Specific Isoforms of Actin-binding Proteins on Distinct
Populations of Golgi-derived Vesicles*
Kirsten
Heimann
,
Justin M.
Percival§,
Ron
Weinberger§,
Peter
Gunning§¶, and
Jennifer L.
Stow
From the
Centre for Molecular and Cellular Biology,
University of Queensland, Brisbane, Queensland 4072 and the
§ Oncology Research Unit, The New Children's Hospital,
Westmead, and Department of Paediatrics and Child Health,
University of Sydney, Sydney, New South Wales 2145, Australia
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ABSTRACT |
Golgi membranes and Golgi-derived vesicles are
associated with multiple cytoskeletal proteins and motors, the
diversity and distribution of which have not yet been defined. Carrier
vesicles were separated from Golgi membranes, using an in
vitro budding assay, and different populations of vesicles were
separated using sucrose density gradients. Three main populations of
vesicles labeled with
-COP,
-adaptin, or p200/myosin II were
separated and analyzed for the presence of actin/actin-binding
proteins.
-Actin was bound to Golgi cisternae and to all populations
of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with
-COP-vesicles, while p200/myosin II
(non-muscle myosin IIA) and non-muscle myosin IIB were found on
different vesicle populations. Isoforms of the Tm5 tropomyosins were
found on selected Golgi-derived vesicles, while other Tm isoforms did
not colocalize with Tm5 indicating the association of specialized actin
filaments with Golgi-derived vesicles. Golgi-derived vesicles were
shown to bind to F-actin polymerized from cytosol with Jasplakinolide.
Thus, newly budded, coated vesicles derived from Golgi membranes can
bind to actin and are customized for differential interactions with
microfilaments by the presence of selective arrays of actin-binding proteins.
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INTRODUCTION |
Protein trafficking into and out of the Golgi complex requires
multiple populations of membrane-bound vesicles or tubules. Each
vesicle population is associated with distinct sets of coat complexes,
G proteins, and SNARE proteins designed to ensure the selective
trafficking of cargo proteins. The Golgi complex is also associated
with diverse cytoskeletal elements, and Golgi-derived vesicles must, it
now appears, carry multiple molecular motors in order to interact with
different cytoskeletal structures (1, 2). Microtubules and
microtubule-based motors participate in positioning of the Golgi
complex within the cell and in vesicular transport between the
endoplasmic reticulum and Golgi as well as in some post-Golgi pathways
(3). Elements of the actin cytoskeleton are also involved in Golgi
trafficking. The p200/myosin II protein, analogous to the heavy chain
of nonmuscle myosin IIA, binds specifically to vesicles budding off the
trans-Golgi network (TGN)1 in
many cells (4-7). Brush border myosin I has been localized to Golgi
membranes, vesicles, and secretory granules (8-11). Dynactin and
myosin I are found on the same carrier vesicles budding off the TGN in
epithelial cells (11). Myosin 5a can interact with actin as well as
with microtubules (12), and diverse roles, ranging from synaptic
vesicle transport (13) to the movement of smooth endoplasmic reticulum
and pigment granules, have been proposed for myosin 5a (14). Recent
studies show that some isoforms of cytoskeletal proteins associate
uniquely with the Golgi, suggesting that the Golgi has its own,
dedicated cytoskeleton. A Golgi-specific kinesin-like protein
(Rab-kinesin; Ref. 15), a Golgi-specific isoform of spectrin (16), and
two isoforms of ankyrin that are concentrated on the Golgi complex (17,
18) have been identified.
The potential roles of actin filaments and their associated proteins in
either maintaining Golgi structure or in vesicular trafficking are not
well understood. In most cells the Golgi complex is closely aligned
with the microtubule-organizing center, but such a morphologically
distinct, stable connection to the Golgi complex is not evident for
microfilaments. The association of actin and actin-binding proteins
with Golgi membranes might therefore be more dynamic. The use of
actin-polymerizing or -depolymerizing drugs has served to generally
implicate the actin cytoskeleton in vesicular trafficking, especially
in endocytic pathways in intact cells (19, 20). Secretory yeast mutants
have provided the most direct evidence for a requirement for actin in
vesicular transport. The temperature-sensitive mutant of Myo2p, a
myosin V gene product of the yeast Saccharomyces cerevisiae,
has defective post-Golgi transport and accumulates vesicles destined
for the vacuole and for exocytosis (21-25). Mutation of the Act-1
genes in yeast block secretion (26). These studies demonstrate that the
actin cytoskeleton, at some level, does participate in vesicle trafficking, but specific roles for actin in various stages of vesicle
budding, transport, or targeting remain unknown.
Proteins participating in vesicular transport interact with highly
specific membrane domains, either on cisternal membranes in different
parts of the Golgi stack or on selected vesicles. The distribution of
individual proteins thus often indicates their sites of action in
different pathways or steps of vesicle transport. The current study was
undertaken to determine the distributions of actin and actin-associated
proteins with Golgi membranes and with Golgi-derived vesicles. Using an
in vitro assay to generate different populations of
Golgi-derived vesicles, we have analyzed the distinct arrays of actin,
myosins II, tropomyosin isoforms, and other proteins associated with
different membrane domains. Probably through the actions of one or more
of these proteins, newly budded vesicles have the ability to bind to
actin filaments.
