EMBO J. 2004 Aug 18;23(16):3237-48. Epub 2004 Aug 5.

Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation.

Kurasawa Y, Earnshaw WC, Mochizuki Y, Dohmae N, Todokoro K.

Source

Cell Fate Signaling Research Unit, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan.

Abstract

A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule-bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP-E, but not to the chromosomal passenger proteins. In KIF4-deficient cells, the central spindle was disorganized, and all midzone-associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1-deficient cells, no midzone was formed, KIF4 and CENP-E did not localize to the disconnected half-spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half-spindle. Thus, PRC1 is required for interaction of the two half-spindles and for localization of KIF4 and CENP-E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation.

PMID:: 15297875; [PubMed - indexed for MEDLINE]
PMCID:: PMC514520

Free PMC Article

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Figure 1

PRC1 specifically binds to KIF4 during late mitosis. (A) Immunoaffinity chromatography. HeLa cell extracts at late mitosis were fractionated by affinity chromatography using anti-PRC1-specific antibody, and bound proteins were separated by SDS–PAGE and silver-stained. (B) Immunoprecipitates with anti-PRC1 antibody contain KIF4. HeLa cell extracts at late mitosis were subjected to immunoprecipitation, with preimmune sera, anti-PRC1 or anti-KIF4, fractionated by SDS–PAGE and immunoblotted with anti-KIF4-specific antibody. The common band in the immunoprecipitates with preimmune serum and anti-KIF4 antibody is antibody-derived protein. (C) Immunoprecipitates with anti-KIF4 antibody include PRC1. (D–E) Pull-down experiments with a series of PRC1 fragments fused to GST. Schematic of PRC1 fragments used for pull-down experiments (D) and their SDS–PAGE pattern (E). The PRC1 fragments were incubated with HeLa cell extracts at late mitosis, and bound proteins separated by SDS–PAGE were probed with anti-KIF4, anti-MKLP1 and CENP-E antibodies (F).

Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation