The Dynactin Complex Maintains the Integrity of Metaphasic Centrosomes to Ensure Transition to Anaphase

Yuko Ozaki,Hirotaka Matsui,Akiko Nagamachi,Hiroya Asou,Daisuke Aki,Toshiya Inaba

doi:10.1074/jbc.m110.167742

Abstract

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

Highlights

Ministry of Education, Culture, Sports, Science and Technology of Japan. □S The on-line version of this article contains supplemental Movies 1–3. 1 To whom correspondence should be addressed: 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
It is noteworthy that overexpression of dynamitin disrupts dynactin structure [7]. The mechanism underlying this disruption is yet to be elucidated, dynamitin overexpression has been the major tool in molecular biology for down-regulation of dynactin function [2]
Dynamitin overexpression was used to verify involvement of the dynactin complex in the spindle checkpoint silencing that induces metaphase arrest/delay [8]

Summary

Introduction

Ministry of Education, Culture, Sports, Science and Technology of Japan. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies 1–3. 1 To whom correspondence should be addressed: 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Similar to previous reports [4], immunostaining of mitotic cells with p24(C) antibody showed p24 localized to kinetochores and centrosomes in prometaphase and metaphase cells with considerable signal remaining in early anaphase (Fig. 1D).

Results

Conclusion