3 D DISTRIBUTION OF CYTOSKELETAL ANTIGENS

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We are continuing our efforts to immunolocalize proteins important for cytoskeletal function in both mammalian cells and in yeast by applying antibodies to thin sections of samples that have been well-fixed by cryopreservation and freeze-substitution and embedded in the Lowicryls or LR-White. Of four antibodies recently obtained from collaborators, two have yielded well-controlled localizations with signals that are above background by statistically rigorous tests. Thus, otherwise excellent antibodies provided by Ahn against active forms of MAP kinase and by Pillus against the telomere-binding protein RAP1 have failed to give good localizations, while two antibodies from Gould have proven successful, yielding one published paper and one manuscript in preparation. Each of these papers describes some new aspect of the relationship between actin and actin-related proteins (ARPs) in fission yeast. We have also had some success with myc-tagged versions of protein, so long as there are enough tags present. One and three mycs have previously been unsuccessful, but with McCollum, we have now been successful using 13 mycs on a gene called sid2+, which is important in specifying the position of the cleavage furrow in fission yeasts, leading to another paper that is now being readied for submission. C10