Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Rodriguez, Adrianna S.; Batac, Joseph; Killilea, Alison N.; Filopei, Jason; Simeonov, Dimitre R.; Lin, Ida; Paluh, Janet L.

doi:10.4161/cc.7.9.5808

Cited by 40 publications

(64 citation statements)

References 81 publications

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“…It has also been shown that Pkl1 is functionally related to ␥-tubulin in the microtubule-organizing center (14), which has been thought to contribute to spindle bipolarity. Most recently, direct association of Pkl1 motor domain with ␥-tubulin has been demonstrated (15). These studies suggest that Pkl1 affects the MT organization via direct interactions with both spindle pole and microtubule-organizing center complexes to regulate spindle bipolarity.…”

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confidence: 85%

Diffusion and Directed Movement

Furuta

Edamatsu

Maeda

et al. 2008

Journal of Biological Chemistry

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Fission yeast Pkl1 is a kinesin-14A family member that is known to be localized at the cellular spindle and is capable of hydrolyzing ATP. However, its motility has not been detected. Here, we show that Pkl1 is a slow, minus end-directed microtubule motor with a maximum velocity of 33 ؎ 9 nm/s. The K m,MT value of steady-state ATPase activity of Pkl1 was as low as 6.4 ؎ 1.1 nM, which is 20 -30 times smaller than that of kinesin-1 and another kinesin-14A family member, Ncd, indicating a high affinity of Pkl1 for microtubules. However, the duty ratio of 0.05 indicates that Pkl1 spends only a small fraction of the ATPase cycle strongly associated with a microtubule. By using total internal reflection fluorescence microscopy, we demonstrated that single molecules of Pkl1 were not highly processive but only exhibited biased one-dimensional diffusion along microtubules, whereas several molecules of Pkl1, probably fewer than 10 molecules, cooperatively moved along microtubules and substantially reduced the diffusive component in the movement. Our results suggest that Pkl1 molecules work in groups to move and generate forces in a cooperative manner for their mitotic functions.The functioning of mitotic spindles involves the coordinated activities of diverse microtubule (MT) 2 -based motor proteins. Kinesin-related proteins play crucial roles in such functions as spindle assembly, maintenance, and chromosome segregation. They exert forces on MTs to translocate a cargo or MTs themselves (i.e. cross-link and slide MTs). Most members of the kinesin family move toward the plus ends of MTs, whereas several members that belong to the kinesin-14A (Kar3/Ncd) family exhibit minus end-directed motility. The minus end-directed kinesins are believed to have two roles for spindle functioning.The first role is to generate counteracting forces against plus end-directed motors to maintain the spindle (1-4). The second role is to organize spindle MTs at MT minus ends and focus them into spindle poles (5-7). However, despite much progress in recent years on spindle functioning of eukaryotic cells, the precise interpretation of the functions of motors remains complicated and controversial.To elucidate the detailed mechanisms of spindle dynamics, it is necessary to characterize the motile properties of these motor proteins at a molecular level and to construct a model explaining the robust and dynamic spindle system. However, during the division of multicellular eukaryotes, it is difficult to specify the roles of each component due to the existence of a large number of potential contributors. In contrast, in the unicellular fission yeast Schizosaccharomyces pombe, there are nine kinesin family members, and among them, only five members are thought to participate in the mitotic functions (8 -11); this offers an ideal model system to study mitotic processes because of its minimal motor constitution and because it has a lot of similarities with the mitotic processes of higher eukaryotes.In the fission yeast, there are two members of the kinesi...

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confidence: 85%

Diffusion and Directed Movement

Furuta

Edamatsu

Maeda

et al. 2008

Journal of Biological Chemistry

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“…We have used biophysical modeling and quantification of spindle assembly in a cell type that is both relatively simple and amenable to experimental modification, the fission yeast Schizosaccharomyces pombe. This organism has previously been studied in enough detail to allow formulation of a realistic model (78,79); it is small enough that detailed three-dimensional simulations are computationally tractable (80); it is amenable to the genetic manipulation and quantitative experiments needed to parameterize the model (5,6,11,19,20,22,(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98); and the cell contains only three chromosomes whose separate motions can be imaged. Unlike the situation in budding yeast, the mitotic spindle of S. pombe shows important similarities to that of metazoans: Spindle assembly begins in mitotic prophase, and kinetochores attach multiple MTs.…”

Section: Introductionmentioning

confidence: 99%

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Betterton

Blackwell

Edelmaier

et al. 2017

Biophysical Journal

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Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective selfassembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assemblythe persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy.

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“…How GCP2 interacts with ctubulin in comparison to Alp4 might also affect its ability to exchange within the complex or be maintained there. The alp4-1891 mutation that lies in the N-terminal domain of Alp4 is suppressed by a mutation in c-tubulin, gtb1-PL302 (Paluh et al, 2000;Tange et al, 2004) that has been shown to result in enhanced binding of Alp4 to c-tubulin (Rodriguez et al, 2008). This means that changes to the N-terminus of Alp4/GCP2 proteins can affect the distal c-tubulin interaction by the Cterminus of these proteins.…”

Section: Discussionmentioning

confidence: 96%

Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3

Riehlman

Olmsted

Branca

et al. 2013

Journal of Cell Science

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SummaryMicrotubule-organizing centers such as the c-tubulin ring complex (c-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast ctubulin small complex (c-TuSC) of the c-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast c-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the c-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human c-TuSC proteins, we found that GCP3 assembles normally into the .2000 kDa fission yeast c-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of c-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast c-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during c-TuRC assembly. Our findings have broad importance for understanding the essential domains of c-TuSC proteins in the c-TuRC mechanism.

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Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Cited by 40 publications

References 81 publications

Diffusion and Directed Movement

Diffusion and Directed Movement

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3

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