RESUMEN
Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.
Asunto(s)
Aneuploidia , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Cromatina/metabolismo , Cromosomas Humanos/genética , ADN/metabolismo , Células Cultivadas , Centrómero/genética , Proteína A Centromérica/genética , Cromatina/genética , Segregación Cromosómica , ADN/genética , Femenino , Humanos , MasculinoRESUMEN
Constriction kinetics of the cytokinetic ring are expected to depend on dynamic adjustment of contractile ring composition, but the impact of ring component abundance dynamics on ring constriction is understudied. Computational models generally assume that contractile networks maintain constant total amounts of components, which is not always true. To test how compositional dynamics affect constriction kinetics, we first measured F-actin, non-muscle myosin II, septin, and anillin during Caenorhabditis elegans zygotic mitosis. A custom microfluidic device that positioned the cell with the division plane parallel to a light sheet allowed even illumination of the cytokinetic ring. Measured component abundances were implemented in a three-dimensional agent-based model of a membrane-associated contractile ring. With constant network component amounts, constriction completed with biologically unrealistic kinetics. However, imposing the measured changes in component quantities allowed this model to elicit realistic constriction kinetics. Simulated networks were more sensitive to changes in motor and filament amounts than those of crosslinkers and tethers. Our findings highlight the importance of network composition for actomyosin contraction kinetics.
Asunto(s)
Citoesqueleto de Actina , Citocinesis , Animales , Cinética , Citocinesis/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Actomiosina/metabolismo , Caenorhabditis elegansRESUMEN
During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Centrómero/genética , Proteínas Cromosómicas no Histona/genética , Recombinación Homóloga/genética , Meiosis/genética , Animales , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Drosophila/genética , Complejo Sinaptonémico , Telómero/genéticaRESUMEN
Under replete growth conditions, abundant nutrient uptake leads to the systemic activation of insulin/IGF-1 signalling (IIS) and the promotion of stem cell growth/proliferation. Activated IIS can stimulate the ERK/MAPK pathway, the activation of which also supports optimal stem cell proliferation in various systems. Stem cell proliferation rates can further be locally refined to meet the resident tissue's need for differentiated progeny. We have recently shown that the accumulation of mature oocytes in the C. elegans germ line, through DAF-18/PTEN, inhibits adult germline stem cell (GSC) proliferation, despite high systemic IIS activation. We show here that this feedback occurs through a novel cryptic signalling pathway that requires PAR-4/LKB1, AAK-1/AMPK and PAR-5/14-3-3 to inhibit the activity of MPK-1/MAPK, antagonize IIS, and inhibit both GSC proliferation and the production of additional oocytes. Interestingly, our results imply that DAF-18/PTEN, through PAR-4/LKB1, can activate AAK-1/AMPK in the absence of apparent energy stress. As all components are conserved, similar signalling cascades may regulate stem cell activities in other organisms and be widely implicated in cancer.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Diferenciación Celular/genética , Longevidad/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP/genética , Envejecimiento/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proliferación Celular/genética , Células Germinativas , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Oocitos/metabolismo , Fosfohidrolasa PTEN/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genética , Células Madre/metabolismoRESUMEN
During development, stem cell populations rapidly proliferate to populate the expanding tissues and organs. During this phase, nutrient status, by systemically affecting insulin/IGF-1 signalling, largely dictates stem cell proliferation rates. In adults, however, differentiated stem cell progeny requirements are generally reduced and vary according to the spatiotemporal needs of each tissue. We demonstrate here that differential regulation of germline stem cell proliferation rates in Caenorhabditis elegans adults is accomplished through localized neutralization of insulin/IGF-1 signalling, requiring DAF-18/PTEN, but not DAF-16/FOXO. Indeed, the specific accumulation of oocytes, the terminally differentiated stem cell progeny, triggers a feedback signal that locally antagonizes insulin/IGF-1 signalling outputs in the germ line, regardless of their systemic levels, to block germline stem cell proliferation. Thus, during adulthood, stem cells can differentially respond within tissues to otherwise equal insulin/IGF-1 signalling inputs, according to the needs for production of their immediate terminally differentiated progeny.