O autor agradece profundamente Dr. Asako Sugimoto (Universidade de Tohoku) para a discussão útil. wyIs251 foi um presente generoso do Dr. Kang Shen (Universidade de Stanford). mnIs17 foi fornecido pelo CGC, que é financiado pelo NIH escritório de programas de infra-estrutura de pesquisa (OD010440 P40). Este trabalho foi apoiado por grant JSPS KAKENHI #17 H 05010 e #16 H 06536 e Daiichi Sankyo foundation, Fundação da ciência do cérebro e Fundação Naito.

1. preparação da amostra <ol><li>gerar um transgénico c. elegans estirpe de interesse usando metodologias transgénicos 18. Como alternativa, obter cepas apropriadas de centro de genética o Caenorhabditis (CGC). Para visualizar o transporte axonal de precursores da vesícula sináptica, use a linha transgênicos wyIs251 [Pmig-13::gfp::rab-3] 7. Para observar o IFT, obter a linha transgênicos mnIs17 [osm-6::gfp] 19. Estas variedades são utilizadas neste protocolo. 
	Nota: Qualquer matriz extracromossômico, integração genômica ou mesmo GFP bater-em obras. Para reduzir os sinais de fundo, usar promotores específicos de célula, ao invés de promotores pan-neuronal. 
	Nota: Manutenção de vermes é descrita em outros protocolos de 20. Cepas foram mantidas nos relvados do alimentador Escherischia coli OP50 no crescimento de nematoides médio chapas (NGM) (agarose 1,7% (p/v), 50 mM de NaCl, 0,25% (p/v) de peptona, 1 mM CaCl 2, 5mg/mL colesterol, 25mm KH 2 PO 4, 1mm MgSO 4 pratos plásticos de 60 mm de diâmetro) a 20 ° C.</li>
	<li>Crescem vermes a 20 ° C em placas NGM para qualquer estágio do ciclo de vida dos vermes. 
	Nota: Pode-se observar tanto o transporte axonal e o IFT em qualquer fase. L4 atrasado para o jovem adulto é um bom controlo positivo porque várias publicações têm usado estes estágios e eventos visualizado tráfico de 14 , 21 , 22 , 23.</li>
</ol> 2. Preparação de 10% Agarose Nota: muitos fornecedores oferecem produtos similares, tais como pó de ágar-ágar, ágar e agarose. Use a electroforese da classe agarose (força do gel &#62; 1200 g/cm 2). Pó de ágar barato não funcionam porque o gel resultante não é forte o suficiente para imobilizar vermes.
<ol><li>Mix 0,4 g de agarose em 4 mL de água destilada (DW) de água em um tubo de vidro (1,5 x 10,5 cm). Coloque uma tampa do tubo de vidro para evitar a evaporação.</li>
	<li>Colocar o tubo de vidro em um bloco de calor de 95 ° C por 90 min. Além disso, preparar um outro tubo de vidro (1,5 cm x 10,5 cm) repleto de DW e mantê-lo em 95 ° C. colocar uma pipeta Pasteur na 95 ° C DW ( Figura 1 A). Será visto um monte de pequenas bolhas no tubo de agarose e a solução deve ser turva. Deixá-lo até que a solução torna-se claro. Em seguida, misture pipetando acima e para baixo até que a solução de agarose torna-se homogênea. 
	Nota: O micro-ondas não podem ser usadas para preparar a solução de agarose de 10% quando feita de DW e agarose. Pequenas bolhas causadas por microondas evitar derretimento de agarose. No entanto, solidificado 10% gel de agarose facilmente pode ser derretido novamente usando um microondas. Tubos de vidro contendo 10% de gel de agarose podem ser preparados antecipadamente e armazenados a 4 ° C durante pelo menos 6 meses. Se fazê-lo, derreter o estoque usando microondas quando necessário e começar a partir do passo 3 neste protocolo. 
	Nota: Agarose perde gradualmente a capacidade de solidificar-se incubada a 95 ° C por um longo tempo ou após repetidas de solidificação e fusão. Se isso acontecer, prepare-se nova 10% agarose.</li>
</ol> 3. Preparação de Agarose Pad <ol><li>usando a pipeta de Pasteur aquecida preparada no passo 2.2, colocar 2 gotas de agarose de 10% em um slide 76 x 22 mm.</li>
	<li>Rapidamente a tampa com um slide de 2 76 x 22 mm segundo antes da queda de agarose solidifica como mostrado na Figura 1 B e empurrar para baixo o segundo slide para achatar a solução de agarose. A espessura deve ser de 0,5-1 mm.</li>
