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Using High Pressure Freezing and Freeze Substitution to Investigate Kinetochore Ultrastructure in Vetebrate Somatic Cells

Published online by Cambridge University Press:  02 July 2020

B.F. McEwen ,
C.E. Hsieh ,
R.M. Barnard  and
C. L. Rieder
B.F. McEwen
Affiliation:
Div. Molecular Medicine, Wadsworth Center, NYS Dept. Health, Albany, NY12201-0509. Dept. Biomedical Medical Sciences, State University of New York, Albany, NY12222.
C.E. Hsieh
Affiliation:
Div. Molecular Medicine, Wadsworth Center, NYS Dept. Health, Albany, NY12201-0509.
R.M. Barnard
Affiliation:
Div. Molecular Medicine, Wadsworth Center, NYS Dept. Health, Albany, NY12201-0509.
C. L. Rieder
Affiliation:
Div. Molecular Medicine, Wadsworth Center, NYS Dept. Health, Albany, NY12201-0509. Dept. Biomedical Medical Sciences, State University of New York, Albany, NY12222.
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Extract

In vertebrate somatic cells, sister kinetochores attach chromosomes to the mitotic spindle by capturing microtubules (Mts) emanating from opposite spindle poles. Kinetochores are also required for chromosome alignment at the spindle equator, stabilizing kinetochore Mts, cell cycle control of anaphase onset, and poleward migration of sister chromatids during anaphase. Thus kinetochores play a critical role in the distribution of genetic information to daughter cells during cell division. Understanding the molecular mechanisms behind kinetochore function requires knowing how the molecular components are arranged relative to each other, and relative to the kinetochore Mts. Currently, however, our knowledge of kinetochore composition is incomplete, and until recently our knowledge of kinetochore ultrastructure was limited to the familiar trilamellar model derived from conventionally fixed and dehydrated specimens(Figures 1a&b).

Highly fibrous structures, such as the kinetochore, are particularly vulnerable to distortions caused by the chemical fixation and dehydration methods used in conventional specimen preparations. Such distortions can be largely avoided by using high pressure freezing and freeze substitution (HPF/FS).

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy II. Cells and Tissues
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 436 - 437
Copyright
Copyright © Microscopy Society of America

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8. Supported by NSF grant MCB 98 08879 and NIH NCPP/BTP grant P41-RR01219Google Scholar