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First published online 13 May 2003
doi: 10.1242/jcs.00469

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A 48 kDa integral membrane phosphoprotein orchestrates the cytoskeletal dynamics that generate amoeboid cell motility in Ascaris sperm

¹ Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA
² MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK

View larger version (203K): [in a new window]   Fig. 1. An MSP fiber grown in vitro. Platinum replicas of MSP fibers show that the fibers are composed of a dense meshwork of MSP filaments. A plasma membrane-derived vesicle (lower left) is located at one end of the fiber and is the site of MSP filament addition. Bar, 500 µm.

View larger version (67K): [in a new window]   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 Mr 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.

View larger version (108K): [in a new window]   Fig. 3. Only fractions that contain the 48 kDa phosphoprotein induce MSP polymerization. (A) Coomassie-stained gels and corresponding western blots probed with antiphosphotyrosine of the vesicle and cytosol fractions of S100. The 48 kDa protein (arrowhead) is detectable only in the vesicle fraction. (B) SEM of filaments assembled on addition of the detergent-solubilized extract of membrane vesicles to cytosol in the presence of ATP. No filaments were observed when the detergent-solubilized extract was omitted. (C) Coomassie-stained gel of fractions 36-42 obtained by gel filtration chromatography of CHAPS-solubilized vesicle components. A western blot probed with antiphosphotyrosine (anti-pY) of the region containing p48 is shown below. Fractions that triggered MSP polymerization when added to cytosol + ATP are indicated by `+'; fractions that yielded no detectable filament formation are designated `-'. (D) SEM of filaments assembled by the addition of fraction 39 to cytosol + ATP. Bars in B and D, 1 µm.

View larger version (101K): [in a new window]   Fig. 4. p48 immunoprecipitated with antiphosphotyrosine induces MSP polymerization when recombined with cytosol. (A) Coomassie-stained gel of S100 (lane 1) and the material obtained by immunoprecipitation with antiphosphotyrosine (lane 2). Lane 3 shows a western blot of the immunoprecipitate probed with antiphosphotyrosine. The two dark bands have Mrs corresponding to IgG heavy (HC) and light chains (LC) and are stained by the secondary antibody used to develop the western blot. To confirm that the band just below HC is p48, S100 was incubated with 32P--ATP, immunoprecipitated with antiphosphotyrosine and analyzed by autoradiography (lane 4). An immunoprecipitated band at Mr 48 kDa is heavily labeled. The fainter band at Mr 45 kDa was not seen on other antiphosphotyrosine western blots (see Fig. 1C and Fig. 2A,C) and is probably a breakdown product of p48. (B) SEM of filaments obtained when immunoprecipitated p48 was combined with cytosol and ATP. Bar, 1 µm.

View larger version (42K): [in a new window]   Fig. 5. Labeling patterns obtained with a polyclonal antibody raised against p48 peptide. (A) SDS-PAGE gel of S100 and corresponding western blots probed with anti-p48 and antiphosphotyrosine. In immunofluorescence assays anti-p48 labels the sperm plasma membrane uniformly in detergent-permeabilized sperm (B) but not in nonpermeabilized cells (C). In addition, the antibody labels organelles in the cell body that correspond in number, size and position to the membranous organelles (MOs). Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 6. Comparison of the distribution of p48 and its phosphorylated form in detergent-permeabilized sperm by confocal fluorescence microscopy. Antiphosphotyrosine (green) labels the plasma membrane primarily at the leading edge of the lamellipod, whereas anti-p48 (red) labels the membrane uniformly. The green labeling in the cell body is due to recognition of fumarate reductase (see also Fig. 1) by antiphosphotyrosine. In this cell, anti-p48 failed to label the MOs. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 7. Effect of pH modulation on phosphorylation of p48. Treatment of spermatozoa with acetate buffer at pH 5.5 (left panels) results in the disassembly of the MSP cytoskeleton. Labeling of the membrane with anti-p48 (red) is uniform but no antiphosphotyrosine labeling can be detected in the membrane. In cells fixed within 4 seconds after washing out the pH 5.5 buffer (middle panels), the cytoskeleton reforms along the lamellipod periphery and discrete spots of antiphosphotyrosine labeling appear along the membrane. By 60 seconds after acid removal (right panels) the cytoskeleton is completely reconstructed and spots of antiphosphotyrosine labeling are located along the lamellipodial leading edge. Bar, 10 µm. (B) Autoradiography of SDS-PAGE gels of S100 following incubation in 32P--ATP at pH 5.5 vs pH 7.0. Labeling of p48 (arrow) is undetectable at the acidic pH.

View larger version (95K): [in a new window]   Fig. 8. Phosphorylation of p48 requires cytosolic components. Coomassie-stained SDS-PAGE gels (left) and autoradiograms (center) of equal aliquots of vesicles incubated with 32P--ATP for the intervals indicated. Labeling of p48 (arrow) is barely detectable after 30 minutes incubation of vesicles alone but increases dramatically by 5 minutes after addition of cytosol. Incubation of cytosol alone in 32P--ATP for 5 minutes yields almost no protein labeling.