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EXPERIMENTAL PROCEDURES |
Antibodies--
The monoclonal antibody (mAb) AD7, which
recognizes p200/myosin II, has previously been characterized (4). Three
antibodies were used to detect tropomyosin isoforms; the CG3 mAb raised
against a peptide encoded by the TmNM5 gene (27) reacts with its
Tm5NM-1, -2, -3, and -4 isoforms. A subset of these isoforms (Tm5NM-1
and 2) is more specifically recognized by the WS5/9d antibody raised against the product of the 9d exon (28). The polyclonal
f9d antiserum recognizes Tms 2, 3, 5a, and 5b encoded by
the
Tmfast gene and Tm1 from the
-Tm gene (29). The
-adaptin antibody was a kind gift from Dr. M. S. Robinson
(University of Cambridge, Cambridge, United Kingdom). mAbs against
-COP, an antibody against the 58-kDa peripheral Golgi protein, and
-smooth muscle actin were obtained from Sigma. Myosin IIA and IIB
antibodies were a kind gift of Dr. R. S. Adelstein (National
Institutes of Health, Bethesda, MD). The p23 antibody was provided by
Dr. J. Gruenberg (University of Geneva, Geneva, Switzerland), the
syntaxin 6 antibody by Dr. D. James (University of Queensland,
Brisbane, Australia), and the Rab 6 antibody by Dr. B. Goud (Institute
Curie, Paris, France). An antibody to rat dynamin I was from
Transduction Laboratories (Lexington, KY). Secondary antibody
conjugates were purchased from Vector Laboratories (Burlingame, CA).
Preparation of Golgi Membranes and Separation of Golgi-derived
Vesicles--
Golgi-rich membranes were prepared from rat liver
homogenates using a procedure modified from Leelavathi et
al. (30). Briefly, a post-nuclear supernatant of homogenized rat
liver was layered onto a discontinuous sucrose gradient consisting of
2.5 ml of 1.3 M and 0.86 M sucrose solutions
overlaid with 2 ml of 0.25 M sucrose. The gradients were
centrifuged at 144,000 × g for 70 min. The supernatant
of the 0.5 M sucrose layer was collected as cytosol, and
the band above the 0.86 M sucrose interface was harvested
as Golgi membranes. Snap-frozen cytosol and membrane samples were
re-ultracentrifuged prior to use.
An in vitro assay for vesicle budding was used (31-33).
Washed Golgi membranes (200 µg) were resuspended in HKM buffer (20 mM KCl, 2.5 mM magnesium acetate, 25 mM Hepes-KOH, pH 7.4) and preincubated with 0.1 mM GTP
S for 5 min at 37 °C, after which precleared
cytosol (14 mg) was added and the reaction was continued for 30 min at
37 °C in the presence of 0.5 mM dithiothreitol and an
ATP-regenerating system (4.6 IU/ml creatine phosphokinase, 81 mM creatine phosphate, and 28.6 mM ATP
(disodium salt) in a 2-ml total volume). The reaction was terminated by
incubation for 10 min in an ice-water slurry. The remnant (unbudded)
Golgi cisternae were separated from the cytosol containing free
vesicles by differential centrifugation (17,500 × g
for 10 min at 4 °C). Vesicles were then separated from cytosol by
discontinuous sucrose gradient centrifugation on 200 µl of 2.1 M sucrose, overlaid with 200 µl of 20% sucrose (w/w),
and 2 ml of the cytosol/vesicle mixture (100,000 × g
for 90 min at 4 °C). All sucrose solutions were prepared in HKM
buffer. The cytosol was collected from the loading zone, and the budded
vesicles were harvested at the 2.1 M/20% interface.
For vesicle separation, free vesicles were resuspended in a total
volume of 400 µl of 20% sucrose and layered onto a discontinuous sucrose gradient consisting of 300 µl of each of the following sucrose solutions: 50%, 45%, 40%, 35%, 30%, and 25% (w/w) (33). The gradient was centrifuged for 18 h at 100,000 × g at 4 °C. The isopycnic gradient was fractionated from
the bottom in 220-µl fractions by syringe puncture. Fractions were
analyzed by SDS-PAGE and immunoblotting.
F-actin Binding Assay--
Rat liver cytosol was precleared by
ultracentrifugation (100,000 × g, 45 min, 4 °C),
and aliquots (1 mg) were incubated in vesicle budding assay buffer (HKM
buffer) supplemented with 0.5 mM dithiothreitol, 0.1 mM GTP
S, in the presence or absence of 5 mM
MgATP at 37 °C for 45 min. Jasplakinolide (JAS, Molecular Probes,
Eugene, OR) was added at a final concentration of 1 µM to
polymerize/stabilize F-actin; we have shown previously that under these
conditions cytosolic G-actin is efficiently converted to F-actin (20).
F-actin was pelleted from the cytosol by low speed centrifugation
(17,500 × g, 30 min, 4 °C), then washed in HKM
buffer, and aliquots representing 100% of the F-actin pellet and 10%
of the remaining cytosol supernatant were loaded onto SDS-PAGE gels and
analyzed by immunoblotting to identify proteins coprecipitated with
F-actin.
