Reconhecemos Dr. Harold Dvorak para discussões úteis, incluindo a sugestão de tentar fixação em 37 ° C PFA. Este trabalho foi financiado pelo NIH conceder P01 CA92644 e por um contrato da Fundação Nacional para Pesquisa do Câncer.

1. Cultura de Células em colágeno revestido Disk Vidro <ol><li> Coloque os discos de vidro (12 mm de diâmetro) em uma jarra de vidro (4 oz) e autoclave para esterilizar os discos. </li><li> Colocar 25 ml de etanol a 70% em um tubo Falcon de 50 ml e coloca-se numa capa de cultura de células. Esta solução pode ser reutilizada várias vezes até que o nível da solução baixa para 20 ml. </li><li> Coloque uma pinça afiada com um ponto extra fino no etanol 70% por 5 minutos antes de usá-lo para lidar com o disco de vidro. </li><li> Cuidadosamente remover a pinça para fora do tubo e fechar a tampa, em seguida, colocar gentilmente a pinça de etanol tratada na parte superior do tubo de ar seco, durante 2 min. Não permita que a ponta da pinça para tocar qualquer coisa no capô. </li><li> Use as pinças esterilizadas para pegar um disco de vidro estéril fora da jarra de vidro e coloque-o em um poço da placa de 24 poços de cultura de células. Repita até que o número desejado de poços foi preenchido com discos. </li><li> Coloque 500 mL de solução de colágeno bovino (50 ng / mL) em cada cavidade que contém um disco de vidro e coloque-a num banho a 37 ° C, 5% de CO 2 célula incubadora de cultura durante pelo menos 30 min. Não há mal nenhum em um período de incubação mais longo. </li><li> Colheita (HUVEC Células Endoteliais da Veia Umbilical Humana) ou PC3 (células de tumor da próstata) que já foram cultivadas em placa de 150 mm de cultura de células por meio de tripsinizao e bloqueiam a actividade da tripsina utilizando o seu meio de cultura completo. Recolher as células para um tubo Falcon de 15 ml. </li><li> Conte o número de celular e certifique-se a viabilidade é superior a 90%. </li><li> Agregar as células por centrifugação a 200 xg durante 10 min a 4 ° C. Remover o sobrenadante. </li><li> Utilizar o meio de cultura para diluir as células a 10 5 células / ml. Coloque as células no gelo. </li><li> Aspirar o colagénio no capuz de cultura de células e colocar suavemente a 500 ul de suspensão celular a cada cavidade em que os discos de vidro foram pré-revestidos com colagénio. 5 x 10 4 células / cavidade da placa de 24 poços, vai dar 60% confluency para HUVEC e 30% de confluência de células PC3. Nota: adicionar mais suspensão de células para uma maior densidade de células. </li><li> Cultura as células em um 37 ° C, 5% CO 2 incubadora de 1 hora, 2 horas, 4 horas, 6 horas, ou 24 horas para rastrear as atividades nanopodia e passagem celular. Normalmente, as células irá anexar ao disco de vidro nos primeiros 30 min, em seguida, começar a se polarizar e migrar na primeira hora, e realizar interações intercelulares e divisão celular nas horas seguintes. </li></ol> 2. A fixação celular <ol><li> Cada poço terá 500 mL de 4% PFA (paraformaldeído) para fixar as células. Quer comercialmente ou fabricado feito recentemente PFA a 4% pode ser usado. Calcular a quantidade de PFA necessário com base no número de poços preparados, e colocar PFA num tubo Falcon de 15 ml. CUIDADO: paraformaldeído é um cancerígeno. </li><li> Coloque o tubo de falcon em um estande de isopor que já estava em vigor em 37 ° C incubadora. Deixe o tubo na incubadora por 30 min para a guerram até o PFA para 37 ° C. </li><li> Coloque uma almofada de aquecimento na capa de cultura e ligá-lo. </li><li> Retire a placa de cultura de células de 24 poços ea PFA pré-aquecido para fora da incubadora e coloque-o em cima da almofada de aquecimento. </li><li> Use uma mão para aspirar o meio de um poço, e imediatamente depois use a outra mão para deslizar suavemente 500 mL PFA no poço através da borda também. Para facilitar a aspiração, inclinar a placa de cultura a cerca de 45 °, encostando-o suporte de isopor de modo que é estável a este ângulo. Isto irá facilitar a aspiração e adição de solução