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1.
J Cell Biol ; 223(10)2024 Oct 07.
Article in English | MEDLINE | ID: mdl-38949648

ABSTRACT

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.


Subject(s)
Anaphase , Drosophila Proteins , Drosophila melanogaster , Dyneins , Microtubules , Animals , Dyneins/metabolism , Dyneins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Microtubules/metabolism , Microtubules/genetics , Molecular Dynamics Simulation , Mutation/genetics , Spindle Apparatus/metabolism , Spindle Apparatus/genetics , Humans , Mutation, Missense
2.
Methods Mol Biol ; 2740: 229-242, 2024.
Article in English | MEDLINE | ID: mdl-38393479

ABSTRACT

Cell division is a conserved process among eukaryotes. It is designed to segregate chromosomes into future daughter cells and involves a complex rearrangement of the cytoskeleton, including microtubules and actin filaments. An additional level of complexity is present in asymmetric dividing stem cells because cytoskeleton elements are also regulated by polarity cues. The neural stem cell system of the fruit fly represents a simple model to dissect the mechanisms that control cytoskeleton reorganization during asymmetric division. In this chapter, we propose to describe protocols that allow accurate analysis of microtubule reorganization during cell division in this model.


Subject(s)
Drosophila Proteins , Neural Stem Cells , Animals , Drosophila , Drosophila Proteins/genetics , Cell Division , Microtubules , Cell Polarity , Asymmetric Cell Division
3.
bioRxiv ; 2023 Aug 04.
Article in English | MEDLINE | ID: mdl-37577480

ABSTRACT

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

4.
J Cell Sci ; 136(11)2023 06 01.
Article in English | MEDLINE | ID: mdl-37288770

ABSTRACT

Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading.


Subject(s)
Cell Cycle Proteins , Chromatids , Histone Methyltransferases , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/metabolism , Chromatin , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Histone Methyltransferases/metabolism , Cohesins
5.
STAR Protoc ; 3(3): 101493, 2022 09 16.
Article in English | MEDLINE | ID: mdl-35776653

ABSTRACT

Drosophila neural stem cells (NSCs) divide asymmetrically to generate siblings of different sizes. This model system has proved helpful in deciphering the contribution of polarity cues and the mitotic spindle in asymmetric cell division. Here, we describe a technique we developed to flatten cultured Drosophila brain explants to accurately image the cytoskeleton in live NCSs. We also describe our approach to efficiently remove centrosomes by laser photo-ablation and to measure daughter cell size after NSC division. For complete details on the use and execution of this protocol, please refer to Thomas et al. (2021).


Subject(s)
Drosophila Proteins , Neural Stem Cells , Animals , Centrosome , Drosophila , Drosophila melanogaster
6.
J Cell Sci ; 135(9)2022 05 01.
Article in English | MEDLINE | ID: mdl-35362526

ABSTRACT

Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 (Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore, Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. This article has an associated First Person interview with the first author of the paper.


Subject(s)
Annexin A2 , Dyneins , Anaphase , Annexin A2/genetics , Annexin A2/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Dynactin Complex/metabolism , Dyneins/metabolism , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Mitosis , Neoplasm Proteins/metabolism , Spindle Apparatus/metabolism
7.
PLoS Genet ; 18(4): e1010145, 2022 04.
Article in English | MEDLINE | ID: mdl-35377889

ABSTRACT

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.


Subject(s)
Drosophila Proteins , Neoplasms , Protein Serine-Threonine Kinases , Animals , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , M Phase Cell Cycle Checkpoints/genetics , Mitosis/genetics , Neoplasms/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
8.
Cell Rep ; 37(4): 109895, 2021 10 26.
Article in English | MEDLINE | ID: mdl-34706235

ABSTRACT

Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.


Subject(s)
Cytokinesis , Microtubules , Neural Stem Cells , Animals , Drosophila melanogaster , Microtubules/genetics , Microtubules/metabolism , Microtubules/ultrastructure , Neural Stem Cells/metabolism , Neural Stem Cells/ultrastructure
9.
Curr Biol ; 31(4): 684-695.e6, 2021 02 22.
Article in English | MEDLINE | ID: mdl-33259793

ABSTRACT

Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. In this study, we found that the Drosophila tubulin-specific chaperone dTBCE is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. Our data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. We propose that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.


