RESUMO
Error-free chromosome segregation requires dynamic control of microtubule attachment to kinetochores, but how kinetochore-microtubule interactions are spatially and temporally controlled during mitosis remains incompletely understood. In addition to the NDC80 microtubule-binding complex, other proteins with demonstrated microtubule-binding activities localize to kinetochores. One such protein is the cytoplasmic linker-associated protein 2 (CLASP2). Here, we show that global GSK3-mediated phosphorylation of the longest isoform, CLASP2α, largely abolishes CLASP2α-microtubule association in metaphase. However, it does not directly control localization of CLASP2α to kinetochores. Using dominant phosphorylation-site variants, we find that CLASP2α phosphorylation weakens kinetochore-microtubule interactions as evidenced by decreased tension between sister kinetochores. Expression of CLASP2α phosphorylation-site mutants also resulted in increased chromosome segregation defects, indicating that GSK3-mediated control of CLASP2α-microtubule interactions contributes to correct chromosome dynamics. Because of global inhibition of CLASP2α-microtubule interactions, we propose a model in which only kinetochore-bound CLASP2α is dephosphorylated, locally engaging its microtubule-binding activity.
Assuntos
Queratinócitos/fisiologia , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Proteína Quinase CDC2 , Linhagem Celular , Segregação de Cromossomos/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas do Citoesqueleto , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas Nucleares/metabolismo , Fosforilação/genética , Ligação Proteica , Engenharia de ProteínasRESUMO
Centrioles are subcellular organelles composed of a ninefold symmetric microtubule array that perform two important functions: (1) They build centrosomes that organize the microtubule cytoskeleton, and (2) they template cilia, microtubule-based projections with sensory and motile functions. We identified HYLS-1, a widely conserved protein, based on its direct interaction with the core centriolar protein SAS-4. HYLS-1 localization to centrioles requires SAS-4 and, like SAS-4, HYLS-1 is stably incorporated into the outer centriole wall. Unlike SAS-4, HYLS-1 is dispensable for centriole assembly and centrosome function in cell division. Instead, HYLS-1 plays an essential role in cilia formation that is conserved between Caenorhabditis elegans and vertebrates. A single amino acid change in human HYLS1 leads to a perinatal lethal disorder termed hydrolethalus syndrome, and we show that this mutation impairs HYLS-1 function in ciliogenesis. HYLS-1 is required for the apical targeting/anchoring of centrioles at the plasma membrane but not for the intraflagellar transport-dependent extension of the ciliary axoneme. These findings classify hydrolethalus syndrome as a severe human ciliopathy and shed light on the dual functionality of centrioles, defining the first stably incorporated centriolar protein that is not required for centriole assembly but instead confers on centrioles the capacity to initiate ciliogenesis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Centríolos/metabolismo , Cílios/fisiologia , Sequência de Aminoácidos , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Divisão Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Humanos , Dados de Sequência Molecular , Mutação/genética , Neurônios/metabolismo , Transporte Proteico , Alinhamento de Sequência , Fatores de Transcrição/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore-microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1-dynein complex. Overexpression of NDEL1-dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1-NDEL1-dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Lisencefalia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Células Cultivadas , Centrossomo , Córtex Cerebral , Segregação de Cromossomos , Células HEK293 , Humanos , Lisencefalia/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Mutação , Neurônios/metabolismo , Estabilidade Proteica , Fuso Acromático/genéticaRESUMO
The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3ß-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3ß-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.
Assuntos
Quinase 3 da Glicogênio Sintase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Colinérgicos/metabolismo , Agrina/fisiologia , Animais , Células COS , Chlorocebus aethiops , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/ultraestrutura , Fibras Musculares Esqueléticas/metabolismo , Fosforilação , Cultura Primária de Células , Transporte ProteicoRESUMO
BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3ß (GSK3ß) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3ß regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3ß activity in neurons. Following spinal cord injury, reduced GSK3ß levels or complete neuronal deletion of GSK3ß led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3ß substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3ß-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3ß-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3ß-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.
Assuntos
Axônios/fisiologia , Quinase 3 da Glicogênio Sintase/genética , Cones de Crescimento/metabolismo , Microtúbulos/metabolismo , Regeneração , Animais , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Ratos , Ratos WistarRESUMO
A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Quinase 3 da Glicogênio Sintase/química , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Simulação de Dinâmica Molecular , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5ß (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5ß) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.
Assuntos
Adesão Celular , Movimento Celular , Exocitose , Adesões Focais/metabolismo , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Matriz Extracelular/metabolismo , Adesões Focais/efeitos dos fármacos , Células HEK293 , Humanos , Queratinócitos/efeitos dos fármacos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , Vesículas Transportadoras/efeitos dos fármacosRESUMO
The palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.
Assuntos
Rastreamento de Células/métodos , Lasers , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Artefatos , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador/métodos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Proteínas/genética , Proteínas/metabolismoRESUMO
The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.