	<li>Rapidamente lave a pipeta Pasteur por pipetagem 95 ° C DW acima e para baixo até que a solução de agarose é lavada para fora. Caso contrário, a pipeta vai ficar obstruída por gel de agarose. Deixar a pipeta de pé na 95 ° C DW.</li>
	<li>Espera por 1 min. A almofada de agarose forma e torna-se turva. Em seguida, deslize os slides separados pela mão ( Figura 1 C).</li>
</ol> 4. Montagem de vermes Nota: mesmo pequenas quantidades de movimento worm impedem boa observação. Levamisole tradicionalmente tem sido utilizada para prevenir o movimento worm a almofada de agarose 24 , 25. No entanto, Levamisole inibe receptores neuronais em c. elegans e, portanto, pode afetar os eventos de tráfico em neurônios 15. Usar o poliestireno microbeads descrito aqui é uma boa alternativa 17.
<ol><li>Sob um microscópio estéreo equipado com uma transiluminação espelho deslizável, transfer ~ 30 vermes transgênicos expressando os marcadores apropriados para um novo NGM placa sem bactérias usando um fio de platina escolher. 
	Nota: Escolher um fio de platina é feita de fio de platina e pipeta Pasteur (5 polegadas), e a ponta do fio de platina foi achatada. A preparação das picaretas fio de platina e manipulação de vermes com estes são descritos no Wormbook (http://www.wormbook.org).</li>
	<li>Deixar o prato para 1 min. bactérias são removidas automaticamente das superfícies de vermes como os vermes se move em gel de agarose NGM sem bactérias.</li>
	<li>Colocar um 0,5-1,0 µ l queda de 2,6% 100 nm de diâmetro poliestireno microbeads em água sobre a placa de ágar-ágar ( Figura 1 D). Transferi 10-20 vermes da placa NGM livre de bactérias para o microbeads. Gota e espalhar os vermes usando o fio de platina escolher para evitar a sobreposição de verme corpos ( Figura 1 E).</li>
	<li>Uma lamela 22 x 40 mm 2 ( Figura 1-F). Empurre suavemente para remover as bolhas de ar, mas evitar o esmagamento de vermes. A amostra está agora pronta para tratamento de imagens. 
	Nota: Porque sem anestesia é administrada, vermes são fixados apenas pela força friccional gerada pelo bloco de ágar e poliestireno microbeads lamela 17. A presença de bactérias do alimentador e/ou demais solução fará com que a força de fricção mais fraco. 
	Nota: Tamanhos diferentes de lamelas (por exemplo, 22 x 22mm 2, 18 x 18 mm 2) de trabalho. No entanto, as lamelas de maiores podem evitar imersão água ou óleo a partir da lente objetiva vazando para amostras durante a observação.</li>
</ol> 5. Observação Nota: parâmetros de imagem apropriados (poder do laser, ganho, binning, etc.) serão diferentes para cada sistema de microscópio e câmera. Aqui, um microscópio widefield equipado com um scanner confocal de disco giratório e uma câmera CCD digital é usado.
<ol><li>Regule a temperatura da fase de 20 ° C, ou ajustar a temperatura ambiente de 20 ° C.</li>
	<li>Colocar o slide de amostra no palco microscópio. Use x 100 (abertura numérica = 1.3 ou superior) da lente objetiva.</li>
	<li>Olha para as áreas indicadas na Figura 2 (para wyIs251 e transporte axonal) ou Figura 2 B (para mnIs17 e IFT) como controles positivos . Outras áreas podem ser analisadas como bem 22.</li>
	<li>Conjunto de parâmetros (CCD ganho = 200, Binning = 1 ou 2, a potência do laser em 488 nm = 1.0-1.3 mW). Executar a gravação de lapso de tempo em 4 frames/s para até 1 min. salvar arquivos como mutli-TIFF formato. 
	Nota: Se a GFP sinais de desaparecer e é difícil continuar a observar GFP::RAB-3 ou OSM-6::GFP por 30 s, a potência do laser é muito forte. Nesse caso, reduza a potência do laser. Por outro lado, se nenhum movimento vesicular é registrado, a potência do laser é muito fraca. Nesse caso, aumentar a potência do laser.</li>
