Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization

doi:10.1083/jcb.147.3.481

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© The Rockefeller University Press, 0021-9525/1999//481 $5.00
The Journal of Cell Biology, Volume 147, Number 3, , 1999 481-492

Original Article

Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization

Aruna Purohit^a^,b, Sharon H. Tynan^b, Richard Vallee^b, and Stephen J. Doxsey^a^,b

^a Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605
^b Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605.(508) 856-4289(508) 856-1613

stephen.doxsey{at}ummed.edu

Pericentrin is a conserved protein of the centrosome involvedin microtubule organization. To better understand pericentrinfunction, we overexpressed the protein in somatic cells andassayed for changes in the composition and function of mitoticspindles and spindle poles. Spindles in pericentrin-overexpressingcells were disorganized and mispositioned, and chromosomes weremisaligned and missegregated during cell division, giving riseto aneuploid cells. We unexpectedly found that levels of themolecular motor cytoplasmic dynein were dramatically reducedat spindle poles. Cytoplasmic dynein was diminished at kinetochoresalso, and the dynein-mediated organization of the Golgi complexwas disrupted. Dynein coimmunoprecipitated with overexpressedpericentrin, suggesting that the motor was sequestered in thecytoplasm and was prevented from associating with its cellulartargets. Immunoprecipitation of endogenous pericentrin alsopulled down cytoplasmic dynein in untransfected cells. To definethe basis for this interaction, pericentrin was coexpressedwith cytoplasmic dynein heavy (DHCs), intermediate (DICs), andlight intermediate (LICs) chains, and the dynamitin and p150^Gluedsubunits of dynactin. Only the LICs coimmunoprecipitated withpericentrin. These results provide the first physiological rolefor LIC, and they suggest that a pericentrin–dynein interactionin vivo contributes to the assembly, organization, and functionof centrosomes and mitotic spindles.

Key Words: pericentrin • centrosomes • mitotic spindle • cytoplasmic dynein light intermediate chains • aneuploidy

1.used in this paper: DAPI, 4',6-diamidino-2-phenylindole; DHC,dynein heavy chain; DIC, dynein intermediate chain; GFP, greenfluorescent protein; HA, hemagglutinin; HA-Pc, hemagglutinin-taggedpericentrin; LIC, dynein light intermediate chain; NuMA, nuclearmitotic apparatus protein

Reprint requests can be made to either S.J. Doxsey or R.B. Vallee.

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