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1.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36052670

RESUMO

In common with other actomyosin contractile cellular machineries, actin turnover is required for normal function of the cytokinetic contractile ring. Cofilin is an actin-binding protein contributing to turnover by severing actin filaments, required for cytokinesis by many organisms. In fission yeast cofilin mutants, contractile rings suffer bridging instabilities in which segments of the ring peel away from the plasma membrane, forming straight bridges whose ends remain attached to the membrane. The origin of bridging instability is unclear. Here, we used molecularly explicit simulations of contractile rings to examine the role of cofilin. Simulations reproduced the experimentally observed cycles of bridging and reassembly during constriction, and the occurrence of bridging in ring segments with low density of the myosin II protein Myo2. The lack of cofilin severing produced ∼2-fold longer filaments and, consequently, ∼2-fold higher ring tensions. Simulations identified bridging as originating in the boosted ring tension, which increased centripetal forces that detached actin from Myo2, which was anchoring actin to the membrane. Thus, cofilin serves a critical role in cytokinesis by providing protection from bridging, the principal structural threat to contractile rings.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Citocinese , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
2.
J Cell Biol ; 222(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36219157

RESUMO

Cytokinesis requires the constriction of an actomyosin-based contractile ring and involves multiple F-actin crosslinkers. We show that partial depletion of the C. elegans cytokinetic formin generates contractile rings with low F-actin levels that constrict but are structurally fragile, and we use this background to investigate the roles of the crosslinkers plastin/PLST-1 and ß-heavy-spectrin/SMA-1 during ring constriction. We show that the removal of PLST-1 or SMA-1 has opposite effects on the structural integrity of fragile rings. PLST-1 loss reduces cortical tension that resists ring constriction and makes fragile rings less prone to ruptures and regressions, whereas SMA-1 loss exacerbates structural defects, leading to frequent ruptures and cytokinesis failure. Fragile rings without SMA-1 or containing a shorter SMA-1, repeatedly rupture at the same site, and SMA-1::GFP accumulates at repair sites in fragile rings and in rings cut by laser microsurgery. These results establish that ß-heavy-spectrin stabilizes the constricting ring and reveals the importance of ß-heavy-spectrin size for network connectivity at low F-actin density.


Assuntos
Citoesqueleto de Actina , Citocinese , Espectrina , Actinas , Actomiosina , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Forminas , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Espectrina/metabolismo
3.
Methods Mol Biol ; 2519: 83-91, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066712

RESUMO

Cytokinesis blocked micronuclei (CBMN) assay is a rapid and sensitive analysis of chromosome aberrations and miss assortments during cell division. Genotoxic agent exposure produces DNA damage and chromosome fragments. Fragmented chromosomes without centromere failed to attach kinetochore which segregates a pair of homologous chromosomes to each daughter cells at cytokinesis, hence leading to form micronuclei. Chromosome or fragments of chromosome can also form micronuclei when they are not accurately sorted to daughter cells. Using cytochalasin B, an actin inhibitor, blocks cytokinesis of which completion leads serration appearance formed with two daughter cells while nuclei segregation is undergoing. As a result, one cell having two daughter nuclei, i.e., binucleated cell, is produced. By analyzing these binucleated cells, chromosome aberrations can be estimated as well as popular chromosome aberration analysis. Frequency of micronuclei formation predicts the testing agents' genotoxicity. By combining use with centromere-specific probes or DNA damage signal probes, the nature of genotoxicity of tested agents can be estimated.


Assuntos
Aberrações Cromossômicas , Citocinese , Divisão Celular , Centrômero , Aberrações Cromossômicas/induzido quimicamente , Sondas de DNA , Humanos , Linfócitos , Testes para Micronúcleos
4.
Nat Commun ; 13(1): 7467, 2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36463216

RESUMO

Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.


