Gostaríamos de agradecer a Patricia Wadsworth para o dom do kinesin-5 construção. Este trabalho foi apoiado por uma bolsa NIH (5 R01 GM107026) para TJM e pela Research Grant No. 5-FY13-205 da March of Dimes Foundation para TJM, bem como o apoio da Charles H. capa Foundation, Inc., Boston, MA. para TJM

1. transfecção de células S2 <ol><li> A partir de um frasco previamente cultivadas 25cm 2, adicionar-GFP ct-expressando-tubulina células S2 10 para um volume final de 2 ml a 100% de confluência, e permitir-lhes semi-aderir ao fundo de uma cultura de 35 mm de tecido do prato para cerca de 1 h. </li><li> Remova a mídia do prato. Transfectar 2 ug da Eg5-mCherry construção 11 com 1 ml de meio de Schneider suplementado com FBS a 10% (a seguir referido como meio de Schneider) e Reagente de Transfecção, seguindo o protocolo do fabricante. Fecha-se o prato com Parafilm e incubar as células a 25 ° C durante 16-18 horas. </li><li> Adicionar 1 ml de meio de Schneider. Retorno células a 25 ° C incubadora. </li><li> No dia 3 após a transfecção, transferir 500 ul de células transfectadas para uma nova placa de cultura de 35 mm de tecido contendo 1,5 ml de meio de Schneider durante um teste de indução. Induzem a expressão de Eg5-mCherry por adição de CuSO4 </sub&gt; até uma concentração final de 500 uM. Selar o prato em Parafilm e incubar a 25 ° CO / N. </li><li> Para fazer lamelas revestidas com concanavalina A pipeta, um volume suficiente de solução de concanavalina A (0,5 mg / mL dissolvido em H 2 O) para revestir uma lamela de ácido lavou-se, remover o excesso, e permitir que as lamelas secar ao ar. </li><li> Coloque um 22 mm x 22 mm concanavalina revestidos por uma lamela limpa numa cultura de 35 mm de tecido do prato, em seguida, 500 ul de sementes a 100% de confluência das células induzidas para a lamela e permitir que as células aderir durante 1 hora à TA. </li><li> Para verificar se há expressão Eg5-mCherry, montar a lamela em uma câmara de rosa (ou vaso de imagem apropriado) com a mídia para imagens em RT em um microscópio de fluorescência. Use fontes de luz adequadas e conjuntos de filtros para visualizar as células. Por exemplo, o microscópio usado no estudo tem uma fonte de luz LED branco e EGFP (ITCF / Cy2) e dsRed (TRITC / Cy3) filtro cubos. Eg5-mCherry localiza aos microtúbulos e é enriquecido near pólos do fuso. </li><li> Depois de garantir que as células expressando tanto são Eg5-mCherry e GFP-α-tubulina, transferir o restante das células transfectadas, não induzidas a 25 cm2 frasco de cultura de tecidos contendo 4 ml de meio de Schneider. </li><li> Adicionar Blasticidina S HCl a uma concentração final de 25 ug / mL ao frasco de células transfectadas e continuar a divisão na presença de 25 ug / ml de Blasticidina S HCl até a morte celular cessa tendo a certeza de verificar a expressão da Eg5-mCherry periodicamente. Incubar as linhas celulares em 25 ° C incubadora. NOTA: A percentagem de células que expressam Eg5-mCherry aumenta ao longo do tempo. </li><li> Uma vez que as células são transfectadas de forma estável, continuam a dividir na ausência de fármaco e congelar as células 12 para uso futuro. </li></ol> 2. RNA Interferência <ol><li> PCR amplificar uma região ~ 500 pb de Klp61F de cDNA (LD15641) utilizando o iniciador com adição de sequência de promotor de T7 (sequência previsto na lista de materiais) para serutilizado como um molde para a síntese de ARNcd. <ol><li> Defina-se quatro reacções de PCR, 50 pi cada uma, contendo 100 ng de ADN molde, 25 pM de cada iniciador, tampão adequado, e a polimerase. Ajuste o protocolo de PCR no termociclador de acordo com o protocolo do fabricante da polimerase. </li><li> Piscina e quatro reacções de PCR limpas usando um PCR limpar kit de acordo com o protocolo do fabricante. Armazenar o molde limpo a -20 ° C. </li></ol></li><li> Prepare ARN de cadeia dupla (ARNcd) de molde usando um kit de transcrição de ARN de T7, seguindo as instruções do fabricante. Esperar ~ concentração de 5-15 mg / ml de ARNdc. ARNcd armazenar a -20 ° C. </li><li> Para knockdown Klp61F com ARNcd, permitem que as células que expressam S2 Eg5-mCherry a 25% de confluência em semi-aderir ao fundo 35 de um prato de cultura de tecido mm durante 1 hora. </li><li> Remova a mídia dos pratos e adicionar 5 ug de dsRNA contra Klp61F (Drosophila kinesin-5) diluído em 1 ml de Schneider isento de soro &quot;s mídia. </li><li> Incubar à temperatura ambiente durante 1 h, em seguida adicionar 1 ml de meio de Schneider com 10% de FBS. Incubar as células a 25 ° C. </li><li> 24 horas após o tratamento ARNcd, induzir a expressão da Eg5-mCherry por adição de CuSO4 até uma concentração final de 500 uM. Incubar as células a 25 ° C durante mais 24 h. </li></ol> 3. Imagens de células vivas <ol><li> Coloque um 22 mm x 22 mm concanavalina A lamela revestida num limpo 35 mm de cultura de tecidos prato. </li><li> Semente de 500 ul de células que foram tratados com ARNdc e induzidas O / N para a concanavalina A lamela revestida e lhes permitem aderir durante cerca de 1 