	<li>Observar um worm diferente e repetir 5.3 e 5.4. As observações podem durar por até 20 min., mas cada verme deve ser registrada apenas uma vez para evitar os efeitos de pAcredita danos. 
	Nota: Worms foram observados pelo menos 20 min sem defeitos significativos de 27 , 7 , 28. Mais observação pode ser possível, mas refúgio &#39; t sido testado ainda. Um sinal claro da morte neuronal é a mudança da morfologia neuronal, tais como inchaço do axônio. Como sinais fracos do GFP podem ser vistos nos axônios e dendritos em wyIs251 e mnIs17, morfologia do axônio pode ser facilmente verificada olhando apenas axônios ou dendritos. Morfologia neuronal é mostrada na Figura 2.</li>
	<li>Gerar arquivos de filme.
	<ol><li>Aberto o TIFF multi arquivo usando software de Fiji. Vá para arquivo &#62; abrir selecione o arquivo TIFF multi salvou na etapa 5.4. 
		Nota: Fiji pode ser baixado em jttps://fiji.sc. Imagem J e plugins também pode ser usado para gerar kymographs. Se fazer isso, siga as instruções relevantes para o plugin escolhido...</li> <li>clique a &#34; seleção retangular &#34; botão ( Figura 3 A, seta) e selecione a área de interesse pelo desenho de um retângulo com um mouse de computador (retângulo amarelo na Figura 3 A). Vá até Image &#62; pilhas &#62; ferramentas &#62; fazer Substacks e selecione os números de fatia que estão incluídos no filme. Um substack é gerado.</li>
		<li>Ativar a janela de substack clicando e vá até Image &#62; ajuste &#62; brilho/contraste. Mostra-se uma janela para ajustar o brilho e contraste. Na janela, deslize a parte superior da barra (mínimo) para a direita para que o sinal de fundo desaparece e o segundo top bar (máximo) para a esquerda assim que o movimento vesículas e partículas podem ser vistas muito bem sobre o fundo.</li>
		<li>Vá até Image &#62; pilhas &#62; tempo Stamper. Como o filme é gravado em 4 frames/s na etapa 5.4, o intervalo de tempo é 0,25 s. Assim, digite &#34; 0.25 &#34; no intervalo.</li>
		<li>Gerar um arquivo de filme, selecionando salvar como &#62; AVI do menu arquivo (filme 1 e 2). 
		Nota: Usando plugins apropriado, você pode salvar o arquivo como outros formatos tais como &#34; mpeg4 &#34; ou &#34; mov &#34; arquivos.</li>
	</ol></li> <li>Gerar kymographs.
	<ol><li>Abrir os arquivos originais do filme usando software de FIji novamente e repita os passos 5.6.2 e 5.6.3 para ajustar o brilho e o contraste.</li>
		<li>Clique &#34; segmentado linha &#34; ( Figura 3 B, seta). Usando um mouse, desenhe uma linha segmentada ao longo dos cílios ou axônio (linha amarela, na Figura 3 B).</li>
		<li>Ir para analisar &#62; Multi Kymograph &#62; Multi Kymograph ( Figura 3 C). Aparece a janela de largura de linha ( Figura 3-D). Tipo &#34; 3 &#34; na janela Linewidth e clique Okey ( Figura 3-D).</li>
		<li>Kymograph janela é criada. Salvar a imagem em formato TIFF ou JPEG.</li>
	</ol>
	</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>Bindels, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Prevo, B., Mangeol, P., Oswald, F., Scholey, J. M., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+cooperating+kinesin-2+motors+orchestrates+cargo+import+and+transport+in+C.+elegans+cilia.">Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.</a> Nat Cell Biol. 17 (12), 1536-1545 (2015).</li><li>Robison, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detyrosinated+microtubules+buckle+and+bear+load+in+contracting+cardiomyocytes.">Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes.</a> Science. 352 (6284), 0659 (2016).</li><li>Hayashi, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+superresolution+fluorescence+imaging+with+spinning+disk+confocal+microscope+optics.">Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.</a> Mol Biol Cell. 26 (9), 1743-1751 (2015).</li></ol>