Assuntos
Cálcio , Pseudópodes , Animais , Membrana Celular , Movimento Celular , Citocinese , Mamíferos
5.
Cells ; 11(21)2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36359734

RESUMO

The midbody is an organelle that forms between the two daughter cells during cytokinesis. It co-ordinates the abscission of the nascent daughter cells and is composed of a multitude of proteins that are meticulously arranged into distinct temporal and spatial localization patterns. However, very little is known about the mechanisms that regulate the localization and function of midbody proteins. Here, we analyzed the temporal and spatial profiles of key midbody proteins during mitotic exit under normal conditions and after treatment with drugs that affect phosphorylation and proteasome-mediated degradation to decipher the impacts of post-translational modifications on midbody protein dynamics. Our results highlighted that midbody proteins show distinct spatio-temporal dynamics during mitotic exit and cytokinesis that depend on both ubiquitin-mediated proteasome degradation and phosphorylation/de-phosphorylation. They also identified two discrete classes of midbody proteins: 'transient' midbody proteins-including Anillin, Aurora B and PRC1-which rapidly accumulate at the midbody after anaphase onset and then slowly disappear, and 'stable' midbody proteins-including CIT-K, KIF14 and KIF23-which instead persist at the midbody throughout cytokinesis and also post abscission. These two classes of midbody proteins display distinct interaction networks with ubiquitylation factors, which could potentially explain their different dynamics and stability during cytokinesis.


Assuntos
Citocinese , Humanos , Citocinese/fisiologia , Células HeLa , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases
6.
Open Biol ; 12(11): 220247, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36416720

RESUMO

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This crucial process requires the assembly and ingression of an actomyosin ring, which must occur with high fidelity to avoid aneuploidy and cell fate changes. Most of our knowledge of mammalian cytokinesis was generated using over-expressed transgenes in HeLa cells. Over-expression can introduce artefacts, while HeLa are cancerous human cells that have lost their epithelial identity, and the mechanisms controlling cytokinesis in these cells could be vastly different from other cell types. Here, we tagged endogenous anillin, Ect2 and RhoA with mNeonGreen and characterized their localization during cytokinesis for the first time in live human cells. Comparing anillin localization in multiple cell types revealed cytokinetic diversity with differences in the duration and symmetry of ring closure, and the timing of cortical recruitment. Our findings show that the breadth of anillin correlates with the rate of ring closure, and support models where cell size or ploidy affects the cortical organization, and intrinsic mechanisms control the symmetry of ring closure. This work highlights the need to study cytokinesis in more diverse cell types, which will be facilitated by the reagents generated for this study.


Assuntos
Actomiosina , Proteínas Contráteis , Citocinese , Proteínas Proto-Oncogênicas , Proteína rhoA de Ligação ao GTP , Humanos , Actomiosina/metabolismo , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Células HeLa , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
7.
Cells ; 11(22)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36429067

RESUMO

Cytokinesis, the conclusive act of cell division, allows cytoplasmic organelles and chromosomes to be faithfully partitioned between two daughter cells. In animal organisms, its accurate regulation is a fundamental task for normal development and for preventing aneuploidy. Cytokinesis failures produce genetically unstable tetraploid cells and ultimately result in chromosome instability, a hallmark of cancer cells. In animal cells, the assembly and constriction of an actomyosin ring drive cleavage furrow ingression, resulting in the formation of a cytoplasmic intercellular bridge, which is severed during abscission, the final event of cytokinesis. Kinase-mediated phosphorylation is a crucial process to orchestrate the spatio-temporal regulation of the different stages of cytokinesis. Several kinases have been described in the literature, such as cyclin-dependent kinase, polo-like kinase 1, and Aurora B, regulating both furrow ingression and/or abscission. However, others exist, with well-established roles in cell-cycle progression but whose specific role in cytokinesis has been poorly investigated, leading to considering these kinases as "minor" actors in this process. Yet, they deserve additional attention, as they might disclose unexpected routes of cell division regulation. Here, we summarize the role of multifunctional kinases in cytokinesis with a special focus on those with a still scarcely defined function during cell cleavage. Moreover, we discuss their implication in cancer.