To analyze binding of Golgi vesicles to F-actin, vesicle fractions
harvested from the vesicle separation gradient were diluted, ultracentrifuged (100,000 × g), and resuspended in HKM
buffer. F-actin, polymerized with JAS from cytosol, was prepared in the presence of MgATP, to avoid saturation with soluble myosin II. Pelleted, washed F-actin was resuspended and incubated with the vesicle
fractions in the presence of GTP
S for 30 min at 37 °C. F-actin
was then repelleted by centrifugation at low speed, and the resulting
pellets and supernatants were analyzed by SDS-PAGE and immunoblotting
to detect actin-bound vesicle-associated proteins and by electron
microscopy to examine actin-bound vesicles.
SDS-PAGE and Immunoblotting--
Samples for SDS-PAGE were
separated on 5-15% gradient gels (34). Proteins were transferred onto
Immobilon-P membrane in 15 mM Tris, 120 mM
glycine, 20% methanol; membranes were routinely stained with 0.1%
Coomassie Brilliant Blue R250 to check protein loading. For
immunoblotting, transfers were blocked in Blotto (20 mM
Tris, 150 mM NaCl, 5% skim milk, 1% Triton X-100),
antibody incubations were carried out in Blotto for 1-2 h at room
temperature or overnight at 4 °C, and bound antibodies were detected
with alkaline phosphatase-conjugated secondary antibodies with
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium or with chemiluminescence.
Immunoelectron Microscopy--
Golgi-enriched membranes and
budded vesicles generated in the in vitro budding assay were
fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at
4 °C, collected on Formvar/carbon-coated copper grids by floating
grids on 5-µl drops of suspensions of fixed budded membranes for 5 min at room temperature, then washed and stained with 1.8%
methylcellulose containing 0.4% uranyl acetate for 10 min on ice.
Negative contrast of whole mounts was obtained by staining with 1%
uranyl acetate for 1 min and removal of excess stain with Whatman
filter paper. Specimens were examined in a Joel 1010 transmission
electron microscope.
Immunofluorescence Staining--
NIH 3T3 fibroblasts grown at
8000 cells/cm2 on poly-L-lysine substrate were
serum-starved for 48 h, causing them to withdraw from the cell
cycle into the G0 (quiescent) phase of the cell cycle.
Quiescent fibroblasts were fixed with 4% paraformaldehyde, permeabilized with chilled (
20 °C) methanol, and then
double-immunostained with the WS5/9d antibody together with the 58K
antibody that detects a peripheral Golgi membrane protein and the AD7
antibody. Goat anti-rabbit-conjugated lissamine rhodamine and goat
anti-mouse-conjugated fluorescein secondary antibodies were used for
detection. Staining patterns were analyzed by confocal scanning laser
microscopy (Wild Leitz Instruments, Heidelberg, Germany).
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RESULTS |
Budding and Separation of Golgi-derived Vesicles--
In vitro
budding assays were performed using rat liver Golgi membranes and
cytosol in the presence of GTP
S. Vesicle budding is demonstrated by
the de novo membrane binding of two cytosolic proteins,
p200/myosin II and
-COP (Fig. 1).
Small amounts (<10%) of the coatomer protein
-COP and of
p200/myosin II are present on freshly isolated, washed Golgi membranes
(Fig. 1). Newly budded vesicles were initially separated from remnant
cisternae and cytosol by centrifugation. The budded vesicle pellet is
enriched in p200/myosin II and
-COP, compared with the original
Golgi membranes, showing that coated vesicles have been generated
during the assay and that these cytosolic proteins have specifically
bound to them (Fig. 1). Some of the bound p200/myosin II and
-COP on
the remnant Golgi cisternae (Fig. 1) represents attachment to
additional budding sites or to budding vesicles that have not yet been
released.

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Fig. 1.
Cytosol and membrane fractions in the vesicle
budding assay. The budding reaction of Golgi-derived vesicles was
monitored by immunoblotting for p200/myosin II and -COP, present in
the cytosol (lane 1) and in small amounts on isolated Golgi
membranes (lane 2). After budding, both proteins
are in a combined cytosol and vesicle fraction (lane
3) and some remained on remnant Golgi membranes
(lane 4). Both proteins are found attached to
vesicles (lane 6) after separation from remnant
cytosol (lane 5). Lanes 1,
3, and 5 represent 10% loading of each fraction
while lanes 2, 4, and 6 represent 100% of each pellet for one budding reaction. The
arrow denotes the 200-kDa p200/myosin II band; a slower
migrating band is sometimes recognized by the AD7 antibody (57). The
lower panel shows the relative proportions of
-COP and p200/myosin II in various fractions. A large excess of both
proteins is contributed by the cytosol, and 20% of both proteins is
attached to budding vesicles.
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Golgi-derived vesicles were then separated on a second gradient, and
fractions were analyzed by immunoblotting and densitometry to
demonstrate the distribution of a range of vesicle-associated proteins
(Fig. 2). The distributions of three
proteins, p200/myosin II,
-COP, and
-adaptin, were found to vary
across the gradient (Fig. 2). Immunogold labeling has previously
confirmed that
-COP (as part of the coatomer or COP I complex),
-adaptin (a component of AP-1 clathrin-coated vesicles), and
p200/myosin II are indeed localized on separate vesicles (5, 35). Thus
the overlapping, but nevertheless distinct, peak distributions of
p200/myosin II,
-COP, and
-adaptin correspond to different
populations of vesicles (Fig. 2).
-COP has a broader distribution
throughout the gradient peaking at fractions F3-F7, and these same
vesicles are labeled for p23, an integral membrane protein of
coatomer-coated vesicles and the cis-Golgi-network (36-39) (Fig.
