High-resolution imaging of microtubule dynamics

I have applied high-resolution imaging to study mechanisms coupling microtubule dynamics to intracellular motility. Combining advanced instrumentation with the Fluorescent Speckle Microscopy (FSM) allowed novel observation of microtubule dynamics. Microtubules produce motility through two general mechanisms; indirectly as a substrate for motor proteins and directly by increasing or decreasing overall length. Using high-resolution imaging of chromosome movements relative to microtubule dynamics in the Xenopus extract and Drosophila embryo systems, I characterized the role of microtubule poleward flux in kinetochore motility during mitosis. These studies revealed that poleward flux is a major contributor to sister kinetochore tension during metaphase as well as chromosome to pole movements during anaphase. Application of high-resolution techniques to the study of microtubule dynamics in the model system S. cerevisiae (budding yeast) revealed that minus ends of cytoplasmic and interpolar spindle microtubules are inert and therefore do not contribute to generating motility. This observation has proven important for understanding the mechanism of proteins that regulate microtubule dynamics in budding yeast. Analysis of microtubule dynamics during a particular stage in the yeast mating cycle identified the shmoo tip as a site of microtubule plus end dynamic attachment. Molecular investigation of this dynamic attachment using single gene knockouts and mutation combined with high-resolution imaging of GFP fusion proteins showed that the minus end directed kinesin, Kar3p is required for maintaining attachment to shortening microtubule plus ends. Thus, a new function for minus end directed kinesins was characterized. Together, this work has served to develop new imaging techniques for microtubule dynamics in Xenopus extract and Drosophila embryonic spindles as well as budding yeast, and identify a novel function for the microtubule motor protein, Kar3p.