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PMID: 8647893 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis.

The Journal of cell biology ·Vol. 132 ·No. 4 ·1996-02-00 ·Pages 617-33

Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB

Abstract

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Cattle Cell Line Chromosomes Cloning, Molecular DNA, Complementary/genetics Dynactin Complex Dyneins/genetics,metabolism Humans Male Microtubule Proteins/genetics,metabolism Microtubule-Associated Proteins Mitosis Molecular Sequence Data Rats Rats, Sprague-Dawley Sequence Analysis
Chemicals
DNA, Complementary Dynactin Complex Microtubule Proteins Microtubule-Associated Proteins Dyneins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Echeverri C J
Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
Paschal B M
Vaughan K T
Vallee R B
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.132.4.617
Published
1996-02-00
Pages
617-33
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2199864
Subset
IM
Grants
NIGMS NIH HHS · GM 15941 · United States
NIGMS NIH HHS · GM 43474 · United States
Databases
GENBANK
U50733
External Links
PubMed source DOI: 10.1083/jcb.132.4.617 PMC Full Text View journal details
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