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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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Immunofluorescence of capping protein in  resting and stimulated Dictyostelium cells at 10°C. AX3  cells were starved for 5 h and  allowed to settle onto coverslips for 30 min at 22°C, and  then transferred to 10°C for  30 min. For whole cell preparations, cells were fixed at 0  (Rest) and 20 s (+cAMP) after  stimulation with 10 μM 2′  deoxy-cAMP. Phase contrast: (a and b); rhodamine-phalloidin (c and d); anti– capping protein-α (e and f).  Images shown are representative of typical cells most  commonly observed in these  experiments. Confocal images  represent 0.8–1.0-μm-thick  optical sections of the stained  cells.
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Figure 3: Immunofluorescence of capping protein in resting and stimulated Dictyostelium cells at 10°C. AX3 cells were starved for 5 h and allowed to settle onto coverslips for 30 min at 22°C, and then transferred to 10°C for 30 min. For whole cell preparations, cells were fixed at 0 (Rest) and 20 s (+cAMP) after stimulation with 10 μM 2′ deoxy-cAMP. Phase contrast: (a and b); rhodamine-phalloidin (c and d); anti– capping protein-α (e and f). Images shown are representative of typical cells most commonly observed in these experiments. Confocal images represent 0.8–1.0-μm-thick optical sections of the stained cells.

Mentions: Previous studies by Wessels et al. (1989) showed that before cAMP treatment, polarized Dictyostelium amebas exhibit an intense accumulation of F-actin in anterior pseudopodia as shown by fluorescein-phalloidin staining. However, 5 s after stimulation at 22°C and concomitant with the peak of actin nucleation/polymerization detected in vitro, the F-actin staining is lost from the pseudopodia and becomes relocalized almost globally throughout the cell cortex, just below the plasma membrane. To determine the in situ localization of capping protein in response to cAMP stimulation, we performed indirect immunofluorescence using capping protein antibodies on glutaraldehyde-fixed whole cells and Triton-insoluble cytoskeletons. F-actin localization by rhodamine-phalloidin staining of both unstimulated whole cells and Triton-insoluble cytoskeletons was primarily in pseudopodia of highly polarized cells, in addition to circumferential staining of the cell cortex in cells displaying a flattened, rounded morphology (Figs. 3 c and 4 c). Capping protein displayed a punctate cytosolic localization in both unstimulated whole cells (Fig. 3 e) and Triton-insoluble cytoskeletons (Fig. 4 e) with a more pronounced staining in areas of F-actin localization, particularly in the cell cortex (Figs. 3 c and 4 c).


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

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

Immunofluorescence of capping protein in  resting and stimulated Dictyostelium cells at 10°C. AX3  cells were starved for 5 h and  allowed to settle onto coverslips for 30 min at 22°C, and  then transferred to 10°C for  30 min. For whole cell preparations, cells were fixed at 0  (Rest) and 20 s (+cAMP) after  stimulation with 10 μM 2′  deoxy-cAMP. Phase contrast: (a and b); rhodamine-phalloidin (c and d); anti– capping protein-α (e and f).  Images shown are representative of typical cells most  commonly observed in these  experiments. Confocal images  represent 0.8–1.0-μm-thick  optical sections of the stained  cells.
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Figure 3: Immunofluorescence of capping protein in resting and stimulated Dictyostelium cells at 10°C. AX3 cells were starved for 5 h and allowed to settle onto coverslips for 30 min at 22°C, and then transferred to 10°C for 30 min. For whole cell preparations, cells were fixed at 0 (Rest) and 20 s (+cAMP) after stimulation with 10 μM 2′ deoxy-cAMP. Phase contrast: (a and b); rhodamine-phalloidin (c and d); anti– capping protein-α (e and f). Images shown are representative of typical cells most commonly observed in these experiments. Confocal images represent 0.8–1.0-μm-thick optical sections of the stained cells.
Mentions: Previous studies by Wessels et al. (1989) showed that before cAMP treatment, polarized Dictyostelium amebas exhibit an intense accumulation of F-actin in anterior pseudopodia as shown by fluorescein-phalloidin staining. However, 5 s after stimulation at 22°C and concomitant with the peak of actin nucleation/polymerization detected in vitro, the F-actin staining is lost from the pseudopodia and becomes relocalized almost globally throughout the cell cortex, just below the plasma membrane. To determine the in situ localization of capping protein in response to cAMP stimulation, we performed indirect immunofluorescence using capping protein antibodies on glutaraldehyde-fixed whole cells and Triton-insoluble cytoskeletons. F-actin localization by rhodamine-phalloidin staining of both unstimulated whole cells and Triton-insoluble cytoskeletons was primarily in pseudopodia of highly polarized cells, in addition to circumferential staining of the cell cortex in cells displaying a flattened, rounded morphology (Figs. 3 c and 4 c). Capping protein displayed a punctate cytosolic localization in both unstimulated whole cells (Fig. 3 e) and Triton-insoluble cytoskeletons (Fig. 4 e) with a more pronounced staining in areas of F-actin localization, particularly in the cell cortex (Figs. 3 c and 4 c).

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