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1.
J Cell Sci ; 136(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-35502670

RESUMO

The precise regulation of microtubule length during mitosis is essential to assemble and position the mitotic spindle and segregate chromosomes. The kinesin-13 Kif2C or MCAK acts as a potent microtubule depolymerase that diffuses short distances on microtubules, whereas the kinesin-8 Kif18b is a processive motor with weak depolymerase activity. However, the individual activities of these factors cannot explain the dramatic increase in microtubule dynamics in mitosis. Using in vitro reconstitution and single-molecule imaging, we demonstrate that Kif18b, MCAK and the plus-end tracking protein EB3 (also known as MAPRE3) act in an integrated manner to potently promote microtubule depolymerization at very low concentrations. We find that Kif18b can transport EB3 and MCAK and promotes their accumulation to microtubule plus ends through multivalent weak interactions. Together, our work defines the mechanistic basis for a cooperative Kif18b-MCAK-EB network at microtubule plus ends, that acts to efficiently shorten and regulate microtubules in mitosis, essential for correct chromosome segregation.


Assuntos
Microtúbulos , Segregação de Cromossomos , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
2.
Mol Cell Proteomics ; 21(1): 100169, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34742921

RESUMO

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number MS-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the "in-cell digest." We combined this with averaged MS1 precursor library matching to quantitatively characterize proteomes from low cell numbers of human lymphoblasts. About 4500 proteins were detected from 2000 cells, and 2500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterized the proteome changes across 16 cell cycle states (CCSs) isolated from an asynchronous TK6 cells, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins that vary across the cell cycle. Clustering of the pseudoperiodic proteins showed abundance patterns consistent with "waves" of protein degradation in late S, at the G2&M border, midmitosis, and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the anaphase-promoting complex/cyclosome. The dataset also identifies putative anaphase-promoting complex/cyclosome substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high-confidence cell cycle-regulated proteins can be used to perform unbiased classification of proteomes into CCSs. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a CCS for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate that this robust cell cycle protein signature will be crucial for classifying cell states in single-cell proteomes.


Assuntos
Peptídeo Hidrolases , Proteômica , Contagem de Células , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Mitose , Proteômica/métodos
3.
Sci Rep ; 12(1): 13160, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35915203

RESUMO

The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.


Assuntos
Proteínas de Ciclo Celular , Ubiquitina-Proteína Ligases , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Células HEK293 , Células HeLa , Humanos , Mitose , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
4.
Science ; 377(6606): 629-634, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35926014

RESUMO

Meiosis, at the transition between diploid and haploid life cycle phases, is accompanied by reprograming of cell division machinery and followed by a transition back to mitosis. We show that, in Arabidopsis, this transition is driven by inhibition of translation, achieved by a mechanism that involves processing bodies (P-bodies). During the second meiotic division, the meiosis-specific protein THREE-DIVISION MUTANT 1 (TDM1) is incorporated into P-bodies through interaction with SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA 7 (SMG7). TDM1 attracts eIF4F, the main translation initiation complex, temporarily sequestering it in P-bodies and inhibiting translation. The failure of tdm1 mutants to terminate meiosis can be overcome by chemical inhibition of translation. We propose that TDM1-containing P-bodies down-regulate expression of meiotic transcripts to facilitate transition of cell fates to postmeiotic gametophyte differentiation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Meiose , Mitose , Corpos de Processamento
5.
Sci Rep ; 12(1): 11210, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35778595

RESUMO

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.


