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

Abstract

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150Glued subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin–dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.

Promoter hijack reveals pericentrin functions in mitosis and the DNA damage response

“…Assembly of pericentrin at centrosomes requires the microtubule motor, cytoplasmic dynein, with which it interacts in the organization of the mitotic spindle. 9,10 Overexpression of pericentrin causes centrosome abnormalities and disrupts mitotic spindle integrity, possibly through its sequestration of dynein. 9,11 However, such overexpression also facilitates the de novo centriole duplication process, which exists in addition to the standard templated mechanism by which centrioles normally duplicate.…”

“…9,10 Overexpression of pericentrin causes centrosome abnormalities and disrupts mitotic spindle integrity, possibly through its sequestration of dynein. 9,11 However, such overexpression also facilitates the de novo centriole duplication process, which exists in addition to the standard templated mechanism by which centrioles normally duplicate. 12,13 Pericentrin overexpression caused expansion of the PCM, allowing the formation of multiple daughter centrioles independently of any spatial or numerical control from the mother centrioles.…”

Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

“…Cell lines (COS-7, SAOS, and U2OS) were grown as described previously (American Type Culture Collection, Manassas, VA) and prepared for transfection experiments as described previously (Purohit et al, 1999). Primary mouse embryonic fibroblasts (MEFs) were obtained from Dr. Geoffrey Wahl (Salk Institute for Biological Studies, La Jolla, CA) and used at less than six passages.…”

“…MEFs were transfected using Superfect (QIAGEN, Valencia, CA). For immunofluorescence, cells were fixed with -20°C MeOH as described previously (Purohit et al, 1999). Data were collected as a Z series for deconvolution with 0.3 m between planes.…”

“…All pericentrin constructs used in this study were cloned into pcDNA vectors (Invitrogen, Carlsbad, CA) with amino terminal hemagglutinin (HA) tags (Purohit et al, 1999;Purohit et al, 2001), except those used in two-hybrid studies (see below). Fragments of pericentrin and other genes were polymerase chain reaction (PCR) amplified from cDNAs by using primers with NotI and XbaI restriction sites.…”