RESUMEN
Kinesin and dynein motors drive bidirectional cargo transport along microtubules and have a critical role in polarized cargo trafficking in neurons [1, 2]. The kinesin-2 family protein KIF17 is a dendrite-specific motor protein and has been shown to interact with several dendritic cargoes [3-7]. However, the mechanism underlying the dendritic targeting of KIF17 remains poorly understood [8-11]. Using live-cell imaging combined with inducible trafficking assays to directly probe KIF17 motor activity in living neurons, we found that the polarized sorting of KIF17 to dendrites is regulated in multiple steps. First, cargo binding of KIF17 relieves autoinhibition and initiates microtubule-based cargo transport. Second, KIF17 does not autonomously target dendrites, but enters the axon where the actin cytoskeleton at the axon initial segment (AIS) prevents KIF17 vesicles from moving further into the axon. Third, dynein-based motor activity is able to redirect KIF17-coupled cargoes into dendrites. We propose a three-step model for polarized targeting of KIF17, in which the collective function of multiple motor teams is required for proper dendritic sorting.
Asunto(s)
Axones/metabolismo , Dendritas/metabolismo , Cinesinas/metabolismo , Animales , Células Cultivadas , Microtúbulos/metabolismo , Transporte de Proteínas , RatasRESUMEN
Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.
Asunto(s)
Anomalías Craneofaciales/metabolismo , Enfermedad de Hirschsprung/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Ratones , Neuronas/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. We find that the velocities of kinesin-based vesicle movements are slower and more homogeneous than those of dynein-based movements. We also find that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. Our study suggests that adaptor proteins can modulate the velocity of dynein-based motility and thereby control the distribution of transport carriers.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Transporte Axonal , Células Cultivadas , Células HeLa , Humanos , Cinesinas/metabolismo , Neuronas/metabolismo , Unión Proteica , Transporte de Proteínas , RatasRESUMEN
In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.
Asunto(s)
Axones/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dendritas/metabolismo , Microtúbulos/metabolismo , Células Piramidales/metabolismo , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Hipocampo/embriología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Humanos , Proteínas Asociadas a Microtúbulos , Microtúbulos/ultraestructura , Células Piramidales/ultraestructura , RatasRESUMEN
Regulation of cargo transport via adaptor molecules is essential for neuronal development. However, the role of PDZ scaffolding proteins as adaptors in neuronal cargo trafficking is still poorly understood. Here, we show by genetic deletion in mice that the multi-PDZ domain scaffolding protein glutamate receptor interacting protein 1 (GRIP1) is required for dendrite development. We identify an interaction between GRIP1 and 14-3-3 proteins that is essential for the function of GRIP1 as an adaptor protein in dendritic cargo transport. Mechanistically, 14-3-3 binds to the kinesin-1 binding region in GRIP1 in a phospho-dependent manner and detaches GRIP1 from the kinesin-1 motor protein complex thereby regulating cargo transport. A single point mutation in the Thr956 of GRIP1 in transgenic mice impairs dendritic development. Together, our results show a regulatory role for GRIP1 during microtubule-based transport and suggest a crucial function for 14-3-3 proteins in controlling kinesin-1 motor attachment during neuronal development.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dendritas/metabolismo , Cinesinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Técnicas de Inactivación de Genes/métodos , Cinesinas/genética , Ratones , Mutación/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bicaudal-D (BICD) belongs to an evolutionary conserved family of dynein adaptor proteins. It was first described in Drosophila as an essential factor in fly oogenesis and embryogenesis. Missense mutations in a human BICD homologue, BICD2, have been linked to a dominant mild early onset form of spinal muscular atrophy. Here we further examine the in vivo function of BICD2 in Bicd2 knockout mice. BICD2-deficient mice develop disrupted laminar organization of cerebral cortex and the cerebellum, pointing to impaired radial neuronal migration. Using astrocyte and granule cell specific inactivation of BICD2, we show that the cerebellar migration defect is entirely dependent upon BICD2 expression in Bergmann glia cells. Proteomics analysis reveals that Bicd2 mutant mice have an altered composition of extracellular matrix proteins produced by glia cells. These findings demonstrate an essential non-cell-autonomous role of BICD2 in neuronal cell migration, which might be connected to cargo trafficking pathways in glia cells.