hora. </li><li> Montar a lamela em uma câmara de rosa (ou recipiente apropriado) para a imagem latente. </li><li> Encontre células mitóticas expressam tanto Eg5-mCherry e GFP-α-tubulina. NOTA: As células mitóticas expressando apenas GFP-α-tubulina fusos monopolares deve ter desde Klp61F, que é necessário para bipolaridade fuso, é derrubado 10,13. </li><li> Coletar imagens de lapso de tempo de eixos bipolares (por exemplo, um quadro por minuto) em células que expressam tanto Eg5-mCherry e GFP-α-tubulina. </li><li> Enquanto imagem, remova a mídia da câmara de rosa, e substituí-la por meios de Schneider contendo 1 mM STLC mais de 3 trocas de mídia consecutivos (~ 5 ml total) para a câmara para visualizar colapso do fuso. </li><li> À lavagem da droga e reverter o colapso do eixo, retire cuidadosamente a mídia contendo STLC da câmara de rosa, e lavar em meio de Schneider 4x (6-8 ml total) antes de reencher a câmara se levantou pela última vez com meio fresco. Continuar imagiologia. </li></ol> 4. Correção de Erros Assay <ol><li> Coloque um 22 mm x 22 mm concanavalina A lamelas revestidas em placas de cultura de 35 mm de tecido limpo. </li><li> Semente 500 ul de células que foram tratados com ARNdc sobre o Klp61F concanavalina A lamela revestida e lhes permitem aderir. </li><li> Depois as células são adherEd, adicionar 1,5 ml de meio de Schneider a cada placa para levar o volume final até 2 ml. </li><li> Para reter as células em mitose, MG132 adicionar a uma concentração final de 10 uM a cada um dos pratos, e incubar durante 1 h. </li><li> Adicionar 1 uM STLC e incubar durante 1 h para permitir que monopoles para formar. </li><li> Lavar a STLC por lavagem 3x lamelas com 2 ml de meio de Schneider frescos cada vez. </li><li> Incubar lamelas com meio de Schneider, além de quaisquer drogas farmacológicas (por exemplo, inibidores de quinase Aurora) ou DMSO, como um controlo. Nota: as concentrações do inibidor variam e concentrações finais adequada deve ser determinada pelo experimentador. As soluções de reserva são normalmente feitas de tal modo que o inibidor é diluída 1: 1.000 em mídia. Neste caso, uma diluição de 1: DMSO (ou num solvente apropriado) 1000 serve como controlo do veículo. Usamos o inibidor de Aurora B Binucleine 2 a uma concentração final de 40 uM. </li><li> Fix células em diferentes pontos no tempo tO observar a progressão da formação do fuso bipolar e para avaliar os estados de ligação cinetócoro. Consertar: <ol><li> Enxaguar rapidamente as lamelas com 2 ml de 1x BRB-80. </li><li> Fixar as células por adição de 2 ml de 10% de paraformaldeído em diluídos 1x BRB-80 durante 10 min. Pipet paraformaldeído cuidadosamente em uma capa química. </li><li> Permeabilizar as células com 2 ml de 1x PBS + 1% de Triton-X durante 8 min. </li><li> Lavagem das lâminas 3x com 2 ml de PBS 1x + 0,1% de Triton-X. </li><li> Transferência lamelas do lado célula viradas para cima para uma folha de Parafilm (use um marcador para rotular Parafilm adequadamente para manter o controle das lâminas) em uma placa de Petri de 150 milímetros. </li><li> Cobrir as lamelas com 150 ul de soro de burro fervida (BDS) e incubar a temperatura ambiente durante 1 h para bloquear o anticorpo não específico de ligação. Nota: Aqui, o protocolo de fixação pode ser parado para ser mantida a um ponto mais tarde. As lamelas podem ser armazenados numa câmara de humidade a 4 ° C durante até 24 h. Para fazer com que a câmara de humidade, a linha do aro de um prato de 150 mmcom um laboratório wipe molhado e cubra com a tampa placa de Petri. </li><li> Remover bloco, e incuba-se as lamelas durante 1 hora à temperatura ambiente com 150 ul de anticorpos primários diluídos apropriadamente em BDS para corar para cinetocoros e microtúbulos até às concentrações finais de 2 ug / ml (ou de acordo com as recomendações do fabricante) e 1 ug / ml, respectivamente . Nota: Aqui, o protocolo de fixação pode ser parado para ser mantida a um ponto mais tarde. As lamelas podem ser armazenados numa câmara de humidade a 4 ° C durante até 24 h. </li><li> Lavar 3x lamelas com 500 ul de 1x PBS + 0,1% de Triton-X. </li><li> Incubar lamelas durante 30-60 min à temperatura ambiente com os anticorpos secundários conjugados com fluoróforos adequados diluídos em BDS. </li><li> Lavar 3x lamelas com 500 ul de 1x PBS + 0,1% de Triton-X. </li><li> Incubar lamelas com 150 ul de BDS contendo 1 ug / ml de concentração final de DAPI, durante 5 min. </li><li> Lavar as lamelas com PBS 1x 2x + 0,1% de Triton-X, C e montaroverslips lado célula para baixo em um slide 3x1 &quot;com 8 mL de meio de montagem. Pinte os cantos com unha polonês para imobilizar as lamelas em lâminas. </li></ol></li><li> Células de imagem com eixos bipolares que estão expressando Eg5-mCherry. Tirar uma série Z constituído por 30 aviões em 0.2 mm intervalos em todos os canais usando um objetivo 100X. Analisar cuidadosamente as cinetócoros e microtúbulos para determinar os estados de fixação (biorientados ou anexos syntelic). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

