Single molecule techniques have uncovered the working principles of classic motor proteins such as myosin, kinesin, and ATP synthase (responsible for muscle contraction, intracellular transport, and energy metabolism, respectively). However, these powerful techniques have scarcely been used to study the mitotic spindle machinery. We are developing in vitro (cell free) motility assays using purified spindle components and bringing advanced biophysical techniques to this new research area. Much of our work is carried out in collaboration with other members of the Seattle Mitosis Group, especially the laboratories of Sue Biggins, Trisha Davis, and Linda Wordeman. Recently, we have also begun collaborating with Eris Duro and Adèle Marston to study meiosis, the specialized division that produces eggs and sperm. We are focusing on many areas fundamental to spindle function:


How is sister co-migration ensured during meiosis? The 'cardinal rule' of normal mitotic cell division is that replicated sister chromatids must always separate from one another to ensure that complete genomes are distributed to each daughter cell. Sophisticated mechanisms have evolved to impose this rule during mitosis. Strikingly, however, the rule is broken during meiosis, the specialized division that produces eggs and sperm. For accurate meiosis the sisters must instead co-migrate together during meiosis I, separating from their homologous partners, and only splitting apart later, in meiosis II. Failures in this process are the cause of Down syndrome and other chromosomal birth defects. Studies of budding yeast have suggested a model for sister co-migration: sister kinetochores, which link sister chromatids to microtubules and drive their movement, may be directly fused by a meiosis I-specific kinetochore-binding factor called monopolin. To date, evidence for monopolin-dependent sister kinetochore fusion has been indirect. Other models, e.g. that monopolin could instead promote co-migration by binding to and inhibiting one of the two sister kinetochores, have not been excluded. To test these ideas directly, we are isolating native meiotic and mitotic yeast kinetochores, reconstituting their function in vitro, and applying advanced tools for manipulating and tracking individual particles.


How are chromosomes coupled to spindle microtubules? Chromosome movements are driven by forces generated at kinetochores, specialized organelles that couple chromosomes to the assembling and disassembling tips of spindle microtubules. Using laser trapping and ultrasensitive fluorescence microscopy, we found that two different kinetochore components, the Dam1 and Ndc80 complexes, can maintain persistent load-bearing attachments to assembling and disassembling microtubule tips, which allows filament shortening to produce tensile force. At least one of these components, the Ndc80 complex, may act through a biased diffusion mechanism that was proposed on purely theoretical grounds more than two decades ago.  Given the wide conservation of the Ndc80 complex, this mechanism could underlie chromosome-microtubule coupling in all eukaryotes.


How are chromosome movements controlled? Isolated microtubules grow and shorten continuously, and they switch randomly between these two behaviors. Kinetochore-attached microtubules in the spindle must therefore be regulated so their otherwise random behavior will bring sister chromatids to the equator of the cell, hold them there until all sisters are aligned, and then separate them toward opposite poles. The kinetochore itself carries out microtubule regulation by stabilizing attached microtubule tips and by promoting tip growth in response to increased tension. We reconstituted this tension-dependent length control in vitro for the first time using individual microtubules, a pure kinetochore component (the Dam1 complex), and a new laser trapping-based assay. Our work demonstrates that the effect does not require complex signaling pathways or microtubule-modifying enzymes. In principle, it could operate wherever dynamic microtubules form load-bearing tip attachments, such as kinetochores, spindle poles, and the cell cortex.


How are aberrant attachments sensed and corrected? When mitosis begins, sister chromatids often attach improperly to spindle microtubules emanating from the same pole, in which case the spindle cannot generate tension to stretch them apart. It is widely believed that kinetochores sense this lack of tension and, in response, trigger their own detachment to give proper connections a chance to form. Error correction in budding yeast requires Ipl1 kinase (Aurora B in higher eukaryotes), which phosphorylates microtubule-bound components within the kinetochore, presumably causing them to release the filament.  Using ultra-sensitive fluorescence microscopy we showed that, indeed, Ipl1 phosphorylation of the Dam1 complex promotes its detachment from microtubules. Whether tension reduces Ipl1 activity at kinetochores or directly controls the rate of detachment of whole kinetochores remains to be demonstrated.


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