Appointment period: 11/1/2009 to 5/14/2010
Characterization of a Novel Spindle “Brake” Mechanism in Yeast and Mammalian Cells
Our previous work had shown that in budding yeast carrying mutations in chromosome passenger subunits (e.g., SLI15 or BIR1) the rate of anaphase spindle elongation is significantly reduced. To explain the mutant phenotype, we hypothesize that either interpolar spindle microtubules are altered or spindle associated proteins have different activities, or both. We further speculate that the spindle brake is active in the presence of unresolved sister chromatids during anaphase, or chromosome bridges.
Our first goal was to characterize interpolar microtubules using fluorescence microscopy in wild type and mutant cells expressing GFP-Tub1 to mark microtubules. I took advantage of previously published methods to analyze fluorescent intensity along the spindle. I successfully created a series of strains expressing GFP-Tub1 in wild type, bim1 Δ, and pGAL1-SLI15 (to deplete Sli15). The bim1 Δ strain served as a control and is published to reduce the length of interpolar microtubules and exhibit a distinct fluorescent intensity profile. I compared wild type, bim1 Δ and cells depleted for the chromosome passenger, Sli15. Interestingly, there was a similar decrease in interpolar microtubules in bim1 Δ and in cells depleted for Sli15. This finding raises the possibility that chromosome passenger mutants change interpolar microtubule dynamics and thus their ability to generate forces. However, bim1Δ alone does not phenocopy the slow spindle elongation rates observed in pGAL1-SLI15, arguing that changes in interpolar microtubule organization are not sufficient to explain the change in spindle elongation rate. My next goal is to determine if the change in interpolar microtubules is required for the slower spindle elongation. I will attempt to rescue the reduction in interpolar microtubule length by over-expressing Bim1 in a chromosome passenger mutant. In addition, I will start to characterize changes in microtubule-associated proteins in chromosome passenger mutants to test the second part of the hypothesis.
My second project is to address the potential interaction between chromosome bridges and the anaphase spindle. One explanation for our unpublished observation that chromosome bridges reduce spindle elongation rates is that unresolved sister chromatids interact directly with anaphase spindle microtubules or associated proteins. To test this hypothesis, I will use high-resolution fluorescence microscopy on mammalian cells and genetic analysis in yeast. I have optimized conditions for detecting chromosome bridges in mammalian cells using anti-sera against the Blooms helicase.