A visualização de correcção de erros é uma técnica valiosa para estudar as etapas envolvidas neste processo celular importante e complexo. Para fazê-lo, os anexos erradas são gerados usando inibidores reversíveis, e correcção de erros é observada mediante lavagem do fármaco. Este ensaio foi inicialmente desenvolvido utilizando células de cultura de tecidos de mamíferos, 5. No entanto, a presença de grande número de cinetocoros em muitos tipos de células de mamíferos modelo coloca um desafio na observação de eventos individuais de correcção de erros. As células S2 de Drosophila possuem tão poucos como 4 pares de cinetocoros, tornando-as numa linha celular mais preferível para a observação de correcção de erros. No entanto, uma grande desvantagem é que muitos inibidores são ineficazes em células S2 de Drosophila. Assim, a capacidade de gerar uma linha de células S2 de Drosophila expressando humana humanizado cinesina-5 fornece uma ferramenta valiosa para estudar a correcção de erros. Embora as células S2 de Drosophila pode ser ummelhor linha celular para estudar a correção de erros, as várias etapas envolvidas na obtenção da linha celular e derrubando genes essenciais coloca alguns desafios para esta técnica. Por exemplo, a eficiência da transfecção pode ser muito baixo. Se menos de 20% das células expressam o Eg5-mCherry, a transfecção deve ser repetido o processo de selecção como irá demorar mais tempo. Além disso, a percentagem de células que expressam ambas as proteínas fluorescentes pode diminuir ao longo do tempo. Isto pode ser superado através da divisão de células na presença de Blasticidina S HCl e higromicina B para seleccionar as células que expressam Eg5-mCherry e GFP-α-tubulina, respectivamente. É também importante notar que as células S2 de Drosophila têm ortólogos para muitas proteínas e humanos; Portanto, é essencial para optimizar as condições de knockdown para as proteínas endógenas. Condições óptimas de knockdown pode variar na quantidade de ARNdc e a duração do tratamento. Considerando-se o surgimento e rápida melhoria das CRISPR-Cas9 Technologies 15-17, a geração de uma linha de células de Drosophila com o gene Klp61F substituído por Eg5 humano apresenta uma alternativa poderosa capaz de superar as limitações da transfecção e abordagem knockdown. Nosso trabalho demonstra que a mosca-a-homem substituição gene deve ser uma opção viável neste caso, embora os reagentes necessários para fazê-lo em células de Drosophila S2 estão sendo desenvolvidos atualmente. Este processo não está limitado a Eg5, mas pode ser aplicado para estudar a função de outras proteínas de interesse. Se o uso de inibidores que tenham sido previamente estabelecidos para ser ineficaz em células de Drosophila S2, este protocolo pode ser modificado para se estudar o efeito direto de inibidores em células vivas, sem preocupações sobre os efeitos alvo fora. A linha celular produzida usando este protocolo pode também ser utilizada no rastreio de alto rendimento para identificar as análises potenciais drogas que visam proteínas envolvidas na correcção de erro.

