(Supported in part by GMS 40198 to C. Rieder and by NSF MCB9420772 to B. McEwen) This project required that methods be developed to tomographically reconstruct small regions of cells followed in the living state by video light microscopy. The general goal was to evaluate several hypothesis concerning how chromosomes move and how the kinetochore/centromere complex on each is assembled. First we used video microscopy to follow a cell in the living state through fixation. Next the embedded cell was serially thick sectioned and the section containing the kinetochore of interest was photographed in a tilt series on the IVEM. The resultant tomogram was then computed and visualized using the SPIDER/WEB image processing software. Different components of the reconstruction (e.g., microtubules and the kinetochore outer plate) were traced using Sterecon. Sterecon also enabled us to make quantitative measurements of microtubule penetration into the outer plate. For illustrations, c omplex surface-rendered and filled-contour models were made with the parallel-processor, reality engine-equipped SGI Onyx computer. During 1997 a large number of tomographic reconstructions were made and analyzed, and parts of this study were published. McEwen, B.F. and A. B. Heagle. (1997) Electron microscopic tomography: A tool for probing the structure and function of subcellular components. Internat. J. Imaging Systems. Technology, 8:175-187. McEwen, B.F., A. B. Heagle and C.L. Rieder. (1997) Probing the mechanisms underlying kinetochore behavior in vertebrate cells using combinations of advanced light and 3-D electron microscopy. Microsc. Microanalysis 3(suppl 2):217-218.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001219-17
Application #
6280675
Study Section
Project Start
1998-01-01
Project End
1998-12-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
17
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204
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McEwen, Bruce F; Dong, Yimin (2010) Contrasting models for kinetochore microtubule attachment in mammalian cells. Cell Mol Life Sci 67:2163-72
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