I have been working on the effects of Cytochalasin D (and other anti-actin agents) on mitosis in crane fly spermatocytes -- a cell from animals I rear. These cells have very favourable cytology, and students and I have studied phalloidin staining in them at selected stages. I have stained spindles in crane-fly spermatocytes with two different monoclonal Abs -- one to tyrosinated tubulin (Ab=YLl/2) and one to acetylated tubulin (Ab = 6-1 lB-I). The YL1/2 stains ALL MTs in the spindle, while the 6-l lB-I stains only the kinetochore-to-pole MTs, and of those the region near the KT does not stain. We have used this differential staining to show that during anaphase the MTs disassemble at the poles, not the Kinetochores. A question we cannot answer is this. We know that the acetylation is only of Kinetochore to pole MTs -- others do not stain. But is this a subset of kinetochore MTs that stain, or do all of them stain? My proposition is to look at this with the photo-oxidation technique - - and in fact this is a good test of the technique, because you would have to see some MTs labelled and others in the same spindle NOT be labelled. And those that are labelled would not be labelled near the kinetochore --only a few micrometres up from the kinetochore.
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