Igual a segregação do genoma durante a divisão celular requer interacções entre as correctas microtúbulos fusiformes ADN e replicados. Os microtúbulos interagir fisicamente com os cromossomas por meio de um conjunto de proteínas que se monta em centrómeros conhecidos como o cinetocoro 1. Distribuição correta dos cromossomos exige que cinetócoros irmãos são biorientado, em que cada irmã está associada a microtúbulos provenientes de pólos opostos do fuso. Kinetochore microtúbulos (KT-MT) anexos que não estão na conformação biorientado são rápida e eficientemente desestabilizada para proporcionar a oportunidade para estabelecer biorientation num processo conhecido como correcção de erros. Um ensaio de correcção de erros que foi previamente estabelecida em células de mamíferos requer montagem 5 fusos monopolares utilizando inibidores de moléculas pequenas reversíveis contra Eg5 (cinesina-5). O tratamento da droga gera uma infinidade de acessórios syntelic errôneas em que botcinetócoros h irmãos anexar ao mesmo pólo do fuso. Uma lavagem subsequente do fármaco, permite a visualização do processo de correcção de erro. O ensaio de correcção de erros pode ser realizada na presença de inibidores de moléculas pequenas ou knockdowns para estudar a contribuição de proteínas candidatas para corrigir erróneos anexos KT-MT. A habilidade de visualizar a correcção de erros em células vivas é uma ferramenta poderosa para melhor compreender o mecanismo molecular envolvido neste processo complexo. No entanto, o grande número de cromossomas presentes na maioria das linhas celulares coloca um desafio na observação anexos KT-MT individuais. As células S2 de Drosophila seria ideal para a aplicação do ensaio de correcção de erro à medida que conter tão poucos como 4 cromossomas 6, mas os inibidores de moléculas pequenas de cinesina 5 tal como S-tritil-L-cisteína (STLC) e monastrol 7-9 não afecta a formação do fuso ou cinesina-5 função motora em células de Drosophila. Nós, portanto, gênerosTed uma linha de células S2 de Drosophila expressando cinesina-5 humana sob um promotor indutível que é sensível aos inibidores da cinesina-5. Este protocolo descreve como o knockdown endógena Drosophila cinesina-5 homólogo, Klp61F, e usar esta linha de células no ensaio de correcção de erros com base em células.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:221px;">Effectine Transfection Reagent</td>
			<td style="width:184px;">Qiagen</td>
			<td style="width:167px;">301425</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:221px;">Klp61F cDNA</td>
			<td style="width:184px;">Drosophila Genomic Resource Center</td>
			<td style="width:167px;">13690</td>
			<td style="width:255px;">Gene Name LD15641</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Schneider&#8217;s Media</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;">21720-024</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:221px;">Fetal Bovine Serum, certified</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;">10082-147</td>
			<td style="width:255px;">Heat Inactivated, US origin</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:221px;">Copper (II) Sulfate (CuSO4)</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:167px;">C8027-500G</td>
			<td style="width:255px;">500mM stock&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">S-trityl-l-cysteine</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:167px;">164739-5G</td>
			<td style="width:255px;">1mM stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Blasticidin HCl</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;">R21001</td>
			<td style="width:255px;">5mg/ml stock in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">Hygromycin B</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;">10687010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:221px;">T7 Large scale RNA Production System</td>
			<td style="width:184px;">Promega</td>
			<td style="width:167px;">P1320</td>
			<td style="width:255px;">The approximate dsRNA concentration from each reaction is about 5-15&#181;g/&#181;l</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:221px;">Klp61F RNAi F primer</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;"></td>
			<td style="width:255px;">TAATACGACTCACTATAGGGTA-TTTGCGCATTATTTTAAAA</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:221px;">Klp61F RNAi R primer</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:167px;"></td>
			<td style="width:255px;">TAATACGACTCACTATAGGGAT-ATTGATCAATTGAAAC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">PCR clean-up kit</td>
			<td style="width:184px;">Mo Bio Laboratories, Inc</td>
			<td style="width:167px;">1250</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:221px;">Concanavalin A</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:167px;">C5275</td>
			<td style="width:255px;">0.5mg/ml solution made by dissolving in 1xPBS.&#160;</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:221px;">Boiled Donkey Serum</td>
