* Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; Division of Signal Transduction, Beth
Israel Hospital, Boston, Massachusetts 02115; and § Division of Experimental Medicine, Brigham and Women's Hospital, Boston,
Massachusetts 02115
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Abstract |
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Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in
Xenopus extracts and examined the relationship of
these pathways. We have found that GTPS stimulates
actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane
vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing
phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol
(3,4,5) trisphosphate can induce actin assembly even in
the absence of GTP
S. RhoGDI, a guanine-nucleotide
dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using
various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that
phosphoinositides may act to facilitate GTP exchange
on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and
Cdc42 are required to induce actin assembly in this cell-free system.
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Introduction |
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THE actin cytoskeleton is involved in most aspects of
cellular morphogenesis where it is dynamically regulated by both extra- and intracellular signals (Stossel, 1993; Fishkind and Wang, 1995
; Mitchison and Cramer,
1996
). In almost all cases, this regulation is posttranslational and the locus of this posttranslational control is not
on actin, but rather on actin binding proteins that control actin dynamics in the cell. There are ~50 actin binding
proteins identified so far that directly interact with actin
and modulate its assembly properties (Kreis and Vale,
1993
). They can be grouped into six functional families:
monomer sequestering, filament severing, capping, nucleating, cross-linking, and bundling. The presence of such a
large number of regulatory proteins makes it very difficult to study the physiological mechanisms controlling actin
functions. Although it is relatively easy to identify the
mechanism by which individual proteins act from in vitro
studies, it is difficult to reveal the network of interactions
that occur in vivo. It is even more challenging to connect
the actin response to extracellular signals, which undoubtedly involve a different assemblage of signaling factors.
One very promising hint of in vivo control that has come
from in vitro studies is the role of phosphoinositides, particularly phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2),1
as potential signaling intermediates (Janmey, 1994). PI(4,5)P2 interacts with several actin binding proteins. For example,
PI(4,5)P2 binds profilin and dissociates actin from profilin/
actin complex (Lassing and Lindberg, 1985
); it also inhibits gelsolin functions, including severing, nucleation, and
actin monomer binding (Janmey and Stossel, 1989
). Studies carried out in intact cells have also implicated phosphoinositides in signaling from activated cell surface receptors
to intracellular actin cytoskeletal responses, making them
attractive places to look for the linkage. The level of phosphoinositide synthesis, for example, has been correlated with the amount of actin assembled in basophilic cells
(Apgar, 1995
) and platelets (Hartwig et al., 1995
). Phosphoinositide 3-kinase (PI 3-kinase), a lipid kinase that
phosphorylates the D-3 position of phosphoinositides, is
known to be required for PDGF-induced membrane ruffling and chemotaxis in fibroblasts (Kundra et al., 1994
;
Wennstrom et al., 1994
; Wymann and Arcaro, 1994
; Nobes
et al., 1995
). Though lipid binding has been studied extensively in individual actin binding proteins, it is not clear
which phosphoinositides are able to modulate actin assembly in cells. Only recently has the specificity been studied
in permeabilized platelets (Hartwig et al., 1995
, 1996
).
Another source of linkage between signal transduction
pathways and actin assembly are the small GTP-binding
proteins (G proteins) of the Rho family (Machesky and
Hall, 1996; Ridley, 1996
; Zigmond, 1996
). This family, including Rac, Rho, and Cdc42, are Ras-like small GTPases
that act as molecular switches cycling from GTP-bound active forms to GDP-bound inactive forms (Bourne et al.,
1991
; Hall, 1992
). The GTP/GDP cycle is mainly controlled by GTPase-activating proteins, guanine-nucleotide
exchange factors (GEFs), and guanine-nucleotide dissociation inhibitors (GDIs) (Boguski and McCormick, 1993
). When injected into fibroblasts in a constitutively active
form, each member of the Rho family elicits distinct morphological changes that involve remodeling of the actin cytoskeleton (Ridley and Hall, 1992
; Ridley et al., 1992
;
Nobes and Hall, 1995
). In yeast cells, Cdc42 is required for
polarized cell growth and bud formation, two actin-dependent processes (Chant, 1994
). Despite the central role postulated for them, it is not clear how these small G proteins are activated or how they regulate actin assembly. Surprisingly, several target-binding domain mutants of these G
proteins do not interfere with their ability to induce actin
assembly (Lamarche et al., 1996
; Tapon and Hall, 1997
). G
proteins are usually activated by GEFs, but nothing is
known about where this occurs. All GEFs for the Rho
family are soluble proteins (Cerione and Zheng, 1996
), but
the function that G proteins execute is membrane associated. It is not clear whether G proteins are activated in the
cytosol before binding to the membrane or whether they are
associated with the membrane first, and then activated there.
Although both phosphoinositides and small G proteins
seem to be involved in regulating the actin cytoskeleton, it
is not clear whether these two signaling pathways are related to each other. Several studies have suggested a role
of G proteins in lipid kinase activation. Phosphoinositide
kinases are associated with activated G proteins in both
cell lysates and tissue homogenates (Tolias et al., 1995). A
phosphatidylinositol 4-phosphate 5-kinase is associated
with Rho in Swiss 3T3 cells (Ren et al., 1996
). Activation of PI 3-kinase requires Rho in platelets (Zhang et al.,
1993
). However, additional evidence suggests that lipid kinases may be involved in controlling G proteins. In fibroblast cells, PI 3-kinase is required for PDGF-stimulated
Rac activation (Hawkins et al., 1995
) and constitutively active PI 3-kinase is able to activate Rac/Rho (Reif et al.,
1996
). In addition, activation of small G proteins appears
to correlate with the increase in phosphoinositide synthesis. The level of PI(4,5)P2 is increased in response to the
activation of Rho in Swiss 3T3 cells (Chong et al., 1994
) and Rac in platelets (Hartwig et al., 1995
). These mixed results make it difficult to draw conclusive lines among the
two signaling molecules and the actin cytoskeleton.
