RESUMEN
Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct ß- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that ß- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that ß-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.
Asunto(s)
Actinas , Proteínas Adaptadoras Transductoras de Señales , Citocinesis , Forminas , Miosina Tipo II , Proteína de Unión al GTP rhoA , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Forminas/metabolismo , Forminas/genética , Actinas/metabolismo , Humanos , Miosina Tipo II/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células HeLa , Animales , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinesis , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Centrosoma/metabolismo , Forminas/genética , Forminas/metabolismo , Células HeLa , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.
Asunto(s)
Actinas/metabolismo , Forminas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Citoesqueleto de Actina/metabolismo , Línea Celular Tumoral , Forminas/antagonistas & inhibidores , Forminas/genética , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Proteínas Activadoras de ras GTPasa/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Rigorous spatiotemporal regulation of cell division is required to maintain genome stability. The final stage in cell division, when the cells physically separate (abscission), is tightly regulated to ensure that it occurs after cytokinetic events such as chromosome segregation. A key regulator of abscission timing is Aurora B kinase activity, which inhibits abscission and forms the major activity of the abscission checkpoint. This checkpoint prevents abscission until chromosomes have been cleared from the cytokinetic machinery. Here we demonstrate that the mitosis-specific CDK11p58 kinase specifically forms a complex with cyclin L1ß that, in late cytokinesis, localizes to the stem body, a structure in the middle of the intercellular bridge that forms between two dividing cells. Depletion of CDK11 inhibits abscission, and rescue of this phenotype requires CDK11p58 kinase activity or inhibition of Aurora B kinase activity. Furthermore, CDK11p58 kinase activity is required for formation of endosomal sorting complex required for transport III filaments at the site of abscission. Combined, these data suggest that CDK11p58 kinase activity opposes Aurora B activity to enable abscission to proceed and result in successful completion of cytokinesis.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Western Blotting , División Celular/genética , División Celular/fisiología , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Citocinesis/genética , Citocinesis/fisiología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Mitosis/genética , Mitosis/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de TiempoRESUMEN
The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin ß2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin ß2 binding does not regulate anillin's function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin's function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.
Asunto(s)
Núcleo Celular/genética , Proteínas de Microfilamentos/metabolismo , Mitosis/fisiología , Señales de Localización Nuclear , beta Carioferinas/metabolismo , Membrana Celular/metabolismo , Citocinesis/fisiología , Citosol/metabolismo , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Interfase/fisiología , Proteínas de Microfilamentos/genética , Membrana Nuclear/metabolismo , Transporte de Proteínas , beta Carioferinas/genéticaRESUMEN
BACKGROUND: The establishment, maintenance, and dissolution of sister chromatid cohesion are sequentially coordinated during the cell cycle to ensure faithful chromosome transmission. This cell-cycle-dependent regulation of cohesion is mediated, in part, by distinct posttranslational modifications of cohesin, a protein complex consisting of the Smc1-Smc3 ATPase, the Mcd1/Scc1 α-kleisin, and Scc3. Although cohesion is established in S phase, cohesins are not sufficient to maintain cohesion as cells progress from G2 to the metaphase-to-anaphase transition. Rather, the cohesin-associated factor Pds5 is also required to keep sisters paired until anaphase onset. How Pds5 maintains cohesion at the molecular level and whether this maintenance involves the regulation of cohesin modifications remains to be defined. RESULTS: In pds5 mutants, we find that Mcd1 is extensively SUMOylated and that premature sister separation requires Siz2-dependent polySUMOylation. Moreover, abrogation of Pds5 function promotes the proteasome-dependent degradation of Mcd1 and a significant loss of cohesin from chromatin independently of anaphase onset. We further demonstrate that inactivation of the Slx5-Slx8 SUMO-targeted ubiquitin ligase, required for targeting polySUMOylated factors for proteasome-mediated destruction, limits Mcd1 turnover and restores both cell growth and cohesion in metaphase cells defective for Pds5 function. CONCLUSIONS: We propose that Pds5 maintains cohesion, at least in part, by antagonizing the polySUMO-dependent degradation of cohesin.
Asunto(s)
Proteínas de Ciclo Celular/uso terapéutico , Cromátides/metabolismo , Proteínas de Saccharomyces cerevisiae/uso terapéutico , Saccharomyces cerevisiae/metabolismo , Sumoilación , Proteínas de Ciclo Celular/genética , Segregación Cromosómica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.
