Fig. 7. Confocal microscopic observations of the invasion site for wild-type Shigella or the virA mutant. (A) HeLa cells were infected with wild-type or the virA null mutant. After a 15 min infection, the actin, tubulin and VirA were visualized by rhodamine–phalloidin (Actin, yellow), a mixture of anti-α- and anti-β-tubulin antibodies followed by Cy5-anti-mouse IgG antibody (MT, red) or the anti-VirA-FL antibody with an Alexa 488–anti-rabbit IgG antibody (VirA, green), respectively. Bar, 10 µm. Arrowheads indicate the bacteria. Upon attachment of the wild-type but not the virA mutant to HeLa cells, membrane protrusions appeared around the invading bacteria; at these points, the local MT networks beneath the protruding ruffles underwent a marked destruction. On wild-type Shigella that provoked local destruction of MT structure, secreted VirA from the bacteria was detected. (B) The local cytoskeletal architecture around the invading bacteria in a high resolution photograph. The actin, tubulin and VirA were visualized by rhodamine–phalloidin (Actin, yellow), a mixture of anti-α- and anti-β-tubulin antibodies followed by Cy5-anti-mouse IgG antibody (MT, red) or the anti-VirA-FL antibody with an Alexa 488–anti-rabbit IgG antibody (VirA, green), respectively. Bar, 10 µm. Arrowheads indicate the bacteria. The boxed portions of the MT network in (MT) and (MT + VirA) are shown enlarged. The VirA secreted from bacteria was detected mostly around the destroyed MTs.