2A).
-Adaptin peaks in fractions F4-F7, along with
syntaxin 6, an integral membrane protein in TGN-derived clathrin-coated
vesicles (40) (Fig. 2A). The peak distribution of
p200/myosin II is in fractions F1-F5, but as yet there is no
endogenous transmembrane marker with which to determine the identity of
these vesicles. To ensure that the protein pattern reflects the
distribution of membrane-bound proteins on vesicles in this gradient,
the gradient was loaded with 1 mg of precleared cytosol and run under
identical conditions. Soluble cytosolic proteins were found to
distribute in fractions F8-F10 (data not shown); in these fractions,
we are therefore unable to distinguish between vesicle-bound and
soluble proteins (Figs. 2A and 3B). However,
proteins in fractions F1-F7 were also analyzed on flotation gradients
and were confirmed as being membrane-bound (data not shown). Thus, this
gradient allows distinction and partial separation of three
Golgi-derived vesicle populations demarked by the vesicle-associated
proteins p200/myosin II,
-COP, and
-adaptin.

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Fig. 2.
Separation of Golgi-derived vesicles on
sucrose density gradients. A, vesicles from the budding
reaction were separated by gradient centrifugation and immunoblotting
detected p200, -COP, p23, -adaptin, syntaxin 6, dynamin, Rab 6, Rab 4, and Rab 5 in fractions. The p200/myosin II and -COP detected
in F10 (loading zone) most likely represent small amounts of remaining
cytosol. B, p200/myosin II, -COP, and -adaptin define
distinct sets of vesicles; peak distributions of each reveal the
separation of three different vesicle populations.
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Other vesicle-associated proteins were also detected on newly budded
vesicles (Fig. 2A). Dynamin was distributed throughout the
vesicle gradient and was not concentrated in any one vesicle population. The dynamin antibody used can recognize both dynamin I and
dynamin II, and the two bands appearing in these gels could represent
two isoforms or splice variants of dynamin. Rab 6, a monomeric G
protein on Golgi membranes, was identified in fractions F3-F8,
corresponding most closely to the distribution of
-COP labeled
vesicles (Fig. 2A). Small amounts of Rab 4 were found in the
gradient codistributing with the peak
-adaptin-labeled vesicles and
barely detectable levels of Rab 5 were found in fractions toward the
top of the gradient. While the bulk of vesicles on this gradient were
generated from Golgi membranes, detection of these early
endosome-associated Rabs indicates a very small contamination by plasma
membrane/early endosome derived vesicles, possibly AP2 vesicles.
Actin and Actin-associated Proteins on Budded Vesicles--
The
gradient separation of vesicle populations provided a basis for
analyzing the differential distribution of vesicle-associated cytoskeletal proteins. By immunoblotting, it was noted that
-actin was routinely present in fractions of Golgi membranes and vesicles (Fig. 3A). While most of the
-actin is found in the cytosol (Fig. 3, Table
I), 7% of the
-actin present is on
newly isolated stacked Golgi membranes. We routinely found that some of
the total membrane-bound
-actin was lost to the cytosol during the
budding assay, possibly due to dissociation or depolymerization of
actin. However, some of the actin remains bound to remnant cisternae
and a consistent proportion (3% of total) of the
-actin binds to
the budded vesicles (Fig. 3, Table I).
-Actin bound to all
populations of vesicles and was evenly distributed across all of the
fractions of the vesicle gradient (Fig. 3B). Each vesicle
fraction contained approximately 0.3% of the total vesicle-associated
-actin. Centractin was detected at low levels throughout the
gradient but was particularly concentrated in the
-COP-labeled
vesicle population (Fig. 3B), suggesting that it does bind
preferentially to these vesicles. p200/myosin II, which has been
identified as nonmuscle myosin IIA, is found on one of the TGN-derived
vesicle populations, and immunoblotting with an antibody raised against
the heavy chain of nonmuscle myosin IIA shows a similar distribution
peaking in fractions F1-F5, as expected. In contrast, the nonmuscle
myosin IIB isoform has a different distribution, peaking in fractions
F6-F8. The distribution of membrane-bound myosin IIB overlaps with
that of
-adaptin vesicles, while some of the myosin IIB, in the
higher fractions, may also be cytosolic (Fig. 3B). These
results now show that more than one population of Golgi-derived
vesicles is associated with a member of the myosin II family and that
myosin II isoforms are targeted separately to specific vesicle-budding
membrane domains.

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Fig. 3.
Distribution of actin and actin binding
proteins. A, -actin was analyzed in fractions from
the in vitro budding reaction by immunoblotting. Lane
1, cytosol; lane 2, isolated Golgi membrane; lane
3, cytosol containing the budded vesicles; lane 4,
remnant Golgi cisternae; lane 5, remnant cytosol; lane
6, total budded vesciles. Total budded vesicles were separated
from the remnant cytosol by ultracentrifugation on a sucrose step
gradient. B, the distribution of actin and actin-binding
proteins in the vesicle gradient was determined by immunoblotting.
Actin distributes evenly across the gradient. p200/myosin II and myosin
IIA are similarly in fractions F1-F5, while myosin IIB has a different
distribution (F5-F8). Centractin peaks in F3-F6 along with -COP.
Tropomyosin isoforms recognized by the f9d antibody are
clustered in fractions F8 and F9, most likely representing a cytosolic
distribution. Tm5 tropomyosins recognized by CG3 are found in fractions
containing cytosol (F8 and F9) and are also in a specific vesicle
population (F1-F4).