de PFA para o bem, sem perturbar as células na cultura. Este é o passo mais importante para preservar a estrutura celular inicial de actividades celulares. </li><li> Repita o processo até que todos os poços com um disco de vidro receberam PFA. Nota: todas as etapas que precisam mudar de solução pré-existente do poço vai aplicar procedimentos mesma aspiração. </li><li> Colocar a placa de 37 ° C durante 5 min. </li&gt; <li> Pegue a placa de volta para a capa de cultura e incline contra isopor como descrito no passo 2.5. Use uma mão para transferir suavemente a PFA do poço em um tubo de coleta; use a outra mão para colocar logo em seguida, pelo menos, 500 temperatura ambiente mL PBS no poço. Depois de todos os poços foram lavados, voltar e repetir a lavagem PBS mais uma vez em todos os poços. Esvazie o PFA coletado em um recipiente de resíduos perigosos. </li><li> Preparar tampão de bloqueio TPI por adição de 2% de soro fetal bovino para PBS. Azida de sódio a 0,04% (diluído a partir de solução-mãe de 20%) é adicionado como conservante. AVISO: A azida de sódio é um composto tóxico. O tampão pode ser armazenado em 4 ° C, durante um longo período de tempo sem a contaminação bacteriana. </li><li> Com a placa de cultura ainda inclinado contra um suporte, uma vez por cada ciclo de aspiração bem para remover PBS e imediatamente depois adição de 500 ul de tampão de bloqueio de ICC. As células estão prontas para a coloração de imunofluorescência. Se necessário, océlulas fixadas podem ser armazenados num refrigerador 4 ° C durante uma semana sem perda significativa da proteína TM4SF1. </li></ol> 3. TM4SF1 Imunofluorescência Coloração de Nanopodia <ol><li> Em cada poço, a substituição de 500 ul de tampão de bloqueio do TPI com um novo tampão de bloqueio contendo 0,01% de Triton X-100 e deixar repousar à temperatura ambiente durante 1 hora. Não utilizar mais elevado do que 0,03% de Triton X-100, uma vez que irá remover TM4SF1 a partir de membranas celulares. PBS células lavadas do passo 2.10 pode ser directamente transferida para o tampão de bloqueio contendo Triton se a coloração está pronto para ser iniciado imediatamente. </li><li> Dilui-se o anticorpo primário anti-TM4SF1 (ou anti-CD9) a 0,5 ug / ml em tampão de bloqueio de ICC. </li><li> Em cada poço, remover o tampão de Triton ICC/0.01% e substituí-la com 300 ml da solução de anti-TM4SF1-anticorpo. Incubar 2 horas à temperatura ambiente ou deixar durante a noite a 4 ° C. </li><li> Lavar cada poço com não menos do que 500 ul de PBS. Repita 3x; cada vez dar a least 5 min do tempo de incubação. </li><li> Prepara-se uma solução de anticorpo secundário e faloidina em ICC tampão de bloqueio, usando uma diluição de 1000 vezes de Alexa 488 anticorpo secundário marcado de burro anti-ratinho (2 ug / ml de concentração final) e 1.000 x diluição de faloidina (50 ng / mL de concentração final). </li><li> Remover PBS e adicionar 300 ul da solução de anticorpo secundário e faloidina a cada poço e incubar durante 2 horas ou deixá-lo durante a noite a 4 ° C. </li><li> Repita os passos de lavagem PBS com pelo menos 5 minutos de incubação por lavagem. Na última lavagem, deixar cada poço em PBS, durante 1 hora. </li><li> Largar uma única gota (~ 10 mL) de montagem anti-fade mídia sobre uma lâmina de vidro. </li><li> Dobre a ponta de um 19 G 1 ½ na agulha da seringa de 90 ° pressionando suavemente sobre uma superfície dura. Use uma mão para segurar a agulha dobrado e gentilmente levante um disco de vidro do bem e utilize a outra mão para segurar o disco usando a pinça afiadas. </li><li> Vire o disco de vidro e# 160; viradas para baixo deixando o contato célula lado os meios de comunicação de montagem na lâmina de vidro. Delicadamente, coloque o disco de vidro e deixe-a secar durante a noite em um lugar escuro à temperatura ambiente. </li><li> Prossiga para a imagem da imunofluorescência manchado nanopodia, ou armazenar os slides em uma caixa de lâminas a -20 ° C. Os slides permanecerá visível por alguns meses em -20 ° C. </li></ol>