Xenopus egg extracts have proven to be a powerful cell-free system for reconstituting heterogeneous and complex
reactions including nuclear assembly and disassembly
(Newport and Spann, 1987), chromosome condensation
(Ohsumi et al., 1993
), spindle assembly (Sawin et al.,
1992
), DNA replication (Hutchison et al., 1989
), and the
control of cell cycle (Murray and Kirschner, 1989
). Recently, the actin-based motility of Listeria has also been reconstituted in egg extracts (Theriot et al., 1994
; Marchand
et al., 1995
), suggesting that the extracts contain all the
necessary components in somatic cells that are required to
modulate actin assembly. However, egg extracts have not
been used to reconstitute the interplay of membrane-based
actin assembly with soluble components. It should be possible to reconstitute membrane-dependent cytoskeletal responses by including endogenous membranes or providing
exogenous membranes to the extracts. We report on the
success of that approach here and its use in unraveling aspects of the physiological control of actin polymerization.
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Materials and Methods |
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Preparation of Extracts
Low speed mitotic extracts from unfertilized eggs were prepared from Xenopus eggs in a buffer (XB) containing 10 mM Hepes, pH 7.7, 100 mM
KCl, 2 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, 50 mM sucrose, 1 mM
DTT, and protease inhibitors as described (Murray, 1991), except that cytochalasin was omitted in all steps. Extracts were either used immediately
or supplemented with 200 mM sucrose and frozen in liquid nitrogen.
High speed supernatants were prepared from low speed extracts according to King et al. (1995). Briefly, low speed extracts were diluted 10-fold in XB and then centrifuged at 400,000 g for 1 h. The clear supernatant
was carefully removed and reconcentrated to its original volume in Centriprep-3 or -10 concentrators (Amicon Corp., Danvers, MA), giving a final concentration of 40-50 mg/ml. The membrane pellet was resuspended
in XB to the same original volume and broken up in a Dounce homogenizer. All extract fractions were supplemented with energy regenerating
mix containing 1 mM ATP, 1.25 mM MgCl2, 7.5 mM creatine phosphate,
and 0.1 mM EGTA, and stored at
80°C. Actin concentration is ~2 mg/
ml in low speed extracts and 1 mg/ml in high speed supernatants as estimated by Western blot.
Preparation of Lipid Vesicles
All phospholipids were obtained from Sigma Chemical Co. (St. Louis,
MO) or Avanti Polar Lipids (Alabaster, AL), except PI(3,4)P2 and
PI(3,4,5)P3, which were gifts from Dr. Ching-Shih Chen (University of
Kentucky, Lexington, KY). Lipids were dissolved in chloroform and
stored at 80°C. Lipid vesicles were prepared as follows. Equal amounts
of phosphatidylcholine (PC) and phosphatidylinositol (PI) were mixed
with or without each phosphoinositide in chloroform and dried in air. The
dried lipid mixture was resuspended in a lipid buffer (20 mM Hepes, pH
7.7, 1 mM EDTA) to a final concentration of 1 mM. The mixture was then
sonicated in a water bath with a W-385 ultrasonic processor (Heat Systems Inc., Farmingdale, NY) at its maximum power (475 Watts) for 5 min.
Video Microscopy and Image Analysis
Extract samples were viewed with a 63×/1.4 NA plan-apochromatic objective on a Zeiss Axiovert 135 microscope (Carl Zeiss, Inc., Thornwood, NY). A multi-band pass dichroic and emission filter set (Chroma Technology Co., Brattleboro, VT) was used for fluorescence observation and a dual filter wheel (Ludl, Hawthorne, NY) was used to choose appropriate excitation and neutral density filters. All images were collected with a cooled charge coupled device (CCD; Photometrics Inc., Tucson, AZ) and stored digitally on a hard disk. Device control, image acquisition, and data analysis were carried out using Metamorph 2.0 (Universal Imaging Corp., West Chester, PA) software running on an IBM-compatible computer. To look for the relative position of lipid vesicles, the nitrobenzoxadiazole image was taken immediately after the rhodamine image with a time delay estimated at ~1 s. The two images were then pseudocolored and overlaid.
Visual Assay for Actin Polymerization
All reactions were carried out at room temperature. For experiments using low speed extracts, 1 µl rhodamine actin (0.2-0.4 mg/ml) was mixed
with 6-µl extracts (either concentrated or diluted twofold with XB).
Rhodamine actin was prepared according to Symons and Mitchison
(1991) using purified rabbit skeletal muscle actin and tetramethylrhodamine-iodoacetamide rhodamine (Molecular Probes, Inc., Eugene,
OR). After the addition of either GTP
S or vanadate (1 µl) to the extract
to a final concentration of 1 mM, 5 µl of the reaction mixture was
squashed between two coverslips. The coverslips were then mounted on
the microscope stage for imaging as described above. For experiments using phosphoinositides, 3 µl high speed supernatants were diluted with 3 µl
XB and supplemented with 1 µl rhodamine actin. 1 µl lipid vesicles (1 mM) were then added.