Assuntos
Actomiosina , Citocinese , Animais , Citocinese/fisiologia , Divisão Celular , Fosforilação , Citoesqueleto de Actina
8.
Proc Natl Acad Sci U S A ; 119(43): e2211431119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36264833

RESUMO

Actomyosin contractile force produced by myosin II molecules that bind and pull actin filaments is harnessed for diverse functions, from cell division by the cytokinetic contractile ring to morphogenesis driven by supracellular actomyosin networks during development. However, actomyosin contractility is intrinsically unstable to self-reinforcing spatial variations that may destroy the actomyosin architecture if unopposed. How cells control this threat is not established, and while large myosin fluctuations and punctateness are widely reported, the full course of the instability in cells has not been observed. Here, we observed the instability run its full course in isolated cytokinetic contractile rings in cell ghosts where component turnover processes are absent. Unprotected by turnover, myosin II merged hierarchically into aggregates with increasing amounts of myosin and increasing separation, up to a maximum separation. Molecularly explicit simulations reproduced the hierarchical aggregation which precipitated tension loss and ring fracture and identified the maximum separation as the length of actin filaments mediating mechanical communication between aggregates. In the final simulated dead-end state, aggregates were morphologically quiescent, including asters with polarity-sorted actin, similar to the dead-end state observed in actomyosin systems in vitro. Our results suggest the myosin II turnover time controls actomyosin contractile instability in normal cells, long enough for aggregation to build robust aggregates but sufficiently short to intercept catastrophic hierarchical aggregation and fracture.


Assuntos
Actinas , Actomiosina , Actomiosina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Miosina Tipo II/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo
9.
Open Biol ; 12(10): 220197, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36196534

RESUMO

Cytokinesis in eukaryotes is regulated by a Polo-like kinase-mediated and Aurora B kinase-mediated signalling pathway that promotes the assembly of the actomyosin contractile ring, a cytokinesis machinery conserved across evolution from yeast to humans. Trypanosoma brucei, an early divergent parasitic protozoan, employs an actomyosin-independent mechanism for its unusual cytokinesis that is controlled by a regulatory pathway comprising the Polo-like kinase TbPLK, the Aurora B kinase TbAUK1 and multiple trypanosomatid-specific regulators. However, whether any of these trypanosomatid-specific regulators function as substrates of TbPLK and/or TbAUK1 and how they cooperate with TbPLK and TbAUK1 to promote cytokinesis remain unknown. Here, we demonstrate that TbPLK and TbAUK1 phosphorylate the cytokinesis regulators CIF1 and CIF2 on multiple sites within their intrinsically disordered regions. We further show that TbPLK localization depends on its interaction with CIF1 from S/G2 phases, that TbPLK maintains CIF1 and CIF2 localization from G2 phase until early mitosis, and that TbAUK1 maintains CIF1 and CIF2 localization from late mitosis. Finally, we demonstrate that the cytokinesis regulators CIF4 and FPRC are not substrates of TbPLK and TbAUK1, and that they function upstream of TbPLK and TbAUK1 in the cytokinesis regulatory pathway. Together, these results provide insights into the functional interplay and the order of actions between the two protein kinases and the trypanosomatid-specific cytokinesis regulators in T. brucei.


Assuntos
Trypanosoma brucei brucei , Actomiosina/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Citocinese/fisiologia , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo
10.
Mol Biol Cell ; 33(14): ar145, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36287824

RESUMO

The contractile ring must anchor to the plasma membrane and cell wall to transmit its tension. F-BAR domain containing proteins including Imp2p and Cdc15p in fission yeast are likely candidate anchoring proteins based on their mutant phenotypes. Cdc15p is a node component, links the actin bundle to the plasma membrane, recruits Bgs1p to the division plane, prevents contractile ring sliding, and contributes to the stiffness of the contractile ring. Less is known about Imp2p. We found that similarly to Cdc15p, Imp2p contributes to the stiffness of the contractile ring and assembles into protein clusters. Imp2p clusters contain approximately eight Imp2p dimers and depend on the actin network for their stability at the division plane. Importantly, Imp2p and Cdc15p reciprocally affect the amount of each other in the contractile ring, indicating that the two proteins influence each other during cytokinesis, which may partially explain their similar phenotypes.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinese , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
11.
Mol Biol Cell ; 33(14): ar134, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36200871

RESUMO

Pkd2 is the fission yeast homologue of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayers of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypoosmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypoosmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 reduced the calcium spikes, accompanying cell separation and the ensuing membrane stretching, by 60%. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.