<ol>
	<li>Chhabra, E. S., Higgs, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+actin:+matching+assembly+factors+with+cellular+structures.">The many faces of actin: matching assembly factors with cellular structures.</a> Nat. Cell Biol. 9, 1110-1121 (2007).</li><li>Shih, S. C., 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=The+L6+protein+TM4SF1+is+critical+for+endothelial+cell+function+and+tumor+angiogenesis.">The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis.</a> Cancer Res. 69, 3272-3277 (2009).</li><li>Zukauskas, A., 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=TM4SF1:+a+tetraspanin-like+protein+necessary+for+nanopodia+formation+and+endothelial+cell+migration.">TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.</a> Angiogenesis. 14, 345-354 (2011).</li><li>Hellstrom, I., Beaumier, P. L., Hellstrom, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effects+of+L6,+an+IgG2a+antibody+that+reacts+with+most+human+carcinomas.">Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 7059-7063 (1986).</li><li>Jang, Y. Y., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+and+stem+cell+research.">Molecular imaging and stem cell research.</a> Mol. Imag. 10, 111-122 (2011).</li><li>Hemler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraspanin+functions+and+associated+microdomains.">Tetraspanin functions and associated microdomains.</a> Nat. Rev. Mol. Cell Biol. 6, 801-811 (2005).</li><li>Vasile, E., Tomita, Y., Brown, L. F., Kocher, O., Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+thymosin+beta-10+by+early+passage+and+senescent+vascular+endothelium+is+modulated+by+VPF/VEGF:+evidence+for+senescent+endothelial+cells+in+vivo+at+sites+of+atherosclerosis.">Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.</a> FASEB. 15, 458-466 (2001).</li><li>Itoh, T. 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Nanopodia são finos canais de membrana celular que ligam firmemente à matriz através TMED e pode se estender mais do que 100 mm a partir de uma célula móvel polarizada para sentir o ambiente e mediar interações intercelulares 2,3. Nanopodia aderir à matriz tão firmemente que os resíduos são deixados para trás, como os movimentos de células de distância (Figuras 1 e 2). Nanopodia assim nos permitem estudar como as células sentir seu ambiente e determinar o camin...