Asunto(s)
Proteínas Contráctiles/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Septinas/metabolismo , Segregación Cromosómica , Proteínas Contráctiles/antagonistas & inhibidores , Proteínas Contráctiles/genética , Citocinesis , Células HeLa , Humanos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Septinas/antagonistas & inhibidores , Septinas/genéticaRESUMEN
Accurate chromosome segregation depends on sister kinetochores making bioriented attachments to microtubules from opposite poles. An essential regulator of biorientation is the Ipl1/Aurora B protein kinase that destabilizes improper microtubule-kinetochore attachments. To identify additional biorientation pathways, we performed a systematic genetic analysis between the ipl1-321 allele and all nonessential budding yeast genes. One of the mutants, mcm21Delta, precociously separates pericentromeres and this is associated with a defect in the binding of the Scc2 cohesin-loading factor at the centromere. Strikingly, Mcm21 becomes essential for biorientation when Ipl1 function is reduced, and this appears to be related to its role in pericentromeric cohesion. When pericentromeres are artificially tethered, Mcm21 is no longer needed for biorientation despite decreased Ipl1 activity. Taken together, these data reveal a specific role for pericentromeric linkage in ensuring kinetochore biorientation.
Asunto(s)
Cromátides/metabolismo , Cinetocoros/metabolismo , Huso Acromático/metabolismo , Aurora Quinasas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Epistasis Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Represoras Lac/genética , Represoras Lac/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The completion of chromosome segregation during anaphase requires the hypercondensation of the approximately 1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Delta cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mitosis/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Tirosina Fosfatasas/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Rb mutants exhibit aneuploidy and aberrant chromosome structure during mitosis. In this issue of Genes & Development, a new paper from Longworth and colleagues (1011-1024) describes both physical and functional interactions between Drosophila Rbf1 and the dCAP-D3 subunit of condensin II. This work directly implicates the Rb family proteins in mitotic chromosome condensation and suggests that a failure in targeting condensin II to chromatin underlies the aneuploidy in rbf1 mutants.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Estructuras Cromosómicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Mitosis , Modelos Biológicos , Proteína de Retinoblastoma/genética , Factores de Transcripción/metabolismoRESUMEN
We describe a novel requirement for the condensin complex in sister chromatid cohesion in Saccharomyces cerevisiae. Strikingly, condensin-dependent cohesion can be distinguished from cohesin-based pairing by a number of criteria. First, condensin is required to maintain cohesion at several chromosomal arm sites but, in contrast to cohesin, is not required at either centromere or telomere-proximal loci. Second, condensin-dependent interlinks are established during mitosis independently of DNA replication and are reversible within a single cell cycle. Third, the loss of condensin-dependent linkages occurs without affecting cohesin levels at the separated URA3 locus. We propose that, during mitosis, robust sister chromatid cohesion along chromosome arms requires both condensinand cohesin-dependent mechanisms, which function independently of each other. We discuss the implications of our results for current models of sister chromatid cohesion.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromátides/metabolismo , Emparejamiento Cromosómico , Proteínas de Unión al ADN/metabolismo , Mitosis , Complejos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , División Celular , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.
Asunto(s)
Proteínas Portadoras/metabolismo , Segregación Cromosómica , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aurora Quinasa B , Aurora Quinasas , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Mitosis , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Proteínas de Unión al ARN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Chromosome condensation plays an essential role in the maintenance of genetic integrity. Using genetic, cell biological, and biochemical approaches, we distinguish two cell-cycle-regulated pathways for chromosome condensation in budding yeast. From G(2) to metaphase, we show that the condensation of the approximately 1-Mb rDNA array is a multistep process, and describe condensin-dependent clustering, alignment, and resolution steps in chromosome folding. We functionally define a further postmetaphase chromosome assembly maturation step that is required for the maintenance of chromosome structural integrity during segregation. This late step in condensation requires the conserved mitotic kinase Ipl1/aurora in addition to condensin, but is independent of cohesin. Consistent with this, the late condensation pathway is initiated during the metaphase-to-anaphase transition, supports de novo condensation in cohesin mutants, and correlates with the Ipl1/aurora-dependent phosphorylation of condensin. These data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.
Asunto(s)
Ciclo Celular/genética , Cromosomas Fúngicos/genética , ADN Ribosómico/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/metabolismo , Aurora Quinasas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas Fúngicos/ultraestructura , Secuencia Conservada , ADN de Hongos/genética , Proteínas de Unión al ADN/metabolismo , Genotipo , Metafase , Mitosis , Complejos Multiproteicos , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de XenopusRESUMEN
The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented.