Protein complexes at the microtubule organizing center regulate bipolar spindle assembly.
Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G(1)/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 cut7-22(ts), encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22(ts) we found that stabilized microtubules or increased bundling can rescue cut7-22(ts) associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on gamma-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 gamma-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/gamma-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22(ts) strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.
Rodriguez AS, Batac J, Killilea AN, Filopei J, Simeonov DR, Lin I, Paluh JL