Durante a polarização para o movimento, as células animais estender uma variedade de dinâmicas, saliências da membrana de suas superfícies, incluindo filopodia, lamellipodia, fibras de retração, e babados 1. Recentemente adicionado a esta lista foram nanopodia, um tipo recém-reconhecido de fina (100-300 m de diâmetro), a projeção da membrana alongada (até 50-100 mM de comprimento) que oferecem canais de membrana para a extensão das estruturas de F-actina como filopodia e fibra de retracção, e que coraram positivamente com TM4SF1 (Transmembrana-4-L e seis-família-1) em células endoteliais e de tumor de células cultivadas 2,3. TM4SF1 é uma proteína com topologia tetraspaninas semelhante que foi originalmente conhecido como um antigénio das células tumorais antes de 4 a descoberta de que a molécula é um biomarcador de células endoteliais, que desempenha um papel essencial na proliferação de células endoteliais e migração de 2,3. Imunofluorescência revelou que TM4SF1 está localizada a perinucvesículas Lear e para a membrana plasmática, e é enriquecido em TM 4SF1 e nriched micro omains d (TMED). TMED âncora nanopodia à matriz e presente em um padrão em faixas regularmente espaçadas de 1-3 comprimento TMED / mM de nanopodia. Nanopodia normalmente se estendem desde principal frente de uma célula e à direita traseira durante a polarização celular para o movimento. Devido à natureza aderente firme de TMED, nanopodia são incapazes de retrair de volta para dentro da célula, uma vez que se move para longe; resíduos nanopodia abandonados traçar, assim, o caminho do movimento celular. Nanopodia fornecer canais de membrana para a extensão F-actina e retração, e são locais de interações intercelulares e comunicações 2,3. Essas características significam que nanopodia proporcionar uma oportunidade única de estudar os mecanismos subjacentes a montagem F-actina durante a polarização celular, sensor de celular do ambiente, a determinação do caminho e direção do movimento celular e interações intercelulares umnd comunicações. Devido à natureza altamente hidrofóbico de TMED ea natureza membranoso fina e frágil de nanopodia, um cuidado especial deve ser tomado a fim de preservar TMED e nanopodia. A destruição de nanopodia e remoção de TMED por métodos comuns de laboratório é uma possível razão para que apenas sessenta e três publicações apareceram na TM4SF1 desde a sua primeira descoberta, em 1986, 1 e para a total falta de conhecimento de TM4SF1 enriquecimento em 100-300 mM microdomínios em superfície das células até que o relatório de TM4SF1 em células endoteliais em 2009 2. Métodos convencionais vulgarmente imunocoloração utilizar solventes orgânicos como o etanol, metanol, acetona ou para fixar as células e utilizar de 0,1% ou mais elevada a concentração de Triton X-100 a permeabilizar as células 5. Estudos descritos aqui implementados três grandes mudanças para o método convencional para revelar TMED e nanopodia: (i) usar 37 ° C PFA 4% e corrigir as células em um 3776; incubadora C, (ii) aplicar suave temperatura ambiente de lavagem PBS, e (iii) utilizar a menos de 0,03% de Triton X-100 apenas brevemente para permeabilizar as células antes da adição do anticorpo primário, tal como Triton X-100 0,03% maior do que irá extrair TM4SF1. Todos tetraspanins formar microdomínios sobre a membrana da célula 6 e alguns colocalize com TMED em endoteliais e células tumorais 2,3. Como tetraspanins como CD9, CD81, CD151 e são ubiquamente expressas, o protocolo de coloração descrito aqui pode ser estendida a muitos tipos de células diferentes que carecem TM4SF1 para os estudos de função nanopodia.

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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass discs&#160;</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:133px;">12-545-82&#160;</td>
			<td style="width:983px;">12-mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass jar</td>
			<td>Fisher Scientific</td>
			<td>02-912-310</td>
			<td>Certified Clean Clear Glass Straight-Sided Jars, 4 oz.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass slide</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">12-544-1</td>
			<td>Fisherfinest Premium Plain Glass Microscope Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50-ml falcon tube&#160;</td>
			<td style="width:160px;">BD Falcon</td>
			<td style="width:133px;">352070</td>
			<td>50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">15-ml falcon tube with Styrofoam rack</td>
			<td style="width:160px;">Corning</td>
			<td style="width:133px;">430790</td>
			<td>Corning 15-ml PP Centrifuge Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sharp forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">13-812-42</td>
			<td>Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24-well cell culture plate</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353047</td>
			<td>24-well Cell Culture Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">150-mm cell culture plate&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353025</td>
			<td>150-mm cell culture plate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">19G 1 &#189; syringe needle&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">309635</td>
			<td>19G 1 &#189;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2701</td>
			<td>Decon&#39;s Pure Ethanol 200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">collagen solution&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">354249</td>
			<td>Collagen I, High Concentration, rat tail, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">trypsin/EDTA</td>
			<td style="width:160px;">Cellgro</td>
			<td style="width:133px;">25-053-CI</td>
			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

