Role of Rho-family proteins in podosome assembly and distribution.

Rho-family proteins control podosome assembly and distribution in normal immature DCs. DCs were fixed 30 to 60 minutes after microinjection with IgG (panel A), N17Cdc42Hs (panel B), V12Cdc42Hs (panel C), N17Rac1 (panel D), V12Rac1 (panel E), or C3 transferase (panel F) and stained with TRITC-phalliodin. In morphology, the DCs injected with IgG (panel A) are indistinguishable from uninjected cells. N17Cdc42Hs results in a loss of polarity, excessive membrane ruffling (arrowheads in panel B), and loss of podosomes (panel B). Microinjection of V12Cdc42Hs (panel C) stimulates filopodial extension around the periphery of the cell (arrows in panel C), resulting in a characteristic stellate morphology. There are generally fewer podosomes (arrowheads in panel C), and clustering of podosomes is inhibited. Vinculin staining was used to confirm the identity of podosomes (data not shown) in such cells. The leading edge of cells injected with N17Rac1 (panel D) show filopodial extensions (arrowhead in panel D) but lack podosomes. In contrast, V12Rac1 injection causes no morphological change, and normal podosome distribution is retained (panel E). Injection of C3 transferase (panel F) results in cell spreading (arrowheads in panel F), and podosomes are absent. In normal DCs, microinjection of control IgG protein causes partial disruption of podosomes and progressive recovery over 60 minutes (panels G-K). Injection of N17Cdc42Hs (panel G), V12Cdc42Hs (panel H), N17Rac1 (panel I), or C3 transferase (panel K) significantly inhibits podosomal recovery. In contrast, recovery occurs normally in V12Rac1-injected cells (panel J). Data were analyzed by means of the chi-square test. Significant differences between IgG and recombinant protein–injected cells are indicated by asterisks. ** indicates P < .01 and *** indicates P < .05. Each graph represents the combined data from 2 experiments, and each bar represents an average of 102 injected cells.