RESUMO
The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process.
Assuntos
Axônios/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Animais , Polaridade Celular/fisiologia , Hipocampo/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ß-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ß-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ß-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ß-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer ß-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/ß-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Anemia de Fanconi/etiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Ubiquitinação , beta Catenina/químicaRESUMO
Ca²âº/calmodulin-dependent kinases (CaMKs) are essential for neuronal development and plasticity, processes requiring de novo protein synthesis. Roles for CaMKs in modulating gene transcription are well established, but their involvement in mRNA translation is evolving. Here we report that activity-dependent translational initiation in cultured rat hippocampal neurons is enhanced by CaMKI-mediated phosphorylation of Ser1156 in eukaryotic initiation factor eIF4GII (4GII). Treatment with bicuculline or gabazine to enhance neuronal activity promotes recruitment of wild-type 4GII, but not the 4GII S1156A mutant or 4GI, to the heterotrimeric eIF4F (4F) complex that assembles at the 5' cap structure (m7GTP) of mRNA to initiate ribosomal scanning. Recruitment of 4GII to 4F is suppressed by pharmacological inhibition (STO-609) of CaM kinase kinase, the upstream activator of CaMKI. Post hoc in vitro CaMKI phosphorylation assays confirm that activity promotes phosphorylation of S1156 in transfected 4GII in neurons. Changes in cap-dependent and cap-independent translation were assessed using a bicistronic luciferase reporter transfected into neurons. Activity upregulates cap-dependent translation, and RNAi knockdown of CaMKIß and γ isoforms, but not α or δ, led to its attenuation as did blockade of NMDA receptors. Furthermore, RNAi knockdown of 4GII attenuates cap-dependent translation and reduces density of dendritic filopodia and spine formation without effect on dendritic arborization. Together, our results provide a mechanistic link between Ca²âº influx due to neuronal activity and regulation of cap-dependent RNA translation via CaMKI activation and selective recruitment of phosphorylated 4GII to the 4F complex, which may function to regulate activity-dependent changes in spine density.
Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bicuculina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Quelantes/farmacologia , Dendritos/genética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião de Mamíferos , Fator de Iniciação Eucariótico 4G/genética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mutação/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Isótopos de Fósforo/farmacocinética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Piridazinas/farmacologia , Capuzes de RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/genética , Bloqueadores dos Canais de Sódio/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Tetrodotoxina/farmacologia , Transfecção/métodosRESUMO
ROS1 fusion proteins resulting from chromosomal rearrangements of the ROS1 gene are targetable oncogenic drivers in diverse cancers. Acquired resistance to targeted inhibitors curtails clinical benefit and response durability. Entrectinib, a NTRK/ROS1/ALK targeted tyrosine kinase inhibitor (TKI), was approved for the treatment of ROS1 fusion-positive non-small cell lung cancer (NSCLC) in 2019. In addition, lorlatinib and repotrectinib are actively being explored in the setting of treatment-naïve or crizotinib-resistant ROS1 fusion driven NSCLC. Here, we employed an unbiased forward mutagenesis screen in Ba/F3 CD74-ROS1 and EZR-ROS1 cells to identify resistance liabilities to entrectinib, lorlatinib, and repotrectinib. ROS1F2004C emerged as a recurrent entrectinib resistant mutation and ROS1G2032R was discovered in entrectinib and lorlatinib-resistant clones. Cell-based and modeling data show that entrectinib is a dual type I/II mode inhibitor, and thus liable to both types of resistant mutations. Comprehensive profiling of all clinically relevant kinase domain mutations showed that ROS1L2086F is broadly resistant to all type I inhibitors, but remains sensitive to type II inhibitors. ROS1F2004C/I/V are resistant to type I inhibitors, entrectinib and crizotinib, and type II inhibitor, cabozantinib, but retain sensitivity to the type I macrocyclic inhibitors. Development of new, more selective type II ROS1 inhibitor(s) or potentially cycling type I and type II inhibitors may be one way to expand durability of ROS1-targeted agents.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Simulação de Acoplamento Molecular/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , TransfecçãoRESUMO
Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca(2+)-signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca(2+)/calmodulin kinase kinase (CaMKK) and its downstream target Ca(2+)/calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the gamma-isoform of CaMKI as shRNA to CaMKIgamma, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKIgamma, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKIgamma as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKIgamma and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKIgamma, which subsequently promote axon formation.
