@article{b88695c927354b539608ce9f91ad63c6,
title = "Protein complexes at the microtubule organizing center regulate bipolar spindle assembly",
abstract = "Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.",
keywords = "Binding site, Bipolar, Kinesin-14, Kinesin-5, MTOC, Spindle, γ-tubulin",
author = "Rodriguez, {Adrianna S.} and Joseph Batac and Killilea, {Alison N.} and Jason Filopei and Simeonov, {Dimitre R.} and Ida Lin and Paluh, {Janet L.}",
note = "Funding Information: in allele cut7-22ts was determined in order to verify its presence in our double and triple mutants. The mutation lies in a MAP kinase consensus phosphorylation site, L/PxS/TP, adjacent to the bimC box changing CCA to TCA to replace the final proline with a serine.50 Mutations in MTOC alleles are described.12Yeast strains generated or sources are listed in Supplementary Table 1. Microscopy and molecular modeling. Procedures for visualizing microtubules, nuclear envelope, γ-tubulin or Pkl1p in living and fixed cells are as described.10,13 Living cells were imaged at room temperature on a Zeiss Axiovert 200 inverted microscope equipped with a Pryor stage, 100X oil 1.45NA PlanFLUAR and 63X Plan- ACHROMAT 1.4 NA oil DIC objectives, Hammamatsu Orca CCD and Metamorph software (Universal Imaging Corp., Downingtown, PA). DIC images were also obtained on the same microscope system using Zeiss LSM 510/Meta confocal acquisition and Zeiss imaging software. Single plane time series images at 2–30 second intervals were obtained. At least 500 cells were imaged for each phenotype. References Molecular modeling of tubulin side chains was done by the program 2.Tassin AM, Bornens M. Centrosome structure and microtubule nucleation in animal cells. 1.Brinkley BR. Microtubule Organizing Centers. Ann Rev Cell Biol 1985; 1:145-72. The authors would like to thank Dr. Osami Niwa and Dr. Takashi Toda for alp and gtb1 alleles and Dr. Elmar Schiebel and Dr. Michael Knop for two hybrid strains and plasmids. We are indebted to Dr. David Pellman, Dr. Rebecca Heald, Dr. W.Z. Cande for insightful discussions during preparation of the manuscript. The authors recognize the research support of RPI{\textquoteright}s CBIS Microscopy and Imaging Core. DS is supported in part by a federal workstudy scholarship. Former undergraduates AR, JB and JF were supported in part by Boston College undergraduate research fellowships. IL is a member of the Columbia High School Science Research Program. This research is supported by NSF grant MCB-0710938. Note Supplementary materials can be found at: www.landesbioscience.com/supplement/Rodr.iguezCC7-9-Sup.pdf",
year = "2008",
month = may,
day = "1",
doi = "10.4161/cc.7.9.5808",
language = "English",
volume = "7",
pages = "1246--1253",
journal = "Cell Cycle",
issn = "1538-4101",
publisher = "Taylor and Francis Ltd.",
number = "9",
}