α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

Incorrect kinetochore–microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination—a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore–microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore–microtubule attachments, thereby promoting mitotic fidelity.

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Microtubule tyrosination/detyrosination specifies a mitotic error code

10.1101/801977 ◽

2019 ◽

Author(s):

Luísa T. Ferreira ◽

Bernardo Orr ◽

Girish Rajendraprasad ◽

António J. Pereira ◽

Carolina Lemos ◽

...

Keyword(s):

Error Correction ◽

Half Life ◽

Chromosomal Instability ◽

Post Translational Modification ◽

Human Cancers

AbstractIncorrect kinetochore-microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination - a post-translational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by microtubule tyrosination/detyrosination remains unknown. Here we found that microtubule detyrosination accumulates on correct, more stable, kinetochore-microtubule attachments, whereas constitutively high microtubule detyrosination near kinetochores compromised efficient error correction. Rescue experiments suggest that mitotic errors due to excessive microtubule detyrosination result from suppression of MCAK activity, without globally affecting kinetochore microtubule half-life. Importantly, MCAK centromeric activity was required and sufficient to rescue mitotic errors due to excessive microtubule detyrosination. Thus, microtubules are not just passive elements during mitotic error correction, and their tyrosination/detyrosination works as a ‘mitotic error code’ that allows centromeric MCAK to discriminate correct and incorrect kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

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Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure

Blood ◽

10.1182/blood.v116.21.880.880 ◽

2010 ◽

Vol 116 (21) ◽

pp. 880-880 ◽

Cited By ~ 1

Author(s):

Katie Lombardo ◽

Jason Stumpff ◽

Susan Parkhurst ◽

Linda Wordeman ◽

Akiko Shimamura

Keyword(s):

Genomic Instability ◽

Mitotic Spindle ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Microtubule Dynamics ◽

Published Data ◽

Hematopoietic Progenitor ◽

Microtubule Stability ◽

Cd34 Cells

Abstract Abstract 880 Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition. Disclosures: No relevant conflicts of interest to declare.

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TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes

Open Biology ◽

10.1098/rsob.130108 ◽

2014 ◽

Vol 4 (6) ◽

pp. 130108 ◽

Cited By ~ 17

Author(s):

Roshan L. Shrestha ◽

Naoka Tamura ◽

Anna Fries ◽

Nicolas Levin ◽

Joanna Clark ◽

...

Keyword(s):

Error Correction ◽

Chromosomal Instability ◽

Spindle Checkpoint ◽

Human Cells ◽

Microtubule Dynamics ◽

Regulatory Pathways ◽

Chromosomal Stability ◽

Microtubule Attachment ◽

Drug Treatments

Chromosomal instability can arise from defects in chromosome–microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1's role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome–microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore–microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.

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Neurofilaments and Paired Helical Filaments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120357 ◽

1985 ◽

Vol 43 ◽

pp. 740-743

Author(s):

J. Metuzals

Keyword(s):

Animal Model ◽

Posttranslational Modifications ◽

Posttranslational Modification ◽

Giant Axon ◽

Paired Helical Filaments ◽

Squid Giant Axon ◽

In Vitro Experiments ◽

Thin Sections ◽

Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

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Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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Stu2p binds tubulin and undergoes an open-to-closed conformational change

The Journal of Cell Biology ◽

10.1083/jcb.200511010 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1009-1022 ◽

Cited By ~ 87

Author(s):

Jawdat Al-Bassam ◽

Mark van Breugel ◽

Stephen C. Harrison ◽

Anthony Hyman

Keyword(s):

Conformational Change ◽

Conformational Transition ◽

Budding Yeast ◽

Microtubule Dynamics ◽

Open Structure ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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Assessment of the genotoxic potential of tetrachlorvinphos insecticide by cytokinesis-block micronucleus and sister chromatid exchange assays

Human & Experimental Toxicology ◽

10.1177/09603271211036126 ◽

2021 ◽

pp. 096032712110361

Author(s):

Hayal Cobanoglu ◽

Akin Cayir

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Exchange ◽

Chromosomal Instability ◽

Human Peripheral Blood ◽

Peripheral Blood Lymphocytes ◽

Proliferation Index ◽

Dna Damages ◽

Genotoxic Potential ◽

Index Level

Tetrachlorvinphos is an organophosphate that is classified as a carcinogen in humans by several authorities. Due to very limited data regarding the genotoxic potential, we aimed to comprehensively investigate in vitro genotoxic potential of tetrachlorvinphos. We performed our study by applying the cytokinesis-block micronucleus cytome and sister chromatid exchange (SCE) assays to human peripheral blood lymphocytes. We evaluated micronucleus (MN) and SCE frequencies and cytokinesis-block proliferation index in both exposed and non-exposed lymphocytes. We also calculated the chromosomal instability level in response to exposure by combining the results of MN and SCE. We found that MN frequency did not increase with exposure to tetrachlorvinphos (0–50 µg/ml). In contrast, we observed that SCE frequencies significantly increased with exposure to ≥5 µg/ml tetrachlorvinphos. Furthermore, exposure to tetrachlorvinphos at concentrations of 50 µg/ml induced a significant increase in chromosomal instability level ( p < 0.05). Cytokinesis-block proliferation index level did not significantly decrease in response to tetrachlorvinphos exposure. Our findings reveal that tetrachlorvinphos resulted in different DNA damages that were measured by two assays. Furthermore, our findings suggested that exposure to tetrachlorvinphos increased chromosomal instability that is a hallmark of many malignancies. We conclude that although tetrachlorvinphos does not significantly increase the MN level, the significant increase of both SCE and CIN frequencies indicates the genotoxic potential of tetrachlorvinphos in human peripheral lymphocytes. Additionally, tetrachlorvinphos is not cytotoxic in the range of tested concentrations.

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Factors affecting the time of formation of the mouse blastocoele

Development ◽

10.1242/dev.41.1.79 ◽

1977 ◽

Vol 41 (1) ◽

pp. 79-92

Author(s):

Rosita Smith ◽

Anne McLaren

Keyword(s):

Cytochalasin B ◽

Critical Factor ◽

Cell Number ◽

Experimental Manipulation ◽

Factors Affecting ◽

Elapsed Time ◽

Developmental Age ◽

Culture Formation

In normal mouse embryos developing in vivo, the first appearance of the blastocyst cavity was found to be associated more closely with developmental age, judged by cell number, than with chronological age, i.e. elapsed time since ovulation. When development was slowed by in vitro culture, formation of the blastocoele was delayed. However, cell number itself was not a critical factor, since the number of cells per embryo could be doubled or tripled or halved by experimental manipulation without substantially affecting the timing of blastocoele formation. Experiments in which one cell division was suppressed with cytochalasin-B, leading to tetraploidy, showed that the number of cell divisions since fertilization was also not critical. A possible role is suggested either for nucleocytoplasmic ratio, or for the number of nuclear or chromosomal divisions or DNA replications since fertilization, all of which increase during cleavage.

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