Figure 9

Gelsolin knockdown inhibits the redistribution and clustering of CD4-CXCR4 and CD4-CCR5 induced by HIV-1 Env-gp120. Confocal microscopy analysis of CD4 and CXCR4 (A) or CCR5 (C) distribution in control CEM.NKR-CCR5 cells transfected with fluorescent scrambled siRNA or siRNA(1 + 2)-GSN. Merged images show the co-localization of CD4-CXCR4 (A) or -CCR5 (C) at the plasma membrane. In (B) and (D), a series of xy midsections shows the HIV-1 Env-gp120-induced redistribution of CD4-CXCR4 and -CCR5, respectively, to one pole of the cell in CEM.NKR-CCR5 cells expressing scrambled Alexa 546 oligos 1 hour after exposure to rs-gp120_IIIB (5 μg/mL) at 37°C (capping, series of images). Merged images show cell surface CD4-CXCR4 (B) or -CCR5 (D) co-localization. In contrast, HIV-1 Env-gp120-induced CD4-CXCR4 and -CCR5 redistribution was not observed in cells transfected with siRNA(1 + 2)-GSN oligos (74% and 54% of cells showing no capping, respective series of images in (B) and (D)). These cells exhibited no CD4-CXCR4 or –CCR5 capping, although aberrant CD4 and CXCR4 or CCR5 redistribution and aberrant pseudopodium formation was evident (see white arrows in (B) or (D), respectively). The CD4-CXCR4 and -CCR5 clustering (35% and 45% of capping, respectively), the no capping and aberrant CD4-CXCR4 and -CCR5 distribution (26% and 46%, respectively) are reflected as a percentage of the 200 cells counted for each experimental condition, the data representing the results of three independent experiments. Scale bar = 5 μm. One representative experiment of three is shown per each experimental condition.