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Table I
Relative proportions of -actin in assay fractions
A reproducible fraction of -actin (7%) is bound to Golgi membranes
in isolated Golgi stacks (G). Most of the -actin in the assay is
present in soluble form in the cytosol (C). Some of the membrane-bound
-actin is present in total budded vesicles (P2, 3%) or remains on
Golgi cisternae (P1). The amounts of -actin in SDS-PAGE gel
fractions were analyzed by immunoblotting and densitometry. These data
are representative of three experiments. S1, cytosol + vesicles;
S2, remnant cytosol.
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Tropomyosin isoforms were analyzed on Golgi membranes and budded
vesicles using two antibodies. Blotting with the
f9d
antibody shows several low and high molecular mass (30-38 kDa)
isoforms of the
Tmfast and
-TM gene families in
fractions at the top of the gradient, which may represent the presence
of cytosolic proteins (Fig. 3B). We found no evidence for
Tmfast and
-TM tropomyosins binding to Golgi
membranes before budding (data not shown) or to newly budded vesicles
(Fig. 3B). Probing with an antibody (CG3) that sees all
isoforms of the Tm5 gene family reveals a single band, which could
represent more than one isoform, and this was present in both Golgi
membrane and cytosol fractions. On the vesicle gradient, the Tm5 band
is seen in fractions at the top of the gradient (F8-F9) which are
likely to represent cytosolic Tm5, and in fractions F1-F4, clearly
representing Tm5 bound to selected vesicles. Tm5 in these lower
fractions of the gradient coincides with the distribution of
p200/myosin II-labeled vesicle population (Fig. 3B).
In order to further investigate whether p200/myosin II and Tm5 are
colocalized on the same vesicles, or are on different populations of
co-migrating vesicles, immunostaining was carried out with the AD7
antibody to p200/myosin II and with an antibody (WS5/9d) that
recognizes a subset of Tm5 gene products. Fig.
4 shows a quiescent 3T3 fibroblast
labeled with the 58K antibody and the WS5/9d antiserum. Two isoforms
(Tm5NM-1 and -2) co-localize identically with the 58K Golgi peripheral
membrane protein in an asymmetric perinuclear fashion. However,
fibroblasts double-stained with p200/myosin II (Fig. 4C) and
WS5/9d (Fig. 4D) showed intense but mutually exclusive
perinuclear staining patterns, suggesting that Tm5 isoforms and
p200/myosin II are enriched in different compartments of the Golgi
complex (Fig. 4, C and D, arrows).
Comparison of p200/myosin II staining with TGN38 showed that both
proteins colocalized and were enriched in the TGN (Fig. 4, E
and F, respectively). This suggests that at least some of
the Tm5 isoforms recognized by CG3 in the liver Golgi fractions are on
separate vesicles from p200/myosin II. These observations demonstrate
an actin-based structural complexity of the Golgi-derived vesicle
network, and suggest that specific microfilament populations may have
unique roles to play in Golgi function.

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Fig. 4.
Tm5NM-1 and -2 do not colocalize with
p200/myosin II in fibroblasts. Quiescent NIH 3T3 fibroblasts were
double-labeled by immunofluorescence with antibodies to p200/myosin II,
Tm5 isoforms, and Golgi markers and analyzed by confocal microscopy.
The staining comparisons shown are within the same optical section.
Panels A and B, cells labeled with the
58K protein antibody and the WS5/9d antibody, respectively, show
identical Golgi staining. Panels C and
D, cells stained with the AD7 antibody and the WS5/9d
antibody, respectively, show different staining patterns in the Golgi
area. Panels E and F, cells stained
with antibodies to p200/myosin II as TGN38, respectively, show the same
staining over the TGN and analyzed by confocal microscopy. Thus,
Tm5NM-1 and -2, recognized by the WS5/9d antibody, colocalize with the
58K protein but not with p200/myosin II in the Golgi region.
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Interaction of Actin-based Motor Proteins with F-actin--
The
presence of actin and actin-binding proteins on Golgi-derived vesicles
led us to investigate whether individual vesicle-associated proteins
and the vesicles themselves could bind to actin filaments (Figs.
5 and 6).
Rat liver cytosol (100,000 × g supernatant) contained mostly G-actin, which was not pelleted by low speed centrifugation (Fig. 5). When rat liver cytosol was incubated with the
actin-polymerizing drug JAS, 50% of the total actin, detected with a
general actin antibody, formed F-actin, which could be pelleted at low
speed. This F-actin was then incubated with cytosol or with the newly budded vesicle fractions to test the actin-binding properties of
soluble proteins (Fig. 5) and vesicles (Fig. 6), respectively. As an
actin-based motor, p200/myosin II in the cytosol would be expected to
bind to actin filaments; accordingly, we found that 50% of the soluble
p200/myosin II in the cytosol was pelleted along with the F-actin,
under these conditions (Fig. 5). In control experiments, neither the
incubation buffer nor the addition of GTP
S to the cytosol was
sufficient to induce polymerization of F-actin or pelleting of
p200/myosin II (Fig. 5). This assay was also carried out in the
presence and absence of MgATP, which typically dissociates motor
proteins from actin (Fig. 6).While 50% of p200/myosin II and myosin
IIB bound to F-actin in the absence of MgATP, both myosins II were
liberated upon treatment with MgATP, no longer appearing in the F-actin
pellet (Fig. 6). These data confirm that both p200/myosin II (myosin
IIA) and myosin IIB in the cytosol can associate with F-actin via their
head domains. In contrast, centractin (30%) was pelleted with F-actin
in a MgATP-independent fashion (Fig. 6). Tm5 tropomyosins also bound to
F-actin in this assay, and this binding was slightly reduced in the
presence of MgATP (Fig. 6).

