This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The S. pombe kinesin-like protein Klp6p is a member of the Kinesin-8 Family. In vivo analyses of Klp6p have suggested that this enzyme has rolls in a variety of processes, including microtubule dynamics, spindle-kinetochore interaction, cell morphogenesis, and meiosis. Despite these results our understanding of the function of this microtubule motor protein is still limited. Fiedler s work has therefore focused on bacterial expression of large segments of Klp6p and characterization of the resulting polypeptides in vitro. We are using cryo-electron microscopy and tomography to complement his biochemical experiments for a better understanding of the protein s structure and function. Our approach has been to image rapidly frozen, taxol-stabilized MTs that are decorated with Klp6p motor domain plus neck (MDN) under a variety of conditions. The results clearly demonstrate that the bacterially expressed Klp6MDN protein binds to microtubules in an ATP-sensitive manner and that the protein exists primarily as a dimer in vitro. MTs decorated with Klp6MDN have a diameter of 36-37nm, while MTs themselves are only ~23nm. Neighboring Klp6MDN heads have a mean spacing of approximately 8 nm, suggesting that one motor binds per tubulin dimer. In addition we have shown that Klp6MDN is able to bundle microtubules in vitro. The distance between neighboring MTs within these bundles ranges from 10-13 nm, and diagonal linkers bridging between neighboring MTs are visible.
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