Assuntos
Cálcio , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Canais de Cátion TRPP , Cálcio/metabolismo , Sinalização do Cálcio , Citocinese , Permeabilidade , Schizosaccharomyces/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
12.
Elife ; 112022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-36093997

RESUMO

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here, we used the ~140 nm resolution of Airyscan super-resolution microscopy to measure the fluorescence intensity of small, single cytokinesis nodes marked with Blt1-mEGFP in live fission yeast cells early in mitosis. The ratio of the total Blt1-mEGFP fluorescence in the broad band of cytokinesis nodes to the average fluorescence of a single node gives about 190 single cytokinesis nodes in wild-type fission yeast cells early in mitosis. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although large diameter rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The Pom1 kinase restricts cytokinesis nodes from the ends of cells, but the surface density of Pom1 on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, when the concentrations of either kinase Pom1 or kinase Cdr2 were varied with the nmt1 promoter, the numbers of cytokinesis nodes increased above a baseline of about ~190 with the total cellular concentration of either kinase.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Tamanho Celular , Citocinese , Interfase , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
13.
J Cell Sci ; 135(18)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36148799

RESUMO

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Anticorpos de Domínio Único , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Citocinese , Corantes Fluorescentes/metabolismo , Mamíferos/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Anticorpos de Domínio Único/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo
14.
Traffic ; 23(10): 478-495, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36068165

RESUMO

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4Δrga6Δ mutants, Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates primary septum formation and only certain membrane trafficking events. As cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity to allow coupling of Cdc42-independent membrane trafficking events with septum formation for proper septum morphology.


Assuntos
Citocinese , Proteínas Ativadoras de GTPase , Proteínas de Schizosaccharomyces pombe , Actomiosina/metabolismo , Citocinese/genética , Citocinese/fisiologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo
15.
Biochem Biophys Res Commun ; 630: 151-157, 2022 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-36155061

RESUMO

The midbody is a transient structure forming out of the central spindle at late telophase. Both the midbody and central spindle have important functions ensuring completion of cytokinesis and defects in this process may lead to genetic diseases, including cancer. Thus, understanding the mechanisms that control cytokinesis during mitosis can reveal the key components taking part in some of the processes that promote accurate cell division. Our previous study showed that overexpression of FLJ25439 causes cytokinesis defect with midbody arrest and induces tetraploids with prolonged cell growth/cell cycle progression (Pan et al., 2015). Here, we extend our investigation with regard to the expression profile/regulation and cellular localization/function of FLJ25439 during mitosis/cytokinesis. Using a monoclonal antibody 2A4 we found that FLJ25439 expression is cell cycle-dependent and subjected to APC/C complex regulation. Furthermore, it is a novel substrate for the APC/C-Cdc20 complex and its degradation is proteasome-dependent through D-box recognition during mitotic exit. Immunofluorescence microscopy showed it is distributed at the central spindle and midbody, two structures considered important for completion of cell division, in telophase and cytokinesis, respectively, during cell cycle progression. Depletion of FLJ25439 expression revealed defects in chromosome alignment/segregation and delayed mitosis/cytokinesis progression. We thus conclude that FLJ25439 is a hitherto undiscovered factor involved in cytokinesis regulation.


Assuntos
Citocinese , Fuso Acromático , Anticorpos Monoclonais/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Mitose , Complexo de Endopeptidases do Proteassoma/metabolismo , Fuso Acromático/metabolismo
16.
Elife ; 112022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36107470

RESUMO

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that is used to recruit essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 recombinant human MIT domains. We measured 228 pairwise interactions, quantified 60 positive interactions, and discovered 18 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: (1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), (2) function in abscission (SPASTIN, KATNA1, and CAPN7), and (3) function in the 'NoCut' abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.


Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte , Citocinese/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Microtúbulos/metabolismo , Espastina/metabolismo
17.
Int J Mol Sci ; 23(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36077482

RESUMO

Air pollution is recognized as one of the most serious public health issues worldwide and was declared to be a leading environmental cause of cancer deaths. At the same time, the cytokinesis-block micronucleus (CBMN) assay serves as a cancer predictive method that is extensively used in human biomonitoring for populations exposed to environmental contamination. The objective of this cross-sectional study is two-fold: to evaluate genomic instability in a sample (N = 130) of healthy, general population residents from Zagreb (Croatia), chronically exposed to different levels of air pollution, and to relate them to air pollution levels in the period from 2011 to 2015. Measured frequencies of CBMN assay parameters were in agreement with the baseline data for the general population of Croatia. Air pollution exposure was based on four factors obtained from a factor analysis of all exposure data obtained for the examined period. Based on the statistical results, we did not observe a significant positive association between any of the CBMN assay parameters tested and measured air pollution parameters for designated time windows, except for benzo(a)pyrene (B[a]P) that showed significant negative association. Our results show that measured air pollution parameters are largely below the regulatory limits, except for B[a]P, and as such, they do not affect CBMN assay parameters' frequency. Nevertheless, as air pollution is identified as a major health threat, it is necessary to conduct prospective studies investigating the effect of air pollution on genome integrity and human health.


