RESUMO
Dendritic spines are small actin-rich protrusions essential for the formation of functional circuits in the mammalian brain. During development, spines begin as dynamic filopodia-like protrusions that are then replaced by relatively stable spines containing an expanded head. Remodeling of the actin cytoskeleton plays a key role in the formation and modification of spine morphology, however many of the underlying regulatory mechanisms remain unclear. Capping protein (CP) is a major actin regulating protein that caps the barbed ends of actin filaments, and promotes the formation of dense branched actin networks. Knockdown of CP impairs the formation of mature spines, leading to an increase in the number of filopodia-like protrusions and defects in synaptic transmission. Here, we show that CP promotes the stabilization of dendritic protrusions, leading to the formation of stable mature spines. However, the localization and function of CP in dendritic spines requires interactions with proteins containing a capping protein interaction (CPI) motif. We found that the CPI motif-containing protein Twinfilin-1 (Twf1) also localizes to spines where it plays a role in CP spine enrichment. The knockdown of Twf1 leads to an increase in the density of filopodia-like protrusions and a decrease in the stability of dendritic protrusions, similar to CP knockdown. Finally, we show that CP directly interacts with Shank and regulates its spine accumulation. These results suggest that spatiotemporal regulation of CP in spines not only controls the actin dynamics underlying the formation of stable postsynaptic spine structures, but also plays an important role in the assembly of the postsynaptic apparatus underlying synaptic function.
RESUMO
The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.
Assuntos
Axônios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Feminino , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/fisiologia , Masculino , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Cultura Primária de Células , Pseudópodes/fisiologia , RatosRESUMO
Oligodendrocytes (OLs) insulate axonal fibers for fast conduction of nerve impulses by wrapping axons of the CNS with compact myelin membranes. Differentiating OLs undergo drastic chances in cell morphology. Bipolar oligodendroglial precursor cells (OPCs) transform into highly ramified multipolar OLs, which then expand myelin membranes that enwrap axons. While significant progress has been made in understanding the molecular and genetic mechanisms underlying CNS myelination and its disruption in diseases, the cellular mechanisms that regulate OL differentiation are not fully understood. Here, we report that developing rat OLs in culture exhibit spontaneous Ca2+ local transients (sCaLTs) in their process arbors in the absence of neurons. Importantly, we find that the frequency of sCaLTs markedly increases as OLs undergo extensive process outgrowth and branching. We further show that sCaLTs are primarily generated through a combination of Ca2+ influx through store-operated Ca2+ entry (SOCE) and Ca2+ release from internal Ca2+ stores. Inhibition of sCaLTs impairs the elaboration and branching of OL processes, as well as substantially reduces the formation of large myelin sheets in culture. Together, our findings identify an important role for spontaneous local Ca2+ signaling in OL development.
Assuntos
Cálcio , Oligodendroglia , Animais , Diferenciação Celular , Bainha de Mielina , Neurogênese , RatosRESUMO
Tissue morphogenesis requires dynamic intercellular contacts that are subsequently stabilized as tissues mature. The mechanisms governing these competing adhesive properties are not fully understood. Using gain- and loss-of-function approaches, we tested the role of p120-catenin (p120) and VE-cadherin (VE-cad) endocytosis in vascular development using mouse mutants that exhibit increased (VE-cadGGG/GGG) or decreased (VE-cadDEE/DEE) internalization. VE-cadGGG/GGG mutant mice exhibited reduced VE-cad-p120 binding, reduced VE-cad levels, microvascular hemorrhaging, and decreased survival. By contrast, VE-cadDEE/DEE mutants exhibited normal vascular permeability but displayed microvascular patterning defects. Interestingly, VE-cadDEE/DEE mutant mice did not require endothelial p120, demonstrating that p120 is dispensable in the context of a stabilized cadherin. In vitro, VE-cadDEE mutant cells displayed defects in polarization and cell migration that were rescued by uncoupling VE-cadDEE from actin. These results indicate that cadherin endocytosis coordinates cell polarity and migration cues through actin remodeling. Collectively, our results indicate that regulated cadherin endocytosis is essential for both dynamic cell movements and establishment of stable tissue architecture.