			<td style="width:184px;">Jackson ImmunoResearch Labs</td>
			<td style="width:167px;">017-000-121</td>
			<td style="width:255px;">5% stock solution in 1X PHEM buffer, bring solution up to boil. Stored in 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:221px;">Mounting Media</td>
			<td style="width:184px;"></td>
			<td style="width:167px;"></td>
			<td style="width:255px;">20mM Tris pH 8.0,&#160; 0.5% N-propyl gallate, 90% Glycerol. Stored in 4&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">1x BRB-80</td>
			<td dir="LTR" style="width:184px;"></td>
			<td style="width:167px;"></td>
			<td dir="LTR" style="width:255px;">80 mM PIPES pH 6.9; 1 mM EGTA; 1 mM MgCl2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">White Light Source</td>
			<td>Lumencor Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">ET EGFP Filter Cube</td>
			<td>Chroma</td>
			<td align="right">49002</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">DSRed Filter Cube</td>
			<td>Chroma</td>
			<td align="right">49005</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">anti-NDC80 antibody&#160;</td>
			<td></td>
			<td></td>
			<td>Custom made by the Maresca Lab</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">DM1&#945; (anti-&#945;-tubulin)</td>
			<td>Sigma</td>
			<td>T6199</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:221px;">anti-CID antibody</td>
			<td>AbCam</td>
			<td>ab10887</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a &#34;humanized&#34; drosophila s2 cell line sensitive to pharmacological inhibition of kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Klp61F é necessário para formar fusos bipolares. O cinesina-5 humana, Eg5, pode resgatar a função de Klp61F em células S2 de Drosophila (Figura 1A e D). Após a adição da cinesina-5 inibidor (STLC), níveis associadas a microtúbulos da gota do motor (Figura 1B e E), e os colapsos do fuso, o que resulta em um monopolo (Figura 1C e F) em células sem Klp61F endógenos sobre o ARNi sucesso (filme Suplementar 1). É digno de nota que fusos bipolares em colapso após a adição de STLC nas células S2 porque cinesina humanizados-5 actividade não é necessária para manter bipolaridade fuso em células humanas. Esta discrepância interessante pode ser um resultado de diferenças na sobreposição de microtúbulos interpolar e / ou estabilidade nos dois sistemas modelo 14. Cinesina-5 de inibição (Figura 2 A e E) pode ser reve<html> rsed por lavagem do fármaco e a recuperação de um fuso bipolar pode ser seguido ao longo do tempo. Após a remoção do inibidor (Figura 2B e F), Eg5-mCherry re-associados com os microtúbulos como um bipolares monta fuso (Figura 2C e G), e as células podem progredir para um anafase bipolar (Figura 2D e H, filme suplementar 2 ). <img alt="figura 1" src="/files/ftp_upload/53594/53594fig1.jpg" /> Figura 1. A adição de 1 mM STLC leva a fusos monopolares em células S2 de Drosophila. Imagens representativas de time-lapse imagem de uma célula de Drosophila S2 expressar Eg5-mCherry (AC) e GFP-α-tubulina (DF) durante a adição de 1 mM STLC. O inibidor de Eg5 foi adicionado em 3 minutos. Barra de escala = 5 uM. Timestamp = min: seg.<html> &quot;target =&quot; _ blank w.jove.com/files/ftp_upload/53594/53594fig1large.jpg &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 2" src="/files/ftp_upload/53594/53594fig2.jpg" /> Figura 2. A reforma do fuso bipolar após a remoção de STLC. Imagens representativas de time-lapse imagem de uma célula de Drosophila expressando 2 Eg5-mCherry (AD) e GFP-α-tubulina (EH) durante um experimento STLC washout. STLC foi lavado para fora em 5 min. A reforma do fuso bipolar dentro de 1 hora de remover o STLC. Barra de escala = 5 uM. Timestamp: min: seg. <a href="https://www.jove.com/files/ftp_upload/53594/53594fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Filme 1" src="/files/ftp_upload/53594/53594mov1.jpg" /> &quot;target =&quot; _ blank ww.jove.com/files/ftp_upload/53594/addin.mov &quot;&gt; Filme Suplementar 1. (Clique direito a download). Widefield imagens de fluorescência de um exemplo representativo de uma célula de Drosophila S2 expressar Eg5-mCherry ( esquerda) e GFP-α-tubulina (à direita). 1 uM STLC foi adicionado aos 2 min, e as formas do fuso um monopolo dentro de 10 min após a adição do inibidor. As imagens foram adquiridas a cada 1 minuto e tocada a uma velocidade de 10 quadros por segundo. Barra de escala = 5 mm. <img alt="Filme 2" src="/files/ftp_upload/53594/53594mov2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/53594/washout.mov" target="_blank">Filme Suplementar 2. (Clique direito a download).</a> Widefield imagens de fluorescência de um exemplo representativo de uma célula de Drosophila S2 expressar Eg5-mCherry (esquerda) e GFP-α-tubulina (à direita). Meios contendo 1 uM foi removido e STLClavado a 5 min. A reforma do fuso bipolar dentro de 1 hora de remover o inibidor. As imagens foram adquiridas a cada 1 min e jogado a uma taxa de 10 quadros por segundo. Barra de escala = 5 uM.

