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PMID: 8896597 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.

The Journal of cell biology ·Vol. 135 ·No. 2 ·1996-10-00 ·Pages 399-414

Gaglio T, Saredi A, Bingham JB, Hasbani MJ, Gill SR, Schroer TA, Compton DA

Abstract

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end-directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.

MeSH Terms
Animals Antibodies, Monoclonal Antigens, Nuclear Cell Cycle Proteins Cell Line Cell-Free System Chickens Chlorocebus aethiops Dynactin Complex Dyneins/metabolism HeLa Cells Humans Kinesins/metabolism Microtubule Proteins/metabolism Microtubule-Associated Proteins Mitosis Models, Biological Nuclear Matrix-Associated Proteins Nuclear Proteins/metabolism Nucleolus Organizer Region/physiology,ultrastructure Recombinant Proteins/metabolism Spindle Apparatus/physiology,ultrastructure Xenopus Proteins
Chemicals
Antibodies, Monoclonal Antigens, Nuclear Cell Cycle Proteins Dynactin Complex KIF11 protein, Xenopus Microtubule Proteins Microtubule-Associated Proteins NUMA1 protein, Xenopus NUMA1 protein, human Nuclear Matrix-Associated Proteins Nuclear Proteins Recombinant Proteins Xenopus Proteins Dyneins Kinesins
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Gaglio T
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Saredi A
Bingham J B
Hasbani M J
Gill S R
Schroer T A
Compton D A
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.135.2.399
Published
1996-10-00
Pages
399-414
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2121053
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.135.2.399 PMC Full Text View journal details
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