16 research outputs found
The outer kinetochore protein KNL-1 contains a defined oligomerization domain in nematodes
The kinetochore is a large, macromolecular assembly that is essential for connecting chromosomes to microtubules during mitosis. Despite the recent identification of multiple kinetochore components, the nature and organization of the higher order kinetochore structure remain unknown. The outer kinetochore KNL-1/Mis12 complex/Ndc80 complex (KMN) network plays a key role in generating and sensing microtubule attachments. Here, we demonstrate that Caenorhabditis elegans KNL-1 exists as an oligomer and we identify a specific domain in KNL-1 responsible for this activity. An N-terminal KNL-1 domain from both C. elegans and the related nematode C. remanei oligomerizes into a decameric assembly that appears roughly circular when visualized by electron microscopy. Based on sequence and mutational analysis, we identify a small hydrophobic region as responsible for this oligomerization activity. However, mutants that precisely disrupt KNL-1 oligomerization did not alter KNL-1 localization or result in the loss of embryonic viability based on gene replacements in C. elegans. In C. elegans, KNL-1 oligomerization may coordinate with other kinetochore activities to ensure the proper organization, function, and sensory capabilities of the kinetochore-microtubule attachment.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776
Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments
Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated if two C. elegans α- and β-tubulin isotypes, identified via mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules (MTs). One isotype, TBB-4, assembles into MTs at the tips of the axoneme core and distal segments, where the MT tip-tracker, EB1, is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, FRAP analysis and modeling suggest that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady state length
Decoding Polo-like kinase 1 signaling along the kinetochore–centromere axis
Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet its spatial coordination is obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection/resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1’s kinetochore functions
Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2
The kinesin Eg5 moves toward minus ends of astral microtubules in early mitosis, switching to plus-end motion in anaphase. Dynein is required for minus-end motion; depletion of TPX2 results in a switch to plus-end motion. On midzone microtubules, Eg5 moves in both directions. Our results explain the redistribution of Eg5 throughout mitosis