The stable propagation of our genomes through cell division depends on the proper organization of complex and dynamic microtubule-based structures, such as the bipolar spindle prior to anaphase and the central spindle (or midzone) during anaphase. Our long-term goal is to determine the molecular mechanisms required for the assembly and function of these cytoskeletal structures. To achieve this goal we take an interdisciplinary approach in which we combine structural, biophysical, chemical and cell biological methods. In the current proposal, which builds upon recent publications and unpublished preliminary data, we focus on the following three Aims: (1) Gain structural insights into antiparallel microtubule crosslinking during cell division. X-ray crystallography, electron microscopy, and TIRF (total internal reflection fluorescence) microscopy assays will be used to examine how PRC1, a widely conserved non-motor microtubule associated protein (MAP) required for central spindle assembly, selectively crosslinks antiparallel microtubules. We will also analyze how PRC1 binds kinesin-4 to control antiparallel microtubule overlap length. (2) Examine how microtubule organization and function are regulated in dividing cells. Structure-guided mutagenesis, in vitro assays, and high-resolution microscopy will be combined with chemical inhibitor and RNAi/add-back based perturbations to examine how central spindle assembly and function are regulated at two different levels: (a) Nanometer-scale features, such as antiparallel microtubule overlap, recognized by PRC1, and (b) Micron-scale spatial activity patterns, such as a spatial phosphorylation gradient that depends on Aurora kinase, a widely conserved regulator of several key processes required for successful cell division. (3) Analyze the mechanical properties of microtubule-based structures essential for cell division. We will use a dual force-calibrated microneedle-based system to examine the viscoelastic properties of the metaphase spindle. Chemical and biochemical perturbations will be used to link the known biochemical and biophysical properties of key proteins to the spindle's micro-mechanics. Together, our findings should advance our understanding of key proteins and regulatory cues (biochemical or mechanical) that contribute to the self-organized assembly of microtubule structures required for the stable propagation of our genomes. Whole chromosome loss, which can arise due to errors in cell division, has been linked to developmental defects and diseases in humans. Our research should provide insight into the molecular mechanisms that ensure cell division is completed without error. Our studies should also contribute to finding new targets for therapeutic agents and impact the development of drugs that target proteins needed for cell division.

Public Health Relevance

Errors in cell division have been linked to developmental defects and disease in humans. We take an interdisciplinary approach to examine the molecular mechanisms needed for accurate cell division. Our findings have the potential to impact 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 #
5R01GM065933-11
Application #
8324563
Study Section
Special Emphasis Panel (ZRG1-CB-G (02))
Program Officer
Deatherage, James F
Project Start
2002-08-15
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
11
Fiscal Year
2012
Total Cost
$404,000
Indirect Cost
$165,652
Name
Rockefeller University
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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
Forth, Scott; Hsia, Kuo-Chiang; Shimamoto, Yuta et al. (2014) Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks. Cell 157:420-32
Hsia, Kuo-Chiang; Wilson-Kubalek, Elizabeth M; Dottore, Alejandro et al. (2014) Reconstitution of the augmin complex provides insights into its architecture and function. Nat Cell Biol 16:852-63
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
Yi, Jason; Wu, Xufeng; Chung, Andrew H et al. (2013) Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage. J Cell Biol 202:779-92
Kleiner, Ralph E; Ti, Shih-Chieh; Kapoor, Tarun M (2013) Site-specific chemistry on the microtubule polymer. J Am Chem Soc 135:12520-3
Subramanian, Radhika; Ti, Shih-Chieh; Tan, Lei et al. (2013) Marking and measuring single microtubules by PRC1 and kinesin-4. Cell 154:377-90
Takagi, Jun; Itabashi, Takeshi; Suzuki, Kazuya et al. (2013) Using micromanipulation to analyze control of vertebrate meiotic spindle size. Cell Rep 5:44-50
Liu, Xin; Kapoor, Tarun M; Chen, James K et al. (2013) Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II. Proc Natl Acad Sci U S A 110:11976-81
Shimamoto, Yuta; Kapoor, Tarun M (2012) Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts. Nat Protoc 7:959-69

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