Agradecemos a todos os membros do laboratório AI por seus comentários e sugestões úteis. Gostaríamos de agradecer ao Prof. Mark Hipp (Instituto Max Planck de Bioquímica) por nos fornecer o vetor shuttle-GFP. Este trabalho foi apoiado por bolsas da Fundação de Ciência Israelense (ISF # 124/14), a Fundação de Ciência Binacional (BSF # 2013325), FP7 Marie Curie Carreira Integração Grant (CIG # 333794), eo Instituto Nacional de Psicobiologia em Israel NIPI # b133-14 / 15).

1. Preparação da Linha Celular <ol><li> Descongelar aproximadamente 1.000.000 células de medula espinhal de neuroblastoma de rato (células NSC-34) que foram armazenadas num recipiente de azoto líquido. Utilize uma incubadora de água a 37 ° C até as células serem completamente descongeladas. </li><li> Adicionar meio fresco e quente (meio DMEM contendo 10% de soro fetal bovino (FBS), 1% de L-glutamina e 1% de antibióticos pen-strep). </li><li> Centrifugar as células descongeladas (SL16R) a 500 xg durante 5 min, formando uma pastilha celular. Descarte o meio e adicione 1 mL de novo meio para a ressuspensão celular. </li><li> Colocar as células ressuspensas num balão de 15 mL e adicionar 5 mL adicionais de meio. Incubar as células durante a noite numa incubadora estéril com 5% de CO2 a 37 ° C. </li><li> Em paralelo, colocar 8 lamínulas não revestidas (tampa de vidro: 12 mm, 0,13-0,17 mm de espessura) em um prato de cultura de 60 mm e esterilizar usando 70% etanol e exposição a 30 minutos de luz UV até que esteja seco. Adicionar meio a cada prato e descartar para lavar o etanol residual.</li><li> Adicionar tampão de tripsina (2,5 g de tripsina e 0,2 g de EDTA em 1 L de Solução de Sal Equilibrada de Hanks) às células depois de atingirem aproximadamente 90% de confluência (cerca de 6 milhões de células por frasco) para permitir que as células aderentes se dissociem. NOTA: As células NSC-34 são sensíveis e requerem apenas 15 s de incubação com tampão de tripsina e 1 minuto adicional de incubação sem tripsina antes da dissociação utilizando o meio. </li><li> Plantar as células nas placas de cultura de 60 mm que já contêm as lamelas com 3 mL de meio, aproximadamente 350.000 por placa. Deixá-los na incubadora por 24 h para permitir a fixação de células para as lamelas. </li></ol> 2. Transfecção celular NOTA: Após aproximadamente 24 h, as células NSC-34 atingem 40% de confluência (aproximadamente 800.000 células) e estarão prontas para transfecção. <ol><li> Preparar meio DMEM puro (300 μL de meio DMEM para cada recipiente de cultura de 60 mm) em uma cuba de microcentrífuga estérilEs. </li><li> Adicionar 2 μg de cada plasmídeo necessário para transfecção a cada tubo de microcentrífuga contendo DMEM. NOTA: O plasmídeo shuttle-GFP é obrigatório em todas as transfecções celulares. </li><li> Adicionar o reagente de transfecção a todos os tubos de microcentrífuga que contêm os seus plasmídeos necessários. Aqui, adicionar 4 μL do reagente de transfecção comercial para cada 2 μg de ADN. </li><li> Vortex os tubos de microcentrífuga contendo DMEM, plasmídeos e o reagente de transfecção e incuba-os à temperatura ambiente durante 25 min. </li><li> Adicionar cada tubo de microcentrífuga a um prato de cultura e agitar suavemente o prato para formar uma solução homogénea. Coloque o prato de volta na incubadora por 48 h. NOTA: Uma vez que a vaivï¿½-GFP deve ser expressa em todas as cï¿½ulas, a eficiï¿½cia de transfecï¿½o de shuttle-GFP pode ser parcialmente examinada e testada previamente utilizando microscopia de fluorescï¿½cia apï¿½ transfecï¿½o durante a noite. Um exame completo de proteínas expressas não fluorescentes pode ser conduzido utilizando uma análise de imunobloté. </li></ol> 3. Microscopia <ol><li> Anexar as