Assuntos
Axônios/fisiologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Membrana Celular/metabolismo , Células Cultivadas , Microdomínios da Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neuritos/fisiologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Canais de Cátion TRPC/genéticaRESUMO
We have investigated the trafficking of two endogenous axonal membrane proteins, VAMP2 and NgCAM, in order to elucidate the cellular events that underlie their polarization. We found that VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2, selective delivery in the case of NgCAM.
Assuntos
Axônios/metabolismo , Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Axônios/ultraestrutura , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Endocitose/fisiologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas R-SNARE , RatosRESUMO
The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.
Assuntos
Espinhas Dendríticas/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Transdução de Sinais/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Ativadoras de GTPase/genética , Homeostase/genética , Homeostase/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Plasticidade Neuronal/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/deficiência , Família de Proteínas da Síndrome de Wiskott-Aldrich/genéticaRESUMO
Neurons develop two types of processes, axons and dendrites, whose growth must be independently controlled. Recent research has identified the small guanosine triphosphatase Rit as a differential regulator of neurite growth. Activation of Rit enhances axonal growth, whereas inhibition of Rit promotes dendritic growth. These results imply that the reciprocal regulation of a single molecule in the same cell can achieve simultaneous regulation of axonal and dendritic growth.
Assuntos
Proteínas ras/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/enzimologia , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/farmacologia , Dendritos/efeitos dos fármacos , Dendritos/enzimologia , Drosophila melanogaster , Ratos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Calcium and calmodulin (CaM) are important signaling molecules that regulate axonal or dendritic extension and branching. The Ca2+-dependent stimulation of neurite elongation has generally been assumed to be mediated by CaM-kinase II (CaMKII), although other members of the CaMK family are highly expressed in developing neurons. We have examined this assumption using a combination of dominant-negative CaMKs (dnCaMKs) and other specific CaMK inhibitors. Here we report that inhibition of cytosolic CaMKI, but not CaMKII or nuclear CaMKIV, dramatically decreases axonal outgrowth and branching in cultured neonatal hippocampal and postnatal cerebellar granule neurons. CaMKI is found throughout the cell cytosol, including the growth cone. Growth cones of neurons expressing dnCaMI or dnCaMKK, the upstream activator of CaMKI, exhibit collapsed morphology with a prominent reduction in lamellipodia. Live-cell imaging confirms that these morphological changes are associated with a dramatic decrease in growth cone motility. Treatment of neurons with 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), an inhibitor of CaMKK, causes a similar change in morphology and reduction in growth cone motility, and this inhibition can be rescued by transfection with an STO-609-insensitive mutant of CaMKK or by transfection with constitutively active CaMKI. These results identify CaMKI as a positive transducer of growth cone motility and axon outgrowth and provide a new physiological role for the CaMKK-CaMKI pathway.
Assuntos
Axônios/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cones de Crescimento/fisiologia , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Axônios/enzimologia , Benzimidazóis/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/enzimologia , Isoquinolinas/farmacologia , Naftalimidas , Inibidores de Proteínas Quinases , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Dendritic spines contain a specialized cytoskeleton composed of dynamic actin filaments capable of producing rapid changes in their motility and morphology. Transient changes in Ca2+ levels in the spine cytoplasm have been associated with the modulation of these effects in a variety of ways. To characterize the contribution of Ca2+ fluxes originating through different pathways to these phenomena, we used time-lapse imaging of cultured hippocampal neurons expressing GFP-actin to follow the influence of postsynaptic neurotransmitter receptors, voltage-activated Ca2+ channels and release from internal Ca2+ stores on spine actin dynamics. Stimulation of AMPA receptors produced a rapid blockade of actin-dependent spine motility that was immediately reversible when AMPA was removed. Stimulation of NMDA receptors also blocked spine motility but in this case suppression of actin dynamics was delayed by up to 30 min depending on NMDA concentration and motility was never seen to recover when NMDA was removed. These effects could be mimicked by depolarizing neurons under appropriate circumstances demonstrating the involvement of voltage-activated Ca2+ channels in AMPA receptor-mediated effects and the receptor associated Ca2+ channel in the effects of NMDA. Caffeine, an agent that releases Ca2+ from internal stores, had no immediate effect on spine actin, a result compatible with the lack of caffeine-releasable Ca2+ in cultured hippocampal neurons under resting conditions. Blocking internal store function by thapsigargin led to a delayed suppression of spine actin dynamics that was dependent on extracellular Ca2+. Together these results indicate the common involvement of changes in Ca2+ levels in modulating actin-dependent effects on dendritic spine motility and morphology through several modes of electrophysiological activation.
