Analysis of anaphase B in saccharomyces cerevisiae using a monoclonal antibody against yeast tubulin.
In Saccharomyces cerevisiae the mitotic spindle is composed of approximately 10 continuous microtubules, that pass uninterrupted between the 2 spindle pole bodies (SPBs), and up to 34 discontinuous microtubules, 17 of which arise from each SPB and terminate in the nucleoplasm [7, 12]. We have previously shown that during spindle elongation (anaphase B) the number of continuous microtubules decreases rapidly until at a length of 4 microns only 1 remains [8]. Further elongation from 4 to 8 microns is achieved with only a single continuous microtubule. Here we have extended our analysis of spindle elongation by investigating the timing of this event by indirect immunofluorescence microscopy using a monoclonal antibody to yeast tubulin. From the frequency distribution of spindle lengths in exponential phase cultures of the haploid wild type strain A364A, the time in mitosis and the velocity of spindle elongation have been calculated. For the first 55 min of mitosis spindle elongation proceeds at a slow rate, 0.36 microns/min. There is then a marked increase to a maximal value of 4.16 microns/min. Correlated with data from previous ultrastructural studies on the decrease in continuous microtubule number during anaphase B, this 12-fold increase in velocity is shown to occur at the single microtubule stage. These experiments provide further evidence to suggest that the role of the spindle is to regulate the rate of anaphase B which is actively achieved by an, as yet, unknown motive force generating system. Antibody staining has also allowed the terminal phenotypes of 2 diploid cell division cycle (cdc) mutants to be more accurately described. This has shown that, although arrested synchronously as judged by phase contrast microscopy, these strains do in fact exhibit considerable heterogeneity in spindle length.
D000705:Anaphase; D000911:Antibodies, Monoclonal; D005455:Fluorescent Antibody Technique; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D008938:Mitosis; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D014404:Tubulin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014404:Tubulin