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Limitação no que diz respeito a métodos existentes O método descrito aqui é otimizado para observar eventos rápidos como transporte axonal e IFT. Assim, a imobilização é mais priorizada de incubação mais longa. Enquanto nós fomos capazes de observar eventos de tráfico pelo menos 20 min sem perturbação significativa, esse método pode não ser sempre adequado para observar eventos lentos, exigindo mais observações, tais como alongamento do axônio e migração celular. Para mais observ...

Imagem de célula viva é uma ferramenta essencial para a análise de transporte intracelular. Em biologia celular neuronal, análises de transporte axonal com imagens ao vivo da célula são essenciais para a compreensão de função e morfogênese neuronal1. Defeitos no transporte axonal fundamentam várias de doenças neurodegenerativas2. Proteínas de superfamília de cinesina e Dineína realizam transporte axonal anterogradely e retrogradely, respectivamente,1,2.
Cílios são outro compartimento celular em que a rede de microtúbulos e maquinaria de tráfico são altamente desenvolvidos3. Maquinaria de síntese de proteína não está localizada em cílios, o que significa que as proteínas ciliares devem ser transportadas do citoplasma para as pontas dos cílios. Específicos de cílios cinesina e Dineína, chamada cinesina-2 e citoplasmática Dineína-2, respectivamente, os componentes de cílios4, em um fenômeno chamado do transporte (IFT)5de transporte. Imparidade de IFT faz com que um espectro de doenças chamadas ciliopathies6. Assim, é necessária uma análise do mecanismo de IFT por imagens de células vivas para entender os mecanismos básicos de formação ciliar e patogênese.
Caenorhabditis elegans (C. elegans) é um bom modelo para estudar o transporte axonal e IFT7,8,9. Para observar o IFT, Chlamydomonas tem sido amplamente utilizado como um organismo modelo5,6. Como Chlamydomonas é um organismo unicelular, a relação do IFT com envelhecimento, função neuronal e comportamento seria difícil de analisar. Além disso, não foram aplicadas técnicas genéticas essenciais tais como CRISPR/Cas9 a Chlamydomonas. Em organismos superiores do modelo, tais como ratos e Drosophila, ruptura do transporte axonal e IFT frequentemente causa fenótipos letais porque transporte axonal e IFT são essenciais para a homeostase dos animais 10e a morfogênese 11. No caso dos ratos, transfection e cultura de células é geralmente necessária para observar o transporte axonal e IFT, que requer muitas habilidades e extenso de tempo12,13. Além disso, um monte de contexto fisiológico importante pode ser perdido em culturas de células e linhas celulares. Porque o sistema nervoso não é essencial para a sobrevivência dos vermes, c. elegans mutantes em que o transporte axonal ou IFT são rompidos muitas vezes não são letais7,9,14. Transporte axonal e IFT podem ser diretamente observado na vivo sem dissecação porque c. elegans é transparente e, portanto, é fácil observar marcadores GFP-etiquetado.
Existem vários protocolos para imobilizar o c. elegans, como o uso de um dispositivo de microfluidic15, almofadas de agarose com anestesia16ou microbeads17. A inclusão da anestesia pode inibir os tráfico de eventos em neurônios15. Uma clara desvantagem do método microfluidic-dispositivo é que preparar um dispositivo microfluidic não é sempre fácil. Em vez disso, imobilização por almofadas de agarose e microbeads é uma maneira fácil e conveniente de realizar imagens de lapso de tempo em c. elegans. Aqui, eu descrevo este protocolo básico para imobilizar o c. elegans e visualizar o transporte axonal e IFT na vivo em c. elegans. Comparado a outros métodos, o método descrito aqui não requer equipamentos especiais e é muito mais barato e mais fácil de realizar.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">C024401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
</table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