Assuntos
Histonas , Mitose , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases , Treonina/metabolismo
6.
Molecules ; 27(13)2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35807551

RESUMO

Acrylamide (AA), is a chemical with multiple industrial applications, however, it can be found in foods that are rich in carbohydrates. Due to its genotoxic and cytotoxic effects, AA has been classified as a potential carcinogen. With the use of spectrophotometry, ICP-OES, fluorescence spectroscopy, and microscopy cell growth, metabolic activity, apoptosis, ROS production, MDA formation, CAT and SOD activity, ionome balance, and chromosome segregation were determined in Schizosaccharomyces pombe. AA caused growth and metabolic activity retardation, enhanced ROS and MDA production, and modulated antioxidant enzyme activity. This led to damage to the cell homeostasis due to ionome balance disruption. Moreover, AA-induced oxidative stress caused alterations in the cell cycle regulation resulting in chromosome segregation errors, as 4.07% of cells displayed sister chromatid non-disjunction during mitosis. Ascorbic acid (AsA, Vitamin C), a strong natural antioxidant, was used to alleviate the negative impact of AA. Cell pre-treatment with AsA significantly improved AA impaired growth, and antioxidant capacity, and supported ionome balance maintenance mainly due to the promotion of calcium uptake. Chromosome missegregation was reduced to 1.79% (44% improvement) by AsA pre-incubation. Results of our multiapproach analyses suggest that AA-induced oxidative stress is the major cause of alteration to cell homeostasis and cell cycle regulation.


Assuntos
Ácido Ascórbico , Schizosaccharomyces , Acrilamida/toxicidade , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Mitose , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Schizosaccharomyces/metabolismo
7.
Cells ; 11(13)2022 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-35805103

RESUMO

In eukaryotes, cyclin-dependent kinases (CDKs) are required for the onset of DNA replication and mitosis, and distinct CDK-cyclin complexes are activated sequentially throughout the cell cycle. It is widely thought that specific complexes are required to traverse a point of commitment to the cell cycle in G1, and to promote S-phase and mitosis, respectively. Thus, according to a popular model that has dominated the field for decades, the inherent specificity of distinct CDK-cyclin complexes for different substrates at each phase of the cell cycle generates the correct order and timing of events. However, the results from the knockouts of genes encoding cyclins and CDKs do not support this model. An alternative "quantitative" model, validated by much recent work, suggests that it is the overall level of CDK activity (with the opposing input of phosphatases) that determines the timing and order of S-phase and mitosis. We take this model further by suggesting that the subdivision of the cell cycle into discrete phases (G0, G1, S, G2, and M) is outdated and problematic. Instead, we revive the "continuum" model of the cell cycle and propose that a combination with the quantitative model better defines a conceptual framework for understanding cell cycle control.


Assuntos
Quinases Ciclina-Dependentes , Ciclinas , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Mitose
8.
J Vis Exp ; (184)2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35815974

RESUMO

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.


Assuntos
Proteínas de Saccharomyces cerevisiae , Fuso Acromático , Animais , Mamíferos/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo
9.
PLoS One ; 17(7): e0268848, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776709

RESUMO

The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed ß propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.


Assuntos
Anticorpos , Fuso Acromático , Actinas/metabolismo , Animais , Mamíferos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
10.
Int J Mol Sci ; 23(15)2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35897652

RESUMO

Haemodynamic wall shear stress varies from site to site within the arterial system and is thought to cause local variation in endothelial permeability to macromolecules. Our aim was to investigate mechanisms underlying the changes in paracellular permeability caused by different patterns of shear stress in long-term culture. We used the swirling well system and a substrate-binding tracer that permits visualisation of transport at the cellular level. Permeability increased in the centre of swirled wells, where flow is highly multidirectional, and decreased towards the edge, where flow is more uniaxial, compared to static controls. Overall, there was a reduction in permeability. There were also decreases in early- and late-stage apoptosis, proliferation and mitosis, and there were significant correlations between the first three and permeability when considering variation from the centre to the edge under flow. However, data from static controls did not fit the same relation, and a cell-by-cell analysis showed that <5% of uptake under shear was associated with each of these events. Nuclear translocation of NF-κB p65 increased and then decreased with the duration of applied shear, as did permeability, but the spatial correlation between them was not significant. Application of an NO synthase inhibitor abolished the overall decrease in permeability caused by chronic shear and the difference in permeability between the centre and the edge of the well. Hence, shear and paracellular permeability appear to be linked by NO synthesis and not by apoptosis, mitosis or inflammation. The effect was mediated by an increase in transport through tricellular junctions.