Subject(s)
Chromatin , Microtubules , Tubulin , Animals , Drosophila , Mitosis , Spindle Apparatus , Tubulin/genetics , Tubulin/metabolism
10.
J Cell Sci ; 133(7)2020 04 06.
Article in English | MEDLINE | ID: mdl-32094264

ABSTRACT

A novel 2,3-benzodiazepine-4 derivative, named 1g, has recently been shown to function as an anti-proliferative compound. We now show that it perturbs the formation of a functional mitotic spindle, inducing a spindle assembly checkpoint (SAC)-dependent arrest in human cells. Live analysis of individual microtubules indicates that 1g promotes a rapid and reversible reduction in microtubule growth. Unlike most anti-mitotic compounds, we found that 1g does not interfere directly with tubulin or perturb microtubule assembly in vitro The observation that 1g also triggers a SAC-dependent mitotic delay associated with chromosome segregation in Drosophila neural stem cells, suggests that it targets a conserved microtubule regulation module in humans and flies. Altogether, our results indicate that 1g is a novel promising anti-mitotic drug with the unique properties of altering microtubule growth and mitotic spindle organization.


Subject(s)
Benzodiazepines , Mitosis , Benzodiazepines/pharmacology , Humans , Microtubules , Spindle Apparatus , Tubulin/genetics
11.
Development ; 146(8)2019 04 17.
Article in English | MEDLINE | ID: mdl-30936181

ABSTRACT

Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro We propose that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Oocytes/metabolism , Animals , Centrosome/metabolism , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , Neurons/cytology , Neurons/metabolism
12.
Biomolecules ; 9(1)2019 01 15.
Article in English | MEDLINE | ID: mdl-30650622

ABSTRACT

Accurate chromosome segregation requires the perfect spatiotemporal rearrangement of the cellular cytoskeleton. Isolated more than two decades ago from Drosophila, Aurora A is a widespread protein kinase that plays key roles during cell division. Numerous studies have described the localisation of Aurora A at centrosomes, the mitotic spindle, and, more recently, at mitotic centromeres. In this review, we will summarise the cytoskeletal rearrangements regulated by Aurora A during cell division. We will also discuss the recent discoveries showing that Aurora A also controls not only the dynamics of the cortical proteins but also regulates the centromeric proteins, revealing new roles for this kinase during cell division.


Subject(s)
Aurora Kinase A/metabolism , Centrosome/metabolism , Animals , Centromere Protein A/metabolism , Humans , Kinetochores/metabolism , Microtubules/metabolism , Protein Kinase C/metabolism , Spindle Apparatus/metabolism
13.
Soins Gerontol ; 22(127): 25-29, 2017.
Article in French | MEDLINE | ID: mdl-28917333

ABSTRACT

The consumption of psychotropic drugs in elderly people remains a concern in France, including in nursing homes. A comparative analysis of prescriptions for psychotropic medication in nursing homes in 2013 and 2015 based on the computer system of the French national health insurance scheme shows a significant reduction in the prescribing of these medications. Example of a nursing home in Dijon.


Subject(s)
Accidental Falls , Alzheimer Disease/drug therapy , Alzheimer Disease/nursing , Mental Disorders/drug therapy , Mental Disorders/nursing , Nursing Homes , Psychotropic Drugs/adverse effects , Psychotropic Drugs/therapeutic use , Accidental Falls/prevention & control , Accidental Falls/statistics & numerical data , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , France , Humans , Male
14.
J Cell Biol ; 216(10): 3029-3039, 2017 10 02.
Article in English | MEDLINE | ID: mdl-28860275

ABSTRACT

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in Drosophila melanogaster oocytes. A critical 14-3-3 target is the minus end-directed motor Ncd (human HSET; kinesin-14), which has well-documented roles in stabilizing a bipolar spindle in oocytes. Phospho docking by 14-3-3 inhibits the microtubule binding activity of the nonmotor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localizes to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to nonspindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.


Subject(s)
14-3-3 Proteins/metabolism , Chromosomes, Insect/metabolism , Drosophila Proteins/metabolism , Kinesins/metabolism , Microtubules/metabolism , Oocytes/metabolism , Spindle Apparatus/metabolism , 14-3-3 Proteins/genetics , Animals , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Chromosomes, Insect/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Kinesins/genetics , Microtubules/genetics , Oocytes/cytology , Protein Transport/physiology , Spindle Apparatus/genetics
15.
Mol Cell Oncol ; 3(3): e1140261, 2016 May.
Article in English | MEDLINE | ID: mdl-27314090

ABSTRACT

Loss of Aurora A in Drosophila neuroblasts promotes loss of cell fate, leading to brain tumors. We showed that these tumor stem cells are delayed during mitosis and efficiently segregate their chromosomes even without the spindle assembly checkpoint. Here, we discuss the possible relevance of our results to human cancers.