Asunto(s)
Movimiento Celular , Cerebelo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/metabolismo , Western Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Cerebelo/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Neuroglía/metabolismo , Neuronas/patología , Ratas , Factores de TiempoRESUMEN
Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.
Asunto(s)
Enfermedades Hereditarias del Ojo/metabolismo , Fibrosis/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Trastornos de la Motilidad Ocular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto , Enfermedades Hereditarias del Ojo/genética , Inhibidores de Crecimiento , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Morfogénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Oftalmoplejía , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Intracellular transport is driven by motor proteins that either use microtubules or actin filaments as their tracks, but the interplay between these transport pathways is poorly understood. Whereas many microtubule-based motors are known to drive long-range transport, several actin-based motors have been proposed to function predominantly in cargo tethering. How these opposing activities are integrated on cargoes that contain both types of motors is unknown. Here we use inducible intracellular transport assays to show that acute recruitment of myosin-V to kinesin-propelled cargo reduces their motility near the cell periphery and enhances their localization at the actin-rich cell cortex. Myosin-V arrests rapid microtubule-based transport without the need for regulated auto- or other inhibition of kinesin motors. In addition, myosin-V, despite being an ineffective long-range transporter, can drive slow, medium-range (1-5 µm), point-to-point transport in cortical cell regions. Altogether, these data support a model in which myosin-V establishes local cortical delivery of kinesin-bound cargos through a combination of tethering and active transport.
Asunto(s)
Actinas/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Miosina Tipo V/metabolismo , Animales , Transporte Biológico Activo , Células COS , Movimiento Celular , Chlorocebus aethiops , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Axones/metabolismo , Proteínas Portadoras/metabolismo , Dendritas/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/ultraestructura , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas Portadoras/genética , Polaridad Celular/genética , Células Cultivadas , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinesinas/metabolismo , Cinesinas/fisiología , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Unión Proteica/genética , Conformación Proteica , Proteínas Quinasas/metabolismo , Transporte de Proteínas/genética , ARN Interferente Pequeño/metabolismo , Ratas , Factores de Tiempo , TransfecciónRESUMEN
Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.
Asunto(s)
Espinas Dendríticas/metabolismo , Hipocampo/fisiología , Microtúbulos/fisiología , Neuronas/citología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/agonistasRESUMEN
Liprin-α proteins are major protein constituents of synapses and are important for the organization of synaptic vesicles and neurotransmitter receptors on their respective sides of the synapse. Although it is becoming apparent that the single liprin-α gene in invertebrates is essential for synapse function, it is not known to what extent the four different liprin-α homologs (liprin-α1-4) in mammals are involved at synapses. We have designed specific antibodies against each of the four liprin-α proteins and investigated their regional and cellular distribution in the brain. Here we show that all four liprin-α proteins are present throughout the mature brain but have different regional distributions, which is highlighted by their differential localization in olfactory bulb, hippocampus, and cerebellar cortex. Double-immunofluorescence staining indicates that different liprin-α proteins are enriched in different synaptic populations but are also present at nonsynaptic sites. In particular, liprin-α2 is preferentially associated with hippocampal mossy fiber endings in the CA3, whereas synapses in the molecular layers of the CA1 and dentate gyrus double-labeled for liprin-α3. The localization of liprin-α2 and liprin-α3 with excitatory synapses was confirmed in cultured primary hippocampal neurons. Liprin-α4, which poorly co-distributed with presynaptic markers in hippocampus, instead strongly co-localized with VGLUT1 in the cerebellar molecular layer, suggesting its presence in parallel fiber-Purkinje cell synapses. Finally, staining of cultured glial cells