Transporte axonal e do transporte (IFT) são essenciais para a função e morfogênese axônio e cílios. Dineína e cinesina superfamília proteínas são motores moleculares que regulam anterógrada e retrógrada, transporte, respectivamente. Estes motores usam redes de microtúbulos como trilhos. Caenorhabditis elegans (C. elegans) é um organismo modelo poderoso para estudar o transporte axonal e IFT na vivo. Aqui, eu descrevo um protocolo para observar o transporte axonal e IFT na vida c. elegans. Carga transportada pode ser visualizada por marcação de proteínas de carga usando proteínas fluorescentes, tais como a proteína verde fluorescente (GFP). C. elegans é transparente e proteínas GFP-etiquetado carga podem ser expressa em células específicas sob promotores específicos de célula. Vermes vivos podem ser corrigidos por microbeads em gel de agarose 10% sem matar ou anestesiar os vermes. Nestas condições, movimentação de carga pode ser diretamente observada nos axônios e cílios de viver c. elegans sem dissecação. Esse método pode ser aplicado para a observação de qualquer molécula de carga em todas as células, modificando as proteínas alvo e/ou as células estão expressos em. As proteínas mais básicas tais como motores moleculares e proteínas do adaptador que estão envolvidas no transporte axonal e IFT são conservadas no c. elegans. Em comparação com outros organismos-modelo, os mutantes podem ser obtidos e mantidos mais facilmente em c. elegans. Combinar este método com vários mutantes de c. elegans pode esclarecer os mecanismos moleculares do transporte axonal e IFT.

Transporte axonal em DA9 neurônio Usando a linha de wyIs251 , o anterógrada e retrógrada transporte axonal de GFP::RAB-3 pode ser gravada simultaneamente no neurônio motor DA9. A velocidade média de anterógrada e retrógrada transporte no axônio do neurônio DA9 dorsal proximal é cerca de 1,8 e 2,6 μm/s, respectivamente22. O número de vesículas em movimento é sobre 0,03 e 0,018 por μm de axônio por s. Assim, para uma obser...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) é um bom modelo para estudar o transporte axonal e intracelular. Aqui, eu descrevo um protocolo para análise de transporte axonal e em c. eleganse in vivo de gravação.

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 ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

immobilization-caenorhabditis-elegans-to-analyze-intracellular

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JoVE Journal

Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

7.9K visualizações.  Tohoku University. The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

Vídeo: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Avaliação Compreensiva de Germline Chemical Toxicity Usando o Nemátodo<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Biologia do Desenvolvimento

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Espaciais e temporais Análise do Active ERK na<em&gt; C. elegans</em&gt; germinativa

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Uso de Immunolabeling para analisar os microtúbulos estáveis, dinâmicos e emergentes no embrião Zebrafish

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Aplicação de RNAi e calor-choque-induzida expressão de fator de transcrição para reprogramar as células germinativas de neurônios em c. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

Avaliação dos lisossomos Alkalinization no intestino de viver elegans de Caenorhabditis

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análise computacional do Germline Caenorhabditis elegans , para estudar a distribuição de núcleos, as proteínas e o citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Estudando estresse oxidativo causado pelo estreptococos grupo Mitis em Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Microscopia de folha de luz isotrópica e análise de linhagem de células automatizadas para catalogar Caenorhabditis elegans Embryogenesis com resolução subcelular

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Medindo a orientação e a motilidade do esperma dentro do intervalo reprodutivo hermafrodita dos elegans de Caenorhabditis

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...