Figure 2.
F-actin organization of cells expressing low levels of WASP. (A) F-actin organization revealed by phalloidin staining in WASPhypo and WASPTK cells under cAMP gradient. Stainings of two cells with representative phenotypes are shown. Arrows indicate the direction of cAMP gradient. Right panel shows ratio of intensity of F-actin staining at the foremost part (front) or rear end (back) of cells divided by the intensity of the center of cells. Intensity value was acquired by linescan of images in the direction of the gradient (n = 10). (B) In vivo actin polymerization assay measuring F-actin assembled in response to the chemoattractant (cAMP) stimulation. Note that F-actin polymerization upon cAMP stimulation is defective in WASPTK cells. (C) F-actin organization revealed with ABP-GFP in live cells. ABP-GFP fusion protein binds specifically and dynamically to the same F-actin structures that phalloidin recognizes in fixed cells. (D) Distribution of free barbed end in cells. Rhodamine-labeled actin (0.4 μM) was incorporated into permeabilized cells to visualize free barbed-ends for actin polymerization in wild-type cells or WASPTK cells. Linescans of a cell from point A to B were shown in bottom panels. Incorporation of Rhodamine-labeled G-actin was quantified by measuring the fluorescence intensity and shown in right panel (n = 6). (E) Axial polarity of wild-type and WASPTK cells. GFP fusion of N-terminal half of PAKa (PAKa-N-GFP) shows specific localization to uropod of polarized cells and was used as a reporter to examine cellular polarity. Aggregation competent wild-type cells are well polarized and showed biased localization of PAKa-N-GFP at the uropod, but WASPTK cells show almost equal distribution of PAKa-N-GFP, indicating lack of axial polarity. Polarity index was calculated by the ratio of PAKa-N-GFP intensity at the leading edge to GFP intensity at the uropod and shown in the right panel (n = 6). The polarity index of wild-type cells is significantly greater than that of WASPTK cells.