Pyrene Actin Assay
Pyrene actin was labeled according to an established protocol (Kouyama
and Mihashi, 1981) using rabbit skeletal muscle actin and N-(1-pyrene)iodoacetamide (Molecular Probes, Inc.). Due to the high background fluorescence in the extracts, high speed supernatants were diluted three- to
fourfold in XB to a final concentration of ~10 mg/ml. 3-5 min after 100 µl
diluted high speed supernatants were mixed with pyrene actin in a quartz
cuvette, 5-µl lipid vesicles (1 mM) were added. Pyrene fluorescence was
measured at 407 nm with excitation at 365 nm in a Aminco-Bowman Series 2 Luminescence Spectrometer (SLM-Aminco Inc., Rochester, NY)
with 4-nm slit width. Kinetics of the reaction was analyzed and plotted in
Origin (Microcal Software, Inc., Northampton, MA).
Purification of Recombinant Proteins
Small G proteins were expressed in COS cells as glutathione-S-transferase
(GST) fusion proteins. COS cells were transfected using the calcium phosphate method and allowed at least 60 h to express fusion proteins. Protein
purification followed (Heyworth et al., 1993) with some modifications.
Briefly, cells were broken by sonication in a buffer with 10 mM HEPES,
pH 7.3, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1 mM EGTA, 1 mM
DTT, and protease inhibitors. Nuclei and cell debris were then removed
by centrifugation at 10,000 g·min. The supernatant was then centrifuged at
200,000 g·h to pellet the membrane. Except for Cdc42V12C189S, the
membrane for all other G proteins was solubilized in an extraction buffer (25 mM Tris-HCl, pH 7.7, 50 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 1% cholate, and protease inhibitors). After a clarifying spin at
200,000 g·h, the supernatant was then incubated with glutathione-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) at 4°C overnight.
After wash, the beads were then eluted with an elution buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2, 5 mM DTT, 0.1% cholate, and 10 mM reduced glutathione). Eluted proteins were
cleaved with thrombin and analyzed by SDS-PAGE. Cdc42V12C189S was
precipitated using glutathione beads directly from the supernatant after
centrifugation at 200,000 g·h. RhoGDI was expressed in Escherichia coli
as a GST fusion protein and was purified using glutathione beads.
To activate them with GTPS, processed G proteins were incubated
with 20 mM EDTA and 40 µM GTP
S for 15 min at 30°C. GTP
S was
then locked in place by adding 20 mM extra MgCl2.
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Results |
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GTPS and Sodium Orthovanadate Each Induce
Localized Actin Polymerization and Vesicle Motility in
Low Speed Egg Extracts
As agonists in several signaling systems, GTPS and sodium orthovanadate were added to low speed extracts
(4,000 g·h) of Xenopus eggs in an attempt to induce actin
assembly. To visualize actin, we used a rhodamine actin-based visual assay, which had been used to study Listeria-induced actin assembly in extracts (Theriot et al., 1994
;
Marchand et al., 1995
). In this assay, a trace amount of
rhodamine actin was added to mark the behavior of endogenous actin. Despite the presence of a large number of
heterogeneous endogenous vesicles in low speed extracts,
the fluorescence of rhodamine actin was uniform across
the field, signifying no apparent assembly (Fig. 1 A). Localized foci of actin assembly could be induced by adding
1 mM GTP
S or sodium orthovanadate to the extracts. Approximately 5 min after the addition of either compound, bright fluorescent foci appeared, some in the shape
of comet tails (Fig. 1). Actin assembly occurred only
around a subset of the phase-dense vesicles, which were
propelled through the cytoplasm along gently curved trajectories (Fig. 1 E). The average velocity was 10.0 µm/min
(SEM = 1.3; measured from the vanadate experiments),
similar to that observed with Listeria (Theriot et al., 1994
;
Marchand et al., 1995
). Cytochalasin D (20 µM) completely inhibited the formation of foci and comet tails, indicating that motility and assembly reflected the accumulation of polymerized actin (data not shown).
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Comet tails appeared as phase-dense structures under
the phase contrast microscope. These comet tails often
had distinct vesicles at one end. When the tails were large
(up to 4 µm in width), rhodamine fluorescence clearly delineated the contour of associated vesicles (Fig. 1 E).
While many phase-dense vesicles showed no association with actin, the majority of comet tails were associated with
visible vesicles of variable sizes. Only when the tails were
small was it difficult to find any vesicles at their ends. After the phase-dense vesicles were removed from extracts
by sedimentation at 400,000 g·h, the remaining high speed
supernatants did not induce actin assembly in the presence
of GTPS or vanadate. When the vesicle fraction was
added back, actin foci and comet tails reformed (data not
shown). The intimate physical association of these phase
dense vesicles with foci and comet tails suggests that actin
assembly is nucleated from the vesicles.
