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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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Match of models to data. The Cdc42-induced filaments  that contribute to Fig. 3 are replotted here as the absolute number of filaments falling in different length categories (bar graph).  Closed triangles, numbers predicted by Model 1 (refer to Materials and Methods), assuming that Cdc42 enhances the rate of profilin-actin mediated elongation by a factor fp = 4.5, compared to  that seen with spectrin-actin seeds, but elongation is terminated  at a rate similar to that of spectrin-actin seeds. The nucleation  rate used was 0.026 nM filaments/s. A chi-square test of goodness  of fit gave P > 0.1. Open circles, numbers predicted by Model 2,  with no acceleration of elongation (fp = 1.0), but an increase in  the duration of elongation by a factor of 4.6 (kcap = 0.025/s) compared to that of spectrin-actin seeds (kcap = 0.115/s). The nucleation rate used was 0.029 nM filaments/s. No meaningful chi-square could be calculated due to the very small predicted  numbers of longer filaments at the earlier times.
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Figure 8: Match of models to data. The Cdc42-induced filaments that contribute to Fig. 3 are replotted here as the absolute number of filaments falling in different length categories (bar graph). Closed triangles, numbers predicted by Model 1 (refer to Materials and Methods), assuming that Cdc42 enhances the rate of profilin-actin mediated elongation by a factor fp = 4.5, compared to that seen with spectrin-actin seeds, but elongation is terminated at a rate similar to that of spectrin-actin seeds. The nucleation rate used was 0.026 nM filaments/s. A chi-square test of goodness of fit gave P > 0.1. Open circles, numbers predicted by Model 2, with no acceleration of elongation (fp = 1.0), but an increase in the duration of elongation by a factor of 4.6 (kcap = 0.025/s) compared to that of spectrin-actin seeds (kcap = 0.115/s). The nucleation rate used was 0.029 nM filaments/s. No meaningful chi-square could be calculated due to the very small predicted numbers of longer filaments at the earlier times.

Mentions: As shown in Fig. 8, the filament length distribution of Cdc42-induced filaments at various times in the absence of phalloidin could be fit by Model 1. The fit required enhancing the rate of polymerization over that expected from homogenous concentrations of G-actin and profilin-actin (see Fig. 8 legend). This demonstrated that the small changes in filament length distribution over time did not require modification of the capping rate. The same parameters allowed a fit of the spectrin-actin seed filament length distribution (refer to Fig. 2 F) if the rate of elongation was assumed to be due to free G-actin alone, without any contribution by profilin-actin (data not shown). In contrast, it was not possible to fit both the early and late Cdc42-induced filament length distributions with Model 2. If the lengths of the filament population at later times was determined by capping, the lengths at earlier times would need to be shorter. In fact, increasing the half-time of capping beyond 6 s resulted in a less good fit to these data. If rapid elongation of Cdc42-induced filaments has a half-life longer than 6 s, a more complicated model is required to generate the distribution.


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)

Match of models to data. The Cdc42-induced filaments  that contribute to Fig. 3 are replotted here as the absolute number of filaments falling in different length categories (bar graph).  Closed triangles, numbers predicted by Model 1 (refer to Materials and Methods), assuming that Cdc42 enhances the rate of profilin-actin mediated elongation by a factor fp = 4.5, compared to  that seen with spectrin-actin seeds, but elongation is terminated  at a rate similar to that of spectrin-actin seeds. The nucleation  rate used was 0.026 nM filaments/s. A chi-square test of goodness  of fit gave P > 0.1. Open circles, numbers predicted by Model 2,  with no acceleration of elongation (fp = 1.0), but an increase in  the duration of elongation by a factor of 4.6 (kcap = 0.025/s) compared to that of spectrin-actin seeds (kcap = 0.115/s). The nucleation rate used was 0.029 nM filaments/s. No meaningful chi-square could be calculated due to the very small predicted  numbers of longer filaments at the earlier times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132886&req=5

Figure 8: Match of models to data. The Cdc42-induced filaments that contribute to Fig. 3 are replotted here as the absolute number of filaments falling in different length categories (bar graph). Closed triangles, numbers predicted by Model 1 (refer to Materials and Methods), assuming that Cdc42 enhances the rate of profilin-actin mediated elongation by a factor fp = 4.5, compared to that seen with spectrin-actin seeds, but elongation is terminated at a rate similar to that of spectrin-actin seeds. The nucleation rate used was 0.026 nM filaments/s. A chi-square test of goodness of fit gave P > 0.1. Open circles, numbers predicted by Model 2, with no acceleration of elongation (fp = 1.0), but an increase in the duration of elongation by a factor of 4.6 (kcap = 0.025/s) compared to that of spectrin-actin seeds (kcap = 0.115/s). The nucleation rate used was 0.029 nM filaments/s. No meaningful chi-square could be calculated due to the very small predicted numbers of longer filaments at the earlier times.
Mentions: As shown in Fig. 8, the filament length distribution of Cdc42-induced filaments at various times in the absence of phalloidin could be fit by Model 1. The fit required enhancing the rate of polymerization over that expected from homogenous concentrations of G-actin and profilin-actin (see Fig. 8 legend). This demonstrated that the small changes in filament length distribution over time did not require modification of the capping rate. The same parameters allowed a fit of the spectrin-actin seed filament length distribution (refer to Fig. 2 F) if the rate of elongation was assumed to be due to free G-actin alone, without any contribution by profilin-actin (data not shown). In contrast, it was not possible to fit both the early and late Cdc42-induced filament length distributions with Model 2. If the lengths of the filament population at later times was determined by capping, the lengths at earlier times would need to be shorter. In fact, increasing the half-time of capping beyond 6 s resulted in a less good fit to these data. If rapid elongation of Cdc42-induced filaments has a half-life longer than 6 s, a more complicated model is required to generate the distribution.

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.

Show MeSH