Assuntos
Antígenos CD/genética , Vasos Sanguíneos/crescimento & desenvolvimento , Caderinas/genética , Cateninas/genética , Desenvolvimento Embrionário/genética , Endotélio Vascular/crescimento & desenvolvimento , Actinas/genética , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Vasos Sanguíneos/metabolismo , Padronização Corporal/genética , Movimento Celular/genética , Polaridade Celular/genética , Embrião de Mamíferos , Endocitose/genética , Endotélio Vascular/metabolismo , Camundongos , Ligação Proteica/genética , delta CateninaRESUMO
Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.
Assuntos
Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Domínios de Homologia de src/genética , Domínios de Homologia de src/fisiologia , Actinas/metabolismo , Animais , Dendritos/ultraestrutura , Espinhas Dendríticas/fisiologia , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Rede Nervosa/citologia , Rede Nervosa/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , RatosRESUMO
Dendritic spines are small postsynaptic compartments of excitatory synapses in the vertebrate brain that are modified during learning, aging, and neurological disorders. The formation and modification of dendritic spines depend on rapid assembly and dynamic remodeling of the actin cytoskeleton in this highly compartmentalized space, but the precise mechanisms remain to be fully elucidated. In this study, we report that spatiotemporal enrichment of actin monomers (G-actin) in dendritic spines regulates spine development and plasticity. We first show that dendritic spines contain a locally enriched pool of G-actin that can be regulated by synaptic activity. We further find that this G-actin pool functions in spine development and its modification during synaptic plasticity. Mechanistically, the relatively immobile G-actin pool in spines depends on the phosphoinositide PI(3,4,5)P3 and involves the actin monomer-binding protein profilin. Together, our results have revealed a novel mechanism by which dynamic enrichment of G-actin in spines regulates the actin remodeling underlying synapse development and plasticity.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal , Fosfatos de Fosfatidilinositol/metabolismo , Sistemas do Segundo Mensageiro , Sinapses/metabolismo , Transmissão Sináptica , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Hipocampo/citologia , Microscopia de Fluorescência , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Profilinas/genética , Profilinas/metabolismo , Interferência de RNA , Ratos , Fatores de Tempo , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
Synapses are the basic unit of neuronal communication and their disruption is associated with many neurological disorders. Significant progress has been made towards understanding the molecular and genetic regulation of synapse formation, modulation, and dysfunction, but the underlying cellular mechanisms remain incomplete. The actin cytoskeleton not only provides the structural foundation for synapses, but also regulates a diverse array of cellular activities underlying synaptic function. Here we will discuss the regulation of the actin cytoskeleton in dendritic spines, the postsynaptic compartment of excitatory synapses. We will focus on a select number of actin regulatory processes, highlighting recent advances, the complexity of crosstalk between different pathways, and the challenges of understanding their precise impact on the structure and function of synapses.
Assuntos
Citoesqueleto de Actina/metabolismo , Espinhas Dendríticas/fisiologia , Neurogênese/fisiologia , Humanos , Plasticidade Neuronal/fisiologia , SinapsesRESUMO
The selective RNA-binding protein Quaking I (QKI) has previously been implicated in RNA localization and stabilization, alternative splicing, cell proliferation, and differentiation. The spontaneously-occurring quakingviable (qkv) mutant mouse exhibits a sharply attenuated level of QKI in myelin-producing cells, including oligodendrocytes (OL) because of the loss of an OL-specific promoter. The disruption of QKI in OLs results in severe hypomyelination of the central nervous system, but the underlying cellular mechanisms remain to be fully elucidated. In this study, we used the qkv mutant mouse as a model to study myelination defects in the cerebellum. We found that oligodendroglial development and myelination are adversely affected in the cerebellum of qkv mice. Specifically, we identified an increase in the total number of oligodendroglial precursor cells in qkv cerebella, a substantial portion of which migrated into the grey matter. Furthermore, these mislocalized oligodendroglial precursor cells retained their migratory morphology late into development. Interestingly, a number of these presumptive oligodendrocyte precursors were found at the Purkinje cell layer in qkv cerebella, resembling Bergman glia. These findings indicate that QKI is involved in multiple aspects of oligodendroglial development. QKI disruption can impact the cell fate of oligodendrocyte precursor cells, their migration and differentiation, and ultimately myelination in the cerebellum. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 972-982, 2016.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Doenças Cerebelares/metabolismo , Cerebelo/fisiologia , Doenças Desmielinizantes/metabolismo , Oligodendroglia/fisiologia , Proteínas de Ligação a RNA/fisiologia , Animais , Cerebelo/crescimento & desenvolvimento , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Quaking , Camundongos Transgênicos , Células de Purkinje/fisiologia , Proteínas de Ligação a RNA/genéticaRESUMO
Activity-dependent dendritic development represents a crucial step in brain development, but its underlying mechanisms remain to be fully elucidated. Here we report that glycogen synthase kinase 3ß (GSK3ß) regulates dendritic development in an activity-dependent manner. We find that GSK3ß in somatodendritic compartments of hippocampal neurons becomes highly phosphorylated at serine-9 upon synaptogenesis. This phosphorylation-dependent GSK3ß inhibition is mediated by neurotrophin signalling and is required for dendritic growth and arbourization. Elevation of GSK3ß activity leads to marked shrinkage of dendrites, whereas its inhibition enhances dendritic growth. We further show that these effects are mediated by GSK3ß regulation of surface GABAA receptor levels via the scaffold protein gephyrin. GSK3ß activation leads to gephyrin phosphorylation to reduce surface GABAA receptor clusters, resulting in neuronal hyperexcitability that causes dendrite shrinkage. These findings thus identify GSK3ß as a key player in activity-dependent regulation of dendritic development by targeting the excitatory-inhibitory balance of the neuron.
