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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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Addition of spectrin-actin seeds to cell supernatants.  (A) Effects of spectrin-actin seeds and/or Cdc42 on actin polymerization. Supernatants were incubated at 37°C with buffer  (open circles), spectrin-actin seeds (open triangles), 100 nM  GTPγS-charged Cdc42 (closed circles) or both spectrin-actin  seeds and 100 GTPγS-Cdc42 (closed triangles), for 2 or 5 min before the F-actin levels were determined by the TRITC-phalloidin  staining of pelletable material (refer to Materials and Methods).  For t = 0, the seeds were added after a 15-fold dilution of supernatant. Data are from a single experiment representative of  three. (B) Spectrin actin seeds become rapidly capped when incubated with supernatant. Spectrin-actin seeds were incubated in  supernatants for 5, 10, or 60 s before the supernatant was diluted  100-fold into 1.5 μM pyrenyl-actin. Polymerization of the pyrenyl- actin was then followed over time from the pyrenyl fluorescence  (refer to Materials and Methods). For the time 0 point, the seeds  were added after supernatant to the pyrenyl-actin. Data shown  are representative samples. (C) Time course of capping was determined from the decrease in initial rate of polymerization. The  initial rate of increase in pyrenyl fluorescence (proportional to  the number of elongating filaments) is plotted versus the duration  of incubation of the seeds with the supernatant. The data, expressed as percent of seed-induced initial rate at the start of incubation, are from supernatants (closed squares) at 3 mg/ml protein  (as used for most experiments) or 0.75 mg/ml (closed triangles).  The half-time of capping, in supernatants at 3 mg/ml, ranged between 3 and 9 s in different supernatants.
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Figure 1: Addition of spectrin-actin seeds to cell supernatants. (A) Effects of spectrin-actin seeds and/or Cdc42 on actin polymerization. Supernatants were incubated at 37°C with buffer (open circles), spectrin-actin seeds (open triangles), 100 nM GTPγS-charged Cdc42 (closed circles) or both spectrin-actin seeds and 100 GTPγS-Cdc42 (closed triangles), for 2 or 5 min before the F-actin levels were determined by the TRITC-phalloidin staining of pelletable material (refer to Materials and Methods). For t = 0, the seeds were added after a 15-fold dilution of supernatant. Data are from a single experiment representative of three. (B) Spectrin actin seeds become rapidly capped when incubated with supernatant. Spectrin-actin seeds were incubated in supernatants for 5, 10, or 60 s before the supernatant was diluted 100-fold into 1.5 μM pyrenyl-actin. Polymerization of the pyrenyl- actin was then followed over time from the pyrenyl fluorescence (refer to Materials and Methods). For the time 0 point, the seeds were added after supernatant to the pyrenyl-actin. Data shown are representative samples. (C) Time course of capping was determined from the decrease in initial rate of polymerization. The initial rate of increase in pyrenyl fluorescence (proportional to the number of elongating filaments) is plotted versus the duration of incubation of the seeds with the supernatant. The data, expressed as percent of seed-induced initial rate at the start of incubation, are from supernatants (closed squares) at 3 mg/ml protein (as used for most experiments) or 0.75 mg/ml (closed triangles). The half-time of capping, in supernatants at 3 mg/ml, ranged between 3 and 9 s in different supernatants.

Mentions: Free barbed ends, in the form of spectrin-actin seeds, added to cell supernatants caused no actin polymerization (Fig. 1 A). The spectrin-actin seeds contain some F-actin (seen as an increase in TRITC-phalloidin staining even at time = 0) which was maintained during incubation, suggesting that the seeds did not depolymerize. In the experiment shown in Fig. 1 A, the concentration of spectrin-actin seeds added had ∼20 times more barbed-end nucleating sites (determined from the initial rate of pyrenyl-actin polymerization) than those present in supernatant incubated for 5 min with 100 nM Cdc42. Addition of 10- or 100-fold lower concentrations of spectrin-actin seeds also did not induce polymerization (data not shown). To rule out the possibility that the failure to induce polymerization was unique to spectrin-actin seeds, we added barbed ends in the form of sheared actin filaments. These also did not induce detectable polymerization. Thus, it appeared that addition of free barbed ends is not sufficient to induce polymerization.