lamelas contendo células NSC-34 transfectadas para um suporte de lamela e lave suavemente com solução salina tamponada com fosfato (PBS) para remover as células mortas destacadas. </li><li> Identificar um adesivo celular adequado utilizando um filtro fluorescente verde com um microscópio invertido. NOTA: Um adesivo de célula adequado é definido como um campo de visão de microscópio mostrando um grande número de células (20-30 células num campo) e em que os núcleos estão claramente definidos nas células. Os núcleos celulares dentro do remendo de células são primeiro identificados por olho e são marcados manualmente pelo software de imagem. </li><li> Adicionar 200 μL de um tampão de leptomicina B (LMB) inibidor da exportina-1 contendo 10 ng / mL de LMB em PBS aos lamínulas por gotejamento do tampão nos lamínulas sob o microscópio. NOTA: Este ponto no tempo é definido como intervalo zero. </li><li> Quantificar a intensidade fluorescente dos núcleos marcados em intervalos de 3 s por cerca de 400-500 s. </li><li&gt; Teste entre 3 e 5 lamínulas para cada grupo de transfecção em cada experiência individual. </li></ol> 4. Análise NOTA: Uma vez que diferentes células expressam diferentes níveis de transferência de GFP fluorescente, é importante normalizar a intensidade fluorescente nuclear de cada célula para a sua própria intensidade fluorescente inicial. <ol><li> Normalize a intensidade fluorescente dos núcleos marcados dividindo o intervalo fluorescente de 3 de cada núcleo com a média dos seis intervalos iniciais desse núcleo. </li><li> Após a normalização de cada núcleo, produzir uma curva de intensidade fluorescente representando a mudança na fluorescência dos núcleos em função do tempo para cada remendo de célula. Para fazer isto, calcule a média de todas as pilhas em cada remendo da pilha (que consiste em aproximadamente 20-30 pilhas em cada remendo). NOTA: A curva de intensidade de fluorescência dos núcleos se assemelha a uma curva de saturação de Michaelis-Menten, que é melhor observadaMe períodos. Isto é devido a uma diminuição nas quantidades citosólicas de shuttle-GFP com o tempo devido ao transporte nuclear. Esta diminuição diminui estatisticamente a probabilidade de uma vaivém-GFP se transportar para o núcleo, atingindo assim a saturação na fluorescência nuclear. A inclinação derivada da taxa inicial linear da curva produz a taxa de entrada de Vmax no núcleo. </li><li> Analise as inclinações de Vmax de cada remendo de célula, derivadas de três a cinco experimentos independentes, usando o software gráfico para produzir uma inclinação que representa cada grupo experimental. Realize a quantificação usando análise estatística ANOVA unidirecional. </li></ol> 5. Validação de Expressão de Proteína <ol><li> Colocar recipientes de cultura de 60 mm contendo as células residuais (não utilizadas na experiência anterior) de cada grupo experimental em gelo. </li><li> Eliminar o meio e lavar as células duas vezes com PBS para diluir e lavar o meio restante. </li><li> Tratar as células com 200-400ML de tampão de lise contendo PBS, 0,5% de Triton e comprimido de cocktail de inibidor de protease (PI). Raspe usando um raspador de células. </li><li> Recolher as células em tampão de lise num tubo de microcentrífuga e homogeneizar com um homogeneizador a 4000 rpm durante 1 min sob gelo. </li><li> Centrifugar as células homogeneizadas a 2.500 xg durante 10 min. Recolher o sobrenadante, quantificar a concentração de proteína utilizando um ensaio de Bradford e armazenar até à utilização a -20 ° C. </li><li> Recolher entre 30 e 70 μg de proteína total (dependendo da proteína) e carregá-lo em um gel de SDS-PAGE para testar a expressão da proteína usando um ensaio de imunoblot. </li></ol>

