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 Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

2003 ◽  
Vol 14 (10) ◽  
pp. 4181-4195 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Ted R. Zarzar ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulatory Mechanism ◽  
Metaphase Plate ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

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 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.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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

scholarly journals Florigen governs shoot regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yaarit Kutsher ◽  
Michal Fisler ◽  
Adi Faigenboim ◽  
Moshe Reuveni
Keyword(s):  
Nitric Oxide ◽  
Shoot Regeneration ◽  
Ectopic Expression ◽  
Flowering Plants ◽  
Regeneration Capacity ◽  
No Donor ◽  
Tobacco Leaves ◽  
Age Related ◽  
Delayed Flowering

AbstractIt is widely known that during the reproductive stage (flowering), plants do not root well. Most protocols of shoot regeneration in plants utilize juvenile tissue. Adding these two realities together encouraged us to study the role of florigen in shoot regeneration. Mature tobacco tissue that expresses the endogenous tobacco florigen mRNA regenerates poorly, while juvenile tissue that does not express the florigen regenerates shoots well. Inhibition of Nitric Oxide (NO) synthesis reduced shoot regeneration as well as promoted flowering and increased tobacco florigen level. In contrast, the addition of NO (by way of NO donor) to the tissue increased regeneration, delayed flowering, reduced tobacco florigen mRNA. Ectopic expression of florigen genes in tobacco or tomato decreased regeneration capacity significantly. Overexpression pear PcFT2 gene increased regeneration capacity. During regeneration, florigen mRNA was not changed. We conclude that florigen presence in mature tobacco leaves reduces roots and shoots regeneration and is the possible reason for the age-related decrease in regeneration capacity.

Bidirectional role of microtubule dynamics in the acquisition and maintenance of temporal information in dorsolateral striatum

2021 ◽  
pp. 107468
Author(s):  
S. Aryana Yousefzadeh ◽  
Anna E. Youngkin ◽  
Nicholas A. Lusk ◽  
Shufan Wen ◽  
Warren H. Meck
Keyword(s):  
Microtubule Dynamics ◽  
Temporal Information ◽  
Dorsolateral Striatum

scholarly journals The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 489-503 ◽  
Author(s):  
Karen E Ross ◽  
Orna Cohen-Fix
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Growth Defects ◽  
Genes Encoding ◽  
Specificity Factor

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

scholarly journals Identification of Autosomal Regions Involved in Drosophila Raf Function

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 763-774 ◽  
Author(s):  
Willis Li ◽  
Elizabeth Noll ◽  
Norbert Perrimon
Keyword(s):  
Limb Development ◽  
Target Gene ◽  
Biological Function ◽  
Genetic Interaction ◽  
Ectopic Expression ◽  
Tor Signaling ◽  
Downstream Effector ◽  
Early Embryos ◽  
Genomic Regions

Abstract Raf is an essential downstream effector of activated p21Ras (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified “Raf activator.” In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing ∼70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf  Su2, causing lethality in Draf  Su2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3ϵ, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf  Su2 and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs.

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response

2021 ◽  
Author(s):  
Zemin Wang ◽  
Darren Chern Jan Wong ◽  
Yi Wang ◽  
Guangzhao Xu ◽  
Chong Ren ◽  
...  
Keyword(s):  
Stress Response ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Cold Treatment ◽  
Ectopic Expression ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Vitis Amurensis ◽  
Mobility Shift

Abstract Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

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