Assuntos
Endotélio Vascular , Mitose , Humanos , Inflamação , Permeabilidade , Estresse Mecânico
11.
PLoS Biol ; 20(7): e3001708, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35849559

RESUMO

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.


Assuntos
Ciliopatias , Citocinese , Anáfase , Centrossomo/metabolismo , Proteínas do Olho/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
12.
Proc Natl Acad Sci U S A ; 119(30): e2203849119, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35867815

RESUMO

Cell proliferation is tightly controlled by inhibitors that block cell cycle progression until growth signals relieve this inhibition, allowing cells to divide. In several tissues, including the liver, cell proliferation is inhibited at mitosis by the transcriptional repressors E2F7 and E2F8, leading to formation of polyploid cells. Whether growth factors promote mitosis and cell cycle progression by relieving the E2F7/E2F8-mediated inhibition is unknown. We report here on a mechanism of cell division control in the postnatal liver, in which Wnt/ß-catenin signaling maintains active hepatocyte cell division through Tbx3, a Wnt target gene. The TBX3 protein directly represses transcription of E2f7 and E2f8, thereby promoting mitosis. This cascade of sequential transcriptional repressors, initiated by Wnt signals, provides a paradigm for exploring how commonly active developmental signals impact cell cycle completion.


Assuntos
Fatores de Transcrição , Via de Sinalização Wnt , Proliferação de Células , Hepatócitos/metabolismo , Mitose , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo
13.
Oncogene ; 41(31): 3859-3875, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35780183

RESUMO

Despite paclitaxel's wide use in cancer treatment, patient response rate is still low and drug resistance is a major clinical obstacle. Through a Phos-tag-based kinome-wide screen, we identified MARK2 as a critical regulator for paclitaxel chemosensitivity in PDAC. We show that MARK2 is phosphorylated by CDK1 in response to antitubulin chemotherapeutics and in unperturbed mitosis. Phosphorylation is essential for MARK2 in regulating mitotic progression and paclitaxel cytotoxicity in PDAC cells. Mechanistically, our findings also suggest that MARK2 controls paclitaxel chemosensitivity by regulating class IIa HDACs. MARK2 directly phosphorylates HDAC4 specifically during antitubulin treatment. Phosphorylated HDAC4 promotes YAP activation and controls expression of YAP target genes induced by paclitaxel. Importantly, combination of HDAC inhibition and paclitaxel overcomes chemoresistance in organoid culture and preclinical PDAC animal models. The expression levels of MARK2, HDACs, and YAP are upregulated and positively correlated in PDAC patients. Inhibition of MARK2 or class IIa HDACs potentiates paclitaxel cytotoxicity by inducing mitotic abnormalities in PDAC cells. Together, our findings identify the MARK2-HDAC axis as a druggable target for overcoming chemoresistance in PDAC.


Assuntos
Histona Desacetilases , Neoplasias Pancreáticas , Animais , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Mitose , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico
14.
J Cell Biol ; 221(9)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-35878017

RESUMO

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.


Assuntos
Proteínas do Citoesqueleto , Cinetocoros , Microtúbulos , Mitose , Aurora Quinases/metabolismo , Proteína Quinase CDC2/metabolismo , Segregação de Cromossomos , Ciclina B1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fosforilação
15.
Nature ; 607(7919): 604-609, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35831506

RESUMO

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.


Assuntos
Aneuploidia , Posicionamento Cromossômico , Segregação de Cromossomos , Cromossomos , Proteína 9 Associada à CRISPR , Linhagem Celular , Linhagem Celular Tumoral , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/metabolismo , Cromotripsia , Crescimento e Desenvolvimento/genética , Humanos , Interfase , Micronúcleos com Defeito Cromossômico , Mitose , Neoplasias/genética , Neoplasias/patologia , Organoides/citologia , Organoides/metabolismo , Análise de Sequência de DNA , Análise de Célula Única
16.
Cells ; 11(14)2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35883570

RESUMO

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.