Assuntos
Actinas/fisiologia , Cálcio/farmacologia , Dendritos/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/fisiologia , Animais , Cafeína/farmacologia , Células Cultivadas , Dendritos/efeitos dos fármacos , N-Metilaspartato/farmacologia , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization. Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Neurônios/ultraestrutura , Vesículas Secretórias/ultraestrutura , Animais , HumanosRESUMO
Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development, including the development of polarity, axonal and dendritic growth, and synapse formation. The cultures are quite homogeneous--â¼90% of the cells are pyramidal neurons--and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. This article describes the cultures and the key features of the system used to image them. It also includes suggestions on labeling cells with GFP-tagged proteins. It concludes with a discussion of the advantages and disadvantages of this culture system.
Assuntos
Técnicas Citológicas/métodos , Hipocampo/citologia , Neurônios/fisiologia , Animais , Células Cultivadas , Camundongos , Microscopia de Fluorescência/métodos , Ratos , Imagem com Lapso de Tempo/métodosRESUMO
Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Studying developmental processes that occur over many hours or days--for example, dendritic branching--involves capturing images of a cell at regular intervals without compromising cell survival. This approach is also useful for studying events of short duration that occur asynchronously across the cell population. For such studies, it is highly desirable to use a computer-controlled microscope with an automated stage, to follow multiple cells at different locations in the culture, moving sequentially from one to the next and capturing an image at each location. A method to correct for focal drift is also required. For long-term imaging, we culture neurons in a medium without phenol red, which is thought to give rise to toxic substances following exposure to light. To label cells with GFP-tagged proteins for long-term imaging, we usually use nucleofection (rather than lipid-mediated transfection); this gives a high transfection efficiency, which makes it easier to find the right cell for imaging. Our protocol for long-term imaging is given here, along with appropriate methods to express GFP-tagged proteins. Examples illustrate how the protocol can be used to image cytoskeletal dynamics during axon specification and to study kinesin motor dynamics in stage 2 neurons (when minor neurites extend).
Assuntos
Técnicas Citológicas/métodos , Hipocampo/citologia , Microscopia de Fluorescência/métodos , Neurônios/fisiologia , Imagem com Lapso de Tempo/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Ratos , Coloração e Rotulagem/métodosRESUMO
Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Because the cultures are essentially two dimensional, there is no need to acquire images at multiple focal planes. For capturing rapid subcellular events at high resolution, as described here, one must maximize weak signals and reduce background fluorescence. Thus, these methods differ in several respects from those used for time-lapse imaging. Lipofectamine-mediated transfection yields a higher level of expression than does transfection with a nucleofection device. Images are usually collected with a spinning-disk confocal microscope, which improves the signal-to-noise ratio. In addition, we use an imaging medium designed to minimize background fluorescence rather than to enhance long-term cell survival. It is also important to select cultures at an appropriate stage of development. In our hands, lipofectamine-based transfection works best on cells between 3 and 10 d after plating. GFP-based fluorescence can be observed as early as 4 h after adding the DNA/lipid complexes to the cells, but expression usually increases over the next â¼12 h and remains steady for days. The ratio of DNA to lipid is critical; to lower expression levels of the tagged construct, we use a combination of expression vector and empty plasmid, keeping the DNA amount constant. An example is included to illustrate the imaging of the microtubule-based vesicular transport of membrane proteins.
