RESUMEN
Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates.
Asunto(s)
Cisteína Endopeptidasas/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Multimerización de Proteína , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Factores de Transcripción/genética , Sitios de Unión , Línea Celular Tumoral , Cisteína Endopeptidasas/metabolismo , Humanos , Microscopía Fluorescente , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína SUMO-1/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , UbiquitinaciónRESUMEN
During mitosis and meiosis in the majority of eukaryotes, centromeric chromatin comprised of CENP-A nucleosomes and their reader CENP-C recruits components of the outer kinetochore to build an interface with spindle microtubules.1,2 One exception is C. elegans oocyte meiosis, where outer kinetochore proteins form cup-like structures on chromosomes independently of centromeric chromatin.3 Here, we show that the nucleoporin MEL-28 (ortholog of human ELYS) and CENP-CHCP-4 act in parallel to recruit outer kinetochore components to oocyte meiotic chromosomes. Unexpectedly, co-inhibition of MEL-28 and CENP-CHCP-4 resulted in chromosomes being expelled from the meiotic spindle prior to anaphase onset, a more severe phenotype than what was observed following ablation of the outer kinetochore.4,5 This observation suggested that MEL-28 and the outer kinetochore independently link chromosomes to spindle microtubules. Consistent with this, the chromosome expulsion defect was observed following co-inhibition of MEL-28 and the microtubule-coupling KNL-1/MIS-12/NDC-80 (KMN) network of the outer kinetochore. Use of engineered mutants showed that MEL-28 acts in conjunction with the microtubule-binding NDC-80 complex to keep chromosomes within the oocyte meiotic spindle and that this function likely involves the Y-complex of nucleoporins that associate with MEL-28; by contrast, the ability to dock protein phosphatase 1, shared by MEL-28 and KNL-1, is not involved. These results highlight nuclear pore-independent functions for a conserved nucleoporin and explain two unusual features of oocyte meiotic chromosome segregation in C. elegans: centromeric chromatin-independent outer kinetochore assembly, and dispensability of the outer kinetochore for constraining chromosomes in the acentrosomal meiotic spindle.
Asunto(s)
Proteínas de Caenorhabditis elegans , Cinetocoros , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis , Proteínas de Complejo Poro Nuclear/metabolismo , Huso Acromático/metabolismoRESUMEN
Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.
Asunto(s)
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Morfogénesis , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Dendritas/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , MitosisRESUMEN
The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.
Asunto(s)
Caenorhabditis elegans/embriología , División Celular , Técnicas Citológicas/métodos , Embrión no Mamífero/citología , Alelos , Animales , Sistemas CRISPR-Cas/genética , Caenorhabditis elegans/genética , Ingeniería Genética , Microtúbulos/metabolismo , Mutación/genética , Interferencia de ARN , Reproducibilidad de los Resultados , TransgenesRESUMEN
During cell division, genome inheritance is orchestrated by microtubule attachments formed at kinetochores of mitotic chromosomes. The primary microtubule coupler at the kinetochore, the Ndc80 complex, is regulated by Aurora kinase phosphorylation of its N-terminal tail. Dephosphorylation is proposed to stabilize kinetochore-microtubule attachments by strengthening electrostatic interactions of the tail with the microtubule lattice. Here, we show that removal of the Ndc80 tail, which compromises in vitro microtubule binding, has no effect on kinetochore-microtubule attachments in the Caenorhabditis elegans embryo. Despite this, preventing Aurora phosphorylation of the tail results in prematurely stable attachments that restrain spindle elongation. This premature stabilization requires the conserved microtubule-binding Ska complex, which enriches at attachment sites prior to anaphase onset to dampen chromosome motion. We propose that Ndc80-tail dephosphorylation promotes stabilization of kinetochore-microtubule attachments via the Ska complex and that this mechanism ensures accurate segregation by constraining chromosome motion following biorientation on the spindle.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Anafase , Animales , Cromosomas/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Eliminación de Gen , Complejos Multiproteicos/química , Fosforilación , Unión Proteica , Polos del Huso/metabolismoRESUMEN
During M-phase entry in metazoans with open mitosis, the concerted action of mitotic kinases disassembles nuclei and promotes assembly of kinetochores-the primary microtubule attachment sites on chromosomes. At M-phase exit, these major changes in cellular architecture must be reversed. Here, we show that the conserved kinetochore-localized nucleoporin MEL-28/ELYS docks the catalytic subunit of protein phosphatase 1 (PP1c) to direct kinetochore disassembly-dependent chromosome segregation during oocyte meiosis I and nuclear assembly during the transition from M phase to interphase. During oocyte meiosis I, MEL-28-PP1c disassembles kinetochores in a timely manner to promote elongation of the acentrosomal spindles that segregate homologous chromosomes. During nuclear assembly, MEL-28 recruits PP1c to the periphery of decondensed chromatin, where it directs formation of a functional nuclear compartment. Thus, a pool of phosphatase activity associated with a kinetochore-localized nucleoporin contributes to two key events that occur during M-phase exit in metazoans: kinetochore disassembly and nuclear reassembly.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Meiosis/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Neuropéptido Y/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN , Humanos , Cinetocoros/metabolismo , Membrana Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Mapas de Interacción de Proteínas/genética , Receptores de Neuropéptido Y/genéticaRESUMEN
Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.
