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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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Association of capping protein with the low and  high speed Triton-insoluble  cytoskeleton after stimulation at 10°C in the presence  and absence of phalloidin.  AX3 cells were starved at  22°C and transferred to 10°C  as described in Materials and  Methods. 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 alone (A and B), or  L buffer containing 20 μM  phalloidin (C and D). Low  speed Triton-insoluble cytoskeleton pellets (A and C)  were recovered by centrifugation in a microfuge for 3  min at 8,700 g. High speed  Triton-insoluble cytoskeleton  pellets (B and D) were recovered by centrifugation for  1 h at 415,000 g in a Beckman  TLA-100.2 rotator, a force  sufficient to pellet individual  actin filaments. The low speed  or high speed cytoskeleton pellets were resuspended on ice in 20% 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. Values represent data from three separate experiments ± the standard deviation.
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Figure 2: Association of capping protein with the low and high speed Triton-insoluble cytoskeleton after stimulation at 10°C in the presence and absence of phalloidin. AX3 cells were starved at 22°C and transferred to 10°C as described in Materials and Methods. 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 alone (A and B), or L buffer containing 20 μM phalloidin (C and D). Low speed Triton-insoluble cytoskeleton pellets (A and C) were recovered by centrifugation in a microfuge for 3 min at 8,700 g. High speed Triton-insoluble cytoskeleton pellets (B and D) were recovered by centrifugation for 1 h at 415,000 g in a Beckman TLA-100.2 rotator, a force sufficient to pellet individual actin filaments. The low speed or high speed cytoskeleton pellets were resuspended on ice in 20% 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. Values represent data from three separate experiments ± the standard deviation.

Mentions: Equilibrating starved Dictyostelium amebas at 10°C before cAMP stimulation allowed changes in the association of capping protein with the low speed Triton-insoluble cytoskeleton during the actin nucleation response to be monitored with higher temporal resolution. At ∼20 s after stimulation, the levels of F-actin increased ∼2.8-fold, relative to unstimulated control levels, whereas the level of capping protein associated with the low speed cytoskeleton increased ∼2.5-fold (Fig. 2 A; Table I). At no time during the initial phase of actin nucleation was capping protein observed to exit the low speed cytoskeleton.


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

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

Association of capping protein with the low and  high speed Triton-insoluble  cytoskeleton after stimulation at 10°C in the presence  and absence of phalloidin.  AX3 cells were starved at  22°C and transferred to 10°C  as described in Materials and  Methods. 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 alone (A and B), or  L buffer containing 20 μM  phalloidin (C and D). Low  speed Triton-insoluble cytoskeleton pellets (A and C)  were recovered by centrifugation in a microfuge for 3  min at 8,700 g. High speed  Triton-insoluble cytoskeleton  pellets (B and D) were recovered by centrifugation for  1 h at 415,000 g in a Beckman  TLA-100.2 rotator, a force  sufficient to pellet individual  actin filaments. The low speed  or high speed cytoskeleton pellets were resuspended on ice in 20% 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. Values represent data from three separate experiments ± the standard deviation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140204&req=5

Figure 2: Association of capping protein with the low and high speed Triton-insoluble cytoskeleton after stimulation at 10°C in the presence and absence of phalloidin. AX3 cells were starved at 22°C and transferred to 10°C as described in Materials and Methods. 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 alone (A and B), or L buffer containing 20 μM phalloidin (C and D). Low speed Triton-insoluble cytoskeleton pellets (A and C) were recovered by centrifugation in a microfuge for 3 min at 8,700 g. High speed Triton-insoluble cytoskeleton pellets (B and D) were recovered by centrifugation for 1 h at 415,000 g in a Beckman TLA-100.2 rotator, a force sufficient to pellet individual actin filaments. The low speed or high speed cytoskeleton pellets were resuspended on ice in 20% 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. Values represent data from three separate experiments ± the standard deviation.
Mentions: Equilibrating starved Dictyostelium amebas at 10°C before cAMP stimulation allowed changes in the association of capping protein with the low speed Triton-insoluble cytoskeleton during the actin nucleation response to be monitored with higher temporal resolution. At ∼20 s after stimulation, the levels of F-actin increased ∼2.8-fold, relative to unstimulated control levels, whereas the level of capping protein associated with the low speed cytoskeleton increased ∼2.5-fold (Fig. 2 A; Table I). At no time during the initial phase of actin nucleation was capping protein observed to exit the low speed cytoskeleton.

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