Fig. 5. Kette regulates F-actin organization. Kette expression (top), the F-actin cytoskeleton (middle) and the merge (bottom) are shown in Schneider S2 cells using anti-Kette antisera. All images represent stacks of confocal sections. (A) In wild-type cells, endogenous Kette protein largely colocalizes with F-actin. (B) Disruption of Kette expression by RNA interference leads to a concomitant disruption of the F-actin cytoskeleton. Forty-eight hours after treatment with kette dsRNA, aggregates of F-actin are found surrounding the nucleus (arrowhead). (C) Depletion of Scar/Wave by treatment with scar/wave dsRNA leads to a marked reduction in F-actin formation and alterations in cell morphology. Interestingly, in the absence of Wave, Kette appears to be uniformly distributed throughout the cell. (D) After RNAi for both kette and scar/wave, the formation of F-actin is not further reduced. (E) Overexpression of high levels of a Myc-tagged Kette protein does not lead to any changes in the organization of the F-actin cytoskeleton. The high dilution of the anti-Kette antiserum (1:50,000 compared with 1:2000 in A does not allow the detection of the endogenous Kette protein). (E) After expression of a membrane-tethered Kette protein, the F-actin cytoskeleton is rearranged. Large clumps of F-actin can be detected close to the membrane at sites that also show high levels of Kette expression. Scale bar: 5 µm.