Limits...
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.

Show MeSH
Time course of filament length distributions formed in  the presence of phalloidin. Supernatants were incubated with  Cdc42 as in Fig. 2 but 1 μM phalloidin was present during the incubation. After negative staining and photographing, filaments  were measured and pooled as in Fig 3. For comparison, the filament number at each time point was normalized to a total of 231  filaments. The number of photographs analyzed and filaments actually measured at each time point was: at 30 s, four photographs  with a total of 137 filaments; at 1 min, seven photographs with  231 filaments; and at 5 min, four photographs with 143 filaments.  The mean length at 30 s was 1.5 μm; at 1 min, 1.5 μm; and at 5  min, 3.7 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132886&req=5

Figure 6: Time course of filament length distributions formed in the presence of phalloidin. Supernatants were incubated with Cdc42 as in Fig. 2 but 1 μM phalloidin was present during the incubation. After negative staining and photographing, filaments were measured and pooled as in Fig 3. For comparison, the filament number at each time point was normalized to a total of 231 filaments. The number of photographs analyzed and filaments actually measured at each time point was: at 30 s, four photographs with a total of 137 filaments; at 1 min, seven photographs with 231 filaments; and at 5 min, four photographs with 143 filaments. The mean length at 30 s was 1.5 μm; at 1 min, 1.5 μm; and at 5 min, 3.7 μm.

Mentions: The length distributions of Cdc42-induced filaments present after 30 s, 1 min, or 5 min of cubation in supernatants containing 100 nM Cdc42 and phalloidin are shown in Fig. 6. As in the absence of phalloidin, the filament length distributions after incubation for 30 s and 1 min were similar. A direct comparison of filament length distributions induced by Cdc42 in the same supernatant with or without phalloidin confirmed that after 1 min of incubation, there was little difference between the length distributions of Cdc42-induced filaments in the presence or absence of phalloidin (data not shown). This indicates that during the first minute, Cdc42-induced filaments, even in the absence of phalloidin, are not depolymerizing (or being cut by cofilin since this is also inhibited by phalloidin). However, after a 5-min incubation, the presence of phalloidin causes a shift toward longer filaments: the mean length went from 1.5 μm (n = 231) at 1 min to 3.6 μm (n = 143) at 5 min. Thus, in the presence of phalloidin, Cdc42-induced filaments did elongate between 1 and 5 min.


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)

Time course of filament length distributions formed in  the presence of phalloidin. Supernatants were incubated with  Cdc42 as in Fig. 2 but 1 μM phalloidin was present during the incubation. After negative staining and photographing, filaments  were measured and pooled as in Fig 3. For comparison, the filament number at each time point was normalized to a total of 231  filaments. The number of photographs analyzed and filaments actually measured at each time point was: at 30 s, four photographs  with a total of 137 filaments; at 1 min, seven photographs with  231 filaments; and at 5 min, four photographs with 143 filaments.  The mean length at 30 s was 1.5 μm; at 1 min, 1.5 μm; and at 5  min, 3.7 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132886&req=5

Figure 6: Time course of filament length distributions formed in the presence of phalloidin. Supernatants were incubated with Cdc42 as in Fig. 2 but 1 μM phalloidin was present during the incubation. After negative staining and photographing, filaments were measured and pooled as in Fig 3. For comparison, the filament number at each time point was normalized to a total of 231 filaments. The number of photographs analyzed and filaments actually measured at each time point was: at 30 s, four photographs with a total of 137 filaments; at 1 min, seven photographs with 231 filaments; and at 5 min, four photographs with 143 filaments. The mean length at 30 s was 1.5 μm; at 1 min, 1.5 μm; and at 5 min, 3.7 μm.
Mentions: The length distributions of Cdc42-induced filaments present after 30 s, 1 min, or 5 min of cubation in supernatants containing 100 nM Cdc42 and phalloidin are shown in Fig. 6. As in the absence of phalloidin, the filament length distributions after incubation for 30 s and 1 min were similar. A direct comparison of filament length distributions induced by Cdc42 in the same supernatant with or without phalloidin confirmed that after 1 min of incubation, there was little difference between the length distributions of Cdc42-induced filaments in the presence or absence of phalloidin (data not shown). This indicates that during the first minute, Cdc42-induced filaments, even in the absence of phalloidin, are not depolymerizing (or being cut by cofilin since this is also inhibited by phalloidin). However, after a 5-min incubation, the presence of phalloidin causes a shift toward longer filaments: the mean length went from 1.5 μm (n = 231) at 1 min to 3.6 μm (n = 143) at 5 min. Thus, in the presence of phalloidin, Cdc42-induced filaments did elongate between 1 and 5 min.

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