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Fig. 5.
Binding of p200/myosin II to F-actin.
Figure shows p200/myosin II and actin in fractions of cytosol
(C). Cytosol was incubated with or without GTP S and JAS
as indicated; resulting F-actin was recovered by low speed
centrifugation, and the actin pellets (P) and supernatants
(S) were analyzed by immunoblotting. p200/myosin II is in
the supernatants and is only co-sedimented with F-actin produced by JAS
(P). S, supernatant, 10% loading of total
fraction on gel; P, pellet, 100% loading on gel.
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Fig. 6.
Binding of cytosolic proteins to
F-actin. Cytosol was incubated with JAS in the presence and
absence of MgATP. Pelleted F-actin (P) and supernatants
(S) were analyzed by immunoblotting. Motor proteins
(p200/myosin II and myosin IIB), centractin, -actin, and tropomyosin
show significant amounts of sediment with F-actin (P)
whereas only insignificant amounts of -COP, -adaptin, and dynamin
pelleted with F-actin. The actin binding of p200/myosin II and myosin
IIB is dramatically reduced in the presence of MgATP. S*,
supernatant, 10% of total fraction loaded on gel; P,
pellet, 100% loading.
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As controls, we also tested whether other cytosolic, vesicle-associated
proteins, which are not known to bind actin, could be pelleted with
F-actin. Soluble forms of
-COP and
-adaptin did not pellet in
significant amounts in either the presence or absence of MgATP and
therefore did not bind specifically to F-actin (Fig. 6). We also found
that, under these conditions, cytosolic dynamin, in its GTP-bound form
in the presence of GTP
S, did not bind appreciably to F-actin (Fig.
6).
Golgi-derived Vesicle Populations Associate with
F-actin--
Having confirmed that some of the vesicle-associated
proteins have the ability to bind to F-actin, we next investigated
whether intact, newly budded vesicles themselves could bind to F-actin (Figs. 7 and
8). Actin was polymerized with JAS from
rat liver cytosol, in the presence of MgATP to avoid saturation of
F-actin with soluble motors such as p200/myosin II, and the F-actin was then pelleted at low speed (Fig. 7, P1). Vesicles were
collected from separated fractions of the vesicle separation gradient.
In order to initially collect vesicles, the sucrose concentration was
reduced, but this did not release any of the membrane-bound
-COP or
p200/myosin II (Fig. 7, S2). The vesicles were resuspended and incubated with cytosol containing F-actin in the presence of
GTP
S (to avoid loss of vesicle coats) in the absence of MgATP, and
F-actin was then pelleted at low speed under conditions that did not
pellet vesicles alone. Nearly all of the vesicles in fractions collected from the p200/myosin II peak and the
-COP peak bound to
the F-actin. Small amounts of
-COP in the supernatant could be due
to saturation of F-actin binding for these vesicles or due to some
uncoating of vesicles. In order to confirm that intact vesicles bind to
F-actin, the pelleted vesicles were also examined by electron
microscopy. Different types of intact, coated vesicles were identified
in the budded vesicle fraction (Fig. 8, A-C). Coated
vesicles and vesicle profiles were found in the F-actin pellet (Fig.
8D), and some vesicle profiles can be seen bound to actin
filaments (Fig. 8, D and E). Negative contrast of
the F-actin/vesicle pellet reveals superpolymerized F-actin (Fig. 8F, arrows) as well as normal F-actin (Fig.
8F, arrowheads) around vesicles. Taken together,
these data show that newly budded Golgi-derived vesicles can bind to
F-actin, presumably through the interaction of one or more of the
actin-binding proteins present on each vesicle population.

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Fig. 7.
Golgi-derived vesicles bind to F-actin.
Actin polymerized from rat liver cytosol with JAS in the presence of
MgATP shows little binding of either soluble p200/myosin II or -COP
(P1). More than 90% of these soluble proteins remain in the
supernatant (S1; 10% loading) upon pelleting F-actin by
differential centrifugation (P1; see Fig. 6). More than 90%
of both proteins remain in the supernatants (S1; see Fig. 6
for loading details). Golgi-derived vesicle fractions (F2-F3, p200
vesicles; and F4-F5, -COP vesicles) from the sucrose density
gradient were diluted and recovered by ultracentrifugation. The
resulting supernatant (S2) and pellet (P2) show
that p200/myosin II and -COP remain bound to the vesicles after
resuspension. Resuspended fractions across the gradient were incubated
with F-actin; the F-actin was then recovered by low speed
centrifugation (P). Under these conditions, both p200/myosin
II and -COP vesicles pelleted with F-actin. Small amounts of -COP
remain in the supernatant (S). S, supernatant,
10% loading of total fraction on gel; P, pellet, 100%
loading.
|
|

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Fig. 8.
