During cell division the mitotic spindle, a multi-component machine, accurately segregates a cell's replicated DNA into two daughter cells. Failure of this process has been linked to numerous developmental defects and oncogenesis. The bipolar organization of the spindle directs movements of sister chromosomes into each daughter cell. The long-term goal of this project is to understand the molecular mechanisms of bipolar spindle assembly. To this end, we have focused our studies on Eg5, an evolutionarily conserved kinesin-related protein required for spindle formation. Loss of Eg5 function during cell division results in monopolar spindles and blocks the cell cycle. Mitosis-specific phosphorylation at a conserved sequence in Eg5 (called the bimC-box) has been proposed to regulate Eg5's spindle targeting. Together with dynein, a minus-end directed microtubule-based motor, and its regulator dynactin, Eg5 organizes spindle microtubules and controls spindle length. We will use Eg5 as a tool to dissect the molecular mechanisms of bipolar spindle formation and force generation in the spindle. Specifically, we will: (1) Examine the influence of microtubule dynamics and organization on the dynamic behavior of Eg5 during spindle assembly, by using fluorescent speckle microscopy and photoactivation of fluorescence. (2) Determine the role of Eg5's motor function in spindle assembly by generating Eg5 mutants with reduced motility and with reduced microtubule affinities, testing these mutants' localization and dynamics in spindles and their ability to rescue spindle formation in Eg5-depleted cell extracts. (3) Analyze the effect of Eg5 phosphorylation on its motor function and structure, by comparing Eg5 phosphorylated at the bimC-box to an Eg5 mutant that cannot be phosphorylated. (4) Determine how dynactin influences Eg5 function in spindles, by examining interactions between dynactin and Eg5 using immunoprecipitation and affinity chromatography, and by comparing the dynamic behavior of dynactin and Eg5 in spindles using fluorescence microscopy. Understanding the mechanism of Eg5 function in spindles should lead to improved inhibitors of Eg5 and could result in better anti-cancer therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CDF-4 (02))
Program Officer
Rodewald, Richard D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rockefeller University
Other Domestic Higher Education
New York
United States
Zip Code
Ti, Shih-Chieh; Alushin, Gregory M; Kapoor, Tarun M (2018) Human ?-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability. Dev Cell 47:175-190.e5
Shimamoto, Yuta; Kapoor, Tarun M (2018) Analyzing the micromechanics of the cell division apparatus. Methods Cell Biol 145:173-190
Forth, Scott; Kapoor, Tarun M (2017) The mechanics of microtubule networks in cell division. J Cell Biol 216:1525-1531
Kapoor, Tarun M (2017) Metaphase Spindle Assembly. Biology (Basel) 6:
Pamula, Melissa C; Ti, Shih-Chieh; Kapoor, Tarun M (2016) The structured core of human ? tubulin confers isotype-specific polymerization properties. J Cell Biol 213:425-33
Kellogg, Elizabeth H; Howes, Stuart; Ti, Shih-Chieh et al. (2016) Near-atomic cryo-EM structure of PRC1 bound to the microtubule. Proc Natl Acad Sci U S A 113:9430-9
Ti, Shih-Chieh; Pamula, Melissa C; Howes, Stuart C et al. (2016) Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends. Dev Cell 37:72-84
Shimamoto, Yuta; Forth, Scott; Kapoor, Tarun M (2015) Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules. Dev Cell 34:669-81
Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya et al. (2014) Micromechanics of the vertebrate meiotic spindle examined by stretching along the pole-to-pole axis. Biophys J 106:735-40
He, Mu; Subramanian, Radhika; Bangs, Fiona et al. (2014) The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment. Nat Cell Biol 16:663-72

Showing the most recent 10 out of 50 publications