RESUMEN
The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.
Asunto(s)
División Celular/fisiología , Sistema Nervioso/embriología , Neuronas/citología , Neuronas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Huso Acromático/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Corteza Cerebral/embriología , Proteína Doblecortina , Quinasas Similares a Doblecortina , Dineínas/fisiología , Desarrollo Embrionario/fisiología , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Mitosis/fisiología , Prometafase/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismoRESUMEN
OBJECTIVE: Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far. RESULTS: Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.
Asunto(s)
Proteínas de Arabidopsis , Proteínas Asociadas a Microtúbulos , Microtúbulos , Animales , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestructura , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructuraRESUMEN
The use of the semiconductor quantum dots (QD) as biolabels for both ensemble and single-molecule tracking requires the development of simple and versatile methods to target individual proteins in a controlled manner, ideally in living cells. To address this challenge, we have prepared small and stable QDs (QD-ND) using a surface coating based on a peptide sequence containing a tricysteine, poly(ethylene glycol) (PEG), and an aspartic acid ligand. These QDs, with a hydrodynamic diameter of 9 +/- 1.5 nm, can selectively bind to polyhistidine-tagged (histag) proteins in vitro or in living cells. We show that the small and monodisperse size of QD-ND allows for the formation of QD-ND/histag protein complexes of well-defined stoichiometry and that the 1:1 QD/protein complex can be isolated and purified by gel electrophoresis without any destabilization in the nanomolar concentration range. We also demonstrate that QD-ND can be used to specifically label a membrane receptor with an extracellular histag expressed in living HeLa cells. Here, cytotoxicity tests reveal that cell viability remains high under the conditions required for cellular labeling with QD-ND. Finally, we apply QD-ND complexed with histag end binding protein-1 (EB1), a microtubule associated protein, to single-molecule tracking in Xenopus extracts. Specific colocalization of QD-ND/EB1 with microtubules during the mitotic spindle formation demonstrates that QD-ND and our labeling strategy provide an efficient approach to monitor the dynamic behavior of proteins involved in complex biological functions.
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Histidina/metabolismo , Sondas Moleculares/química , Péptidos/química , Polietilenglicoles/química , Proteínas/metabolismo , Puntos Cuánticos , Animales , Supervivencia Celular , Espacio Extracelular/metabolismo , Células HeLa , Humanos , Luz , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Sondas Moleculares/metabolismo , Movimiento , Estabilidad Proteica , Transporte de Proteínas , Proteínas/análisis , Proteínas/química , Dispersión de Radiación , Espectrometría de Fluorescencia , Huso Acromático/metabolismo , Coloración y Etiquetado , Especificidad por SustratoRESUMEN
+TIPs (plus-end tracking proteins) are an increasing group of molecules that localize preferentially to the end of growing microtubules. +TIPs regulate microtubule dynamics and contribute to the organization of the microtubular network within the cell. Thus they participate in a wide range of cellular processes including cell division, motility and morphogenesis. EB1 (end-binding 1) is a highly conserved key member of the +TIP group that has been shown to modulate microtubule dynamics both in vitro and in cells. EB1 is involved in accurate chromosome segregation during mitosis and in the polarization of the microtubule cytoskeleton in migrating cells. Here, we review recent in vitro studies that have started to reveal a regulating activity of EB1, and its yeast orthologue Mal3p, on microtubule structure. In particular, we examine how EB1-mediated changes in the microtubule architecture may explain its effects on microtubule dynamics.
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Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos , Conformación Proteica , Animales , Polaridad Celular , Segregación Cromosómica , Citoesqueleto/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Neoplasias de la Úvea/metabolismo , Citoesqueleto de Actina/metabolismo , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Movimiento Celular , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación con Pérdida de Función , Melanoma/genética , Melanoma/patología , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Fosfatasas/genética , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patologíaRESUMEN
Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules.This article has an associated First Person interview with the first author of the paper.
RESUMEN
CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.
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Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Células COS , Complejo Dinactina , Células HeLa , Humanos , Interfase , Cinetocoros/metabolismo , Microscopía Fluorescente , Microtúbulos/metabolismo , Proteínas de Neoplasias , Unión Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Dedos de ZincRESUMEN
In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.
Asunto(s)
Corteza Cerebelosa/citología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Benzopiranos/farmacología , Citoplasma/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Red Nerviosa/efectos de los fármacos , Propionatos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
BACKGROUND: Doublecortin (DCX) domains serve as protein-interaction platforms. Mutations in members of this protein superfamily are linked to several genetic diseases. Mutations in the human DCX gene result in abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, and DCDC2 has been associated with dyslectic reading disabilities. RESULTS: The DCX-repeat gene family is composed of eleven paralogs in human and in mouse. Its evolution was followed across vertebrates, invertebrates, and was traced to unicellular organisms, thus enabling following evolutionary additions and losses of genes or domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. Developmental in situ hybridization data for nine genes was generated. In addition, a novel co-expression analysis for most human and mouse DCX superfamily-genes was performed using high-throughput expression data extracted from Unigene. We performed an in-depth study of a complete gene superfamily using several complimentary methods. CONCLUSION: This study reveals the existence and conservation of multiple members of the DCX superfamily in different species. Sequence analysis combined with expression analysis is likely to be a useful tool to predict correlations between human disease and mouse models. The sub-specialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution.
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Proteínas Asociadas a Microtúbulos/genética , Familia de Multigenes , Neuropéptidos/genética , Animales , Bovinos , Pollos/genética , Análisis por Conglomerados , Perros , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Evolución Molecular , Perfilación de la Expresión Génica , Genes Fúngicos , Genes de Plantas , Humanos , Hibridación in Situ , Macaca mulatta/genética , Ratones , Modelos Genéticos , Zarigüeyas/genética , Pan troglodytes/genética , Filogenia , Estructura Terciaria de Proteína/genética , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
Cryo-electron tomography of vitrified specimens allows visualization of thin biological samples in three-dimensions. This method can be applied to study the interaction of proteins that show disorder and/or bind in a nonregular fashion to microtubules. Here, we describe the protocols we use to observe microtubules assembled in vitro in the presence of XMAP215, a large and flexible protein that binds to discrete sites on the microtubule lattice. Gold particles are added to the mix before vitrification to facilitate image acquisition in low-dose mode and their subsequent alignment before tomographic reconstruction. Three-dimensional reconstructions are performed using the IMOD software, processed with ImageJ and visualized in UCSF Chimera. Extraction of features of interest is performed using a patch-based algorithm (CryoSeg) developed in the laboratory. All the software used in this procedure is freely available or can be obtained on request, and run on most operating systems.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Microtúbulos/ultraestructura , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/química , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructuraRESUMEN
End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos , Tubulina (Proteína)/metabolismo , Animales , Ratones , Microscopía por Video , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Biológicos , Conformación Proteica , PorcinosRESUMEN
The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.
Asunto(s)
Proteínas Asociadas a Microtúbulos/fisiología , Familia de Multigenes/fisiología , Neuropéptidos/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis/fisiología , Neuropéptidos/química , Neuropéptidos/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Transducción de Señal/fisiologíaRESUMEN
Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.