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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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Generation of Capg−/− mice. (A) Diagram of the murine Capg gene and the gene-targeting construct. The probe used for Southern blot analysis and expected fragment sizes are indicated. (B and C) Southern blot analysis of BamHI-digested ES cell DNAs. The shifted 6.8-kb band (*) indicates that homologous recombination has occurred. (D) Southern blot analysis of DNAs derived from an intercross of mice each having a targeted Capg allele. One mouse is homozygous for the targeted allele. Immunoblot analysis of tissue extracts from wild-type and CapG- mice using a rabbit IgG anti-CapG polyclonal antibody (ab). No CapG was detected in any tissue from the CapG- mice. (E) Immunoblot analysis of macrophage extracts from wild-type, gelsolin-, and CapG- mice using antibodies against CapZ, gelsolin, and the NH2 terminus of CapG. There is no significant difference in the concentrations of CapZ and gelsolin between wild-type and CapG- cells, and no CapG signal of any size.
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fig1: Generation of Capg−/− mice. (A) Diagram of the murine Capg gene and the gene-targeting construct. The probe used for Southern blot analysis and expected fragment sizes are indicated. (B and C) Southern blot analysis of BamHI-digested ES cell DNAs. The shifted 6.8-kb band (*) indicates that homologous recombination has occurred. (D) Southern blot analysis of DNAs derived from an intercross of mice each having a targeted Capg allele. One mouse is homozygous for the targeted allele. Immunoblot analysis of tissue extracts from wild-type and CapG- mice using a rabbit IgG anti-CapG polyclonal antibody (ab). No CapG was detected in any tissue from the CapG- mice. (E) Immunoblot analysis of macrophage extracts from wild-type, gelsolin-, and CapG- mice using antibodies against CapZ, gelsolin, and the NH2 terminus of CapG. There is no significant difference in the concentrations of CapZ and gelsolin between wild-type and CapG- cells, and no CapG signal of any size.

Mentions: An 11-kb HindIII fragment of the murine CAPG gene was identified that contained exons 5–8. To generate an inactivating mutation, exons 7 and 8 were deleted and replaced by a neomycin resistance cassette (neor). The herpes simplex virus thymidine kinase cassette was then positioned on the 3′ side of the construct for negative selection (Fig. 1 A). After electroporation into J1 embryonic stem (ES)* cells, 135 clones were selected by resistance to G418 and 1-2′-deoxy-2′-fluoro-β-d-arabinofuranosyl-5-iodouracil. Southern blot analysis using a flanking probe showed that homologous recombination with the native CapG locus had occurred in a single ES cell clone (Fig. 1 B). This ES cell clone was injected into BalbC blastocysts, chimeras were obtained, and transmission of the targeted allele in successive breedings was confirmed by Southern blot analysis (Fig. 1 C). Offspring generated by intercrosses of mice bearing the targeted Capg allele demonstrated Mendelian segregation, indicating that mice homozygous for the targeted allele were viable (see below).


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)

Generation of Capg−/− mice. (A) Diagram of the murine Capg gene and the gene-targeting construct. The probe used for Southern blot analysis and expected fragment sizes are indicated. (B and C) Southern blot analysis of BamHI-digested ES cell DNAs. The shifted 6.8-kb band (*) indicates that homologous recombination has occurred. (D) Southern blot analysis of DNAs derived from an intercross of mice each having a targeted Capg allele. One mouse is homozygous for the targeted allele. Immunoblot analysis of tissue extracts from wild-type and CapG- mice using a rabbit IgG anti-CapG polyclonal antibody (ab). No CapG was detected in any tissue from the CapG- mice. (E) Immunoblot analysis of macrophage extracts from wild-type, gelsolin-, and CapG- mice using antibodies against CapZ, gelsolin, and the NH2 terminus of CapG. There is no significant difference in the concentrations of CapZ and gelsolin between wild-type and CapG- cells, and no CapG signal of any size.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196452&req=5

fig1: Generation of Capg−/− mice. (A) Diagram of the murine Capg gene and the gene-targeting construct. The probe used for Southern blot analysis and expected fragment sizes are indicated. (B and C) Southern blot analysis of BamHI-digested ES cell DNAs. The shifted 6.8-kb band (*) indicates that homologous recombination has occurred. (D) Southern blot analysis of DNAs derived from an intercross of mice each having a targeted Capg allele. One mouse is homozygous for the targeted allele. Immunoblot analysis of tissue extracts from wild-type and CapG- mice using a rabbit IgG anti-CapG polyclonal antibody (ab). No CapG was detected in any tissue from the CapG- mice. (E) Immunoblot analysis of macrophage extracts from wild-type, gelsolin-, and CapG- mice using antibodies against CapZ, gelsolin, and the NH2 terminus of CapG. There is no significant difference in the concentrations of CapZ and gelsolin between wild-type and CapG- cells, and no CapG signal of any size.
Mentions: An 11-kb HindIII fragment of the murine CAPG gene was identified that contained exons 5–8. To generate an inactivating mutation, exons 7 and 8 were deleted and replaced by a neomycin resistance cassette (neor). The herpes simplex virus thymidine kinase cassette was then positioned on the 3′ side of the construct for negative selection (Fig. 1 A). After electroporation into J1 embryonic stem (ES)* cells, 135 clones were selected by resistance to G418 and 1-2′-deoxy-2′-fluoro-β-d-arabinofuranosyl-5-iodouracil. Southern blot analysis using a flanking probe showed that homologous recombination with the native CapG locus had occurred in a single ES cell clone (Fig. 1 B). This ES cell clone was injected into BalbC blastocysts, chimeras were obtained, and transmission of the targeted allele in successive breedings was confirmed by Southern blot analysis (Fig. 1 C). Offspring generated by intercrosses of mice bearing the targeted Capg allele demonstrated Mendelian segregation, indicating that mice homozygous for the targeted allele were viable (see below).

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