Lipid Vesicles Containing Purified Phosphoinositides Can Induce Localized Actin Polymerization in Vesicle-free Extracts
Membrane vesicles seem indispensable for actin polymerization in this system since GTPS is unable to induce actin assembly in high speed supernatants lacking endogenous vesicles. Though it is possible that these membrane
vesicles simply provide a hydrophobic docking surface for
activated G proteins, some specific constituents of the
membrane, such as modified phospholipids or membrane-associated proteins, might be required for the induction of
actin assembly. To examine the role of specific phospholipids in regulating actin assembly, we prepared synthetic
lipid vesicles from purified phospholipids and tested their
ability to induce actin assembly in vesicle-free supernatants.
Lipid vesicles of diameter estimated to be <200 nm
(Janmey and Stossel, 1989) were constructed from PC and
PI. When added to high speed supernatants containing labeled actin, they did not induce localized actin assembly
(Fig. 2 A). However, inclusion of 33% phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3) in PC/PI vesicles
stimulated the formation of actin foci and comet tails (Fig.
2 B). In a typical field of 10,000 µm2, there were ~41.2 ± 10 foci and 4.0 ± 0.5 comet tails with an average length of
4.3 ± 0.2 µm. These comet tails moved at a rate of 5.4 µm/
min (SEM = 1.1) (Fig. 2 G). PI(3,4,5)P3-containing lipid
vesicles themselves could be visualized by including in them nitrobenzoxadiazole (NBD)-tagged PC (4%). The
vast majority of rhodamine signals were associated with
the tagged vesicles (data not shown). Strikingly, each
comet tail had only one NBD-vesicle attached to its end
(Fig. 2 H). All assembly could be blocked completely by
cytochalasin D.
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The induction of actin foci was dependent on the type of phosphoinositide used. Vesicles containing PI(4,5)P2 produced both foci (19.7 ± 4.4) and comet tails (1.5 ± 0.6) (Fig. 2 C) as did vesicles containing PI(3,4,5)P3. However, vesicles containing phosphatidylinositol (3,4) bisphosphate or phosphatidylinositol (3) phosphate (PI(3)P) produced fewer foci (6.5 ± 0.8 or 9.5 ± 0.7 per 10,000 µm2 field) and no comet tails (Fig. 2, D and F). Vesicles containing phosphatidylinositol (4) phosphate (PI(4)P) induced foci (35.9 ± 7.5 per field) but fewer tails (1.2 ± 0.6 per field) (Fig. 2 E).
Quantitative Pyrene Actin Assays for Assembly Induced by Phosphoinositides
Although the visual assay gave a reproducible qualitative
picture of the effectiveness of different phosphoinositides,
it was not readily quantifiable. Vesicles varied in size, as
did the amount of actin associated with them and there
was no simple measure of the volume in the field to be
used for comparison. To give a more accurate measurement of the polymerized actin, we used a well-established
pyrene actin assay (Cooper et al., 1983). Pyrene actin has
increased fluorescence when it is incorporated into filamentous actin (F-actin). Therefore, an increase in fluorescence intensity is a direct measure of actin polymerization and
the kinetics can be easily monitored in a spectrofluorometer.
Like purified actin, pyrene actin assembles spontaneously into filaments in high salt buffers, with a lag phase
followed by a nearly linear increase (Cooper et al., 1983).
After the addition of 1 µM pyrene actin to high speed supernatants containing physiological salts, there was no appreciable increase in fluorescence for at least 1 h (data not
shown). The magnitude of pyrene signal in the supernatant did not differ much from that in a low salt buffer, indicating that most of the actin in the extract remained unpolymerized. Under the condition used in the experiments,
nearly 90% of the basal fluorescence signal came from the
extracts diluted to 10 mg/ml, and 10% was due to pyrene
actin (1 µM final concentration).
When vesicles containing only PC/PI were added to high speed supernatants, the pyrene signal again remained unchanged after an immediate 5-10% increase due to the light scattering of the lipids (Fig. 3 A). However, the addition of vesicles containing only 4% PI(3,4,5)P3 or PI(4,5)P2 to the supernatant caused a rapid increase in fluorescence (Fig. 3 A) during the first 3-5 min. Within 5-10 min, the fluorescence was at 10× the original signal. At the peak, we estimated that ~10% of total actin in the extract had been incorporated into F-actin, after normalizing to the fluorescence intensity from extracts treated with 2.5 µM phalloidin. The kinetics and the magnitude of the response were similar for both phosphoinositides. The increase in pyrene signal could be completely inhibited by 5 µM cytochalasin D (Fig. 3 C).
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The role of these specific phosphoinositides to induce
actin assembly was confirmed by incubation of extracts
with a lipid-binding peptide (QRLFQVKGRR) derived
from gelsolin. This peptide has been shown to bind phosphoinositides and can compete for phosphoinositide-binding with gelsolin (Janmey et al., 1992). The peptide completely inhibited actin assembly induced by PI(4,5)P2 at
20 µM with a half inhibitory concentration of 5-10 µM
(Fig. 3 C), consistent with its estimated micromolar affinity with PI(4,5)P2 micelles (Janmey et al., 1992
).
Vesicles containing three other phosphoinositides, 4% PI(3,4,)P2, (an isomer of PI(4,5)P2), 4% PI(4)P, or 4% PI(3)P induced actin assembly at very slow rates and to a much lower extent when compared with PI(4,5)P2 (Fig. 3 B). The rate of actin assembly for PI(3,4)P2 was only 5% that for PI(4,5)P2; the total amount of F-actin after 5 min was 29% that of PI(4,5)P2. These results are consistent with those from the visual assay.