Assuntos
Técnicas Citológicas/métodos , Hipocampo/citologia , Neurônios/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Ratos , Coloração e Rotulagem/métodosRESUMO
Polarized transport by microtubule-based motors is critical for neuronal development and function. Selective translocation of the Kinesin-1 motor domain is the earliest known marker of axonal identity, occurring before morphological differentiation. Thus, Kinesin-1-mediated transport may contribute to axonal specification. We tested whether posttranslational modifications of tubulin influence the ability of Kinesin-1 motors to distinguish microtubule tracks during neuronal development. We detected no difference in microtubule stability between axons and minor neurites in polarized stage 3 hippocampal neurons. In contrast, microtubule modifications were enriched in a subset of neurites in unpolarized stage 2 cells and the developing axon in polarized stage 3 cells. This enrichment correlated with the selective accumulation of constitutively active Kinesin-1 motors. Increasing tubulin acetylation, without altering the levels of other tubulin modifications, did not alter the selectivity of Kinesin-1 accumulation in polarized cells. However, globally enhancing tubulin acetylation, detyrosination, and polyglutamylation by Taxol treatment or inhibition of glycogen synthase kinase 3beta decreased the selectivity of Kinesin-1 translocation and led to the formation of multiple axons. Although microtubule acetylation enhances the motility of Kinesin-1, the preferential translocation of Kinesin-1 on axonal microtubules in polarized neuronal cells is not determined by acetylation alone but is probably specified by a combination of tubulin modifications.
Assuntos
Cinesinas/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Polaridade Celular , Células Cultivadas , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/citologia , Cinesinas/genética , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Paclitaxel/farmacologia , Isoformas de Proteínas/genética , Transporte Proteico/fisiologia , Ratos , Transdução de Sinais/fisiologia , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologiaRESUMO
Epac1 (exchange protein directly activated by cyclic AMP [cAMP]) couples intracellular cAMP to the activation of Rap1, a Ras family GTPase that regulates cell adhesion, proliferation, and differentiation. Using mass spectrometry, we identified the small G protein Ran and Ran binding protein 2 (RanBP2) as potential binding partners of Epac1. Ran is a small G protein best known for its role in nuclear transport and can be found at the nuclear pore through its interaction with RanBP2. Here we demonstrate that Ran-GTP and Epac1 interact with each other in vivo and in vitro. This binding requires a previously uncharacterized Ras association (RA) domain in Epac1. Surprisingly, the interaction of Epac1 with Ran is necessary for the efficient activation of Rap1 by Epac1. We propose that Ran and RanBP2 anchor Epac1 to the nuclear pore, permitting cAMP signals to activate Rap1 at the nuclear envelope.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membrana Nuclear/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Sequência de Bases , AMP Cíclico/metabolismo , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Técnicas In Vitro , Modelos Biológicos , Poro Nuclear/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.
Assuntos
Técnicas de Cultura de Células/métodos , Hipocampo/citologia , Neurônios , Animais , Astrócitos , Feminino , Camundongos , Polilisina , Gravidez , RatosRESUMO
The adenomatous polyposis coli (APC) tumour suppressor protein is a component of the Wnt signalling pathway in which it plays a major role in controlling nuclear accumulation of beta-catenin and hence in the modulation of beta-catenin-regulated gene transcription. APC also associates with microtubules at the ends of cytoplasmic extensions in epithelial cells, a distribution that can be reproduced in COS cells ectopically expressing APC. To examine the effect of APC on microtubule properties, we monitored directly the behaviour of APC and of APC-decorated microtubules by time-lapse imaging of cytoplasmic extensions in live COS cells expressing APC tagged with a green fluorescent protein. On the proximal part of microtubules, APC was visualised as particulate material moving unidirectionally towards the plus end of microtubules. The distal parts of microtubules were uniformly decorated by APC and were animated by a motile behaviour in the form of aperiodic bending. This behaviour is likely to be the consequence of compression forces acting on microtubules encountering obstacles while elongating. The majority of APC-decorated microtubules in transfected COS cells was sensitive to depolymerisation by nocodazole, but they contained detyrosinated and acetylated alpha-tubulin, suggesting a reduction in the rate of subunit exchange at their growing end. Taken together, these results demonstrate that microtubule domains uniformly decorated by APC display dynamic and motile properties that may be significant for the postulated role of APC in targeting microtubules to specialised membrane sites.