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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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Length distribution of filaments induced by Cdc42. Supernatant was incubated in 100 nM Cdc42 for 15 s (top), 30 s  (middle), or 5 min (bottom) before negative staining. Filament  lengths are displayed as described in Fig. 2. For comparison between times the total number of filaments at each time was normalized to 76; actual number of filaments measured at 15 s was 76  in six photos, at 30 s was 64 filaments in three photos, and at 5  min was 406 filaments in four photos. The mean filament length  at 15 s was 1.4 μm; at 30 s, 1.7 μm; and at 5 min, 1.8 μm.
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Figure 3: Length distribution of filaments induced by Cdc42. Supernatant was incubated in 100 nM Cdc42 for 15 s (top), 30 s (middle), or 5 min (bottom) before negative staining. Filament lengths are displayed as described in Fig. 2. For comparison between times the total number of filaments at each time was normalized to 76; actual number of filaments measured at 15 s was 76 in six photos, at 30 s was 64 filaments in three photos, and at 5 min was 406 filaments in four photos. The mean filament length at 15 s was 1.4 μm; at 30 s, 1.7 μm; and at 5 min, 1.8 μm.

Mentions: To estimate the rate of Cdc42-induced filament elongation, the lengths of filaments present in supernatants incubated for various times with Cdc42 were determined (Fig. 3). Cdc42-induced filaments elongated rapidly, reaching a mean length of 1.4 μm at 15 s. An effective G-actin concentration (i.e., the sum of all free actin and actin complexes able to contribute to elongation) of ∼3.3 μM would be required for a filament to elongate to 1.4 μm in 15 s (given an actin barbed-end on rate of 107 M−1s−1 and assuming no depolymerization or capping). This estimate is conservative since it assumes that all filaments were present at time 0 and continued to elongate throughout the 15 s. A higher concentration would be required if there is a lag before Cdc42 produces free barbed ends and if new filaments are produced continuously during incubation (see below). A higher concentration would also be required if elongation were transient, e.g. if some filaments were capped within 15 s. A more realistic estimate of effective G-actin concentration is at least double this, i.e., 6.6 μM.


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)

Length distribution of filaments induced by Cdc42. Supernatant was incubated in 100 nM Cdc42 for 15 s (top), 30 s  (middle), or 5 min (bottom) before negative staining. Filament  lengths are displayed as described in Fig. 2. For comparison between times the total number of filaments at each time was normalized to 76; actual number of filaments measured at 15 s was 76  in six photos, at 30 s was 64 filaments in three photos, and at 5  min was 406 filaments in four photos. The mean filament length  at 15 s was 1.4 μm; at 30 s, 1.7 μm; and at 5 min, 1.8 μm.
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Related In: Results  -  Collection

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Figure 3: Length distribution of filaments induced by Cdc42. Supernatant was incubated in 100 nM Cdc42 for 15 s (top), 30 s (middle), or 5 min (bottom) before negative staining. Filament lengths are displayed as described in Fig. 2. For comparison between times the total number of filaments at each time was normalized to 76; actual number of filaments measured at 15 s was 76 in six photos, at 30 s was 64 filaments in three photos, and at 5 min was 406 filaments in four photos. The mean filament length at 15 s was 1.4 μm; at 30 s, 1.7 μm; and at 5 min, 1.8 μm.
Mentions: To estimate the rate of Cdc42-induced filament elongation, the lengths of filaments present in supernatants incubated for various times with Cdc42 were determined (Fig. 3). Cdc42-induced filaments elongated rapidly, reaching a mean length of 1.4 μm at 15 s. An effective G-actin concentration (i.e., the sum of all free actin and actin complexes able to contribute to elongation) of ∼3.3 μM would be required for a filament to elongate to 1.4 μm in 15 s (given an actin barbed-end on rate of 107 M−1s−1 and assuming no depolymerization or capping). This estimate is conservative since it assumes that all filaments were present at time 0 and continued to elongate throughout the 15 s. A higher concentration would be required if there is a lag before Cdc42 produces free barbed ends and if new filaments are produced continuously during incubation (see below). A higher concentration would also be required if elongation were transient, e.g. if some filaments were capped within 15 s. A more realistic estimate of effective G-actin concentration is at least double this, i.e., 6.6 μM.

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