Figure 8

Expression of Vrp1 in the FCM population rescues fusion and lethality in Vrp1^f06715mutants. (A) Overview of the transgenic constructs generated for UAS-Gal4 fly experiments and for cell culture overexpression experiments. Dark grey boxes represent the WH2 domains, light grey box denotes the proline rich domain (Pro), black box is the WASP-binding domain (WBD). Myc-tag is indicated by an oval. Various domains of the Vrp1 protein were deleted as shown. Transgenes containing the Wasp-binding domain were able to rescue the Vrp1^f06715mutant lethality when specifically expressed in the FCMs using a sns-GAL4 driver, while those transgenes lacking the Wasp-binding domains were unable to rescue the lethality of the Vrp1^f06715mutant as indicated in the table. (B) Ectopic expression of the full length Vrp1 transgene, but not the truncated forms, induced a dramatic reorganization of the actin filament system in form of the assembly of thick bundles and the formation of actin dots, resulting in loss of stress fibers. Actin dots (accumulation of actin in foci, red arrowhead) and thick bundles (thick actin filaments, red arrow) are known to be formed upon ectopic expression of actin reorganizing proteins, such as mammalian Vrp1, at the expense of the stress fibers. A detailed description of the phenotypes are given in [Additional file 2: Supplemental Figure 2E]. Filamentous actin was visualized by TRITC-labeled phalloidin (red). Vrp1-expressing cells were detected by co-transfecting an EGFP- and Vrp1-expressing plasmids. Bar represents 20 μm. (C) Quantification of the effects on the actin organization caused by ectopic expression of the Vrp1 transgenes in PAE cells was performed; the percentage of cells displaying extensive stress fiber loss, thick bundles and actin dots were counted manually employing a 63x immersion oil objective. The values represent triplicates of analyzes of at least 100 transfected cells.