16.
Nat Commun ; 6: 8879, 2015 Nov 16.
Article in English | MEDLINE | ID: mdl-26568519

ABSTRACT

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.


Subject(s)
Aurora Kinase A/genetics , Brain Neoplasms/genetics , Carcinogenesis/genetics , Cyclin B/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/metabolism , Neuroblastoma/genetics , Animals , Blotting, Western , Brain Neoplasms/metabolism , Cell Proliferation , Chromosome Segregation , Drosophila , Fluorescent Antibody Technique , Microtubule-Associated Proteins , Mitosis , Mutation , Neural Stem Cells , Neuroblastoma/metabolism , Neurons
17.
PLoS One ; 10(2): e0117418, 2015.
Article in English | MEDLINE | ID: mdl-25658757

ABSTRACT

Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.


Subject(s)
Holoprosencephaly/genetics , Intracellular Signaling Peptides and Proteins/genetics , Microcephaly/genetics , Animals , Brain/diagnostic imaging , Cell Line , Centrioles , Chick Embryo , Chickens/metabolism , Child , Child, Preschool , DNA Mutational Analysis , Female , Holoprosencephaly/pathology , Homozygote , Humans , In Situ Hybridization , Intracellular Signaling Peptides and Proteins/metabolism , Magnetic Resonance Imaging , Male , Microcephaly/pathology , Mutation, Missense , Prosencephalon/metabolism , Radiography , Siblings
18.
J Cell Biol ; 204(7): 1111-21, 2014 Mar 31.
Article in English | MEDLINE | ID: mdl-24687279

ABSTRACT

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.


Subject(s)
Centrosome/metabolism , Drosophila melanogaster/cytology , Microtubule-Associated Proteins/physiology , Microtubules/metabolism , Neural Stem Cells/physiology , Animals , Cells, Cultured , Chromosome Segregation , Drosophila Proteins/metabolism , Interphase , Kinesins/metabolism , Microscopy, Fluorescence , Mitosis , Protein Multimerization , Spindle Apparatus/metabolism , Time-Lapse Imaging
19.
Chromosome Res ; 22(3): 267-76, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24436071

ABSTRACT

The cyclin-dependent kinase CDK11(p58) is specifically expressed at G2/M phase. CDK11(p58) depletion leads to different cell cycle defects such as mitotic arrest, failure in centriole duplication and centrosome maturation, and premature sister chromatid separation. We report that upon CDK11 depletion, loss of sister chromatid cohesion occurs during mitosis but not during G2 phase. CDK11(p58) depletion prevents Bub1 and Shugoshin 1 recruitment but has no effect on the dimethylation of histone H3 lysine 4 at centromeres. We also report that a construct expressing a kinase dead version of CDK11(p58) fails to prevent CDK11 depletion-induced sister chromatid separation, showing that CDK11(p58) kinase activity is required for protection of sister chromatid cohesion at centromeres during mitosis. Thus, CDK11(p58) kinase activity appears to be involved in early events in the establishment of the centromere protection machinery.


Subject(s)
Centromere/metabolism , Chromatids/metabolism , Cyclin D3/metabolism , Mitosis , Sister Chromatid Exchange , Cell Cycle Proteins/metabolism , G2 Phase , HeLa Cells , Humans , Protein Serine-Threonine Kinases/metabolism
20.
J Cell Sci ; 125(Pt 12): 2844-52, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22454512

ABSTRACT

MNK1 is a serine/threonine kinase identified as a target for MAP kinase pathways. Using chemical drug, kinase-dead expression or knockdown by RNA interference, we show that inhibition of MNK1 induces the formation of multinucleated cells, which can be rescued by expressing a form of MNK1 that is resistant to RNA interference. We found that the active human form of MNK1 localises to centrosomes, spindle microtubules and the midbody. Time-lapse recording of MNK1-depleted cells displays cytokinesis defects, as daughter cells fuse back together. When MNK1 activity was inhibited, no microtubule defect at the midbody was detected, however, anchorage of the membrane vesicle at the midbody was impaired as lumenal GFP-positive vesicles did not accumulate at the midbody. At the molecular level, we found that centriolin localisation was impaired at the midbody in MNK1-depleted cells. As a consequence, endobrevin - a v-SNARE protein implicated in the abscission step - was not properly localised to the midbody. Altogether, our data show that MNK1 activity is required for abscission.


Subject(s)
Cells/cytology , Cells/enzymology , Cytokinesis , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Centrosome/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Microtubules/metabolism , Mitosis , Protein Serine-Threonine Kinases/genetics
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