Accurate cell division requires the proper assembly and function of microtubule-based structures such as the bipolar spindle needed to segregate chromosomes, and the spindle midzone (or central spindle) required to keep segregated chromosomes apart and help position the site of cell cleavage. These micron-sized structures require both motor and non-motor proteins to move and organize microtubules, as well as to regulate the formation of new filaments. Our long-term goal is to decipher the molecular mechanisms underlying the assembly and function of these essential structures. To achieve this goal we take a multidisciplinary approach that combines biochemical, structural, biophysical, chemical and cell biological methods. We will build on recent publications and our unpublished preliminary data to focus on the following three Aims: (1) Analyze centrosome-independent microtubule formation. In particular, we will examine how augmin, a recently discovered hetero-octameric protein complex, contributes to microtubule formation within the metaphase spindle. We will employ biochemical, electron microscopy-based, and TIRF (total internal reflection fluorescence) microscopy-based approaches to elucidate how augmin nucleates branched arrays of parallel microtubules. (2) Examine mechanisms regulating microtubule organization. Specifically, we will dissect how PRC1, a non-motor protein that selectively crosslinks antiparallel microtubules, and kinesin-4, a microtubule plus-end directed motor protein that can suppress microtubule growth and disassembly, contribute to the assembly of the spindle midzone, a specialized array of overlapping microtubules that is rapidly assembled during anaphase. We will combine structural, TIRF-microscopy-based, cell biological, and real-time confocal microscopy-based approaches to examine how these two proteins, which bind each other to form a stable complex in solution, regulate microtubule dynamics and antiparallel crosslinking in dividing human cells. (3) Analyze the micromechanics of microtubule arrays. In particular, we will measure how much force is generated to slide microtubules relative to each other by ensembles of motor proteins. TIRF- and optical trap-based assays will be used to determine how the magnitude of forces generated by motor proteins that can crosslink and slide two microtubules apart depends on filament orientation and overlap length. Together, our findings should advance our understanding of how conserved nanometer-sized proteins build the micron-sized structures needed for the stable propagation of our genomes through multiple cell divisions. Errors in cell division have been linked to developmental defects and diseases in humans. Our research should shed light on the molecular mechanisms that ensure the fidelity of chromosome segregation and cytokinesis. Blocking cell division with chemotherapeutic agents is a mainstay of cancer treatments. New anti-cancer drugs are being developed that inhibit cell division by targeting the proteins we study. It is possible that our proposed research will help decipher how these drugs work and may also suggest new targets for therapeutic agents.

Public Health Relevance

Errors in cell division have been linked to developmental defects and disease in humans. We take a multi- disciplinary approach to decipher the molecular mechanisms required for accurate cell division. Our findings have the potential to facilitate the development of therapeutic strategies based on targeting cell division.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065933-14
Application #
8970320
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2002-08-15
Project End
2019-07-31
Budget Start
2015-09-01
Budget End
2016-07-31
Support Year
14
Fiscal Year
2015
Total Cost
$509,480
Indirect Cost
$177,480
Name
Rockefeller University
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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

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