Watch this Scientific Journal Video about Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5 at JoVE.com

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Gerando um &quot;humanizado&quot;<em&gt; Drosophila</em&gt; S2 linha de células sensíveis a inibição farmacológica de cinesina-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

generating-humanized-drosophila-s2-cell-line-sensitive-to

Research

JoVE Journal

Biology

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

7.2K visualizações.  University of Massachusetts, Amherst. This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined. 

Vídeo: Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Um dispositivo de geração de Gradient microfluídicos de Biologia Celular

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biologia

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Ensaios Neurocircuit para Apreensões em mutantes de Epilepsia Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Toxicidade celular de Nanogenomedicine em MCF-7 Linhagem Celular: MTT

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

Preparação de Drosophila Células S2 para microscopia de luz

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Farmacológica e funcional Ensaios genética para manipular Regeneração da planária Dugesia japonica</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

O Utilitário de Estágio específico-Mid-de-final<em&gt; Drosophila</em&gt; Folículo Isolamento

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Um método de quantificação sensível e específico para Determinação do Serum miosina cardíaca Binding Protein-C por Imunoensaio electroquimioluminescência

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Transporte Organelle em Cultivadas<em&gt; Drosophila</em&gt; Células: Linha celular S2 e primário neurônios.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Uma estratégia para sensíveis, Large Scale quantitativos Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura de Drosophila Pupal Testículo e único macho da linha germinal Cistos: Dissection, Imaging, eo tratamento farmacológico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...