Murine CENP-F regulates centrosomal microtubule nucleation and interacts with Hook2 at the centrosome.
The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F(-/-) cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F(-/-) cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.
Stahlman Cardiovascular Research Laboratories, Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA.
Katherine L
KL
Moynihan
Ryan
R
Pooley
Paul M
PM
Miller
Irina
I
Kaverina
David M
DM
Bader
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D050257:Tubulin Modulators / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D002868:Chromosomal Proteins, Non-Histone; C521349:Hook2 protein, human; C546999:Hook2 protein, mouse; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D050257:Tubulin Modulators; C094772:centromere protein F; D015739:Nocodazole