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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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The barbed ends of actin filaments point toward foci of  polymerization. Cdc42 was incubated in supernatant for 1 min  before dilution into buffer containing phalloidin followed by incubation in S-1 fragment of myosin (refer to Materials and Methods). Samples were rinsed in water before negative staining. Arrows parallel to some of the filaments indicate the orientation of  the arrowheads on the filaments. The orientation of 143 filaments  was determined in 34 photographs of foci from two different experiments. 75% of the filaments emanating from a focus and  whose orientation could be determined had their barbed ends at  the foci. Bar, 0.25 μm.
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Figure 7: The barbed ends of actin filaments point toward foci of polymerization. Cdc42 was incubated in supernatant for 1 min before dilution into buffer containing phalloidin followed by incubation in S-1 fragment of myosin (refer to Materials and Methods). Samples were rinsed in water before negative staining. Arrows parallel to some of the filaments indicate the orientation of the arrowheads on the filaments. The orientation of 143 filaments was determined in 34 photographs of foci from two different experiments. 75% of the filaments emanating from a focus and whose orientation could be determined had their barbed ends at the foci. Bar, 0.25 μm.

Mentions: The fact that enhanced elongation occurred selectively on the Cdc42-induced filaments suggested that Cdc42 or some Cdc42 target might remain associated with the barbed end during elongation. Geranylgeranylated Cdc42 often aggregates and then both the Cdc42 (as assayed by Western blots) and ability to induce polymerization are lost upon pelleting the Cdc42 preparation at high speed (Zigmond et al., 1997). Actin polymerization induced by Cdc42, when viewed by either fluorescence or electron microscopy occasionally emanated from foci. By S-1 labeling, the filaments emanating from these foci were observed to have their barbed ends preferentially (∼75% of the filaments) associated with the focus (Fig. 7). Although we have no direct evidence that Cdc42 is in these clumps, this filament orientation is similar to that of filaments induced at the leading edge of cells and by cytoplasmic Listeria and is consistent with the observation that, like Listeria, Cdc42 can mediate movement of lipid vesicles (Ma et al., 1998). Since filaments elongate at their barbed ends, it appears that the barbed end might for a time remain associated with factors that could affect their rate of elongation and/ or capping.


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)

The barbed ends of actin filaments point toward foci of  polymerization. Cdc42 was incubated in supernatant for 1 min  before dilution into buffer containing phalloidin followed by incubation in S-1 fragment of myosin (refer to Materials and Methods). Samples were rinsed in water before negative staining. Arrows parallel to some of the filaments indicate the orientation of  the arrowheads on the filaments. The orientation of 143 filaments  was determined in 34 photographs of foci from two different experiments. 75% of the filaments emanating from a focus and  whose orientation could be determined had their barbed ends at  the foci. Bar, 0.25 μm.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: The barbed ends of actin filaments point toward foci of polymerization. Cdc42 was incubated in supernatant for 1 min before dilution into buffer containing phalloidin followed by incubation in S-1 fragment of myosin (refer to Materials and Methods). Samples were rinsed in water before negative staining. Arrows parallel to some of the filaments indicate the orientation of the arrowheads on the filaments. The orientation of 143 filaments was determined in 34 photographs of foci from two different experiments. 75% of the filaments emanating from a focus and whose orientation could be determined had their barbed ends at the foci. Bar, 0.25 μm.
Mentions: The fact that enhanced elongation occurred selectively on the Cdc42-induced filaments suggested that Cdc42 or some Cdc42 target might remain associated with the barbed end during elongation. Geranylgeranylated Cdc42 often aggregates and then both the Cdc42 (as assayed by Western blots) and ability to induce polymerization are lost upon pelleting the Cdc42 preparation at high speed (Zigmond et al., 1997). Actin polymerization induced by Cdc42, when viewed by either fluorescence or electron microscopy occasionally emanated from foci. By S-1 labeling, the filaments emanating from these foci were observed to have their barbed ends preferentially (∼75% of the filaments) associated with the focus (Fig. 7). Although we have no direct evidence that Cdc42 is in these clumps, this filament orientation is similar to that of filaments induced at the leading edge of cells and by cytoplasmic Listeria and is consistent with the observation that, like Listeria, Cdc42 can mediate movement of lipid vesicles (Ma et al., 1998). Since filaments elongate at their barbed ends, it appears that the barbed end might for a time remain associated with factors that could affect their rate of elongation and/ or capping.

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