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Células Germinativas/citología , Insulina/metabolismo , Oocitos/citología , Transducción de Señal , Células Madre/citología , Envejecimiento/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Diferenciación Celular , Proliferación Celular , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Modelos Biológicos , Espermatozoides/citología , Nicho de Células Madre , Células Madre/metabolismoRESUMEN
Centromeres are chromosomal loci that direct segregation of the genome during cell division. The histone H3 variant CENP-A (also known as CenH3) defines centromeres in monocentric organisms, which confine centromere activity to a discrete chromosomal region, and holocentric organisms, which distribute centromere activity along the chromosome length. Because the highly repetitive DNA found at most centromeres is neither necessary nor sufficient for centromere function, stable inheritance of CENP-A nucleosomal chromatin is postulated to propagate centromere identity epigenetically. Here, we show that in the holocentric nematode Caenorhabditis elegans pre-existing CENP-A nucleosomes are not necessary to guide recruitment of new CENP-A nucleosomes. This is indicated by lack of CENP-A transmission by sperm during fertilization and by removal and subsequent reloading of CENP-A during oogenic meiotic prophase. Genome-wide mapping of CENP-A location in embryos and quantification of CENP-A molecules in nuclei revealed that CENP-A is incorporated at low density in domains that cumulatively encompass half the genome. Embryonic CENP-A domains are established in a pattern inverse to regions that are transcribed in the germline and early embryo, and ectopic transcription of genes in a mutant germline altered the pattern of CENP-A incorporation in embryos. Furthermore, regions transcribed in the germline but not embryos fail to incorporate CENP-A throughout embryogenesis. We propose that germline transcription defines genomic regions that exclude CENP-A incorporation in progeny, and that zygotic transcription during early embryogenesis remodels and reinforces this basal pattern. These findings link centromere identity to transcription and shed light on the evolutionary plasticity of centromeres.
Asunto(s)
Caenorhabditis elegans/genética , Centrómero/genética , Cromatina/genética , Células Germinativas/metabolismo , Transcripción Genética , Animales , Autoantígenos/metabolismo , Evolución Biológica , Caenorhabditis elegans/embriología , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/genética , Femenino , Fertilización , Regulación del Desarrollo de la Expresión Génica , Genoma de los Helmintos , Gónadas/citología , Gónadas/metabolismo , Organismos Hermafroditas , Masculino , MeiosisRESUMEN
The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora/metabolismo , División Celular Asimétrica/fisiología , Polaridad Celular/genética , Endocitosis/genética , Células Madre Hematopoyéticas/citología , Células Madre Neoplásicas/patología , Células Madre/citología , Complejo 2 de Proteína Adaptadora/antagonistas & inhibidores , Complejo 2 de Proteína Adaptadora/genética , Subunidades alfa de Complejo de Proteína Adaptadora/antagonistas & inhibidores , Subunidades alfa de Complejo de Proteína Adaptadora/genética , Animales , Biomarcadores/metabolismo , Western Blotting , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Citometría de Flujo , Perfilación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Leucemia/metabolismo , Leucemia/patología , Ratones , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/fisiologíaRESUMEN
The Centromere is a unique chromosomal locus where the kinetochore is formed to mediate faithful chromosome partitioning, thus maintaining ploidy during cell division. Centromere identity is inherited via an epigenetic mechanism involving a histone H3 variant, called centromere protein A (CENP-A) which replaces H3 in centromeric chromatin. In spite of extensive efforts in field of centromere biology during the past decade, controversy persists over the structural nature of the CENP-A-containing epigenetic mark, both at nucleosomal and chromatin levels. Here, we review recent findings and hypotheses regarding the structure of CENP-A-containing complexes.