Assuntos
Poluição do Ar , Citocinese , Poluição do Ar/efeitos adversos , Croácia , Estudos Transversais , Dano ao DNA , Humanos , Linfócitos , Testes para Micronúcleos/métodos , Estudos Prospectivos
18.
PLoS Biol ; 20(9): e3001599, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36170207

RESUMO

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.


Assuntos
Citocinese , Esfingomielinas , Actomiosina/metabolismo , Animais , Citocinese/genética , Drosophila/genética , Endossomos/metabolismo , Masculino , Meiose , Fosfatos de Fosfatidilinositol/metabolismo
19.
ACS Synth Biol ; 11(10): 3120-3133, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36164967

RESUMO

One of the major challenges of bottom-up synthetic biology is rebuilding a minimal cell division machinery. From a reconstitution perspective, the animal cell division apparatus is mechanically the simplest and therefore attractive to rebuild. An actin-based ring produces contractile force to constrict the membrane. By contrast, microbes and plant cells have a cell wall, so division requires concerted membrane constriction and cell wall synthesis. Furthermore, reconstitution of the actin division machinery helps in understanding the physical and molecular mechanisms of cytokinesis in animal cells and thus our own cells. In this review, we describe the state-of-the-art research on reconstitution of minimal actin-mediated cytokinetic machineries. Based on the conceptual requirements that we obtained from the physics of the shape changes involved in cell division, we propose two major routes for building a minimal actin apparatus capable of division. Importantly, we acknowledge both the passive and active roles that the confining lipid membrane can play in synthetic cytokinesis. We conclude this review by identifying the most pressing challenges for future reconstitution work, thereby laying out a roadmap for building a synthetic cell equipped with a minimal actin division machinery.


Assuntos
Actomiosina , Células Artificiais , Animais , Actomiosina/metabolismo , Actinas/metabolismo , Citocinese , Lipídeos
20.
Biochem Biophys Res Commun ; 631: 32-40, 2022 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-36162327

RESUMO

Dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) activate two members of Rho family GTPases, Rac1/Cdc42, to exert diverse cellular processes, including cell migration. As DOCK GEFs have been critically implicated in tumour cell migration, understanding their function and specificity is imperative for designing anti-metastatic drugs. Based on their GTPase specificity they have been classified as Rac, Cdc42 and dual specific GEFs. Despite extensive structural studies, the factors that determine GTPase specificity of DOCK GEFs have remained elusive. Here, we show that subtle dynamical coupling between GEF and GTPase structures modulate the binding interface to generate mutual specificity. To cluster the dynamically coupled residues in GEF-GTPase complexes a novel intra-residue backbone-torsion-angles based mutual information (TMI) technique was employed. TMI was calculated from 4500 trajectories obtained from a total of 4.5µs molecular dynamics simulations performed on members of all the three clades of DOCK GEFs. The obtained clusters suggest a specificity generation mechanism that involves optimization of the binding pocket for the crucial divergent residue at the 56th position of Rac/Cdc42 (FCdc42/WRac1). These clusters encompass five residues from the structural segment lobe C - α10 helix of the DOCK proteins and functional SWI region of GTPase, which induce orchestrated structural modulations to generate the specificity. Even the conserved residues from SWI region are seen to augment the specificity defining dynamical rearrangements. Furthermore, the proposed dynamical GTPase- DOCK GEF specificity model was verified using mutagenesis studies on Rac1 and dual GTPase specific Dock2 and Dock6, respectively. Thus the current study provides the generic substrate specificity determinants of DOCK GEFs, which are not apparent from the conventional structural analysis.


Assuntos
Citocinese , Fatores de Troca do Nucleotídeo Guanina , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutagênese , Especificidade por Substrato
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