CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.
Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment
G. Géraud (Institut Jacques Monod, Paris) contributed to this work with his excellent expertise with confocal imaging. We are very grateful to UK Laemmli (University of Geneva) for the gift of purified chromosomes and the help with the chromosome labeling. T. Kreis, P. Pierre, K. Pfister, D. Meyer, and H. Ponstingl are thanked for their encouragement and generous gifts of antibodies and plasmids. Mark Eckley and Manfred Lohka are thanked for their helpful remarks on the manuscript. We are very grateful to D. Compton (Dartmouth Medical School, Hanover, NH) for allowing us to cite unpublished results from his lab.
Received for publication 6 October 1997 and in revised form 2 April 1998.
Address all correspondence to Dr. Jan De Mey, Institut Jacques Monod, CNRS-University of Paris VI & VII, 2, place Jussieu, Tour 43, F-75251 Paris Cedex 05, France. Tel.: 33 1 44 27 77 64. Fax: 33 1 44 27 59 94. E-mail: [email protected]
Denis Dujardin, U. Irene Wacker, Anne Moreau, Trina A. Schroer, Janet E. Rickard, Jan R. De Mey; Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment . J Cell Biol 18 May 1998; 141 (4): 849–862. doi: https://doi.org/10.1083/jcb.141.4.849
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