This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Electron tomography of rapidly frozen, freeze-substitution fixed samples will be used to examine a significant fraction of the volume of metaphase spindles from U2OS cells, followed by comparable work on cells frozen and fixed during spindle formation. Our structural studies will employ improved methods for sample preparation (TR&D 3A) and both large-scale montages and multiple serial sections, assembled into 3D reconstructions (TR&D 5D). The trajectories of spindle microtubules (MTs) will initially be determined by hand modeling, though in subsequent work we hope to have software for automatic segmentation. The resulting models of MT trajectories will be studied with cluster analysis and several application-specific software tools to compare MT organization with the predictions from four distinct and popular models for spindle formation: the classic, centrosome-initiation model, the idea that spindle MTs self-organize as a result of MT-associated motor enzymes, the idea of MT initiation and/or stabilization in response to gradients that emanate from chromosomes, and the concept that a non/MT """"""""matrix"""""""" guides the formation and organization of MTs. The data should eliminate or at least erode one or more of these models and will certainly provide baseline structural information for the interpretation of functional experiments on spindle formation and function that are being carried out in our labs and elsewhere.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000592-41
Application #
8362545
Study Section
Special Emphasis Panel (ZRG1-CB-J (40))
Project Start
2011-05-01
Project End
2012-04-30
Budget Start
2011-05-01
Budget End
2012-04-30
Support Year
41
Fiscal Year
2011
Total Cost
$31,913
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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