Golgi-derived vesicles and F-actin-bound
vesicles. Vesicles budded from rat liver Golgi membranes (Fig. 1,
lane 6) and Golgi-derived vesicles bound to
F-actin were examined as whole mounts by transmission electron
microscopy. The total vesicle population contains coated vesicles of
distinct appearance and size (A-C). Vesicles with typical
clathrin coats (A and B) and smaller coats
(C) were recognized. Intact coated vesicles
(arrowhead) were recovered in the F-actin pellet
(D). Actin filaments (arrows) could be seen in
these samples, often aligned with vesicles (D and
E). Negative contrast of whole mounts reveals the
association of different lipid vesicles with fine actin filaments
(arrowheads) as well as with long, superpolymerized F-actin
(arrows, F). Scale bar, 200 nm.
|
|
 |
DISCUSSION |
The budding assay described in this paper yields multiple
populations of intact, coated vesicles derived from Golgi membranes (Fig. 9). When the full complement of
vesicles was further separated by gradient centrifugation, three
distinct populations were harvested and identified by the presence of
the vesicle-associated proteins p200/myosin II,
-COP, and
-adaptin as markers. Each of these proteins from the cytosol bound
to Golgi membranes in a GTP-dependent fashion during the
budding assay, and each was then recovered in distinct peak fractions
of the vesicle separation gradient. The separation of these vesicles is
consistent with results obtained by immunoelectron microscopy showing
that
-COP and p200/myosin II (5) and
-adaptin and p200/myosin II
(35) are on separate populations of Golgi-derived vesicles. In
addition, there are undoubtedly other types of Golgi-derived vesicles
present. For instance,
-COP has also been found on
non-clathrin-coated vesicles budding off other parts of the Golgi
complex, including the TGN (41). Two other types of vesicles that bud
off the TGN are potentially also present within this gradient: vesicles
labeled with the p230 protein, which are distinct from those bearing
p200/myosin II (42); and those defined by the AP-3 adaptor complex,
which bud off the TGN for transit to late endosomes and lysosomes (43). Our data also suggest that fractions F1-F4 on the gradient contain separate populations of vesicles labeled for either p200/myosin II or
for Tm5 isoforms.

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Fig. 9.
Schematic summary of cytoskeletal proteins
associated with different vesicles. Different Golgi-derived
vesicle populations, depicted as budding profiles, are distinguished by
the presence of different cytoskeletal proteins and vesicle markers.
F-actin is associated with all newly budded vesicles. Myosins IIA and
B, and Tm5 are, however, found on distinct vesicles consistent with
there being customized or unique actin filaments on different vesicle
populations. It has been shown that coating actin with different Tm
isoforms has the demonstrated potential to influence the specificity of
their interaction with different myosin isoforms (61).
|
|
Other proteins were found on one or more populations of vesicles. The
Rab family of monomeric G proteins bind to different classes of
transport vesicles and commonly serve as compartment markers. Rab 6 was
found to codistribute with
-COP, consistent with it being on
vesicles involved in trafficking in early parts of the Golgi stack,
most likely those involved in retrograde transport (44). Dynamin is a
GTPase implicated in the scission of clathrin-coated vesicles at the
plasma membrane (45, 46). Dynamin II has been localized on the TGN,
where it appears to be involved in the budding of at least two vesicle
populations: clathrin-coated vesicles and constitutive secretory
vesicles labeled for pIgA-R (47). Our data similarly indicate a
GTP-dependent binding of dynamin II to Golgi membranes and
additionally show that dynamin is distributed across the whole gradient
and is therefore associated with multiple populations of vesicles and
that dynamin does stay attached to the membranes on newly budded
vesicles after their release. The presence of dynamin on all budded
vesicles suggests that it may form part of a generalized budding
machinery, perhaps by aiding vesicle interactions with both
microtubules and microfilaments, through binding to profilin and
amphiphysin (48, 49).
A dedicated spectrin/ankyrin network is assembled on Golgi membranes
(16, 18) and, although the functions of this network are not yet
understood, it has potential roles in maintaining Golgi structure, in
marshaling transmembrane proteins within the plane of Golgi membranes
and/or in vesicular transport (50). Based on the equivalent spectrin
structure at the plasma membrane, it is reasonable to assume that short
actin filaments or actin-like molecules are also associated with the
Golgi complex. Centractin (ARP1) is an actin-related protein shown to
associate with spectrin on plasma membranes and on Golgi membranes
(51). Our data show that centractin is on newly budded vesicles and,
unlike actin itself, it is concentrated on COP I vesicles. As part of
the dynactin complex (52), centractin has the potential to link Golgi
membranes or vesicles with microtubules. COP I vesicles are known to
associate with microtubules (53) and have recently been shown to
associate with spectrin/ankyrin (50), suggesting several possible roles for centractin on these vesicles. Our results also indicate that small
amounts of actin itself, as the
-actin isoform (<10%), is stably
associated with Golgi membranes and that some
-actin is bound to
budded vesicles. Unlike the other vesicle-associated proteins,
-actin was slightly de-enriched in the budded vesicle fraction
compared with the Golgi stack, suggesting that relatively more of the
actin is retained on non-budding membrane domains of Golgi cisternae.
