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Figure 2. Induction of actin assembly in high speed supernatants by lipid vesicles containing different phosphoinositides. (AâF) Rhodamine images show the difference of actin assembly. High speed supernatants were mixed with PC/PI vesicles containing no phosphoinositides (A), 33% PI(3,4,5)P3 (B), 33% PI(4,5)P2 (C), 33% PI(3,4)P2 (D), 33% PI(4)P (E), or 33% PI(3)P (F). (G) Two moving comet tails induced by PI(3,4,5)P3 are shown in sequential frames 30 s apart. (H) Four examples of color-overlaid images. Note that NBD-vesicles (green) containing PI(3,4,5)P3 are associated with rhodamine-actin comet tails (red). Bars: (AâG) 10 μm; (H) 5 μm.
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Figure 3. Characterization of phosphoinositide-induced actin assembly in high speed supernatants using pyrene actin. (A) Time course of change in pyrene fluorescence in high speed supernatants treated with PC/PI alone (dotted line), or PC/PI vesicles containing 4% PI(3,4,5)P3 (solid line), or 4% PI(4,5)P2 (dashed line). The lipid concentration was 50 μM. The arrow indicates the time when the lipid was added. (B) Comparison of the inductive activity among different phosphoinositides in PC/PI vesicles (4% phosphoinositides in 50 μM total lipids) as measured by the initial rate of actin assembly and the maximum F-actin at the peak. All activities are normalized to that of PI(4,5)P2. (C) Inhibition of PI(4,5)P2-induced actin assembly in high speed supernatants by cytochalasin D or a peptide (QRLFQVKGRR) derived from gelsolin. Each reaction included 50-μM lipid vesicles containing 4% PI(4,5)P2. Inhibition of PI(3,4,5)P3-induced activity is similar and is not shown here. (D) Dose response of actin assembly to phosphoinositides. (a) F-actin induced by 50-μM PC/PI vesicles containing different percentages of PI(3,4,5)P3 (solid line) or PI(4,5)P2 (dashed line). (b) F-actin induced by vesicles containing 4% PI(4,5)P2 in high speed supernatants at various total lipid concentrations (expressed as total PI(4,5)P2). The data fit into a linear curve (dashed line). The initial rate has very similar doseâ response profiles (not shown). All activities are normalized to that of phosphoinositides at 4% in 50 μM total lipids. All data in BâD are expressed as mean ± SEM calculated from at least two sets of normalized data.
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Figure 4. Effects of GTPγS on phosphoinositide-induced actin assembly in high speed supernatants. (A) Time course of actin assembly induced by lipid vesicles containing 25% PI(3,4)P2 or PI(4,5)P2 followed by pyrene fluorescence. High speed supernatants were treated with (solid line) or without (dashed line) 0.1 mM GTPγS for 5 min, and then stimulated with lipid vesicles (50 μM). (B) Comparison of the effect of GTPγS on different lipid vesicles. Experiments were done as in A and lipid vesicles contained 25% of each phosphoinositide. All activities were normalized to that of PI(4,5)P2 in the absence of GTPγS. (C) Effects of GTPγS dose on phosphoinositide-induced actin assembly. Again, experiments were done as in A.
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Figure 5. Inhibition of actin assembly by RhoGDI. (A) Inhibition of vesicle-dependent actin assembly in low speed extracts as viewed with rhodamine actin. Endogenous vesicles were stimulated with 100 μM GTPγS in extracts that were preincubated without (a) or with (b) 2.5 μM recombinant RhoGDI for 5 min. (B) Inhibition of phosphoinositide-induced actin assembly. Actin assembly induced by vesicles containing 25% PI(3,4)P2 (a and c) or 25% PI(4,5)P2 (b and d) in the absence (solid line) or presence (dashed line) of 2 μM RhoGDI. In a and c, high speed supernatants were preincubated with 10 μM GTPγS and RhoGDI for 5 min. In b and d, GTPγS was omitted. Lipid vesicles were then added to 50 μM final concentration. a and b are time courses of actin assembly monitored with pyrene actin. c and d show the dose effect of RhoGDI.
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Figure 6. Involvement of Cdc42 in PI(4,5)P2-induced actin assembly in high speed supernatants. (A) Cdc42V12 rescues the inhibition of PI(4,5)P2-induced actin assembly by RhoGDI. Cdc42V12 without GTPγS was added to high speed supernatants along with RhoGDI (0.4 μM). After a 5-min incubation, vesicles containing 25% PI(4,5)P2 were added to a final concentration of 60 μM. (B) Inhibition of PI(4,5)P2-induced actin assembly in high speed supernatants by the dominant negative form of Cdc42, but not that of Rac. High speed supernatants were preincubated with either Cdc42N17 or RacN17 for 5 min, and then stimulated with 60 μM PI(4,5)P2-containing vesicles (12%). (C) Partial inhibition of GTPγS-stimulated actin assembly by Cdc42N17 in high speed supernatants. Extracts were incubated with or without 0.34 μM Cdc42N17 for 5 min before the addition of 10 μM GTPγS and 60 μM phosphoinositide-containing vesicles (12% for PI(4,5)P2 and 25% for PI(3,4)P2). In all experiments, actin assembly was monitored using pyrene actin and the activities were normalized to the controls.
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Figure 7. Actin assembly induced by GTPγS-charged Cdc42 in high speed supernatants. (A) Time course of actin assembly in high speed supernatants incubated with 0.17 μM Cdc42V12-GTPγS (solid line), 0.83 μM RacV12-GTPγS (dashed line), buffer (dotted line) or 0.17 μM Cdc42V12C189S-GTPγS (dotted and dashed line). G proteins were preloaded with 40 μM GTPγS, and then added to high speed supernatants containing pyrene actin. (B) As in A, but viewed with rhodamine actin.
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Figure 8. A schematic diagram of Cdc42 activation by phosphoinositides. GDP-Cdc42 is complexed with RhoGDI in the cytosol. Phosphoinositides of broad specificity dissociate RhoGDI from the complex and bring Cdc42 to the membrane through the prenyl group. Cdc42 is then activated in the presence of specific phosphoinositides (i.e., PI(4,5)P2 or PI(3,4,5)P3), which either recruits a GEF or directly acts as a GEF. Alternatively, GTPγS can activate Cdc42 at this step (not shown here). In both cases, activated Cdc42 leads to actin assembly at the cell membrane.
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