Asunto(s)
Autoantígenos/genética , Centrómero/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Nucleosomas/genética , Autoantígenos/química , División Celular/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/química , Epigénesis Genética , Histonas/química , Histonas/genética , Humanos , Cinetocoros/químicaRESUMEN
Polo-like kinases (PLKs) are evolutionarily conserved kinases essential for cell cycle regulation. These kinases are characterized by the presence of a C-terminal phosphopeptide-interaction domain, the polo-box domain (PBD). How the functional domains of PLKs work together to promote cell division is not understood. To address this, we performed a genetic screen to identify mutations that independently modulate the kinase and PBD activities of yeast PLK/Cdc5. This screen identified a mutagenic hotspot in the F-helix region of Cdc5 kinase domain that allows one to control kinase activity in vivo. These mutations can be systematically engineered into other major eukaryotic cell cycle kinases to similarly regulate their activity in live cells. Here, using this approach, we show that the kinase activity of Cdc5 can promote the execution of several stages of mitosis independently of PBD activity. In particular, we observe that the activation of Cdc14 and execution of mitotic exit are uniquely sensitive to the modulation of Cdc5 kinase activity. In contrast, PBD-defective mutants are capable of completing mitosis but are unable to maintain spindle pole body integrity. Consistent with this defect, PBD-deficient cells progressively double the size of their genome and ultimately lose genome integrity. Collectively, these results highlight the specific contributions of Cdc5 functional domains to cell division and reveal unexpected mechanisms controlling spindle pole body behavior and genome stability.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inestabilidad Genómica/fisiología , Mitosis/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Proteínas de Ciclo Celular/aislamiento & purificación , Electroforesis , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Mitosis/genética , Mutación/genética , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Huso Acromático/metabolismo , Huso Acromático/fisiologíaRESUMEN
Chromosome segregation is the one of the great problems in biology with complexities spanning from biophysics and polymer dynamics to epigenetics. Here, we summarize the current knowledge and highlight gaps in understanding of the mechanisms controlling epigenetic regulation of chromosome segregation.
Asunto(s)
Autoantígenos/fisiología , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Epigénesis Genética , Cinetocoros/metabolismo , Animales , Centrómero/genética , Proteína A Centromérica , Cromatina/genética , Cromatina/metabolismo , Humanos , Mitosis , Neoplasias/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).
RESUMEN
Septins, a conserved family of filament-forming proteins, contribute to eukaryotic cell division, polarity, and membrane trafficking. Septins are thought to act in these processes by scaffolding other proteins to the plasma membrane. The mechanisms by which septins associate with the plasma membrane are not well understood but can involve two polybasic domains and/or an amphipathic helix. We discovered that the genomes of organisms throughout phylogeny, but not most commonly used model organisms, encode one or more septins predicted to have transmembrane domains. The nematode Caenorhabditis elegans, which was thought to express only two septin proteins, UNC-59 and UNC-61, translates multiple isoforms of UNC-61, and one isoform, UNC-61a, is predicted to contain a transmembrane domain. UNC-61a localizes specifically to the apical membrane of the C. elegans vulva and is important for maintaining vulval morphology. UNC-61a partially compensates for the loss of the other two UNC-61 isoforms, UNC-61b and UNC-61c. The UNC-61a transmembrane domain is sufficient to localize a fluorophore to membranes in mammalian cells, and its deletion from UNC-61a recapitulates the phenotypes of unc-61a null animals. The localization and loss-of-function phenotypes of UNC-61a and its transmembrane domain suggest roles in cell polarity and secretion and help explain the cellular and tissue biological underpinnings of C. elegans septin null alleles' enigmatically hypomorphic phenotypes. Together, our findings reveal a novel mechanism of septin-membrane association with profound implications for the dynamics and regulation of this association.
RESUMEN
Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. Here we describe a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which we propose is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry.
