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Comparisons of CapG and gelsolin- macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing.

Witke W, Li W, Kwiatkowski DJ, Southwick FS - J. Cell Biol. (2001)

Bottom Line: However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function.These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages.These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a Capg allele engineered into mice. Both CapG- mice and CapG/gelsolin double- mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG- macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

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Quantitation of the ruffling responses of wild-type and Capg−/− macrophages. (A) Bar graphs comparing the ruffling indexes of wild-type and Capg−/− macrophages before and after exposure to MCSF. The ruffling index was determined based on analysis of phalloidin staining by a blinded observer (Materials and methods). In the first four bars, brackets represent the SEM for 80–100 determinations/condition. Wild-type cells have a higher spontaneous ruffling index and nearly double their ruffling activity in response to MCSF, whereas Capg−/− macrophages have a lower spontaneous ruffling activity and fail to respond to MCSF. The far right bar quantifies the ruffling activity of Capg−/− cells stimulated with MCSF after introduction of recombinant CapG by microinjection. Bracket represents the SEM of n = 34 cells. CSF, MCSF. (B) Bar graphs comparing the ruffling responses of wild-type and Capg−/− macrophages after exposure to Salmonella (Salm.). Unlike MCSF which failed to stimulate ruffling in Capg−/− macrophages, exposure to Salmonella resulted in a significant increase in ruffling activity (P < 0.0001). Brackets represent the SEM of n = 80–100 measurements. Cells were scored as described in A. (C and D) Phase micrographs of wild-type (C) and Capg−/− (D) macrophages after 30-min exposure to Salmonella. Note the giant phagolysosomes in both the wild-type and Capg−/− cells induced by exposure to the bacteria. Arrows point to individual bacteria contained in giant phagolysosomes. Bar, 10 mm.
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fig4: Quantitation of the ruffling responses of wild-type and Capg−/− macrophages. (A) Bar graphs comparing the ruffling indexes of wild-type and Capg−/− macrophages before and after exposure to MCSF. The ruffling index was determined based on analysis of phalloidin staining by a blinded observer (Materials and methods). In the first four bars, brackets represent the SEM for 80–100 determinations/condition. Wild-type cells have a higher spontaneous ruffling index and nearly double their ruffling activity in response to MCSF, whereas Capg−/− macrophages have a lower spontaneous ruffling activity and fail to respond to MCSF. The far right bar quantifies the ruffling activity of Capg−/− cells stimulated with MCSF after introduction of recombinant CapG by microinjection. Bracket represents the SEM of n = 34 cells. CSF, MCSF. (B) Bar graphs comparing the ruffling responses of wild-type and Capg−/− macrophages after exposure to Salmonella (Salm.). Unlike MCSF which failed to stimulate ruffling in Capg−/− macrophages, exposure to Salmonella resulted in a significant increase in ruffling activity (P < 0.0001). Brackets represent the SEM of n = 80–100 measurements. Cells were scored as described in A. (C and D) Phase micrographs of wild-type (C) and Capg−/− (D) macrophages after 30-min exposure to Salmonella. Note the giant phagolysosomes in both the wild-type and Capg−/− cells induced by exposure to the bacteria. Arrows point to individual bacteria contained in giant phagolysosomes. Bar, 10 mm.

Mentions: Dynamic changes in intracellular [Ca2+] are associated with macrophage ruffling, and CapG's affinity for actin is regulated in a similar [Ca2+] range; therefore, we used time-lapse microscopy to examine macrophage ruffling. A qualitative decrease in spontaneous ruffling was observed in Capg −/− macrophages (Fig. 2) . To provide a more quantitative score of ruffling activity we used rhodamine-phalloidin staining and fluorescence microscopy. It has previously been shown that localized increases in actin filaments occur at the site of ruffling that appear as a serpentine pattern of rhodamine-phalloidin staining (Cox et al., 1997; Heidemann et al., 1999). A reduction in the spontaneous ruffling activity of compared with wild-type macrophages was apparent (compare Fig. 3, A with E), and this difference was markedly accentuated by a 5-min exposure to macrophage colony stimulating factor (MCSF) (compare Fig. 3, B–D with F–H). These differences were quantitated to calculate a ruffling index for each set of cells by a blinded observer (see Materials and methods). Capg−/− macrophages had a ruffling index that was <1/2 the ruffling index of wild-type cells (P < 0.001) (Fig. 4 A). After exposure to MCSF, the ruffling index of Capg−/− macrophages did not change, whereas that of wild-type cells increased by nearly 70% (Fig. 4 A). The lack of response to MCSF was a consistent finding, seen in four of four separate experiments.


Comparisons of CapG and gelsolin- macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing.