Dose Response of Actin Assembly to Phosphoinositides.To ascertain whether the effective levels of phosphoinositides in vesicles were physiologically meaningful, we varied the phosphoinositide concentration in the vesicles and compared the initial rate of actin polymerization and the total amount of F-actin at the peak. Fig. 3 D, a, shows the dose response to PI(3,4,5)P3 and PI(4,5)P2 at a total lipid concentration of 50 µM. Vesicles containing only 1% of PI(3,4,5)P3 or 1% PI(4,5)P2 gave measurable stimulation of assembly. The extent of assembly increased with the percentage of phosphoinositides in lipid vesicles to a maximum at 12% for PI(3,4,5)P3 and 4% for PI(4,5)P2. Above 4%, the activity induced by PI(4,5)P2 plateaued. Interestingly, a higher concentration of PI(3,4,5)P3 (33%) reduced the activity to about one third of that at 12%.
These results suggested that there might be a limiting component in the extract, depleted above 12% PI(3,4,5)P3 or 4% PI(4,5)P2 at 50 mM total lipids. We therefore varied the concentration of total lipids, holding the phosphoinositide composition at 4%. F-actin increased linearly with increasing total lipids, from 3.8% of the total actin in the extract at 20 µM total lipids to 15.8% at 100 µM lipids (Fig. 3 D, b). The initial rate also increased linearly (data not shown). This argues against a shortage of cytoplasmic factors and suggests that the plateau of effects at 12% or more PI(4,5)P2 or PI(3,4,5)P3 reflects limits on each vesicle.
GTPS Broadens the Specificity of Phosphoinositides
for Actin Assembly
The effect of GTPS in stimulating actin assembly off endogenous vesicles suggested that it might be able to overcome unfavorable lipid or protein composition. We therefore retested the lipid specificity for actin assembly in the
presence of GTP
S. Although PI(3,4)P2-containing vesicles did not induce actin assembly in vesicle-free supernatants, incubation with 100 µM GTP
S before the addition
of PI(3,4)P2 stimulated these vesicles to induce actin assembly. In this case, there was a noticeable lag of ~100 s
between the addition of lipids and the beginning of increase in fluorescence (Fig. 4). Actin assembly was sensitive to the GTP
S concentration. Concentrations below
1 µM GTP
S had no stimulatory effect, but at 10 µM GTP
S,
PI(3,4)P2 was as effective as PI(4,5)P2 (Fig. 4 B). In control
experiments, GTP
S (100 µM) alone did not stimulate actin assembly (data not shown).
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The stimulation by GTPS was not limited to PI(3,4)P2-containing vesicles (Fig. 4 B). Both the rate and amount of
F-actin formation increased several fold in the presence of
GTP
S for vesicles containing PI(4)P or PI(3)P. For vesicles containing PI(4,5)P2 or PI(3,4,5)P3, the rate was almost doubled by 10 µM GTP
S, and the effect was dependent on the dose of GTP
S (Fig. 4 C). The effect of GTP
S
was specific for phosphoinositides, since there was very little stimulatory effect of GTP
S on PC/PI vesicles (Fig. 4
B). These results suggest that some but not all of the lipid
specificity can be suppressed by GTP
S.
It is worth noting that GTPS, at concentrations >100 µM,
also induced a delayed fluorescence increase followed by a
drop to almost the basal line (Fig. 4 A, right). This was due to
the aggregation of the added lipid vesicles because we noticed a visible lipid aggregate in the reaction mixture and
the loss of NBD-PC signal after the spike appeared. We
speculated that an actin-based contraction and/or gelation
system was activated by a G protein at high GTP
S doses.
Involvement of the Rho Family Small G Proteins in Phosphoinositide-induced Actin Assembly
GTPS is an agonist with a broad spectrum and can activate
any GTP-binding proteins in the extracts. To determine
whether the Rho family small G proteins were required for
GTP
S-induced actin assembly, we tested whether the activity was sensitive to RhoGDI, a protein that binds preferentially to GDP-bound proteins and prevents nucleotide
exchange (Boguski and McCormick, 1993
; Takai et al., 1993
).
To test whether RhoGDI could inhibit actin assembly
from the endogenous vesicles stimulated by GTPS, recombinant RhoGDI was purified from E. coli and incubated in high speed supernatants supplemented with endogenous vesicles before the addition of GTP
S. Actin
assembly was completely abolished in the presence of
RhoGDI at concentrations between 0.5 and 2.5 µM (Fig.
5 A). Since RhoGDI interacts with all three members of
the family (Takai et al., 1993
), we can only conclude that
some or all G proteins of the Rho family are involved in
GTP
S-induced actin assembly in egg extracts.
|
We further tested whether RhoGDI could block GTPS-
stimulated actin assembly by a variety of phosphoinositides
in high speed supernatants. RhoGDI (2 µM) abolished the
ability of GTP
S to stimulate PI(3,4)P2-induced actin assembly (Fig. 5 B, a). The dose for half maximum inhibition by
RhoGDI was between 80 and 400 nM (Fig. 5 B, c).
Surprisingly, RhoGDI inhibited not only the enhancement of PI(4,5)P2-dependent activity by GTPS, but the
basal activity as well. Fig. 5 B, b shows that F-actin-inducing activity of PI(4,5)P2 in the absence of GTP
S was
blocked by RhoGDI (2 µM). The inhibition was dependent on the concentration of RhoGDI, with similar effective doses for the GTP
S-stimulated assembly of PI(3,4)P2
vesicles (Fig. 5 B, d). This result suggests that the Rho
family small G proteins may play a role downstream of all
phosphoinositides in regulating actin assembly.