indicated that liprin-α1 and liprin-α3 are also associated with astrocytes. We conclude that liprin-α family proteins might perform independent and specialized synaptic and nonsynaptic functions in different regions of the brain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Encéfalo/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Encéfalo/anatomía & histología , Células Cultivadas , Células HeLa , Hipocampo/citología , Humanos , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/genética , Ratas , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Dendritic arbors are compartments of neurons dedicated to receiving synaptic inputs. Their shape is an outcome of both the intrinsic genetic program and environmental signals. The microtubules and actin cytoskeleton are both crucial for proper dendritic morphology, but how they interact is unclear. The present study demonstrates that microtubule plus-end tracking protein CLIP-170 and actin-binding protein IQGAP1 regulate dendrite morphology of rat neurons by coordinating the interaction between microtubules and the actin cytoskeleton. Moreover, we show that mTOR kinase interacts with CLIP-170 and is needed for efficient formation of a protein complex containing CLIP-170 and IQGAP1. Dynamic microtubules, CLIP-170, and IQGAP1 are required for proper dendritic arbor morphology and PI3K-mTOR-induced increase in dendritic arbor complexity. Moreover, CLIP-170 and IQGAP1 knockdown modulates dendritic arbor growth via regulation of the actin cytoskeleton. We postulate that mTOR controls dendritic arbor morphology by enhancing cross talk between dynamic microtubules and actin through CLIP-170 and IQGAP1.
Asunto(s)
Dendritas/ultraestructura , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Activadoras de ras GTPasa/fisiología , Actinas/metabolismo , Animales , Biotinilación , Células Cultivadas , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , ADN/genética , Dendritas/fisiología , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Hipocampo/citología , Hipocampo/fisiología , Procesamiento de Imagen Asistido por Computador , Indicadores y Reactivos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Ratas , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins.
Asunto(s)
Bioensayo/métodos , Espacio Intracelular/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Transporte Biológico , Línea Celular , Polaridad Celular , Humanos , Quimografía , Peroxisomas/metabolismoRESUMEN
Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Centrosoma/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Neuronas/patología , Proteínas de Unión al GTP rab/metabolismo , Animales , Encéfalo/metabolismo , Células COS , Diferenciación Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Riñón/metabolismo , Cinesinas/química , Modelos Biológicos , Neuronas/metabolismo , Pez CebraRESUMEN
BACKGROUND: To establish and maintain their polarized morphology, neurons employ active transport driven by molecular motors to sort cargo between axons and dendrites. However, the basic traffic rules governing polarized transport on neuronal microtubule arrays are unclear. RESULTS: Here we show that the microtubule minus-end-directed motor dynein is required for the polarized targeting of dendrite-specific cargo, such as AMPA receptors. To directly examine how dynein motors contribute to polarized dendritic transport, we established a trafficking assay in hippocampal neurons to selectively probe specific motor protein activity. This revealed that, unlike kinesins, dynein motors drive cargo selectively into dendrites, governed by their mixed microtubule array. Moreover, axon-specific cargos, such as presynaptic vesicle protein synaptophysin, are redirected to dendrites by coupling to dynein motors. Quantitative modeling demonstrated that bidirectional dynein-driven transport on mixed microtubules provides an efficient mechanism to establish a stable density of continuously renewing vesicles in dendrites. CONCLUSIONS: These results demonstrate a powerful approach to study specific motor protein activity inside living cells and imply a key role for dynein in dendritic transport. We propose that dynein establishes the initial sorting of dendritic cargo and additional motor proteins assist in subsequent delivery.
Asunto(s)
Dendritas/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Animales , Transporte Biológico Activo/fisiología , Células COS , Chlorocebus aethiops , Hipocampo/citología , Inmunohistoquímica , Cinesinas/metabolismo , Peroxisomas/metabolismo , Receptores AMPA/metabolismo , Sinaptofisina/metabolismoRESUMEN
The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.