Witke W, Li W, Kwiatkowski DJ, Southwick FS - J. Cell Biol. (2001)

Quantitation of the ruffling responses of wild-type and Capg−/− macrophages. (A) Bar graphs comparing the ruffling indexes of wild-type and Capg−/− macrophages before and after exposure to MCSF. The ruffling index was determined based on analysis of phalloidin staining by a blinded observer (Materials and methods). In the first four bars, brackets represent the SEM for 80–100 determinations/condition. Wild-type cells have a higher spontaneous ruffling index and nearly double their ruffling activity in response to MCSF, whereas Capg−/− macrophages have a lower spontaneous ruffling activity and fail to respond to MCSF. The far right bar quantifies the ruffling activity of Capg−/− cells stimulated with MCSF after introduction of recombinant CapG by microinjection. Bracket represents the SEM of n = 34 cells. CSF, MCSF. (B) Bar graphs comparing the ruffling responses of wild-type and Capg−/− macrophages after exposure to Salmonella (Salm.). Unlike MCSF which failed to stimulate ruffling in Capg−/− macrophages, exposure to Salmonella resulted in a significant increase in ruffling activity (P < 0.0001). Brackets represent the SEM of n = 80–100 measurements. Cells were scored as described in A. (C and D) Phase micrographs of wild-type (C) and Capg−/− (D) macrophages after 30-min exposure to Salmonella. Note the giant phagolysosomes in both the wild-type and Capg−/− cells induced by exposure to the bacteria. Arrows point to individual bacteria contained in giant phagolysosomes. Bar, 10 mm.
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Related In: Results  -  Collection

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fig4: Quantitation of the ruffling responses of wild-type and Capg−/− macrophages. (A) Bar graphs comparing the ruffling indexes of wild-type and Capg−/− macrophages before and after exposure to MCSF. The ruffling index was determined based on analysis of phalloidin staining by a blinded observer (Materials and methods). In the first four bars, brackets represent the SEM for 80–100 determinations/condition. Wild-type cells have a higher spontaneous ruffling index and nearly double their ruffling activity in response to MCSF, whereas Capg−/− macrophages have a lower spontaneous ruffling activity and fail to respond to MCSF. The far right bar quantifies the ruffling activity of Capg−/− cells stimulated with MCSF after introduction of recombinant CapG by microinjection. Bracket represents the SEM of n = 34 cells. CSF, MCSF. (B) Bar graphs comparing the ruffling responses of wild-type and Capg−/− macrophages after exposure to Salmonella (Salm.). Unlike MCSF which failed to stimulate ruffling in Capg−/− macrophages, exposure to Salmonella resulted in a significant increase in ruffling activity (P < 0.0001). Brackets represent the SEM of n = 80–100 measurements. Cells were scored as described in A. (C and D) Phase micrographs of wild-type (C) and Capg−/− (D) macrophages after 30-min exposure to Salmonella. Note the giant phagolysosomes in both the wild-type and Capg−/− cells induced by exposure to the bacteria. Arrows point to individual bacteria contained in giant phagolysosomes. Bar, 10 mm.
Mentions: Dynamic changes in intracellular [Ca2+] are associated with macrophage ruffling, and CapG's affinity for actin is regulated in a similar [Ca2+] range; therefore, we used time-lapse microscopy to examine macrophage ruffling. A qualitative decrease in spontaneous ruffling was observed in Capg −/− macrophages (Fig. 2) . To provide a more quantitative score of ruffling activity we used rhodamine-phalloidin staining and fluorescence microscopy. It has previously been shown that localized increases in actin filaments occur at the site of ruffling that appear as a serpentine pattern of rhodamine-phalloidin staining (Cox et al., 1997; Heidemann et al., 1999). A reduction in the spontaneous ruffling activity of compared with wild-type macrophages was apparent (compare Fig. 3, A with E), and this difference was markedly accentuated by a 5-min exposure to macrophage colony stimulating factor (MCSF) (compare Fig. 3, B–D with F–H). These differences were quantitated to calculate a ruffling index for each set of cells by a blinded observer (see Materials and methods). Capg−/− macrophages had a ruffling index that was <1/2 the ruffling index of wild-type cells (P < 0.001) (Fig. 4 A). After exposure to MCSF, the ruffling index of Capg−/− macrophages did not change, whereas that of wild-type cells increased by nearly 70% (Fig. 4 A). The lack of response to MCSF was a consistent finding, seen in four of four separate experiments.

Bottom Line: However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function.These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages.These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a Capg allele engineered into mice. Both CapG- mice and CapG/gelsolin double- mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG- macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

Show MeSH
Related in: MedlinePlus