No abstract

Summary During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process, and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end directed motors are balanced by forces from minus-end directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive Kinesin-14 minusend directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast Kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end directed Kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity.

Microtubules are structural polymers inside of cells that are subject to posttranslational modifications. These posttranslational modifications create functionally distinct subsets of microtubule networks in the cell, and acetylation is the only modification that takes place in the hollow lumen of the microtubule. Although it is known that the α-tubulin acetyltransferase (αTAT1) is the primary enzyme responsible for microtubule acetylation, the mechanism for how αTAT1 enters the microtubule lumen to access its acetylation sites is not well understood. By performing biochemical assays, fluorescence and electron microscopy experiments, and computational simulations, we found that αTAT1 enters the microtubule lumen through the microtubule ends, and through bends or breaks in the lattice. Thus, microtubule structure is an important determinant in the acetylation process. In addition, once αTAT1 enters the microtubule lumen, the mobility of αTAT1 within the lumen is controlled by the affinity of αTAT1 for its acetylation sites, due to the rapid rebinding of αTAT1 onto highly concentrated α-tubulin acetylation sites. These results have important implications for how acetylation could gradually accumulate on stable subsets of microtubules inside of the cell.microtubule | acetylation | biophysics | microscopy | modeling

UNC-45A, a highly conserved member of the UCS (UNC45A/CRO1/SHE4P) protein family of cochaperones, plays an important role in regulating cytoskeletal-associated functions in invertebrates and mammalian cells, including cytokinesis, exocytosis, cell motility, and neuronal development. Here, for the first time, UNC-45A is demonstrated to function as a mitotic spindle-associated protein that destabilizes microtubules (MT) activity. Using in vitro biophysical reconstitution and total internal reflection fluorescence microscopy analysis, we reveal that UNC-45A directly binds to taxol-stabilized MTs in the absence of any additional cellular cofactors or other MT-associated proteins and acts as an ATP-independent MT destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles, and its depletion causes severe defects in chromosome congression and segregation. UNC-45A is overexpressed in human clinical specimens from chemoresistant ovarian cancer and that UNC-45A-overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and restores sensitivity to paclitaxel. Implications: These findings reveal novel and significant roles for UNC-45A in regulation of cytoskeletal dynamics, broadening our understanding of the basic mechanisms regulating MT stability and human cancer susceptibility to paclitaxel, one of the most widely used chemotherapy agents for the treatment of human cancers.

Microtubules are structural polymers that participate in a wide range of cellular functions. The addition and loss of tubulin subunits allows the microtubule to grow and shorten, as well as to develop and repair defects and gaps in its cylindrical lattice. These lattice defects act to modulate the interactions of microtubules with molecular motors and other microtubule-associated proteins. Therefore, tools to control and measure microtubule lattice structure will be invaluable for developing a quantitative understanding of how the structural state of the microtubule lattice may regulate its interactions with other proteins. In this work, we manipulated the lattice integrity of in vitro microtubules to create pools of microtubules with common nucleotide states, but with variations in structural states. We then developed a series of novel semi-automated analysis tools for both fluorescence and electron microscopy experiments to quantify the type and severity of alterations in microtubule lattice integrity. These techniques will enable new investigations that explore the role of microtubule lattice structure in interactions with microtubule-associated proteins.