The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle ofbudding yeast

Background: Two genetic 'pathways' contribute to the fidelity of nuclear se gregation during the process of budding in the yeast Saccharomyces cerevisi ae. An early pathway, involving Kar9p and other proteins, orients the mitot ic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasm ic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is le thal. Here, to visualize the functional steps leading to correct spindle or ientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. Results: Transport of Kar9p into the bud was found to require the myosin My o2p. Kar9p interacted with microtubules through the microtubule-binding pro tein Bim1p and facilitated microtubule penetration into the bud. Once micro tubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shorteni ng in the bud, suggesting that Kar9p couples microtubule shortening to nucl ear migration. Conclusions: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. La ter, Kar9p couples microtubule disassembly with nuclear migration.