Evans JP et al. (1990), Talin and vinculin in the oocytes, eggs, and ea...

XB-ART-26113

Dev Biol 1990 Feb 01;1372:403-13. doi: 10.1016/0012-1606(90)90264-j.

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Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution.

Evans JP , Page BD , Kay BK .

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We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.

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Species referenced: Xenopus laevis
Genes referenced: tln1 tln2 vcl

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	FIG. 1. Immunofluorescent staining of A6 cells, sectioned Xenqpus oviduct tissue, and a whole mount of Xenopus tadpole tail muscle with anti-talin and anti-vinculin antibodies. Panels A and B show corresponding views of fixed permeabilized cultured Xaopus A6 kidney epithelial cells double-labeled with rabbit anti-talin polyclonal antibodies and mouse anti-vinculin monoclonal antibodies, respectively. Panels C and D show a section of oviduct double-labeled with mouse anti-talin monoclonal antibodies and rabbit anti-vinculin polyclonal antibodies, respectively. Panels E and F show a piece of whole-mounted tadpole tail muscle double-labeled with rabbit anti-talin polyclonal antibodies and mouse anti-vinculin monoclonal antibodies. Arrows in panels E and F indicate the termini of a single muscle fiber where postsynaptic neuromuscular junctions are. Bars = ‘7 pm for panels A and B, 26 pm for panels C and D, and 64 pm for panels E and F, respectively.
	FIG. 2. Immunoblot analysis of chick embryo fibroblast, A6, and Xenopus oviduct proteins. Mixtures of various chick embryo fibroblast, A6 cell, and oviduct proteins were resolved on a 6% SDS-polyacrylamide gel, blotted to Immobilon transfer membrane, and reacted with antibodies. Blot A was reacted to the rabbit anti-talin polyclonal antibodies; blot B was reacted to the mouse anti-vinculin monoclonal antibodies. Lanes l-4 correspond to chick embryo fibroblast whole lysate, A6 cell whole lysate, oviduct detergent-solubilized extracts, and oviduct cytoskeletal extracts, respectively. Protein standards are myosin H-chain (206 kDa), phosphorylase B (106 kDa), bovine serum albumin (68 kDa), and ovalbumin (42 kDa); these molecular weight standards are used throughout the paper.
	FIG. 3. Immunofluorescent staining of sectioned Xenopus oocyte with anti-talin and anti-vinculin antibodies. Panels A and B show a sectioned Xenopus oocyte double-labeled with rabbit anti-talin polyclonal antibodies and mouse anti-vinculin monoclonal antibodies, respectively. Panel C shows a similar section treated with nonimmune antibodies. Bar = 28 pm.
	FIG. 4. Immunoblot analysis of Xenqpus ovary, oocytes, and ovarian sheathes. Mixtures of detergent-solubilized and cytoskeletal extracts from ovary, oocytes, and ovarian sheathes were resolved on a 6% SDS-polyacryamide gel, blotted to Immobilon transfer membrane, and reacted with antibodies. Blot A was reacted to rabbit anti-talin polyclonal antibodies; blot B was reacted to mouse anti-vinculin monoclonal antibodies. Lanes l-3 correspond to detergent-solubilized extracts of ovary, oocyte, and ovarian sheathes; lanes 4-6 correspond to cytoskeletal extracts of the same tissues. The arrow on the left points out the bands corresponding to talin; the arrow on the right points out the bands corresponding to vinculin.
	FIG. 5. Immunofluorescent staining of sectioned Xenom egg with anti-talin and anti-vinculin antibodies. Panels A and B show a sectioned Xenopus egg double-labeled with rabbit anti-talin polyclonal antibodies and mouse anti-vinculin monoclonal antibodies, respectively. Panel C shows the corresponding phase-contrast image; panel D shows a similar section treated with nonimmune serum. Bar = 28 pm.
	FIG. 6. Immunofluorescent staining of sectioned Xen0pu.s two-cell embryo with anti-talin and anti-vinculin antibodies. Panel A shows a sectioned Xenopus two-cell embryo labeled with rabbit anti-talin polyclonal antibodies. Panel B shows an adjacent section reacted with mouse anti-vineulin monoclonal antibodies. The region shown is from the vegetal hemisphere; first cleavage is nearly complete. Bar = 17 pm.
	FIG. 7. Immunoblot analysis of Xenqnus oocyte, egg, and embryo proteins. Mixtures of cytoskeletal extracts from oocytes, eggs, and embryos 15 min after fertilization were resolved on 6% SDS-polyacrylamide gels, blotted to Immobilon transfer membrane, and reacted with antibodies. Blot A was reacted to rabbit anti-talin polyelonal antibodies; blot B was reacted to mouse anti-vinculin monoclonal anti-bodies. Blot B was run farther to separate vinculin (116 kDa) from the yolk proteins (110 kDa). The broad bands of yolk proteins are occasionally prevalent in our cytoskeletal extracts and react with immune and nonimmune serum. The arrow on blot A points out the bands corresponding to talin; the arrow in blot B points out the bands corresponding to vinculin.