RESUMO
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Assuntos
Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP , Humanos , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/metabolismo , Endossomos/genética , Endossomos/metabolismoRESUMO
During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.
Assuntos
Cinetocoros , Microtúbulos , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Ligação Proteica , AnimaisRESUMO
Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation.
Assuntos
Citoesqueleto , Microtúbulos , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microscopia de Fluorescência , Microtúbulos/metabolismoRESUMO
Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona-localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona-MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.
Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Cinetocoros/metabolismo , Mitose , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Células HeLa , Humanos , Mutação Puntual , Domínios ProteicosRESUMO
In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants (calponin homology (CH) domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.
Assuntos
Cinetocoros , Proteínas Nucleares , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Segregação de CromossomosRESUMO
The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eµ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.
Assuntos
Diversidade de Anticorpos/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Genes de Cadeia Pesada de Imunoglobulina , Região Variável de Imunoglobulina/genética , Células Precursoras de Linfócitos B/imunologia , Animais , Sequência de Bases , Sítios de Ligação , Fator de Ligação a CCCTC , Mapeamento Cromossômico , Rearranjo Gênico , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX5/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Fator de Transcrição YY1/metabolismoRESUMO
Cohesin is a ring-shaped protein complex that plays a crucial role in sister chromatid cohesion and gene expression. The dynamic association of cohesin with chromatin is essential for these functions. However, the exact nature of cohesin dynamics, particularly cohesin translocation, remains unclear. We evaluated the dynamics of individual cohesin molecules on DNA and found that the cohesin core complex possesses an intrinsic ability to traverse DNA in an adenosine triphosphatase (ATPase)-dependent manner. Translocation ability is suppressed in the presence of Wapl-Pds5 and Sororin; this suppression is alleviated by the acetylation of cohesin and the action of mitotic kinases. In Xenopus laevis egg extracts, cohesin is translocated on unreplicated DNA in an ATPase- and Smc3 acetylation-dependent manner. Cohesin movement changes from bidirectional to unidirectional when cohesin faces DNA replication; otherwise, it is incorporated into replicating DNA without being translocated or is dissociated from replicating DNA This study provides insight into the nature of individual cohesin dynamics and the mechanisms by which cohesin achieves cohesion in different chromatin contexts.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Movimento , Processamento de Proteína Pós-Traducional , Proteínas de Xenopus/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Xenopus laevis , CoesinasRESUMO
The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Transcrição Gênica , Fator de Ligação a CCCTC , Humanos , Proteínas Repressoras/metabolismo , Imagem Individual de Molécula , Fatores de Tempo , CoesinasRESUMO
The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising â¼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Cromossômicas não Histona/química , Humanos , Proteínas dos Microfilamentos/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Quaternária de Proteína , Transporte Proteico , Especificidade por SubstratoRESUMO
Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Família Multigênica/genética , Complexos Multiproteicos/genética , Engenharia de Proteínas/métodos , Transfecção/métodos , Clonagem Molecular/métodos , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida/métodos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin's subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Animais , Aurora Quinase B , Aurora Quinases , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Ciclo Celular/farmacologia , Clonagem Molecular , Humanos , Proteínas Nucleares/antagonistas & inibidores , Fosforilação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Xenopus , CoesinasRESUMO
The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Cinetocoros/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transdução de Sinais , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fuso Acromático/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Células HeLaRESUMO
Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.
Assuntos
Mitose , Proteínas , Proteínas/genética , Adenosina Trifosfatases/metabolismo , DNA , Cromátides/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Replicação do DNA/genética , Dano ao DNARESUMO
As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).
Assuntos
Proteína Quinase CDC2 , Mitose , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Humanos , FosforilaçãoRESUMO
The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2-dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese/efeitos dos fármacos , Citocinese/efeitos da radiação , Hidroxiureia/toxicidade , Pressão Osmótica , Schizosaccharomyces/fisiologia , Raios Ultravioleta , Proteínas Mad2 , Proteínas Quinases Ativadas por Mitógeno , Proteínas Nucleares , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosfatases cdc25RESUMO
Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.
Assuntos
Proteínas Cdc20/metabolismo , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Mad2/metabolismo , Complexos Multiproteicos/metabolismo , Biocatálise , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Serina-Treonina Quinases , Fuso Acromático/metabolismoRESUMO
Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Proteínas do Citoesqueleto/genética , Humanos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de Proteína , Fuso Acromático/metabolismoRESUMO
Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules' residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Mitose , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Polimerização , Ligação Proteica , Multimerização ProteicaRESUMO
Stable kinetochore-microtubule attachment is essential for cell division. It requires recruitment of outer kinetochore microtubule binders by centromere proteins C and T (CENP-C and CENP-T). To study the molecular requirements of kinetochore formation, we reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and CENP-T. Whereas CENP-C recruits a single MIS12:NDC80 complex, we show here that CENP-T binds one MIS12:NDC80 and two NDC80 complexes upon phosphorylation by the mitotic CDK1:Cyclin B complex at three distinct CENP-T sites. Visualization of reconstituted complexes by electron microscopy supports this model. Binding of CENP-C and CENP-T to MIS12 is competitive, and therefore CENP-C and CENP-T act in parallel to recruit two MIS12 and up to four NDC80 complexes. Our observations provide a molecular explanation for the stoichiometry of kinetochore components and its cell cycle regulation, and highlight how outer kinetochore modules bridge distances of well over 100 nm.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Proteínas do Citoesqueleto , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore-microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.