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Este protocolo demonstra um ensaio altamente sensível e quantitativo novo para avaliar mudanças minuciosas no transporte nucleocytoplasmic. Utilizando este sistema, é possível observar e medir o transporte nucleocitoplasmático e sua disfunção em tempo real, não só grandes defeitos que resultam em mudanças dramáticas na distribuição de proteínas. Este ensaio não só tem uma vantagem de sensibilidade, mas também requer pouca preparação, é barato e é um ensaio muito fácil e de baixo engajamento. <p...

O complexo de poros nucleares (NPC) controla a importação e exportação de grandes moléculas dentro e fora do núcleo da célula. Em contraste com pequenas moléculas, que podem entrar no núcleo sem a regulação 1 , o transporte de moléculas maiores é fortemente controlado pelo NPC. Essas grandes moléculas, como proteínas e RNA, associam-se a fatores de transporte, incluindo importações e exportações, para serem importadas e exportadas do núcleo 2 . Para serem importados, as proteínas devem conter um pequeno motivo peptídico, geralmente chamado de sinal de localização nuclear (NLS), que é limitado por importinas 2 . Estas sequências de aminoácidos actuam como um marcador e são diversas na composição 3 , 4 . As proteínas podem ser exportadas do núcleo para o citoplasma devido à sua associação com aS, que ligam uma seqüência de sinal, geralmente chamada de sinal de exportação nuclear (NES) 2 . Ambos importins e exportins são capazes de transportar sua carga devido à regulação da pequena proteína nuclear relacionada Ras GTPase (Ran) 2 . Ran demonstrou-se que existem em diferentes estados conformacionais dependendo se está ligado a GTP ou GDP. Quando ligado a GTP, Ran pode vincular a importins ou exportins. Após a vinculação ao RanGTP, importins liberar sua carga, enquanto exportins deve vincular RanGTP para formar um complexo com sua carga de exportação. O estado de ligação de nucleótidos dominante de Ran depende da sua localização no núcleo (RanGTP) ou do citoplasma (RanGDP). Recentemente, vários estudos têm demonstrado uma ligação entre deficiências na via nucleocitoplasmática e tanto a esclerose lateral amiotrófica (ELA) quanto a demência frontotemporal (FTD) 5 , 6 </sup7 , 8 , 9 , 10 , 11 , 12 . A ELA é uma doença neurodegenerativa progressiva e fatal que afeta os neurônios motores superiores e inferiores (MNs) 13 e resulta em paralisia e, em última instância, na morte, em média de 2 a 5 anos. A maioria dos casos de ELA é classificada como esporádica (SALS), sem qualquer ligação genética aparente, mas 10% são herdados de forma dominante (ALS familiar, FALS). Recentemente, foram identificadas as expansï¿½s de repetiï¿½o de hexanucleï¿½idos (HREs) no gene 72 de leitura aberta do cromossoma 9 (C9orf72) 14 , 15 como uma causa genï¿½ica de ALS e FTD. Esses mutantes respondem por 30-40% dos casos FALS 16 , e diferentes estudos alegaramQue causam toxicidade por afetar o transporte nucleocitoplasmático 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . Estes estudos mostram principalmente os resultados finais e manifestações de HREs sobre o transporte nucleocytoplasmic, mas eles não demonstram disrupção nucleocytoplasmic em tempo real. Como resultado, apenas a deficiência grave de transporte nucleocitoplasmático foi avaliada 11 , 17 , 18 . Este protocolo descreve um novo eSensível para avaliar e quantificar a disfunção nucleocitoplasmática de transporte em tempo real. A taxa de importação de uma proteína NLS-NES-GFP (shuttle-GFP) pode ser quantificada em tempo real utilizando microscopia de fluorescência. Isto é feito utilizando um inibidor de exportação, tal como descrito anteriormente 18 , permitindo assim que a vaivém-GFP apenas entre no núcleo. Usando a microscopia fluorescente e um software capaz de quantificar alterações fluorescentes em tempo real, é possível quantificar mudanças graduais na intensidade de fluorescência da nave-GFP, localizada no núcleo. Como resultado, pode ser feita uma curva de saturação tipo Michaelis-Menten do eixo de fluorescência ao tempo, representando a quantidade de GFP nuclear de transporte em qualquer momento dado. Usando a taxa linear inicial das curvas, é possível gerar uma inclinação, que representa a taxa de entrada no núcleo antes da saturação de fluorescência. Para validar o ensaio, oForam utilizadas repetições de dipeptídeos C9orf72, poli (GR) e poli (PR), que foram previamente relatados como perturbadores do transporte nucleocitoplasmático, 5 , 7 , 8 , 10 , 12 . Utilizando este ensaio, observou-se uma diminuição de 50% na taxa de importação nuclear em comparação com o controlo. Usando este sistema, podem ser examinadas pequenas alterações no transporte nucleocitoplasmático. Além disso, a capacidade de diferentes factores para resgatar um defeito de transporte nucleocitoplasmático pode ser determinada.

<table><tbody><tr><td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td><td>tebu-bio</td><td>CLU140-A</td><td></td></tr><tr><td>DMEM 4.5 g/L L-glucose</td><td>Biological Industries</td><td>01-055-1A</td><td></td></tr><tr><td>FBS European Grade</td><td>Biological Industries</td><td>04-007-1A</td><td></td></tr><tr><td>SL 16R Centrifuge</td><td>Thermo Scientific</td><td>75004030</td><td></td></tr><tr><td>Thermo Forma Series ii Water Jacketed CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>3111</td><td></td></tr><tr><td>Cover glass 12 mm dia., 0.13-0.17 mm thick</td><td>Bar Naor LTD</td><td></td><td></td></tr><tr><td>TurboFect Transfection Reagent</td><td>Thermo Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/R0531&quot;&gt;R0531&lt;/a&gt;</td><td></td></tr><tr><td>Axiovert 100 inverted microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Imaging Workbench 2</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Leptomycin B</td><td>Sigma</td><td>L2913</td><td></td></tr><tr><td>PBS</td><td>Biological Industries</td><td>02-023-1A</td><td></td></tr><tr><td>Homogenizer motor/control/chuck/90 degree clamp</td><td>Glas-Col</td><td>099C K5424CE</td><td></td></tr><tr><td>MicroCL 17R Centrifuge, Refrigerated</td><td>Thermo Scientific</td><td>75002455</td><td></td></tr></tbody></table>