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)

Addition of spectrin-actin seeds to cell supernatants.  (A) Effects of spectrin-actin seeds and/or Cdc42 on actin polymerization. Supernatants were incubated at 37°C with buffer  (open circles), spectrin-actin seeds (open triangles), 100 nM  GTPγS-charged Cdc42 (closed circles) or both spectrin-actin  seeds and 100 GTPγS-Cdc42 (closed triangles), for 2 or 5 min before the F-actin levels were determined by the TRITC-phalloidin  staining of pelletable material (refer to Materials and Methods).  For t = 0, the seeds were added after a 15-fold dilution of supernatant. Data are from a single experiment representative of  three. (B) Spectrin actin seeds become rapidly capped when incubated with supernatant. Spectrin-actin seeds were incubated in  supernatants for 5, 10, or 60 s before the supernatant was diluted  100-fold into 1.5 μM pyrenyl-actin. Polymerization of the pyrenyl- actin was then followed over time from the pyrenyl fluorescence  (refer to Materials and Methods). For the time 0 point, the seeds  were added after supernatant to the pyrenyl-actin. Data shown  are representative samples. (C) Time course of capping was determined from the decrease in initial rate of polymerization. The  initial rate of increase in pyrenyl fluorescence (proportional to  the number of elongating filaments) is plotted versus the duration  of incubation of the seeds with the supernatant. The data, expressed as percent of seed-induced initial rate at the start of incubation, are from supernatants (closed squares) at 3 mg/ml protein  (as used for most experiments) or 0.75 mg/ml (closed triangles).  The half-time of capping, in supernatants at 3 mg/ml, ranged between 3 and 9 s in different supernatants.
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Related In: Results  -  Collection

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Figure 1: Addition of spectrin-actin seeds to cell supernatants. (A) Effects of spectrin-actin seeds and/or Cdc42 on actin polymerization. Supernatants were incubated at 37°C with buffer (open circles), spectrin-actin seeds (open triangles), 100 nM GTPγS-charged Cdc42 (closed circles) or both spectrin-actin seeds and 100 GTPγS-Cdc42 (closed triangles), for 2 or 5 min before the F-actin levels were determined by the TRITC-phalloidin staining of pelletable material (refer to Materials and Methods). For t = 0, the seeds were added after a 15-fold dilution of supernatant. Data are from a single experiment representative of three. (B) Spectrin actin seeds become rapidly capped when incubated with supernatant. Spectrin-actin seeds were incubated in supernatants for 5, 10, or 60 s before the supernatant was diluted 100-fold into 1.5 μM pyrenyl-actin. Polymerization of the pyrenyl- actin was then followed over time from the pyrenyl fluorescence (refer to Materials and Methods). For the time 0 point, the seeds were added after supernatant to the pyrenyl-actin. Data shown are representative samples. (C) Time course of capping was determined from the decrease in initial rate of polymerization. The initial rate of increase in pyrenyl fluorescence (proportional to the number of elongating filaments) is plotted versus the duration of incubation of the seeds with the supernatant. The data, expressed as percent of seed-induced initial rate at the start of incubation, are from supernatants (closed squares) at 3 mg/ml protein (as used for most experiments) or 0.75 mg/ml (closed triangles). The half-time of capping, in supernatants at 3 mg/ml, ranged between 3 and 9 s in different supernatants.
Mentions: Free barbed ends, in the form of spectrin-actin seeds, added to cell supernatants caused no actin polymerization (Fig. 1 A). The spectrin-actin seeds contain some F-actin (seen as an increase in TRITC-phalloidin staining even at time = 0) which was maintained during incubation, suggesting that the seeds did not depolymerize. In the experiment shown in Fig. 1 A, the concentration of spectrin-actin seeds added had ∼20 times more barbed-end nucleating sites (determined from the initial rate of pyrenyl-actin polymerization) than those present in supernatant incubated for 5 min with 100 nM Cdc42. Addition of 10- or 100-fold lower concentrations of spectrin-actin seeds also did not induce polymerization (data not shown). To rule out the possibility that the failure to induce polymerization was unique to spectrin-actin seeds, we added barbed ends in the form of sheared actin filaments. These also did not induce detectable polymerization. Thus, it appeared that addition of free barbed ends is not sufficient to induce polymerization.

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