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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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Incorporation of exogenous rhodamine-labeled G-actin  and localization of capping protein in resting and stimulated Dictyostelium at 10°C. AX3 cells were starved as described in Fig. 3  and stimulated with 10 μM 2′ deoxy-cAMP at 10°C for 0 (rest)  and 20 s (+cAMP). Cells were then permeablized with 0.1% Triton X-100 in the presence of 20 μM phalloidin, rinsed, and incubated with 0.45 μM rhodamine-labeled G-actin, prepared as described (Chan et al., 1997), for 5 min. Polymerization was  terminated by fixation and stained for capping protein as described. Rhodamine-labeled G-actin (a and b); anti–capping protein-α (c and d). Images shown are representative of typical cells  most commonly observed in these experiments.
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Figure 5: Incorporation of exogenous rhodamine-labeled G-actin and localization of capping protein in resting and stimulated Dictyostelium at 10°C. AX3 cells were starved as described in Fig. 3 and stimulated with 10 μM 2′ deoxy-cAMP at 10°C for 0 (rest) and 20 s (+cAMP). Cells were then permeablized with 0.1% Triton X-100 in the presence of 20 μM phalloidin, rinsed, and incubated with 0.45 μM rhodamine-labeled G-actin, prepared as described (Chan et al., 1997), for 5 min. Polymerization was terminated by fixation and stained for capping protein as described. Rhodamine-labeled G-actin (a and b); anti–capping protein-α (c and d). Images shown are representative of typical cells most commonly observed in these experiments.

Mentions: The localization of capping protein mirrors changes in F-actin localization in response to stimulation, suggesting that capping protein binds to the barbed ends of actin filaments. To investigate this further, the localization of capping protein relative to regions of the cell containing barbed ends after stimulation was determined using a rhodamine-labeled G-actin polymerization assay. To visualize sites of actin nucleation and, therefore, free barbed filament ends in situ, we incubated permeabilized, phalloidin-stabilized Dictyostelium cells with exogenous rhodamine-labeled G-actin under polymerizing conditions. This technique has been used to visualize the distribution of free barbed ends in permeabilized fibroblasts (Symons and Mitchison, 1991), polymorphonuclear leukocytes (Redmond and Zigmond, 1993), and mammary adenocarcinoma cells (Chan et al., 1997). Unstimulated cells that possessed a polarized morphology contained sites of rhodamine-labeled G-actin incorporation primarily in the anterior pseudopod (Fig. 5 a). No incorporation of rhodamine-labeled G-actin was observed in the presence cytochalasin D (data not shown) indicating that sites of rhodamine-labeled G-actin incorporation represent free barbed ends. The pattern of rhodamine-labeled G-actin incorporation colocalized with capping protein staining (Fig. 5, a and c). At 20 s after cAMP stimulation at 10°C, there was a redistribution of the incorporation of rhodamine-labeled G-actin and capping protein staining to the entire cell cortex (Fig. 5, b and d), consistent with the redistribution of F-actin globally throughout the cell cortex after stimulation (Figs. 3 c and d, and 4 c and d), and as observed by Wessels et al. (1989). These results demonstrate that sites of increased free barbed ends, F-actin, and capping protein are all colocalized to the cell cortex in response to cAMP stimulation.


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

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

Incorporation of exogenous rhodamine-labeled G-actin  and localization of capping protein in resting and stimulated Dictyostelium at 10°C. AX3 cells were starved as described in Fig. 3  and stimulated with 10 μM 2′ deoxy-cAMP at 10°C for 0 (rest)  and 20 s (+cAMP). Cells were then permeablized with 0.1% Triton X-100 in the presence of 20 μM phalloidin, rinsed, and incubated with 0.45 μM rhodamine-labeled G-actin, prepared as described (Chan et al., 1997), for 5 min. Polymerization was  terminated by fixation and stained for capping protein as described. Rhodamine-labeled G-actin (a and b); anti–capping protein-α (c and d). Images shown are representative of typical cells  most commonly observed in these experiments.
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Related In: Results  -  Collection

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Figure 5: Incorporation of exogenous rhodamine-labeled G-actin and localization of capping protein in resting and stimulated Dictyostelium at 10°C. AX3 cells were starved as described in Fig. 3 and stimulated with 10 μM 2′ deoxy-cAMP at 10°C for 0 (rest) and 20 s (+cAMP). Cells were then permeablized with 0.1% Triton X-100 in the presence of 20 μM phalloidin, rinsed, and incubated with 0.45 μM rhodamine-labeled G-actin, prepared as described (Chan et al., 1997), for 5 min. Polymerization was terminated by fixation and stained for capping protein as described. Rhodamine-labeled G-actin (a and b); anti–capping protein-α (c and d). Images shown are representative of typical cells most commonly observed in these experiments.
Mentions: The localization of capping protein mirrors changes in F-actin localization in response to stimulation, suggesting that capping protein binds to the barbed ends of actin filaments. To investigate this further, the localization of capping protein relative to regions of the cell containing barbed ends after stimulation was determined using a rhodamine-labeled G-actin polymerization assay. To visualize sites of actin nucleation and, therefore, free barbed filament ends in situ, we incubated permeabilized, phalloidin-stabilized Dictyostelium cells with exogenous rhodamine-labeled G-actin under polymerizing conditions. This technique has been used to visualize the distribution of free barbed ends in permeabilized fibroblasts (Symons and Mitchison, 1991), polymorphonuclear leukocytes (Redmond and Zigmond, 1993), and mammary adenocarcinoma cells (Chan et al., 1997). Unstimulated cells that possessed a polarized morphology contained sites of rhodamine-labeled G-actin incorporation primarily in the anterior pseudopod (Fig. 5 a). No incorporation of rhodamine-labeled G-actin was observed in the presence cytochalasin D (data not shown) indicating that sites of rhodamine-labeled G-actin incorporation represent free barbed ends. The pattern of rhodamine-labeled G-actin incorporation colocalized with capping protein staining (Fig. 5, a and c). At 20 s after cAMP stimulation at 10°C, there was a redistribution of the incorporation of rhodamine-labeled G-actin and capping protein staining to the entire cell cortex (Fig. 5, b and d), consistent with the redistribution of F-actin globally throughout the cell cortex after stimulation (Figs. 3 c and d, and 4 c and d), and as observed by Wessels et al. (1989). These results demonstrate that sites of increased free barbed ends, F-actin, and capping protein are all colocalized to the cell cortex in response to cAMP stimulation.

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