Assuntos
Proteínas de Drosophila , Cinetocoros , Animais , Demecolcina/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo
17.
Cells ; 11(14)2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35883622

RESUMO

The spindle position checkpoint (SPOC) of budding yeast delays mitotic exit in response to misaligned spindles to ensure cell survival and the maintenance of genomic stability. The GTPase-activating protein (GAP) complex Bfa1-Bub2, a key SPOC component, inhibits the GTPase Tem1 to induce mitotic arrest in response to DNA and spindle damage, as well as spindle misorientation. However, previous results strongly suggest that Bfa1 exerts a GAP-independent function in blocking mitotic exit in response to misaligned spindles. Thus, the molecular mechanism by which Bfa1 controls mitotic exit in response to misaligned spindles remains unclear. Here, we observed that overexpression of the N-terminal domain of Bfa1 (Bfa1-D16), which lacks GAP activity and cannot localize to the spindle pole body (SPB), induced cell cycle arrest along with hyper-elongation of astral microtubules (aMTs) as Bfa1 overexpression in Δbub2. We found that Δbub2 cells overexpressing Bfa1 or Bfa1-D16 inhibited activation of Mob1, which is responsible for mitotic exit. In anaphase-arrested cells, Bfa1-D16 overexpression inhibited Tem1 binding to the SPB as well as Bfa1 overexpression. Additionally, endogenous levels of Bfa1-D16 showed minor SPOC activity that was not regulated by Kin4. These results suggested that Bfa1-D16 may block mitotic exit through inhibiting Tem1 activity outside of SPBs. Alternatively, Bfa1-D16 dispersed out of SPBs may block Tem1 binding to SPBs by physically interacting with Tem1 as previously reported. Moreover, we observed hyper-elongated aMTs in tem1-3, cdc15-2, and dbf2-2 mutants that induce anaphase arrest and cannot undergo mitotic exit at restrictive temperatures, suggesting that aMT dynamics are closely related to the regulation of mitotic exit. Altogether, these observations suggest that Bfa1 can control the SPOC independent of its GAP activity and SPB localization.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Mitose , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo
18.
Cells ; 11(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35883632

RESUMO

Cyclin-dependent kinases (CDKs) are presumed to control the cell cycle by phosphorylating a large number of proteins involved in S-phase and mitosis, two mechanistically disparate biological processes. While the traditional qualitative model of CDK-mediated cell cycle control relies on differences in inherent substrate specificity between distinct CDK-cyclin complexes, they are largely dispensable according to the opposing quantitative model, which states that changes in the overall CDK activity level promote orderly progression through S-phase and mitosis. However, a mechanistic explanation for how such an activity can simultaneously regulate many distinct proteins is lacking. New evidence suggests that the CDK-dependent phosphorylation of ostensibly very diverse proteins might be achieved due to underlying similarity of phosphorylation sites and of the biochemical effects of their phosphorylation: they are preferentially located within intrinsically disordered regions of proteins that are components of membraneless organelles, and they regulate phase separation. Here, we review this evidence and suggest a mechanism for how a single enzyme's activity can generate the dynamics required to remodel the cell at mitosis.


Assuntos
Quinases Ciclina-Dependentes , Mitose , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Fosforilação , Proteínas/metabolismo
19.
PLoS One ; 17(7): e0270923, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35797385

RESUMO

Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Mitose , Software
20.
Curr Biol ; 32(13): R744-R746, 2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35820385

RESUMO

During mitosis, chromosomes must bind spindle microtubules via kinetochores in a stable yet dynamic manner to ensure rapid frictionless movements. A recent study identifies the first complex that specifically reduces friction in the kinetochore-microtubule interface to ensure efficient chromosome segregation.


Assuntos
Cinetocoros , Mitose , Segregação de Cromossomos , Fricção , Microtúbulos/metabolismo
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