Fig. 2. Tyrosine phosphorylation is required for MSP polymerization. (A) Confocal fluorescence micrographs of a sperm (left) and a fiber assembled in vitro (right). Antiphosphotyrosine (green) labels the plasma membrane at the tips of fiber complexes in vivo and the vesicles of MSP fibers grown in vitro; both are sites of MSP cytoskeletal polymerization. The fiber complexes in the lamellipod and the fiber are stained with anti-MSP antibody (red). The antiphosphotyrosine fluorescence in the cell body is due to labeling of a mitochondrial enzyme, fumerate reductase, which contains phosphotyrosine residues. Bar, 1 µm. (B) The effect of the tyrosine phosphatase YOP on fiber assembly. The upper panel shows a phase contrast micrograph of fibers grown for 10 minutes in untreated S100. Addition of YOP to S100 (center panel) blocks assembly so that no fibers are detectable after 60 minutes. By contrast, the addition of YOP in the presence of sodium orthovanadate (bottom panel), a potent inhibitor of tyrosine phosphatases, restores the capacity of S100 to construct fibers. Bar, 10 µm. (C) The effect of YOP on protein tyrosine phosphorylation in S100 cell-free extract. The left lane shows a Coomassie-stained gel of S100. The right lanes show corresponding western blots probed with antiphosphotyrosine antibody. A single M_r 48 kDa band is labeled in S100. This band is unlabeled in YOP-treated S100 but restored in S100 treated with YOP in the presence of sodium orthovanadate.