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Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium.

Eddy RJ, Han J, Condeelis JS - J. Cell Biol. (1997)

Bottom Line: The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell.We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34.An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.

ABSTRACT
The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

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(A) Association of capping protein with the low speed  Triton-insoluble cytoskeleton after cAMP stimulation at 22°C.  Dictyostelium AX3 cells (107 cells/ml) in 20 mM phosphate buffer, pH 6.6, were starved in suspension for 5.5 h at 22°C, and then  treated with 3 mM caffeine for 30 min. At various times after  stimulation with 10 μM 2′ deoxy-cAMP, 2 × 106 cells/ml (final)  were lysed in L buffer containing 0.5% Triton X-100. Lysates  were immediately microfuged for 3 min at 8,700 g and the low  speed Triton-insoluble cytoskeleton pellets were resuspended to  20% of lysate volume and Western blotted using anti–capping  protein-α antibodies followed by densitometry. Actin levels in cytoskeleton pellets were determined by densitometry of Coomassie blue staining of the 42-kD actin band after SDS-PAGE.  The 5-s time point value represents data from three separate determinations. (B) Relative actin nucleation rate of Dictyostelium  lysates after stimulation at 22°C and 10°C. AX3 cells were starved  at 4–6 × 106 cells/ml at 22°C and transferred to either 22° or 10°C  as described in Materials and Methods. At various times after  stimulation with 10 μM 2′ deoxy-cAMP, 106 cells/ml (final) were  lysed in L buffer containing 0.5% Triton X-100. 2 μM G-actin  (30% pyrene labeled) was immediately added to the lysate, and  the initial rate of actin polymerization was monitored as an increase in pyrene fluorescence. Relative nucleation rate is defined  as the ratio of initial rate in stimulated lysates to initial rate in  resting lysates. Points represent data from three separate experiments ± the standard deviation.
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Figure 1: (A) Association of capping protein with the low speed Triton-insoluble cytoskeleton after cAMP stimulation at 22°C. Dictyostelium AX3 cells (107 cells/ml) in 20 mM phosphate buffer, pH 6.6, were starved in suspension for 5.5 h at 22°C, and then treated with 3 mM caffeine for 30 min. At various times after stimulation with 10 μM 2′ deoxy-cAMP, 2 × 106 cells/ml (final) were lysed in L buffer containing 0.5% Triton X-100. Lysates were immediately microfuged for 3 min at 8,700 g and the low speed Triton-insoluble cytoskeleton pellets were resuspended to 20% of lysate volume and Western blotted using anti–capping protein-α antibodies followed by densitometry. Actin levels in cytoskeleton pellets were determined by densitometry of Coomassie blue staining of the 42-kD actin band after SDS-PAGE. The 5-s time point value represents data from three separate determinations. (B) Relative actin nucleation rate of Dictyostelium lysates after stimulation at 22°C and 10°C. AX3 cells were starved at 4–6 × 106 cells/ml at 22°C and transferred to either 22° or 10°C as described in Materials and Methods. At various times after stimulation with 10 μM 2′ deoxy-cAMP, 106 cells/ml (final) were lysed in L buffer containing 0.5% Triton X-100. 2 μM G-actin (30% pyrene labeled) was immediately added to the lysate, and the initial rate of actin polymerization was monitored as an increase in pyrene fluorescence. Relative nucleation rate is defined as the ratio of initial rate in stimulated lysates to initial rate in resting lysates. Points represent data from three separate experiments ± the standard deviation.

Mentions: To test whether the uncapping of actin filaments by capping protein is a viable mechanism for the exposure of free barbed ends, low speed cytoskeletons were prepared from Triton X-100 lysates prepared at various times after cAMP stimulation of 6-h starved Dictyostelium at 22°C. The low speed Triton-insoluble fraction has been shown previously to contain all of the nucleation activity present in stimulated cells (Hall et al., 1989). The levels of both F-actin and capping protein in the low speed cytoskeleton were analyzed by densitometry of Coomassie blue–stained gels and immunoblots, respectively. At ∼5 s after stimulation with cAMP, the levels of F-actin increased ∼2.3-fold relative to unstimulated control levels, whereas the level of capping protein associated with the low speed cytoskeleton increased ∼2.2-fold (Fig. 1 A). To eliminate the possibility that the association of capping protein with Triton-insoluble cytoskeletons may be nonspecific due to the presence of detergent, cells were lysed by forced passage through 3-μm nucleopore membranes (Millipore Corp.) in the absence of Triton X-100. Low speed pellet fractions prepared from nucleopore lysates of Dictyostelium cells are enriched in F-actin and membrane sheets (Das and Henderson, 1983). The low speed cytoskeleton fraction prepared by nucleopore lysis displayed a peak increase of ∼2.1- and ∼2.35-fold in F-actin and capping protein, respectively, relative to prestimulatory levels at ∼5 s following cAMP stimulation. These results suggest that capping protein is not lost from the cross-linked cytoskeleton in response to stimulation as predicted in an uncapping mechanism, but rather shows an increased association with the cross-linked cytoskeleton during the peak of actin nucleation.


Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium.

