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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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Effect of Cdc42 on nucleation sites for pyrenyl actin.  (A) Dose response of Cdc42-induced increase of nucleation sites.  The supernatant was incubated for 5 min at 37°C with varying  concentrations of GTPγS-charged Cdc42 before dilution into 1.5  μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to  Materials and Methods). Data shown is from a representative experiment. The nucleation sites increase with concentration eventually reaching a plateau. The absolute levels of nucleation and  the concentration of Cdc42 at the plateau vary somewhat with  different supernatants and Cdc42 preparations. (B) Time course  of Cdc42-induced increase in nucleation sites. The supernatant  was incubated at 37°C with 100 nM GTPγS-charged Cdc42 for  various times before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined  from the pyrenyl fluorescence (refer to Materials and Methods).  Data shown is from a representative experiment.
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Figure 5: Effect of Cdc42 on nucleation sites for pyrenyl actin. (A) Dose response of Cdc42-induced increase of nucleation sites. The supernatant was incubated for 5 min at 37°C with varying concentrations of GTPγS-charged Cdc42 before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to Materials and Methods). Data shown is from a representative experiment. The nucleation sites increase with concentration eventually reaching a plateau. The absolute levels of nucleation and the concentration of Cdc42 at the plateau vary somewhat with different supernatants and Cdc42 preparations. (B) Time course of Cdc42-induced increase in nucleation sites. The supernatant was incubated at 37°C with 100 nM GTPγS-charged Cdc42 for various times before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to Materials and Methods). Data shown is from a representative experiment.

Mentions: The increase in filament number as a function of concentration and duration of incubation with Cdc42 was also detected as an increase in barbed-end nucleation sites for pyrenyl actin. Nucleation sites increased with Cdc42 concentration until a plateau level was reached (Fig. 5 A). The number of nucleation sites induced by 100 nM Cdc42 increased with time reaching a peak between 3 and 5 min (Fig. 5 B). With 100 nM Cdc42, there often was a lag before nucleation sites could be detected.


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)

Effect of Cdc42 on nucleation sites for pyrenyl actin.  (A) Dose response of Cdc42-induced increase of nucleation sites.  The supernatant was incubated for 5 min at 37°C with varying  concentrations of GTPγS-charged Cdc42 before dilution into 1.5  μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to  Materials and Methods). Data shown is from a representative experiment. The nucleation sites increase with concentration eventually reaching a plateau. The absolute levels of nucleation and  the concentration of Cdc42 at the plateau vary somewhat with  different supernatants and Cdc42 preparations. (B) Time course  of Cdc42-induced increase in nucleation sites. The supernatant  was incubated at 37°C with 100 nM GTPγS-charged Cdc42 for  various times before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined  from the pyrenyl fluorescence (refer to Materials and Methods).  Data shown is from a representative experiment.
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Figure 5: Effect of Cdc42 on nucleation sites for pyrenyl actin. (A) Dose response of Cdc42-induced increase of nucleation sites. The supernatant was incubated for 5 min at 37°C with varying concentrations of GTPγS-charged Cdc42 before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to Materials and Methods). Data shown is from a representative experiment. The nucleation sites increase with concentration eventually reaching a plateau. The absolute levels of nucleation and the concentration of Cdc42 at the plateau vary somewhat with different supernatants and Cdc42 preparations. (B) Time course of Cdc42-induced increase in nucleation sites. The supernatant was incubated at 37°C with 100 nM GTPγS-charged Cdc42 for various times before dilution into 1.5 μM pyrenyl-actin. The initial rate of polymerization of the pyrenyl-actin was determined from the pyrenyl fluorescence (refer to Materials and Methods). Data shown is from a representative experiment.
Mentions: The increase in filament number as a function of concentration and duration of incubation with Cdc42 was also detected as an increase in barbed-end nucleation sites for pyrenyl actin. Nucleation sites increased with Cdc42 concentration until a plateau level was reached (Fig. 5 A). The number of nucleation sites induced by 100 nM Cdc42 increased with time reaching a peak between 3 and 5 min (Fig. 5 B). With 100 nM Cdc42, there often was a lag before nucleation sites could be detected.

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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