Assuntos
Dendritos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Hipocampo/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dendritos/ultraestrutura , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/citologia , Hipocampo/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disabilities. BRAG1 is a guanine nucleotide exchange factor for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif; however, the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors, depresses AMPA receptor (AMPA-R)-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic XLID.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Região CA1 Hipocampal/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Glicoproteínas de Membrana/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores de AMPA/metabolismo , Sulfotransferases/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Motivos de Aminoácidos/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Calmodulina/metabolismo , Quelantes/farmacologia , Proteína 4 Homóloga a Disks-Large , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ionomicina/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Glicoproteínas de Membrana/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/fisiologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Ratos , Receptores de AMPA/genética , Sulfotransferases/genética , Transfecção , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)-IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP-dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.
Assuntos
Citoplasma/metabolismo , Dineínas/metabolismo , Endossomos/enzimologia , Neurônios/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Citoplasma/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Cinética , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Células PC12 , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Receptor trkA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Small GTPases of the Arf family are best known for their role in vesicular transport, wherein they nucleate the assembly of coat proteins at sites of carrier vesicle formation. However, accumulating evidence indicates that the Arfs are also important regulators of actin cytoskeleton dynamics and are involved in a variety of actin-based processes, including cell adhesion, migration and neurite outgrowth. The mechanisms of this regulation are remarkably diverse, ranging from the integration of vesicular transport with cytoskeleton assembly to the direct regulation of Rho-family GTPase function. Here, we review recent progress in our understanding of how Arfs and their interacting proteins function to integrate membrane and cytoskeletal dynamics.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Regulação Enzimológica da Expressão Gênica , Animais , Transporte Biológico , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Humanos , Modelos Biológicos , Fosfatidilinositóis/química , Transporte Proteico , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Cytoplasmic dynein 1 is a multi-subunit motor protein responsible for microtubule minus end-directed transport in axons. The cytoplasmic dynein intermediate chain subunit has a scaffold-like role in the dynein complex; it directly binds to four of the other five subunits, the heavy chain and the three light chains. The intermediate chain also binds the p150 subunit of dynactin, a protein that is essential for many dynein functions. We reexamined the generation of rat cytoplasmic dynein intermediate chain isoforms by the alternative splicing of the two genes that encode this subunit and identified an additional splicing site in intermediate chain gene 1. We reinvestigated the expression of the intermediate chain 1 isoforms in cultured cells and tissues. The Loa mouse, which is homozygote lethal, contains a missense mutation in the region of the cytoplasmic dynein heavy chain gene that binds the intermediate chain. Protein binding studies showed that all six intermediate chains were able to bind to the mutated heavy chain. GFP-tagged intermediate chains were constructed and PC12 cell lines with stable expression of the fusion proteins were established. Live cell imaging and comparative immunocytochemical analyses show that dynein is enriched in the actin rich region of growth cones.
Assuntos
Transporte Axonal/fisiologia , Citoplasma/metabolismo , Dineínas/metabolismo , Subunidades Proteicas/metabolismo , Animais , Diferenciação Celular/fisiologia , Citoplasma/efeitos dos fármacos , Diagnóstico por Imagem/métodos , Dineínas/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/fisiologia , Células PC12 , Ligação Proteica/fisiologia , Isoformas de Proteínas/metabolismo , RatosRESUMO
Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.