles already present in the subapical region which are rapidly discharged following CCH stimulation, as indicated by the depletion of these vesicles in resting acini exposed to nocodazole or dynamitin overexpression (Fig. 11). Evidence for direct dynein association with these vesicles includes the finding that traces of p150Glued and rab3D labeling are colocalized in resting and CCH-stimulated acini (Fig. 4) and the identification of a small amount of rab3D in peak j (Fig. 7), which contains major stores of dynein and VAMP2. Rab3D-enriched secretory vesicles in lacrimal acini may use small amounts of dynein to maintain their subapical localization by anchoring to MTs. Alternatively, rab3D-enriched vesicles may be formed and dynamically maintained from precursors (similar to VAMP2-enriched transport vesicles) transported by dynein.
The recruitment of dynein and its cargo to the subapical region in response
to CCH stimulation suggests that dynein-driven secretory traffic is
physiologically regulated through signaling pathways triggered by activation
of the M3 muscarinic receptor, the target of CCH action. One potential target
of regulation may be the dynactin complex, which exhibits a small but
significant increase in its relative abundance in the Tx-100 insoluble, SDS
soluble protein pool from CCH-stimulated acini relative to resting acini
(Fig. 3). This change in
partitioning behavior could reflect association with a new membrane cargo
enriched in Tx-100-resistant membranes or alternatively a tighter binding to
MTs either alone or in association with membrane cargo. Dynactin recruitment
to MTs has previously been proposed as a mechanism for assembly of the
holo-complex required for dynein-driven vesicle transport
(Vaughan et al., 1999).
Interestingly, the content of dynein in the Tx-100 insoluble, SDS soluble
protein pool is not increased by CCH in parallel with the dynactin complex.
Failure to observe increased dynein in this pool may reflect a more labile
association, even in its activated state. Data obtained by dynamitin
overexpression are also consistent with the concept that physiological
regulation of dynein in acinar secretory traffic may occur through modulation
of the dynactin complex. The observation of increased punctate Arp1
fluorescence throughout the cytoplasm of Ad-Dynt-transduced acini stimulated
with CCH (Fig. 10), in
parallel with the lack of inhibition of CCH-induced recruitment of Arp1 to the
SDS-soluble fraction (Table 2),
suggest that one mechanism for recruitment of dynactin to membranes may
involve regulation of the Arp1 filament.
However, data obtained by fractionation of membranes from resting and CCH-stimulated acini (Figs 6A,6B and 7) also show that although dynein and dynactin are largely associated with the same membranes, their relative abundances vary. The abundance of dynein in higher density regions of the gradient (fractions 11, 12 and P) is relatively greater than that of the dynactin complex. These observations indicate that the factors governing assembly of an active dynein-dynactin holo-complex may be even more complicated than has previously been anticipated.
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 lacrimal acinar protein secretion are associated with a variety of diseases ranging from keratoconjunctivitis sicca to the severe dry eye associated with immune-mediated disorders such as Sjögren's syndrome, AIDS and graft versus host disease. Further exploration of the contributions of dynein and other effectors to stimulated protein release in lacrimal acini may shed insights into the etiology of these processes.
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Acknowledgments |
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