As células aderentes em cultura manter um estado polarizado para apoiar o movimento e interações intercelulares. Nanopodia são finas, alongadas, em grande parte de actina F-negativo-projeções de membrana em células endoteliais e de câncer que podem ser visualizadas através de TM4SF1 (Transmembrane-4-L-seis-família-1) técnica de imunofluorescência. Cachos TM4SF1 em 100-300 diâmetro mM TMED (TM 4SF1 e nriched micro d omains) contendo 3 a até 14 individuais TM4SF1 moléculas. TMED estão dispostas de forma intermitente ao longo nanopodia com um espaçamento regular de 1 a 3 TMED por μ m e ancorar firmemente nanopodia a matriz. Isso permite nanopodia estender mais de 100 μ m do líder frente ou à direita traseira de células endoteliais ou tumorais polarizadas, e faz com que os resíduos de membrana para ser deixado para trás em matrix quando a célula move-se para longe. TMED e nanopodia ter sido ignorado por causa de sua extrema fragilidade e sensibilidade à temperatura. Lavagem de rotina e fixação romper a estrutura. Nanopodia são preservadas por meio da fixação directa de paraformaldeído (PFA) a 37 ° C, seguida por uma breve exposição a 0,01% de Triton X-100 antes da coloração. Nanopodia abrir novas perspectivas na biologia de células: eles prometem para reformular a nossa compreensão de como as células perceber seu ambiente, detectar e identificar outras células à distância, iniciar interações intercelulares em contacto estreito, e dos mecanismos de sinalização envolvidos no movimento, proliferação e celular -celulares comunicações. Os métodos que são desenvolvidos para estudar nanopodia TM4SF1 derivados podem ser úteis para estudos de nanopodia que se formam em outros tipos de células através da agência de tetraspanins clássicos, nomeadamente a CD9 onipresente expressa, CD81 e CD151.

Para o Passo 1: Se as células (como HUVEC e PC3 utilizado neste estudo) são capazes de crescer normalmente, as células irá anexar o disco revestido de colágeno dentro de 30 minutos depois que eles são semeadas, polarizar e tornar-se móvel logo depois, e estender nanopodia à frente do seu caminho de movimento. Figuras 1A e 1B mostram, respectivamente, um polarizado e em proliferação de HUVEC. Figura 2A mostra células PC3 polarizado nu...

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia são canais de membrana fina, mas frágeis, que se estendem até 100 mm de líder frente ou à direita traseira de um celular e sentir o ambiente celular. Fixação directa a 37 ° C, lavagem suave, e evitar os solventes orgânicos como o etanol, metanol, ou de acetona e de Triton X-100 a concentrações mais elevadas são necessárias para observar estas estruturas celulares.

Nanopodia - finas, Projeções frágil membrana com papéis em Movimento Celular e intercelulares Interações

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Research

JoVE Journal

Biology

10.2K visualizações.  Harvard Medical School.  Nanopodia são canais de membrana fina, mas frágeis, que se estendem até 100 mm de líder frente ou à direita traseira de um celular e sentir o ambiente celular. Fixação directa a 37 ° C, lavagem suave, e evitar os solventes orgânicos como o etanol, metanol, ou de acetona e de Triton X-100 a concentrações mais elevadas são necessárias para observar estas estruturas celulares. 

Vídeo: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Estudando Biogenesis de membrana com um ensaio de luciferase Gene-Based Reporter

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biologia

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Viva Imagem Ensaio para Avaliação das Funções de Ca<sup&gt; 2 +</sup&gt; E esfingomielinase na reparação de Pore-formando feridas Tóxicas

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Análise de partícula única open-source para Microscopia Super-resolução com VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flash-e-Freeze: uma nova técnica para capturar Membrane Dynamics com Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

Puxando a membrana nanotubos de vesículas Unilamellar gigante

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

Avaliando interações proteicas em células vivas com emissão sensibilizada por FRET

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Espectroscopia de Eco de Rotação de Nêutrons como uma sonda única para dinâmicas de membrana lipídica e interações membrana-proteína

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Isolamento de células musculares lisas cardíacas e vasculares de zebrafish adulto, juvenil, larval e embrionário para estudos eletrofisiológicos

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...