In addition,
-actin was present on all newly budded vesicles and,
when tested, vesicles from all fractions appeared to be equally
competent to bind to F-actin. The presence of membrane-associated actin
with both the Golgi cisternae and with budding vesicles suggests that
actin filaments could be both a structural component of the Golgi stack
as well as participating in trafficking. By electron microscopy, under
normal conditions of the budding reaction, we did not observe prominent
actin filaments associated with budded vesicles. Polymerized actin
produced in the presence of JAS could, however, be seen associated
directly with vesicles. Thus, actin present on Golgi membranes and/or
on vesicles under physiological conditions may be composed primarily of
short filaments, or actin filaments may associate with the membranes
only transiently. Actin on budded vesicles could be transferred from
the donor Golgi membranes, or new short filaments could arise from
local nucleation sites on the surface of vesicles.
Tropomyosins associate closely with actin filaments and serve to
regulate filament stability and length. Cells typically express several
types and isoforms of tropomyosins, and there is growing evidence to
show that specific isoforms can be differentially localized on actin
filaments throughout cells (54). Tropomyosins of the Tm5 family,
recognized by the CG3 antibody, were detected here on rat liver Golgi
membranes and were on at least one specific vesicle population. This
correlates well with the demonstration that Tm5NM-1 and Tm5NM-2
isoforms of the Tm5 family are localized on Golgi membranes in
fibroblasts, as shown by immunofluorescence. In contrast, tropomyosins
detected by the
f9d antibody, were not found on
vesicles. These findings indicate that tropomyosin isoforms mark
specific populations of microfilaments associated with discrete
cellular subcompartments. It also provides compelling evidence that
these different filament populations have specific cellular functions
(55). Actin filament heterogeneity may be regulated throughout cells
and even within the Golgi milieu by the differential placement of
tropomyosins associated with membranes and vesicles. One of the two
tropomyosin isoforms in S. cerevisiae has been implicated in
post-Golgi trafficking (56) by the finding that temperature-sensitive
mutants of the TPM1 gene accumulate Golgi-derived transport
vesicles. A functional interaction of tropomyosin with a myosin V in
this pathway is further suggested, since the TPM1 gene shows
synthetic lethality with the MYO2 gene (56). An important
role of tropomyosins in vesicle trafficking could potentially be to
liaise with myosin motors in coordinating filament stability and
vesicle movement.
Myosins of multiple families appear in association with Golgi membranes
and on newly budded vesicles in cells (1, 35). p200/myosin II is
homologous to the heavy chain of non-muscle myosin IIA (MHCIIA) and is
on a specific subset of TGN-derived vesicles (35, 57). It is
interesting to note that non-muscle myosin IIB is on different
Golgi-derived vesicles. In several cell types, nonmuscle myosins IIA
and IIB have been found on distinct membrane domains and have been
ascribed to different cellular functions such as cytokinesis and cell
motility (58, 59). The current data, however, provide the first
indication that both non-muscle myosin II isoforms could be involved in
trafficking and that there is highly specific targeting of each protein
to distinct vesicle-budding domains of Golgi membranes. Myosin I is
also found on vesicles budding off Golgi membranes, where it is
proposed to provide motor activity for vesicle movement along actin
filaments at the cell periphery (10). The non-muscle myosins IIA and B,
both in the cytosol and on vesicles, are oriented for actin binding in
a MgATP-dependent motor-like configuration. p200/myosin II
binds to the vesicles in a GTP-dependent fashion during
budding, and it dissociates from vesicles soon after
budding,2 suggesting it may
function only in the locale of the Golgi. However, there are
conflicting reports about whether p200/myosin II is an essential
participant in the vesicle budding reaction (6, 60). Thus, as with
actin/membrane interfaces in other parts of the cell, the surfaces of
Golgi-derived vesicles appear to harbor multiple classes of motors,
perhaps for a series of temporal interactions with microfilaments.
Possible mechanisms for binding vesicles to actin filaments, and their
associated proteins, include direct binding via myosins I or II or
indirect association of actin with the vesicle surface through
spectrin/ankyrin assemblies. Membrane binding through any of the known
mechanisms for vesicle-associated proteins, for instance through
different actions of ARF-GTP (50), is likely to result in dynamic or
temporary binding of microfilaments to vesicles.
These data add to the emerging evidence that the Golgi complex is
associated with the actin cytoskeleton through multiple families of
actin-binding proteins or actin-based motors. Moreover, we show that
specific isoforms or members of these families are placed strategically
and specifically on specific membrane domains, or on specific vesicle
populations. The binding of specific isoforms of Tm5 tropomyosins to
selected vesicles suggests a mechanism for customizing actin filament
stability during vesicle budding or trafficking. Isoforms of myosin II
on different vesicles could also provide for different motor activity,
vesicle movement, or attachment to actin filaments during budding. A
key issue to now resolve is how individual actin-binding proteins and
actin filaments bind to vesicles and the relationships of these
proteins during budding or subsequent trafficking.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Adelstein, Robinson, Gruenberg,
and Goud for kindly providing antibodies, and Darren Brown for
technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Health and
Medical Research Council (NHMRC) grants (to J. L. S., P. G., and R. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Senior Research Fellow of the NHMRC.
Senior Medical Research Fellow of the Wellcome Trust. To whom
correspondence should be addressed. Tel.: 61-7-3365-4985; Fax: 61-7-3365-4388; E-mail: j.stow{at}cmcb.uq.edu.au.
2
K. Heimann and J. L. Stow, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
TGN, trans-Golgi
network;
mAb, monoclonal antibody;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
PAGE, polyacrylamide gel
electrophoresis;
JAS, Jasplakinolide.
 |
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