Special Role of Cdc42
Cdc42 Is Required for PI(4,5)P2-induced Actin Assembly.To characterize the small G proteins that were required
for PI(4,5)P2-induced activity, we first tested the dominant
active forms of small G proteins of the Rho family and
asked whether they could rescue RhoGDI inhibition.
Dominant active Cdc42V12 was purified from COS cell
membranes and added directly to the extracts without
loading with GTPS. Fig. 6 A shows that Cdc42V12 restored F-actin, inducing activity of PI(4,5)P2 in a dose-
dependent manner. In the absence of GTP
S, Cdc42V12
itself did not induce any actin assembly in the extracts. We
further tested whether Cdc42 was necessary using Cdc42N17, a dominant negative form. As shown in Fig. 6 B,
0.17 µM Cdc42N17 blocked PI(4,5)P2-induced actin assembly. In contrast, the dominant negative form RacN17 was not able to do so, even when present at a much higher
concentration (1.67 µM). These results suggest that Cdc42
may be both necessary and sufficient for PI(4,5)P2-induced
actin polymerization. Interestingly, Cdc42N17 only partially inhibited PI(3,4)P2- and PI(4,5)P2-induced actin assembly in the presence of GTP
S (Fig. 6 C). This suggests that GTP
S may activate other small G proteins in this
system.
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To test whether
Cdc42 alone could induce actin assembly, both dominant
active Cdc42V12 and RacV12 were added to the extracts. Neither had an effect on actin assembly (data not shown).
We reasoned that there might not be sufficient exchange
activities present in the extracts to activate the proteins
with GTP. We then loaded Cdc42V12 and RacV12 with
GTPS in the presence of EDTA before adding them to
high speed supernatants. As shown in Fig. 7 A, GTP
S-Cdc42 induced a significant amount of F-actin in a lipid- independent manner, with an assembly rate more than
half of that induced by PI(4,5)P2. In contrast, GTP
S-RacV12 did not generate any noticeable amount of F-actin.
The free GTP
S carried over from the loading reaction
was diluted to <4 µM and did not have any effect on its
own (Fig. 7 A, Control). In the visual assay using rhodamine
actin, vesicle-free extracts supplemented with GTP
S-Cdc42V12 contained many rhodamine foci as well as tail structures similar to those observed in the low speed extract (Fig.
7 B). These structures were completely absent in GTP
S-RacV12-treated extracts. Actin assembly stimulated by
activated Cdc42V12 requires prenylated proteins. Cdc42V12C189S, a CAAX box mutant devoid of prenylation,
was unable to stimulate actin assembly even when charged
with GTP
S (Fig. 7 A).
|
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Discussion |
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Molecular and pharmacological studies in intact cells have
suggested many signaling pathways that regulate the actin
cytoskeleton under physiological conditions (Machesky
and Hall, 1996; Ridley, 1996
; Zigmond, 1996
). Recently,
permeabilized cells have also been used to study actin assembly (Hartwig et al., 1995
; Tardif et al., 1995
; Barkalow et
al., 1996
). Due to the intimate association of actin assembly with the plasma membrane (Symons and Mitchison, 1991
), it is difficult to use conventional biochemical approaches, which have been developed to study the cytoskeleton in soluble systems, to study membrane-dependent regulation of the actin cytoskeleton.
In this report, we have demonstrated the use of Xenopus
egg extracts to reconstitute membrane-dependent actin assembly and to study both actin assembly and aspects of
signal transduction. Endogenous membranes can be stimulated to nucleate actin assembly in the presence of GTPS
or sodium orthovanadate. We have been able to define
some of the important functional membrane components and distinguish them from the soluble factors contributing
to actin assembly. We also demonstrate a key role for
Cdc42 in mediating phosphoinositide-stimulated actin assembly in this cell-free system. Finally, as described below,
we suggest a regulatory pathway involving phosphoinositides in both the catalysis of guanine-nucleotide exchange
(directly or indirectly) and the localization of activated small G proteins.
We have shown that PI(4,5)P2 and PI(3,4,5)P3 are able
to induce actin assembly in high speed supernatants. Since
other phosphoinositide isoforms and phospholipids are ineffective, it cannot be simply charge that is important for
PI(4,5)P2 and PI(3,4,5)P3 in regulating actin assembly. The
specificity of phosphoinositides is likely to be universally
applicable because high speed extracts contain a physiological mixture of well balanced actin binding proteins under which actin polymerization is globally inhibited but
locally activatable. For this reason, we propose that
PI(4,5)P2 and PI(3,4,5)P3 are important membrane signals
for actin assembly in vivo. At present we are unable to determine whether the apparently equal effectiveness of
PI(3,4,5)P3 and PI(4,5)P2 in inducing actin assembly is due
to the fact that one may be converted to another in the egg
extract. Wortmannin, a potent PI 3-kinase inhibitor (Arcaro and Wymann, 1993), does not interfere with PI(4,5)P2-
induced actin assembly and we have not seen phosphate
incorporation into PI(4,5)P2 in the extracts (data not shown).
