scholarly journals Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast

2016 ◽  
Vol 27 (22) ◽  
pp. 3490-3514 ◽  
Author(s):  
Zachary R. Gergely ◽  
Ammon Crapo ◽  
Loren E. Hough ◽  
J. Richard McIntosh ◽  
Meredith D. Betterton
Keyword(s):  
Fission Yeast ◽  
Microtubule Dynamics ◽  
Chromosome Loss ◽  
Mitotic Spindles ◽  
Aberrant Chromosome ◽  
Large Fluctuations ◽  
Spindle Function ◽  
Chromosome Movements ◽  
Sliding Force

Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen.

scholarly journals EB1 Targets to Kinetochores with Attached, Polymerizing Microtubules

2002 ◽  
Vol 13 (12) ◽  
pp. 4308-4316 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Julie C. Canman ◽  
E.D. Salmon ◽  
Timothy J. Mitchison
Keyword(s):  
Spindle Checkpoint ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Microtubule Polymerization ◽  
Spindle Microtubules ◽  
Chromosome Movements

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end–kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

scholarly journals The dual role of fission yeast Tbc1/cofactor C orchestrates microtubule homeostasis in tubulin folding and acts as a GAP for GTPase Alp41/Arl2

2013 ◽  
Vol 24 (11) ◽  
pp. 1713-1724 ◽  
Author(s):  
Risa Mori ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Molecular Level ◽  
Dual Role ◽  
Small Gtpase ◽  
Microtubule Dynamics ◽  
Regulatory Proteins ◽  
Dependent Manner ◽  
Continuous Cycling ◽  
Β Tubulin

Supplying the appropriate amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably elaborate at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. This sequential step is performed by the tubulin-folding cofactor pathway, comprising a specific set of regulatory proteins: cofactors A–E. We identified the fission yeast cofactor: the orthologue of cofactor C, Tbc1. In addition to its roles in tubulin folding, Tbc1 acts as a GAP in regulating Alp41/Arl2, a highly conserved small GTPase. Of interest, the expression of GDP- or GTP-bound Alp41 showed the identical microtubule loss phenotype, suggesting that continuous cycling between these forms is important for its functions. In addition, we found that Alp41 interacts with Alp1D, the orthologue of cofactor D, specifically when in the GDP-bound form. Intriguingly, Alp1D colocalizes with microtubules when in excess, eventually leading to depolymerization, which is sequestered by co-overproducing GDP-bound Alp41. We present a model of the final stages of the tubulin cofactor pathway that includes a dual role for both Tbc1 and Alp1D in opposing regulation of the microtubule.

scholarly journals Chp1-Tas3 Interaction Is Required To Recruit RITS to Fission Yeast Centromeres and for Maintenance of Centromeric Heterochromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2154-2166 ◽  
Author(s):  
Jennifer L. DeBeauchamp ◽  
Arian Moses ◽  
Victoria J. P. Noffsinger ◽  
Dagny L. Ulrich ◽  
Godwin Job ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Feedback Loop ◽  
Histone H3 ◽  
Adaptor Protein ◽  
Centromeric Heterochromatin ◽  
Chromosome Loss ◽  
Methyltransferase Activity ◽  
Interaction Domain ◽  
Positive Feedback Loop

ABSTRACT The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3Δ 10-24) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.

scholarly journals Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

1998 ◽  
Vol 143 (7) ◽  
pp. 1775-1787 ◽  
Author(s):  
Pascal Bernard ◽  
Kevin Hardwick ◽  
Jean-Paul Javerzat
Keyword(s):  
Fission Yeast ◽  
Genomic Instability ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Chromosome Loss ◽  
Checkpoint Response ◽  
Chromosome Missegregation ◽  
Centromere Function

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1+ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1+ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.

scholarly journals APC and EB1 Function Together in Mitosis to Regulate Spindle Dynamics and Chromosome Alignment

2005 ◽  
Vol 16 (10) ◽  
pp. 4609-4622 ◽  
Author(s):  
Rebecca A. Green ◽  
Roy Wollman ◽  
Kenneth B. Kaplan
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Functional Relationship ◽  
Microtubule Dynamics ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Spindle Dynamics ◽  
Chromosome Alignment ◽  
Spindle Function ◽  
Insight Into

