The goals of this study are to determine how S. cerevisiae kinetochores bind to microtubules and how them forces required for chromosome movement are generated. This will be accomplished by linking systematic experiments to quantitative analysis. A mechanical model of the spindle will be developed combining explicit numerical representations of cellular mechanics with molecular detail. Chromosome segregation is the process by which replicated DNA molecules are pulled into daughter cells by attaching to and moving along the microtubules of the mitotic spindle. Chromosome-microtubule attachment is mediated by kinetochores, multi-component protein complexes that form on centromeric DNA. S. cerevisiae is an attractive organism in which to study kinetochores because it contains the simplest of all known centromeres. In addition, the powerful molecular genetics of budding yeast make it straightforward to engineer specific lesions into spindle and kinetochores proteins and to monitor the phenotypic consequences. The conservation of kinetochore proteins from yeast to man suggests that principles learned from the study of simple yeast kinetochores will be applicable to complex kinetochores in higher cells. The following specific aims will be addressed:
Aim 1 : Systematic analysis of chromosome movement will be performed at different phases of the cell cycle and in cells in which spindle geometry has been perturbed genetically.
Aim 2 : Machine vision algorithms incorporating biological prior knowledge will be developed for superresolution tracking of chromosome movement in living cells Aim 3: Numerical models of spindle mechanics will be formulated in which various force-generating mechanisms can be explored through simulation and subsequent experimentation.
Aim 4 : Mutations will be introduced into kinetochores genes implicated in force generation and phenotypes analyzed with reference to computational models of spindle function.
