Spatiotemporal analysis of the behavior of the small GTPase, Rho

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We screened combinations of anti-Rho antibodies and fixation protocols for precise localization of Rho within cells. Several commercial antibodies and antibodies produced by ourselves were found to be reliable with appropriate fixation protocols such as TCA fixation. For this screening, we doubly stained Myc-RhoA-expressing cultured cells with an anti-myc antibody and an anti-Rho antibody. Reliable anti-Rbo antibodies should stain the cells as anti-Myc antibody does. Using valid antibodies, we localized Rho within cells and tissues. In cultured epithelial cells, Rho was accumulated on lateral membranes. In fibroblastic cells, Rho was distributed almost evenly throughout the cytoplasm. During cytokinesis, Rho was generally concentrated at the cleavage furrow. In tissues, although Rho concentration at microvilli was obvious, Rho localization within cells differed depending on cell type, probably reflecting the functional difference of the cells in tissues. Biochemical data indicated that Rho translocates from the cytoplasm to the plasma membrane on its activation. We visualized this translocation for the first time using cultured cells stimulated with LPA or EGF. On stimulatioh with these factors, Rho was rapidly recruited from the cytoplasm to the plasma membrane within 30 seconds.