Asunto(s)
Dendritas/metabolismo , Endosomas/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Animales , Transporte Biológico , Células COS , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Chlorocebus aethiops , Dendritas/ultraestructura , Escherichia coli/genética , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiología , Ratones , Plasticidad Neuronal , Proteínas Qa-SNARE/metabolismo , Ratas , Receptores de Glutamato/metabolismo , Porcinos , Proteínas de Unión al GTP rab4/análisis , Proteínas de Unión al GTP rab4/fisiologíaRESUMEN
Dendritic spines are the major sites of excitatory synaptic input, and their morphological changes have been linked to learning and memory processes. Here, we report that growing microtubule plus ends decorated by the microtubule tip-tracking protein EB3 enter spines and can modulate spine morphology. We describe p140Cap/SNIP, a regulator of Src tyrosine kinase, as an EB3 interacting partner that is predominantly localized to spines and enriched in the postsynaptic density. Inhibition of microtubule dynamics, or knockdown of either EB3 or p140Cap, modulates spine shape via regulation of the actin cytoskeleton. Fluorescence recovery after photobleaching revealed that EB3-binding is required for p140Cap accumulation within spines. In addition, we found that p140Cap interacts with Src substrate and F-actin-binding protein cortactin. We propose that EB3-labeled growing microtubule ends regulate the localization of p140Cap, control cortactin function, and modulate actin dynamics within dendritic spines, thus linking dynamic microtubules to spine changes and synaptic plasticity.
Asunto(s)
Espinas Dendríticas/ultraestructura , Microtúbulos/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Cortactina/metabolismo , Citoesqueleto/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nocodazol/farmacología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/ultraestructura , Moduladores de Tubulina/farmacologíaRESUMEN
Exocytic events are tightly regulated cellular processes in which rab GTPases and their interacting proteins perform an important function. We set out to identify new binding partners of rab3, which mediates regulated secretion events in specialized cells. We discovered Zwint-1 as a rab3 specific binding protein that bound preferentially to rab3c. The interaction depends on a critical residue in rab3c that determines the binding efficiency of Zwint-1, which is immaterial for interaction with rabphilin3a. Rab3c and Zwint-1 are expressed highly in brain and colocalized extensively in primary hippocampal neurons. We also found that SNAP25 bound to the same region in Zwint-1 as rab3c, suggesting a new role for the kinetochore protein Zwint-1 in presynaptic events that are regulated by rab3 and SNAP25.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Serina/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Secuencia Conservada , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismo , Ratas , Serina/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteínas de Unión al GTP rab3/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by progressive motor neuron degeneration and muscle paralysis. Genetic evidence from man and mouse has indicated that mutations in the dynein/dynactin motor complex are correlated with motor neuron degeneration. In this study, we have generated transgenic mice with neuron-specific expression of Bicaudal D2 N-terminus (BICD2-N) to chronically impair dynein/dynactin function. Motor neurons expressing BICD2-N showed accumulation of dynein and dynactin in the cell body, Golgi fragmentation and several signs of impaired retrograde trafficking: the appearance of giant neurofilament swellings in the proximal axon, reduced retrograde labelling by tracer injected in the muscle and delayed expression of the injury transcription factor ATF3 after axon transection. Despite these abnormalities, BICD2-N mice did not develop signs of motor neuron degeneration and motor abnormalities. Interestingly, the BICD2-N transgene increased lifespan in 'low copy' SOD1-G93A ALS transgenic mice. Our findings indicate that impaired dynein/dynactin function can explain several pathological features observed in ALS patients, but may be beneficial in some forms of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Portadoras/metabolismo , Modelos Animales de Enfermedad , Dineínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas Motoras/metabolismo , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Transporte Biológico , Proteínas Portadoras/genética , Células Cultivadas , Complejo Dinactina , Dineínas/genética , Femenino , Expresión Génica , Aparato de Golgi/metabolismo , Humanos , Esperanza de Vida , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Ratas , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , SobrevidaRESUMEN
Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.