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Mechanism of Cdc42-induced actin polymerization in neutrophil extracts.

Zigmond SH, Joyce M, Yang C, Brown K, Huang M, Pring M - J. Cell Biol. (1998)

Bottom Line: Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s.There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number.These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania. Philadelphia, Pennsylvania 19104-6018, USA. szigmond@sas.upenn.edu

ABSTRACT
Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

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Comparison of filaments induced by 800 versus  100 nM Cdc42. Supernatants  were incubated with 800 or  100 nM Cdc42 for 30 s, 1 min,  or 5 min before negative  staining. Photographed filaments were measured and  pooled as in Fig. 2. For comparison, the filament number  at each time point was normalized to a total of 100 filaments. The number of photographs analyzed and filaments  actually measured at each  time point was: for 800 nM  Cdc42 at 30 s, two photographs with total of 127 filaments; for 1 min, four photographs with 127 filaments;  and for 5 min, three photographs with 198 filaments.  The mean length at 30 s was  1.7 μm; at 1 min, 1.4 μm; and  at 5 min, 1.6 μm. For 100 nM  Cdc42 at 30 s, there were  three photographs with total  of 47 filaments; at 1 min,  three photographs with 102  filaments; and at 5 min, four  photographs with 196 filaments. The mean length at 30 s  was 1.7 μm; at 1 min, 2.1 μm;  and at 5 min, 1.9 μm.
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Figure 4: Comparison of filaments induced by 800 versus 100 nM Cdc42. Supernatants were incubated with 800 or 100 nM Cdc42 for 30 s, 1 min, or 5 min before negative staining. Photographed filaments were measured and pooled as in Fig. 2. For comparison, the filament number at each time point was normalized to a total of 100 filaments. The number of photographs analyzed and filaments actually measured at each time point was: for 800 nM Cdc42 at 30 s, two photographs with total of 127 filaments; for 1 min, four photographs with 127 filaments; and for 5 min, three photographs with 198 filaments. The mean length at 30 s was 1.7 μm; at 1 min, 1.4 μm; and at 5 min, 1.6 μm. For 100 nM Cdc42 at 30 s, there were three photographs with total of 47 filaments; at 1 min, three photographs with 102 filaments; and at 5 min, four photographs with 196 filaments. The mean length at 30 s was 1.7 μm; at 1 min, 2.1 μm; and at 5 min, 1.9 μm.

Mentions: The rate of polymerization is a function of the Cdc42 concentration (Zigmond et al., 1997). Increasing the concentration of Cdc42 from 100 to 800 nM increased the number of filaments observed on the grid at early times; however, increasing the Cdc42 concentration had little effect on the distribution of filament lengths (Fig. 4). Some decrease in filament length after longer incubation in 800 nM Cdc42 might reflect the fact that a limited amount of G-actin must now be distributed in a larger number of filaments.


Mechanism of Cdc42-induced actin polymerization in neutrophil extracts.

Zigmond SH, Joyce M, Yang C, Brown K, Huang M, Pring M - J. Cell Biol. (1998)

Comparison of filaments induced by 800 versus  100 nM Cdc42. Supernatants  were incubated with 800 or  100 nM Cdc42 for 30 s, 1 min,  or 5 min before negative  staining. Photographed filaments were measured and  pooled as in Fig. 2. For comparison, the filament number  at each time point was normalized to a total of 100 filaments. The number of photographs analyzed and filaments  actually measured at each  time point was: for 800 nM  Cdc42 at 30 s, two photographs with total of 127 filaments; for 1 min, four photographs with 127 filaments;  and for 5 min, three photographs with 198 filaments.  The mean length at 30 s was  1.7 μm; at 1 min, 1.4 μm; and  at 5 min, 1.6 μm. For 100 nM  Cdc42 at 30 s, there were  three photographs with total  of 47 filaments; at 1 min,  three photographs with 102  filaments; and at 5 min, four  photographs with 196 filaments. The mean length at 30 s  was 1.7 μm; at 1 min, 2.1 μm;  and at 5 min, 1.9 μm.
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Figure 4: Comparison of filaments induced by 800 versus 100 nM Cdc42. Supernatants were incubated with 800 or 100 nM Cdc42 for 30 s, 1 min, or 5 min before negative staining. Photographed filaments were measured and pooled as in Fig. 2. For comparison, the filament number at each time point was normalized to a total of 100 filaments. The number of photographs analyzed and filaments actually measured at each time point was: for 800 nM Cdc42 at 30 s, two photographs with total of 127 filaments; for 1 min, four photographs with 127 filaments; and for 5 min, three photographs with 198 filaments. The mean length at 30 s was 1.7 μm; at 1 min, 1.4 μm; and at 5 min, 1.6 μm. For 100 nM Cdc42 at 30 s, there were three photographs with total of 47 filaments; at 1 min, three photographs with 102 filaments; and at 5 min, four photographs with 196 filaments. The mean length at 30 s was 1.7 μm; at 1 min, 2.1 μm; and at 5 min, 1.9 μm.
Mentions: The rate of polymerization is a function of the Cdc42 concentration (Zigmond et al., 1997). Increasing the concentration of Cdc42 from 100 to 800 nM increased the number of filaments observed on the grid at early times; however, increasing the Cdc42 concentration had little effect on the distribution of filament lengths (Fig. 4). Some decrease in filament length after longer incubation in 800 nM Cdc42 might reflect the fact that a limited amount of G-actin must now be distributed in a larger number of filaments.

Bottom Line: Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s.There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number.These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania. Philadelphia, Pennsylvania 19104-6018, USA. szigmond@sas.upenn.edu

ABSTRACT
Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

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