A fundamental, but poorly understood, problem in cell biology is how the sizes of organelles are controlled. The lengths of mitotic spindles and axonemes, for example, vary by as little as a few per cent between cells of the same type. Furthermore, the correct size and morphology are essential for function-mitotic spindles for cell division and axonemes for motility. Cells regulate the sizes of these organelles by tightly controlling the lengths of their constituent microtubules. In the absence of a molecular ruler that templates microtubule length, it is thought that length control results from a delicate balance between polymerization and depolymerization of the microtubules. How this is achieved is not known. ! Based on our previous work in which we showed that the motor kinesin-8 Kip3 is a length-dependent microtubule depolymerase, we hypothesize that motor proteins, in conjunction with other microtubule- associated proteins (MAPs), can provide feedback between length and dynamics that tightly regulates the lengths of microtubules. ! The general aim of this grant is to use single-molecule techniques, together with mathematical modeling, to understand how two additional proteins-the yeast kinesin Kip2 and the yeast homolog of the vertebrate polymerase XMAP215, Stu2-together with Kip3, regulate the lengths of yeast microtubules. We have devised a novel purification scheme for native budding-yeast tubulin and this allows us to employ yeast as our model system, which has distinct advantages due to the small number of tubulin isoforms and the absence of potentially confounding post-translational modifications found in vertebrate, and in particular brain, tubulin. ! Our specific aims are to (1) characterize the acceleration of growth of yeast microtubules by Stu2, (ii) determine how Kip2 promotes microtubule assembly, and (iii) examine the precision with which Kip3, in combination with Kip2 and Stu2, controls microtubule lengths. These studies will provide important insight into the assembly and function of the mitotic spindle and establish principles of length regulation that will be applicable to other biomedically relevant organellar systems such axonemes, microvilli, stereocilia and filopodia.

Public Health Relevance

The high fidelity of chromosome segregation ensures that the two daughters of a dividing cell equally inherit the mother's genes;errors in segregation lead to aneuploidy, which causes birth defects, and which is a property of cancer cells. We are studying the protein machinery, called the mitotic spindle that accurately segregates the chromosomes. Our studies on the mechanisms and principles by which these proteins build and localize the spindle will be directly relevant to the better understanding of cell division and its errors in human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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Yale University
Schools of Medicine
New Haven
United States
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Howard, Jonathon; Garzon-Coral, Carlos (2017) Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces: Mechanics of mitotic spindle positioning. Bioessays 39:
Coombes, Courtney; Yamamoto, Ami; McClellan, Mark et al. (2016) Mechanism of microtubule lumen entry for the ?-tubulin acetyltransferase enzyme ?TAT1. Proc Natl Acad Sci U S A 113:E7176-E7184
Borisy, Gary; Heald, Rebecca; Howard, Jonathon et al. (2016) Microtubules: 50 years on from the discovery of tubulin. Nat Rev Mol Cell Biol 17:322-8
Xiao, Xun; Geyer, Veikko F; Bowne-Anderson, Hugo et al. (2016) Automatic optimal filament segmentation with sub-pixel accuracy using generalized linear models and B-spline level-sets. Med Image Anal 32:157-72
Bowne-Anderson, Hugo; Hibbel, Anneke; Howard, Jonathon (2015) Regulation of Microtubule Growth and Catastrophe: Unifying Theory and Experiment. Trends Cell Biol 25:769-779
Hibbel, Anneke; Bogdanova, Aliona; Mahamdeh, Mohammed et al. (2015) Kinesin Kip2 enhances microtubule growth in vitro through length-dependent feedback on polymerization and catastrophe. Elife 4:
Podolski, Marija; Mahamdeh, Mohammed; Howard, Jonathon (2014) Stu2, the budding yeast XMAP215/Dis1 homolog, promotes assembly of yeast microtubules by increasing growth rate and decreasing catastrophe frequency. J Biol Chem 289:28087-93
Howard, Jonathon (2014) Quantitative cell biology: the essential role of theory. Mol Biol Cell 25:3438-40