Eddy RJ, Han J, Condeelis JS - J. Cell Biol. (1997)

(A) Association of capping protein with the low speed  Triton-insoluble cytoskeleton after cAMP stimulation at 22°C.  Dictyostelium AX3 cells (107 cells/ml) in 20 mM phosphate buffer, pH 6.6, were starved in suspension for 5.5 h at 22°C, and then  treated with 3 mM caffeine for 30 min. At various times after  stimulation with 10 μM 2′ deoxy-cAMP, 2 × 106 cells/ml (final)  were lysed in L buffer containing 0.5% Triton X-100. Lysates  were immediately microfuged for 3 min at 8,700 g and the low  speed Triton-insoluble cytoskeleton pellets were resuspended to  20% of lysate volume and Western blotted using anti–capping  protein-α antibodies followed by densitometry. Actin levels in cytoskeleton pellets were determined by densitometry of Coomassie blue staining of the 42-kD actin band after SDS-PAGE.  The 5-s time point value represents data from three separate determinations. (B) Relative actin nucleation rate of Dictyostelium  lysates after stimulation at 22°C and 10°C. AX3 cells were starved  at 4–6 × 106 cells/ml at 22°C and transferred to either 22° or 10°C  as described in Materials and Methods. At various times after  stimulation with 10 μM 2′ deoxy-cAMP, 106 cells/ml (final) were  lysed in L buffer containing 0.5% Triton X-100. 2 μM G-actin  (30% pyrene labeled) was immediately added to the lysate, and  the initial rate of actin polymerization was monitored as an increase in pyrene fluorescence. Relative nucleation rate is defined  as the ratio of initial rate in stimulated lysates to initial rate in  resting lysates. Points represent data from three separate experiments ± the standard deviation.
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Figure 1: (A) Association of capping protein with the low speed Triton-insoluble cytoskeleton after cAMP stimulation at 22°C. Dictyostelium AX3 cells (107 cells/ml) in 20 mM phosphate buffer, pH 6.6, were starved in suspension for 5.5 h at 22°C, and then treated with 3 mM caffeine for 30 min. At various times after stimulation with 10 μM 2′ deoxy-cAMP, 2 × 106 cells/ml (final) were lysed in L buffer containing 0.5% Triton X-100. Lysates were immediately microfuged for 3 min at 8,700 g and the low speed Triton-insoluble cytoskeleton pellets were resuspended to 20% of lysate volume and Western blotted using anti–capping protein-α antibodies followed by densitometry. Actin levels in cytoskeleton pellets were determined by densitometry of Coomassie blue staining of the 42-kD actin band after SDS-PAGE. The 5-s time point value represents data from three separate determinations. (B) Relative actin nucleation rate of Dictyostelium lysates after stimulation at 22°C and 10°C. AX3 cells were starved at 4–6 × 106 cells/ml at 22°C and transferred to either 22° or 10°C as described in Materials and Methods. At various times after stimulation with 10 μM 2′ deoxy-cAMP, 106 cells/ml (final) were lysed in L buffer containing 0.5% Triton X-100. 2 μM G-actin (30% pyrene labeled) was immediately added to the lysate, and the initial rate of actin polymerization was monitored as an increase in pyrene fluorescence. Relative nucleation rate is defined as the ratio of initial rate in stimulated lysates to initial rate in resting lysates. Points represent data from three separate experiments ± the standard deviation.
Mentions: To test whether the uncapping of actin filaments by capping protein is a viable mechanism for the exposure of free barbed ends, low speed cytoskeletons were prepared from Triton X-100 lysates prepared at various times after cAMP stimulation of 6-h starved Dictyostelium at 22°C. The low speed Triton-insoluble fraction has been shown previously to contain all of the nucleation activity present in stimulated cells (Hall et al., 1989). The levels of both F-actin and capping protein in the low speed cytoskeleton were analyzed by densitometry of Coomassie blue–stained gels and immunoblots, respectively. At ∼5 s after stimulation with cAMP, the levels of F-actin increased ∼2.3-fold relative to unstimulated control levels, whereas the level of capping protein associated with the low speed cytoskeleton increased ∼2.2-fold (Fig. 1 A). To eliminate the possibility that the association of capping protein with Triton-insoluble cytoskeletons may be nonspecific due to the presence of detergent, cells were lysed by forced passage through 3-μm nucleopore membranes (Millipore Corp.) in the absence of Triton X-100. Low speed pellet fractions prepared from nucleopore lysates of Dictyostelium cells are enriched in F-actin and membrane sheets (Das and Henderson, 1983). The low speed cytoskeleton fraction prepared by nucleopore lysis displayed a peak increase of ∼2.1- and ∼2.35-fold in F-actin and capping protein, respectively, relative to prestimulatory levels at ∼5 s following cAMP stimulation. These results suggest that capping protein is not lost from the cross-linked cytoskeleton in response to stimulation as predicted in an uncapping mechanism, but rather shows an increased association with the cross-linked cytoskeleton during the peak of actin nucleation.

Bottom Line: The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell.We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34.An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.

ABSTRACT
The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

Show MeSH
Related in: MedlinePlus