These data suggest that PI(4,5)P2 itself is sufficient. However, the presence of a wortmannin-insensitive PI 3-kinase
could not be ruled out, and the conversion of a small
amount of PI(4,5)P2 into PI(3,4,5)P3 would be hard to detect. On the other hand, PI(3,4,5)P3 could be converted
back to PI(4,5)P2 if a 3-phosphatase were present. It is also
possible that both phosphoinositides are equally potent in
the extract.
The levels of PI(3,4,5)P3 and PI(4,5)P2 needed to generate actin assembly in egg extracts are surprisingly low. As
little as 1% will induce polymerization. The overall level
of PI(4,5)P2 and PI(3,4,5)P3 in cells is ~0.4% and <0.01%,
respectively (Auger et al., 1989). Since it is likely that their
amount is not uniformly distributed in the cell, our results
suggest that local concentrations of PI(3,4,5)P3 and PI(4,5)P2
could be high enough to drive actin assembly in cells. Actin assembly induced by these phosphoinositides is saturable at relatively low levels of PI(3,4,5)P3 and PI(4,5)P2 and
this is not due to saturation of downstream factors. Saturation has also been observed for the inhibition of gelsolin
(Janmey and Stossel, 1989
) and the activation of
-adrenergic receptor kinase (Pitcher et al., 1995
). This is possibly
due to the limited surface area of the vesicle. Alternatively, phosphoinositides in a PC/PI bilayer might form microdomains or boundaries within the bilayer, affecting
their interaction with downstream proteins. The extracts
also contain excess downstream factors because actin polymerization increases linearly with the concentration of
lipid vesicles up to 100 µM. If the lipid/protein ratio used
in our experiments was close to that in cells, we would conclude that Xenopus eggs and perhaps somatic cells would
never exhaust their downstream machinery for nucleating
actin polymerization.
Small G proteins of the Rho family are also able to induce
actin assembly in Xenopus extracts. GTPS can stimulate
both endogenous membranes and purified lipid vesicles
to assemble actin. RhoGDI blocks not only the GTP
S-
induced actin assembly, but also phosphoinositide-induced activity. All these results can be explained by the participation of the Rho family G proteins. Cdc42V12 rescues
RhoGDI inhibition (Fig. 6 A). Dominant negative Cdc42N17,
but not RacN17, inhibits phosphoinositide-induced actin
assembly (Fig. 6 B). These results suggest that Cdc42 is
involved in actin assembly induced by PI(3,4,5)P3 and PI(4,5)P2. Cdc42 also appears to be the target for GTP
S,
since Cdc42N17 inhibits GTP
S-stimulated phosphoinositide-dependent actin assembly (Fig. 6). However, the
inhibition is partial, suggesting that other members of the
Rho family also mediate GTP
S-induced assembly. More
strikingly, exogenous Cdc42, when charged with GTP
S, is
sufficient to induce actin assembly.
Actin polymerization induced by GTPS and Cdc42 has
been observed in cell lysates from polymorphonuclear leukocytes and Dictyostelium (Zigmond et al., 1997
). Bundles
and meshworks of F-actin are observed using phalloidin in
the low speed lysates from these cells. In our extracts when
rhodamine actin is used, the morphology of F-actin induced
by GTP
S is similar but has additional features, possibly due to different extract concentrations and assay methods.
F-actin does not only assemble into localized foci around
endogenous vesicles, but also incorporates into moving
comet tails. The comet tails and vesicle movements bear
striking similarity to Listeria motility (Theriot et al., 1994
;
Marchand et al., 1995
), endosome rocketing induced by
lanthanides in macrophages (Heuser, J.E., and J.H. Morisaki. 1992. Mol. Biol. Cell. 3:172a) and "inductopodia"
observed in Aplysia growth cones (Forscher et al., 1992
).
Conceivably, the mechanism for these actin-based motilities might be very similar.
The effects of phosphoinositides and Rho-family small
G proteins on actin assembly in Xenopus egg extracts appear to be dependent on each other. First, phosphoinositide-containing membranes are required for localized actin assembly: (a) GTPS induces actin assembly only in
low speed extracts, but not in extracts depleted of endogenous membranes, presumably containing phosphoinositides; (b) exogenous lipid vesicles containing phosphoinositides induce actin assembly in these vesicle-depleted
extracts; (c) only in the presence of exogenous phosphoinositide-containing vesicles does GTP
S induce actin assembly. Second, phosphoinositide-induced assembly requires
small G proteins: (a) RhoGDI inhibits GTP
S-induced actin assembly; (b) RhoGDI inhibits phosphoinositide-
induced assembly; (c) dominant negative Cdc42 blocks
phosphoinositide-induced assembly.
The requirement of phosphoinositides for GTPS-stimulated actin assembly can be explained by two scenarios:
(a) they stimulate an GEF activity to activate G proteins,
or (b) they promote translocation of G proteins to the
membrane and serve as docking surfaces for activated G
proteins to nucleate actin assembly. It has been shown recently that GEF activities are required for GTP
S-stimulated actin assembly in high speed supernatants from polymorphonuclear leukocytes, and anionic phospholipids
allow GTP
S to induce actin assembly (Zigmond et al.,
1997
). However, the latter scenario would explain our results better because we have observed different lipid specificity depending on the nucleotides used (see below). Small G proteins contain a CAAX box at their COOH terminals and are modified by prenylation. Although they
may interact with other proteins, prenyl groups are known
to be important for protein-membrane association. In
the cytosol, the attached prenyl group is embedded in
RhoGDI, which keeps G proteins in GDP forms (Boguski
and McCormick, 1993
). Phosphoinositides have recently
been shown to dissociate RhoGDI from the inactive Rac-GDI complex (Chuang et al., 1993
). Therefore, the requirement of phosphoinositides in our system is consistent
with the model that phosphoinositides translocate G protein to membranes. Endogenous small G proteins are
likely to be brought to the membrane by phosphoinositides and activated by nonhydrolyzable GTP
S through
basal nucleotide exchange. Actin assembly then occurs
from membrane-associated GTP
S-activated G proteins.