Asunto(s)
Anafase , Autoantígenos/metabolismo , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/metabolismo , Meiosis , Metafase , Separasa/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteína A Centromérica , Segregación Cromosómica , Cromosomas/metabolismo , Duplicación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Oocitos/citología , Oocitos/metabolismo , Separasa/antagonistas & inhibidoresRESUMEN
The kinetochore is a large, macromolecular assembly that is essential for connecting chromosomes to microtubules during mitosis. Despite the recent identification of multiple kinetochore components, the nature and organization of the higher-order kinetochore structure remain unknown. The outer kinetochore KNL-1/Mis12 complex/Ndc80 complex (KMN) network plays a key role in generating and sensing microtubule attachments. Here we demonstrate that Caenorhabditis elegans KNL-1 exists as an oligomer, and we identify a specific domain in KNL-1 responsible for this activity. An N-terminal KNL-1 domain from both C. elegans and the related nematode Caenorhabditis remanei oligomerizes into a decameric assembly that appears roughly circular when visualized by electron microscopy. On the basis of sequence and mutational analysis, we identify a small hydrophobic region as responsible for this oligomerization activity. However, mutants that precisely disrupt KNL-1 oligomerization did not alter KNL-1 localization or result in the loss of embryonic viability based on gene replacements in C. elegans. In C. elegans, KNL-1 oligomerization may coordinate with other kinetochore activities to ensure the proper organization, function, and sensory capabilities of the kinetochore-microtubule attachment.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Caenorhabditis/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Caenorhabditis/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Supervivencia Celular/genética , Segregación Cromosómica/genética , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Imagen de Lapso de Tiempo/métodosRESUMEN
Recruitment of Mad1-Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1-Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1-MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/citología , Proteínas de Ciclo Celular/fisiología , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Nucleares/fisiología , Anafase/fisiología , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas/metabolismo , Cinetocoros/fisiología , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Proteínas Mad2/fisiología , Meiosis/fisiología , Modelos Biológicos , Mutagénesis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Estructura Terciaria de Proteína , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Promyelocytic leukemia protein (PML) is the core component of PML-nuclear bodies (PML NBs). The small ubiquitin-like modifier (SUMO) system (and, in particular, SUMOylation of PML) is a critical component in the formation and regulation of PML NBs. SUMO protease SENP6 has been shown previously to be specific for SUMO-2/3-modified substrates and shows preference for SUMO polymers. Here, we further investigate the substrate specificity of SENP6 and show that it is also capable of cleaving mixed chains of SUMO-1 and SUMO-2/3. Depletion of SENP6 results in accumulation of endogenous SUMO-2/3 and SUMO-1 conjugates, and immunofluorescence analysis shows accumulation of SUMO and PML in an increased number of PML NBs. Although SENP6 depletion drastically increases the size of PML NBs, the organizational structure of the body is not affected. Mutation of the catalytic cysteine of SENP6 results in its accumulation in PML NBs, and biochemical analysis indicates that SUMO-modified PML is a substrate of SENP6.
Asunto(s)
Estructuras del Núcleo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Dominio Catalítico , Estructuras del Núcleo Celular/ultraestructura , Supervivencia Celular , Cisteína/genética , Cisteína Endopeptidasas/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Multimerización de Proteína , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Factores de Transcripción/química , Proteínas Supresoras de Tumor/química , UbiquitinaciónRESUMEN
In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.
Asunto(s)
Arsénico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína SUMO-1/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Proteína SUMO-1/genética , Alineación de Secuencia , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.