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC1–1450) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC1–1450 acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC1–1450 compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

scholarly journals How Essential Kinesin-5 Becomes Non-Essential in Fission Yeast: Force Balance and Microtubule Dynamics Matter

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1154 ◽  
Author(s):  
Masashi Yukawa ◽  
Yasuhiro Teratani ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Force Balance ◽  
Fine Tuning ◽  
Mitotic Spindles ◽  
Clinical Implementation ◽  
Bipolar Spindle ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

The bipolar mitotic spindle drives accurate chromosome segregation by capturing the kinetochore and pulling each set of sister chromatids to the opposite poles. In this review, we describe recent findings on the multiple pathways leading to bipolar spindle formation in fission yeast and discuss these results from a broader perspective. The roles of three mitotic kinesins (Kinesin-5, Kinesin-6 and Kinesin-14) in spindle assembly are depicted, and how a group of microtubule-associated proteins, sister chromatid cohesion and the kinetochore collaborate with these motors is shown. We have paid special attention to the molecular pathways that render otherwise essential Kinesin-5 to become non-essential: how cells build bipolar mitotic spindles without the need for Kinesin-5 and where the alternate forces come from are considered. We highlight the force balance for bipolar spindle assembly and explain how outward and inward forces are generated by various ways, in which the proper fine-tuning of microtubule dynamics plays a crucial role. Overall, these new pathways have illuminated the remarkable plasticity and adaptability of spindle mechanics. Kinesin molecules are regarded as prospective targets for cancer chemotherapy and many specific inhibitors have been developed. However, several hurdles have arisen against their clinical implementation. This review provides insight into possible strategies to overcome these challenges.

scholarly journals Mammalian CLASP1 and CLASP2 Cooperate to Ensure Mitotic Fidelity by Regulating Spindle and Kinetochore Function

2006 ◽  
Vol 17 (10) ◽  
pp. 4526-4542 ◽  
Author(s):  
Ana L. Pereira ◽  
António J. Pereira ◽  
Ana R.R. Maia ◽  
Ksenija Drabek ◽  
C. Laura Sayas ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Ectopic Expression ◽  
Microtubule Dynamics ◽  
Turnover Rates ◽  
Primary Fibroblasts ◽  
Mitotic Spindle Assembly ◽  
Spindle Function ◽  
Partial Redundancy ◽  
Kinetochore Function

CLASPs are widely conserved microtubule plus-end–tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.

scholarly journals Myosin-10 and actin filaments are essential for mitotic spindle function

2008 ◽  
Vol 182 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Sarah Woolner ◽  
Lori L. O'Brien ◽  
Christiane Wiese ◽  
William M. Bement
Keyword(s):  
Mitotic Spindle ◽  
Actin Filaments ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Unconventional Myosin ◽  
Spindle Poles ◽  
Chromosome Partitioning ◽  
Meiotic Spindles ◽  
Spindle Function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.

scholarly journals Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3Alp6

2013 ◽  
Vol 24 (21) ◽  
pp. 3337-3349 ◽  
Author(s):  
Deepsharan K. Dhani ◽  
Benjamin T. Goult ◽  
Gifty M. George ◽  
Daniel T. Rogerson ◽  
Danny A. Bitton ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Essential Gene ◽  
Direct Interaction ◽  
Mitotic Spindles ◽  
Amino Acid Peptide ◽  
Cell Extracts ◽  
Complex Protein ◽  
Ring Complex ◽  
Two Hybrid

In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64–amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3Alp6. Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3Alp6.

scholarly journals Role of cofilin in tau/microtubule dynamics and tauopathy

IBRO Reports ◽  
2019 ◽  
Vol 6 ◽  
pp. S366
Author(s):  
Junga Alexa Woo ◽  
Tian Liu ◽  
Cenxiao Fang ◽  
Sara Cazzaro ◽  
Teresa Kee ◽  
...  
Keyword(s):  
Microtubule Dynamics
Sign in / Sign up

Export Citation Format

Share Document