Chromosome segregation during mitosis requires chromosomes to undergo bipolar attachment on spindle microtubules (MTs) and subsequent silencing of the spindle checkpoint. Here, we describe the identification and characterisation of a novel spindle and kinetochore (KT)-associated complex that is required for timely anaphase onset. The complex comprises at least two proteins, termed Ska1 (Spindle and KT Associated 1) and Ska2. Ska1 associates with KTs following MT attachment during prometaphase. Ska1 and Ska2 interact with each other and Ska1 is required for Ska2 stability in vivo. Depletion of either Ska1 or Ska2 by small interfering RNA results in the loss of both proteins from the KT. The absence of Ska proteins does not disrupt overall KT structure, but KT fibres show an increased cold-sensitivity. Most strikingly, Ska-depleted cells undergo a prolonged checkpointdependent delay in a metaphase-like state. This delay is characterised by the recruitment of Mad2 protein to a few KTs and the occasional loss of individual chromosomes from the metaphase plate. These data suggest that the Ska1/2 complex plays a critical role in the maintenance of the metaphase plate and/or spindle checkpoint silencing.
The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. During mitosis, the two newly forming daughter cells must receive one copy of each chromosome. To accomplish this task, the mitotic spindle pulls sister chromatids toward opposite poles of the dividing cell. This microtubule-based structure comprises dynamic polymers made of ␣-tubulin heterodimers, associated with a large variety of microtubuleassociated proteins (1-5). At the transition from interphase to mitosis, the microtubule network undergoes a profound morphological change. In particular, the microtubule organizing centers of animal cells, the centrosomes, position to opposite sides of the nucleus and increase their microtubule nucleation capacity. Following nuclear envelope breakdown, microtubules emanating from the centrosomes capture each chromosome at the kinetochore, a protein complex assembled on centromeric DNA (6). Appropriate bipolar attachment of sister chromatids is monitored by a surveillance mechanism, the spindle checkpoint (7). Once all kinetochores are attached to microtubules emanating from opposite poles, the connection between sister chromatids is severed and chromatids are pulled apart. In addition to its central role in chromosome segregation, the mitotic spindle also determines the positioning and orientation of the cleavage plane. Therefore, proper positioning of the mitotic spindle is of particular importance for asymmetric cell divisions during development (8,9).A large number of proteins associate with the mitotic spindle and regulate its dynamic formation and function. Stabilizing and destabilizing proteins control the high turnover rate of mitotic microtubules, which have a half-life of less than 60 s (3). Furthermore, motor proteins of the kinesin and dynein families play crucial roles in the formation of a bipolar mitotic spindle (10,11). By interacting with microtubules during early mitosis, they push the spindle poles apart, then play important roles in chromosome-capture by microtubules and power chromosome movement throughout mitosis. Finally, the spindle harbors several regulatory proteins, notably protein kinases and phosphatases (12), which coordinate spindle function in time and space.Although our understanding of microtubule dynamics and spindle formation has greatly advanced during the last two decades, the complexity of the spindle continues to hamper its investigation. A more complete inventory of the mitotic spindle may thus contribute to a better understanding of how exactly the spindle is assembled, what role it plays in th...
Polo-like kinase 1 (Plk1) has multiple important functions during M-phase progression. In addition to a catalytic domain, Plk1 possesses a phosphopeptide-binding motif, the polo-box domain (PBD), which is required for proper localization. Here, we have explored the importance of correct Plk1 subcellular targeting for its mitotic functions. We either displaced endogenous Plk1 through overexpression of the PBD or introduced the catalytic domain of Plk1, lacking the PBD, into Plk1-depleted cells. Both treatments resulted in remarkably similar phenotypes, which were distinct from the Plk1 depletion phenotype. Cells depleted of Plk1 mostly arrested with monoastral spindles, because of inhibition of centrosome maturation and separation. In contrast, these functions were not impaired in cells with mislocalized Plk1. Instead, these latter cells showed a checkpoint-dependent mitotic arrest characterized by impaired chromosome congression. Thus, whereas chromosome congression requires localized Plk1 activity, other investigated Plk1 functions are less dependent on correct PBD-mediated targeting. This opens the possibility that PBD-directed drugs might be developed to selectively interfere with a subset of Plk1 functions.
Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at ∼1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.