Asunto(s)
Centrosoma/fisiología , Microtúbulos/fisiología , Mitosis , Membrana Nuclear/genética , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Aurora Quinasas , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Centrosoma/metabolismo , Cromosomas/genética , Cromosomas/fisiología , Dineínas/fisiología , Embrión no Mamífero , Activación Enzimática , Modelos Genéticos , Membrana Nuclear/metabolismo , Membrana Nuclear/fisiología , Permeabilidad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Huso AcromáticoRESUMEN
Two distinct chromosome architectures are prevalent among eukaryotes: monocentric, in which localized centromeres restrict kinetochore assembly to a single chromosomal site, and holocentric, in which diffuse kinetochores form along the entire chromosome length. During mitosis, both chromosome types use specialized chromatin, containing the histone H3 variant CENP-A, to direct kinetochore assembly. For the segregation of recombined homologous chromosomes during meiosis, monocentricity is thought to be crucial for limiting spindle-based forces to one side of a crossover and to prevent recombined chromatids from being simultaneously pulled towards both spindle poles. The mechanisms that allow holocentric chromosomes to avert this fate remain uncharacterized. Here, we show that markedly different mechanisms segregate holocentric chromosomes during meiosis and mitosis in the nematode Caenorhabditis elegans. Immediately prior to oocyte meiotic segregation, outer-kinetochore proteins were recruited to cup-like structures on the chromosome surface via a mechanism that is independent of CENP-A. In striking contrast to mitosis, both oocyte meiotic divisions proceeded normally following depletion of either CENP-A or the closely associated centromeric protein CENP-C. These findings highlight a pronounced difference between the segregation of holocentric chromosomes during meiosis and mitosis and demonstrate the potential to uncouple assembly of outer-kinetochore proteins from CENP-A chromatin.
Asunto(s)
Autoantígenos/fisiología , Caenorhabditis elegans/genética , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Meiosis , Mitosis , Animales , Caenorhabditis elegans/citología , Proteína A Centromérica , Cromatina , Estructuras Cromosómicas , Cinetocoros/química , Oocitos , Transporte de ProteínasRESUMEN
Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.
Asunto(s)
Autoantígenos/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Autoantígenos/genética , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/química , Centrómero/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Genómica , Histonas/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myb/química , Interferencia de ARN , Alineación de SecuenciaRESUMEN
Work over the last several decades has shown that kinetochores play an active part in chromosome segregation, while the chromatin and, more to the point, the DNA have gathered little attention. In two intriguing papers, the Bloom and Khodjakov groups show that intercentromeric chromatin plays a much more active part in chromosome segregation than previously suspected.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Centrómero/fisiología , Cromatina/fisiología , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica/fisiología , Proteínas Nucleares/fisiología , Animales , CohesinasRESUMEN
The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Delta cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Replicación del ADN , Factores de Transcripción Forkhead/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Aumento de la Célula , Polaridad Celular , Quinasa de Punto de Control 2 , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Deficiency for telomerase results in transgenerational shortening of telomeres. However, telomeres have no known role in transgenerational epigenetic inheritance. C. elegans Protection Of Telomeres 1 (Pot1) proteins form foci at the telomeres of germ cells that disappear at fertilization and gradually accumulate during development. We find that gametes from mutants deficient for Pot1 proteins alter levels of telomeric foci for multiple generations. Gametes from pot-2 mutants give rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci throughout development, which persists for six generations. In contrast, gametes from pot-1 mutants or pot-1; pot-2 double mutants induce diminished Pot1 foci for several generations. Deficiency for MET-2, SET-25, or SET-32 methyltransferases, which promote heterochromatin formation, results in gametes that induce diminished Pot1 foci for several generations. We propose that C. elegans POT-1 may interact with H3K9 methyltransferases during pot-2 mutant gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that causes constitutively high levels of heterochromatic Pot1 foci.
Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/deficiencia , Gametogénesis , Células Germinativas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/genética , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Herencia , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Masculino , Telómero/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.
Asunto(s)
Polaridad Celular , Mitosis , Anafase/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Cromosomas/efectos de los fármacos , Cromosomas/fisiología , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Mitosis/efectos de los fármacos , Pirimidinas/farmacología , Huso Acromático/efectos de los fármacos , Huso Acromático/fisiología , Tionas/farmacologíaRESUMEN
Microtubule plus ends dynamically attach to kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of kinetochores in Xenopus extract spindles. During metaphase, kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5-2.5 micro m/min. Because kinetochore microtubules polymerize at metaphase kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a "slip-clutch" mechanism that prevents detachment at high tension. At anaphase onset, kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule-kinetochore interface and for how flux regulates kinetochore function.