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Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion.
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MedLine Citation:
PMID:  1541637     Owner:  NLM     Status:  MEDLINE    
During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.
E Shelden; P Wadsworth
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  The Journal of cell biology     Volume:  116     ISSN:  0021-9525     ISO Abbreviation:  J. Cell Biol.     Publication Date:  1992 Mar 
Date Detail:
Created Date:  1992-04-06     Completed Date:  1992-04-06     Revised Date:  2010-09-07    
Medline Journal Info:
Nlm Unique ID:  0375356     Medline TA:  J Cell Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1409-20     Citation Subset:  IM    
Department of Zoology, University of Massachusetts, Amherst 01003.
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MeSH Terms
Cell Line
Centromere / metabolism*,  ultrastructure
Chromosomes / physiology*
Microscopy, Immunoelectron
Microtubules / metabolism*,  ultrastructure
Tubulin / metabolism*
Reg. No./Substance:
0/Tubulin; 58-85-5/Biotin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): J Cell Biol
Journal ID (publisher-id): J. Cell Biol.
ISSN: 0021-9525
ISSN: 1540-8140
Publisher: The Rockefeller University Press
Article Information
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Print publication date: Day: 2 Month: 3 Year: 1992
Volume: 116 Issue: 6
First Page: 1409 Last Page: 1420
ID: 2289371
Publisher Id: 92176239
PubMed Id: 1541637

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to- pole motion

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