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

O transporte nucleocitoplasmático refere-se à importação e exportação de grandes moléculas do núcleo celular. Recentemente, vários estudos têm mostrado uma ligação entre a esclerose lateral amiotrófica (ELA) e deficiências na via nucleocitoplasmática. ALS é uma doença neurodegenerativa que afeta os neurônios motores e resultando em paralisia e, finalmente, na morte, em média 2-5 anos. A maioria dos casos de ELA são esporádicos, sem qualquer ligação genética aparente, mas 10% são herdados de forma dominante. Recentemente, as expansï¿½s de repetiï¿½o de hexanucleï¿½idos (HREs) no gene 72 de leitura aberta do cromossoma 9 (C9orf72) foram identificadas como uma causa genï¿½ica de ALS e demï¿½cia frontotemporal (FTD). É importante notar que diferentes grupos propuseram recentemente que estes mutantes afectam o transporte nucleocitoplasmático. Esses estudos mostram principalmente o resultado final e manifestações causadas por HREs sobre o transporte nucleocitoplasmático, mas eles não demonstram disfunção de transporte nuclear emtempo real. Como resultado, apenas a deficiência grave de transporte nucleocitoplasmático pode ser determinada, principalmente devido à alta sobreexpressão ou inserção de proteína exógena. Este protocolo descreve um ensaio novo e muito sensível para avaliar e quantificar a disfunção nucleocitoplasmática de transporte em tempo real. A taxa de importação de uma proteína NLS-NES-GFP (shuttle-GFP) pode ser quantificada em tempo real utilizando microscopia de fluorescência. Isto é realizado utilizando um inibidor de exportação, permitindo assim que a GFP de transporte apenas entre no núcleo. Para validar o ensaio, utilizaram-se as repetições de dipeptídeos traduzidos por HRE de C9orf72, poli (GR) e poli (PR), que anteriormente foram mostrados como perturbadores do transporte nucleocitoplasmático. Utilizando o ensaio descrito, observou-se uma diminuição de 50% na taxa de importação nuclear em comparação com o controlo. Usando este sistema, podem ser examinadas pequenas alterações no transporte nucleocitoplasmático e a capacidade de diferentes fatores para resgatar (mesmo parcialmente) um trato nucleocitoplasmáticoPode ser determinado.

Utilizando o procedimento aqui apresentado, as células NSC-34 do tipo neurónio motor foram transfectadas com uma proteína NLS-NES-GFP (shuttle-GFP). Esta proteína, que tem NLS e NES sinal seqüências ( Figura 1 ), pode shuttle entre o núcleo eo citoplasma. A GFP genï¿½ica pode entrar ou sair do nï¿½leo na ausï¿½cia de qualquer marca de sinal de localizaï¿½o apenas por difusï¿½ e, por conseguinte, ï¿½ distribuï¿½a uniformemente entre o nï¿½le...

Watch this Scientific Journal Video about Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells at JoVE.com

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Este protocolo descreve um método para medir sensivelmente a taxa de transporte nucleocitoplasmático dentro de células NSC-34 do tipo neurônio motor, quantificando a mudança em tempo real na importação nuclear de uma proteína NLS-NES-GFP.

Ensaio para Medir o Transporte Nucleocitoplasmático em Tempo Real dentro de Células NSC-34 do tipo Motor-Neuron

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

assay-to-measure-nucleocytoplasmic-transport-real-time-within-motor

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.6K visualizações.  Ben-Gurion University of the Negev.  Este protocolo descreve um método para medir sensivelmente a taxa de transporte nucleocitoplasmático dentro de células NSC-34 do tipo neurônio motor, quantificando a mudança em tempo real na importação nuclear de uma proteína NLS-NES-GFP. 

Vídeo: Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Imobilização de Caenorhabditis elegans , para analisar o transporte intracelular nos neurônios

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Biologia do Desenvolvimento

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Caracterizar a composição de motores moleculares sobre Movimentação axonal Carga Utilizando Análise &quot;Mapeamento Cargo&quot;

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Neurociência

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Única molécula-Imaging dos Transportes Nuclear

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Biologia

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Um ensaio de alto teor para monitoramento Leandro tráfico de Receptor

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imagem e Análise do Transporte de Neurofilamento em Nervo Tibial de Camundongo Excised

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Medição fluorescente em tempo real de funções sinápticas em modelos de esclerose lateral amiotrófica

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Expansão do Kit de Ferramentas para Imagens In Vivo do Transporte Axonal

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.