Further studies will be necessary to investigate the mechanism of this protein translocation and determine whether other cytosolic factors are required.
The requirement of phosphoinositides for actin assembly has another interesting aspect. We have observed very
different lipid specificities depending on the nucleotide
used. In the presence of endogenous GTP (but in the absence of GTPS), only extremely specific phosphoinositides, PI(4,5)P2 and PI(3,4,5)P3, induce actin assembly
(Fig. 3 B). In contrast, the specificity is broadened when
GTP
S is added and PI(3,4)P2, PI(4)P, and PI(3)P are now able to induce actin assembly (Fig. 4 B). A simple explanation for the difference in specificity is that only PI(3,4,5)P3
and PI(4,5)P2, but not other phosphoinositides, stimulate a
GEF activity, a physiological means to activate G proteins.
In the presence of GTP
S, the GEF activity is no longer
required for G protein activation. Lipid specificity is relaxed because phosphoinositides are only needed for
the G protein-membrane interaction (Fig. 8). All known
GEFs for the Rho family contain the pleckstrin homology (PH) domain (Cerione and Zheng, 1996
), a 120 amino
acid-long sequence thought to be important for interaction with phosphoinositides (Shaw, 1996
). Three related
proteins, cytohesin-1, ARNO, and Grp1, which all have an
amino-terminal PH domain and a sequence highly similar
to an exchange factor have recently been identified (Chardin et al., 1996
; Kolanus et al., 1996
; Klarlund et al., 1997
).
In addition, some PH domains have been shown to preferentially bind to PI(4,5)P2 and PI(3,4,5)P3 compared with
PI(3,4)P2 or other phosphoinositides (Rameh et al., 1997
).
These data suggest that PI(3,4,5)P3 and PI(4,5)P2 may recruit a GEF to the membrane and activate G proteins in
the vicinity. Alternatively, PI(4,5)P2 may itself activate G
proteins since in vitro studies have indicated that PI(4,5)P2
can function as an exchange factor (Zheng et al., 1996
).
|
Our data suggest that actin assembly through small G
proteins is dependent on phosphoinositides at two steps:
association with membranes and activation by GEF activities. However, the dependence on phosphoinositides
seems to be bypassed when exogenous GTPS-activated
Cdc42 is added to the extracts, as long as it contains an intact CAAX motif for prenylation. In this nonphysiological situation, Cdc42 is activated by GTP
S instead of GEFs,
and actin assembly is associated with Cdc42 aggregates instead of lipid membranes. We believe that the aggregation
of Cdc42 is essential for actin polymerization, since soluble
forms of Cdc42 (Cdc42V12C189S) are completely ineffectual in stimulating actin assembly in the absence of phosphoinositides (Fig. 7 A). However, since prenylation at the
CAAX box may have other functions, we can not rule out that soluble Cdc42 proteins are also able to stimulate actin
assembly. The establishment of quantitative assays for actin assembly in response to Cdc42 and specific phosphoinositides offers us an opportunity to identify and purify
factors regulating actin assembly at the cell membrane.
![]() |
Footnotes |
---|
Received for publication 11 September 1997 and in revised form 29 December 1997.
Address all correspondence to Marc Kirschner, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Tel.: 617-432-2250; Fax: 617-432-0420. E-mail: marc{at}hms.harvard.eduWe thank Dr. Ching-Shih Chen of University of Kentucky for synthesizing PI(3,4)P2 and PI(3,4,5)P3, Dr. Rolands Vegners of the Latvian Institute of Organic Synthesis (Riga, Latvia) for providing the gelsolin peptide, Drs. Julie Theriot and Rong Li for protocols. We also thank Margaret Chou and Kimberly Tolias for plasmid DNAs. We are grateful to Drs. James Sabry, Tim Mitchison, Rong Li, John Hartwig, Jack Taunton, Chris Carpenter, and members of the Kirschner lab for helpful discussion and critical reading of the manuscript. We especially thank Sally Zigmond for discussing her results on the role of Cdc42 in actin assembly before publication.
This work was supported by grants from the National Institutes of Health (GM26875 to M.W. Kirschner, P50-HL 56993 and R01-36622 to L.C. Cantley, and AR38910 to P.A. Janmey).
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Abbreviations used in this paper |
---|
F-actin, filamentous actin; G proteins, GTP-binding proteins, GDI, guanine-nucleotide dissociation inhibitor; GEF, guanine-nucleotide exchange factor; NBD, nitrobenzoxadiazole; PC, phosphatidylcholine; PI, phosphatidyinositol; PI(4,5)P2, PI (4,5) bisphosphate; PI(3,4)P2, PI (3,4) bisphosphate; PI(3,4,5)P3, PI (3,4,5) trisphosphate.
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