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© The Rockefeller University Press,
0021-9525/1998//1519 $5.00
The Journal of Cell Biology, Volume 142, Number 6,
, 1998 1519-1532
Regular Articles |
Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells
Laboratoire des Protéines du Cytosquelette, Institut de Biologie Structurale J.-P. Ebel (CEA/CNRS), 38027 Grenoble cedex 1, France; Centre National de la Recherche Scientifique UPR8402, CRBM, 34033 Montpellier cedex 5, France; and || Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany
Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
Key Words: microtubule • turnover • reconstitution • permeabilized cells • treadmilling
Abbreviations used in this paper: CD, colchicine–tubulin dimer complexes; cdk, cyclin-dependent kinase; TTL, tubulin tyrosine ligase.
Dr. L. Lafanechère kindly provided the TTL– cell clone A607 and Glu-tubulin antibody. We thank Dr. C. Bosc and Dr. L. Guillaud for computer assistance. We are grateful to Dr. A. Lieuvin and to Dr. A. Andrieux for critical reading of this manuscript.
Address all correspondence to Didier Job, CEA-Grenoble, Département de Biologie Moléculaire et Structurale, Laboratoire du Cytosquelette, INSERM Unité 366, 17 rue des Martyrs, 38054 Grenoble cedex 9, France. Tel.: 33-476883801. Fax: 33-476885057. E-mail: job{at}dsvgre.cea.fr
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