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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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Electron microscopy of Cdc42-induced and  spectrin-actin seed-induced  filaments. Representative  electron micrograph of negative-stained sample (left column) for: (A) 100 nM Cdc42  incubated in supernatant for  1 min; (B) spectrin-actin  seeds (1.5 nM) incubated for  2 min in supernatant; (C)  spectrin-actin seeds incubated for 30 s in 1.5 μM pure  actin; (D) spectrin-actin  seeds (1.5 nM) incubated in  supernatant for 5 min in the  presence of 1 μM phalloidin.  Filament length distributions  (right column) E–H measured for samples illustrated  in A–D, respectively. The  lengths of all filaments  present longer than 0.25 μm  were measured from photographs (equal to 4.5 mm on  the photo). The data are expressed as the number of filaments on the y axis with a  length equal to the value ±  0.25 μm on the x axis. Thus,  all filaments with lengths between 0.25 to 0.75 μm are  represented by the bar labeled 0.5, those with lengths  0.75–1.25 μm are represented  by the bar labeled 1.0, etc. In  each case the total filament  number has been normalized  to 100. Actual counts for each  sample were: E, 102; F, 59; G,  175; H, 67. The mean lengths  were: E, 2.1 μm; F, 0.4 μm;  G, 1.1 μm; and H, 0.5 μm.
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Figure 2: Electron microscopy of Cdc42-induced and spectrin-actin seed-induced filaments. Representative electron micrograph of negative-stained sample (left column) for: (A) 100 nM Cdc42 incubated in supernatant for 1 min; (B) spectrin-actin seeds (1.5 nM) incubated for 2 min in supernatant; (C) spectrin-actin seeds incubated for 30 s in 1.5 μM pure actin; (D) spectrin-actin seeds (1.5 nM) incubated in supernatant for 5 min in the presence of 1 μM phalloidin. Filament length distributions (right column) E–H measured for samples illustrated in A–D, respectively. The lengths of all filaments present longer than 0.25 μm were measured from photographs (equal to 4.5 mm on the photo). The data are expressed as the number of filaments on the y axis with a length equal to the value ± 0.25 μm on the x axis. Thus, all filaments with lengths between 0.25 to 0.75 μm are represented by the bar labeled 0.5, those with lengths 0.75–1.25 μm are represented by the bar labeled 1.0, etc. In each case the total filament number has been normalized to 100. Actual counts for each sample were: E, 102; F, 59; G, 175; H, 67. The mean lengths were: E, 2.1 μm; F, 0.4 μm; G, 1.1 μm; and H, 0.5 μm.

Mentions: Filaments induced by Cdc42 can be observed after negative staining in the electron microscope. Incubation of 100 nM Cdc42 with supernatant for 1 min resulted in the appearance of many filaments (Fig. 2 A). The lengths of the filaments were measured (refer to Materials and Methods) and the distribution of their lengths is shown in Fig 2 E. The mean length of filaments was 2 μm. Consistent with their inability to induce polymerization, spectrin-actin seeds incubated with supernatant produced only few short filaments (Fig. 2, B and F). This was not a result of the seeds being ineffective nucleators since after a 30-s incubation with 1.5 μM pure actin, they produced many filaments with a mean length of 1.1 μm (Fig. 2, C and G).


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)

Electron microscopy of Cdc42-induced and  spectrin-actin seed-induced  filaments. Representative  electron micrograph of negative-stained sample (left column) for: (A) 100 nM Cdc42  incubated in supernatant for  1 min; (B) spectrin-actin  seeds (1.5 nM) incubated for  2 min in supernatant; (C)  spectrin-actin seeds incubated for 30 s in 1.5 μM pure  actin; (D) spectrin-actin  seeds (1.5 nM) incubated in  supernatant for 5 min in the  presence of 1 μM phalloidin.  Filament length distributions  (right column) E–H measured for samples illustrated  in A–D, respectively. The  lengths of all filaments  present longer than 0.25 μm  were measured from photographs (equal to 4.5 mm on  the photo). The data are expressed as the number of filaments on the y axis with a  length equal to the value ±  0.25 μm on the x axis. Thus,  all filaments with lengths between 0.25 to 0.75 μm are  represented by the bar labeled 0.5, those with lengths  0.75–1.25 μm are represented  by the bar labeled 1.0, etc. In  each case the total filament  number has been normalized  to 100. Actual counts for each  sample were: E, 102; F, 59; G,  175; H, 67. The mean lengths  were: E, 2.1 μm; F, 0.4 μm;  G, 1.1 μm; and H, 0.5 μm.
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Figure 2: Electron microscopy of Cdc42-induced and spectrin-actin seed-induced filaments. Representative electron micrograph of negative-stained sample (left column) for: (A) 100 nM Cdc42 incubated in supernatant for 1 min; (B) spectrin-actin seeds (1.5 nM) incubated for 2 min in supernatant; (C) spectrin-actin seeds incubated for 30 s in 1.5 μM pure actin; (D) spectrin-actin seeds (1.5 nM) incubated in supernatant for 5 min in the presence of 1 μM phalloidin. Filament length distributions (right column) E–H measured for samples illustrated in A–D, respectively. The lengths of all filaments present longer than 0.25 μm were measured from photographs (equal to 4.5 mm on the photo). The data are expressed as the number of filaments on the y axis with a length equal to the value ± 0.25 μm on the x axis. Thus, all filaments with lengths between 0.25 to 0.75 μm are represented by the bar labeled 0.5, those with lengths 0.75–1.25 μm are represented by the bar labeled 1.0, etc. In each case the total filament number has been normalized to 100. Actual counts for each sample were: E, 102; F, 59; G, 175; H, 67. The mean lengths were: E, 2.1 μm; F, 0.4 μm; G, 1.1 μm; and H, 0.5 μm.
Mentions: Filaments induced by Cdc42 can be observed after negative staining in the electron microscope. Incubation of 100 nM Cdc42 with supernatant for 1 min resulted in the appearance of many filaments (Fig. 2 A). The lengths of the filaments were measured (refer to Materials and Methods) and the distribution of their lengths is shown in Fig 2 E. The mean length of filaments was 2 μm. Consistent with their inability to induce polymerization, spectrin-actin seeds incubated with supernatant produced only few short filaments (Fig. 2, B and F). This was not a result of the seeds being ineffective nucleators since after a 30-s incubation with 1.5 μM pure actin, they produced many filaments with a mean length of 1.1 μm (Fig. 2, C and G).

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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