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>
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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.

<table><tbody><tr><td>Glass disks</td><td>Fisher Scientific</td><td>12-545-82</td><td>12 mm diameter</td></tr><tr><td>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><td>Glass slide</td><td>Fisher Scientific</td><td>12-544-1</td><td>Fisherfinest Premium Plain Glass Microscope Slides</td></tr><tr><td>50 ml Falcon tube</td><td>BD Falcon</td><td>352070</td><td>50 ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td></tr><tr><td>15 ml Falcon tube with Styrofoam rack</td><td>Corning</td><td>430790</td><td>Corning 15 ml PP Centrifuge Tubes</td></tr><tr><td>Sharp forceps</td><td>Fisher Scientific</td><td>13-812-42</td><td>Dissecting Extra Fine Pointed Splinter Forceps</td></tr><tr><td>24-well Cell culture plate</td><td>BD Bioscience</td><td>353047</td><td>24-well Cell Culture Plate</td></tr><tr><td>150 mm Cell culture plate</td><td>BD Bioscience</td><td>353025</td><td>150 mm cell culture plate</td></tr><tr><td>19 G 1 &amp;frac12; Syringe needle</td><td>BD Bioscience</td><td>309635</td><td>19 G 1 &amp;frac12;</td></tr><tr><td>Ethanol</td><td>Decon Laboratories, Inc.</td><td>2701</td><td>Decon&#039;s Pure Ethanol 200 Proof</td></tr><tr><td>Collagen solution</td><td>BD Bioscience</td><td>354249</td><td>Collagen I, High Concentration, rat tail, 100 mg</td></tr><tr><td>Trypsin/EDTA</td><td>Cellgro</td><td>25-053-CI</td><td>0.25% Trypsin-EDTA 1x</td></tr><tr><td>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><td>10x PBS</td><td>Affymetrix</td><td>75889 5 LT</td><td>PBS, 10x Solution, pH 7.4</td></tr><tr><td>PFA (paraformaldehyde)</td><td>Affymetrix</td><td>19943 1 LT</td><td>4% in PBS</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F4135-500ML</td><td>Fetal Bovine Serum</td></tr><tr><td>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 Supplement &amp;amp; Growth Factors</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Triton X-100</td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S2002-25G</td><td>Solubilized in water. 4% stock solution and working concentration is 0.4%.</td></tr><tr><td>Mouse anti-human TM4SF1 antibody&amp;nbsp;</td><td>Millipore</td><td>MAB3127</td><td>Epitope is located in extracellular domain</td></tr><tr><td>Mouse anti-human CD9 antibody&amp;nbsp;</td><td>BD Bioscience</td><td>555370</td><td>Epitope is located in extracellular domain</td></tr><tr><td>Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td><td>Life Technologies</td><td>A-21202</td><td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td></tr><tr><td>Phalloidin</td><td>Chemicon</td><td>90324</td><td>Rhodamine-conjugated Phalloidin</td></tr><tr><td>Anti-fade mounting media&amp;nbsp;</td><td>Life Technologies</td><td>P-36931</td><td>ProLong Gold Antifade Reagent with DAPI</td></tr><tr><td>70% Ethanol&amp;nbsp;</td><td></td><td></td><td>17.5 ml of ethanol (200 proof)&lt;br/&gt; 7.5 ml of double-deionized water</td></tr><tr><td>10x PBS (make 200 ml)</td><td></td><td></td><td>20 ml of 10x PBS&lt;br/&gt; 180 ml of double-deionized water</td></tr><tr><td>20% Triton X-100 (make 20 ml)&amp;nbsp;</td><td></td><td></td><td>4 ml Triton X-100&lt;br/&gt; 16 ml double-deionized water</td></tr><tr><td>4% NaN&lt;sub&gt;3&lt;/sub&gt; (make 25 ml)</td><td></td><td></td><td>1 g of NaN&lt;sub&gt;3&lt;/sub&gt;&lt;br/&gt; 25 ml of double-deionized water</td></tr><tr><td>50 ng/ml Bovine collagen solution in PBS (make 5 ml)</td><td></td><td></td><td>Stock solution of 50 &amp;mu;g/ml (50 ml)&lt;br/&gt; 830 &amp;mu;l of Collagen I (3 mg)&lt;br/&gt; 50 ml PBS</td></tr><tr><td>ICC Blocking Buffer (50 ml)</td><td></td><td></td><td>49 ml PBS&lt;br/&gt; 2% FBS (add 1 ml)&lt;br/&gt; 0.04% NaN&lt;sub&gt;3&lt;/sub&gt; (add 100 &amp;mu;l of 4% NaN&lt;sub&gt;3&lt;/sub&gt;)</td></tr><tr><td>ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td><td></td><td></td><td>50 ml of ICC Blocking Buffer&lt;br/&gt; 25 &amp;mu;l of 20% Triton X-100</td></tr><tr><td>HUVEC&amp;nbsp;</td><td>Lonza</td><td>C2517A</td><td>&lt;a href=&quot;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&quot;&gt;www.lonza.com&lt;/a&gt;</td></tr><tr><td>PC3</td><td>ATCC</td><td>CRL-1435</td><td>&lt;a href=&quot;http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7&quot;&gt;www.atcc.org&lt;/a&gt;</td></tr><tr><td>Cell culture hood</td><td>NuAIRE</td><td>Nu-425-600</td><td>NU-425 (Series 60) Biological Safety Cabinet&amp;nbsp;</td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; Cell culture incubator&amp;nbsp;</td><td>CellStar</td><td>QWJ300DABA</td><td>Cellstar CO&lt;sub&gt;2&lt;/sub&gt; Water Jacketed Incubator</td></tr><tr><td>Heating pad&amp;nbsp;</td><td>K&amp;amp;H Manufacturing</td><td>1020</td><td>K&amp;amp;H Lectro Kennel Heated Pad with Free Fleece Cover (&lt;a href=&quot;http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y&quot;&gt;www.amazon.com&lt;/a&gt;)</td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>T6000B</td><td>Sorvall T6000 (B) Benchtop Centrifuge</td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>Bright-Line Hemocytometer</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

Universidad de Cartagena UDEC

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

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Visualizando dinâmica difusional de nanorods de ouro na membrana celular usando microscopia de campo escuro de nanopartícula única

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Bioquímica

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

Microscopia de força de protrusão: Um método para quantificar as forças desenvolvidas pela célula saliências

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Biologia

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Análise quantitativa da dinâmica da borda celular durante a disseminação celular

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after &#945;-catenin depletion in HaCaT keratinocytes.

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

Medindo a dinâmica de saliência da borda celular durante a disseminação usando microscopia de células vivas

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Types of Membrane Protrusions

The protrusion of the cell surface is an initial step for several cellular processes, including cell migration, phagocytosis, and neurite outgrowth. These membrane protrusions are a result of cytoskeletal rearrangement. The most&#160; widely observed cell protrusions include lamellipodia, pseudopodia, filopodia, microvilli, invadopodia, and podosomes. These protrusions can be of two types &#8212; static or dynamic.
The microvilli, an example of stable protrusions, are finger-like projections with a generally stable actin architecture. These brush-like extensions are found on the apical surface of epithelial cells lining internal organs, such as the intestine. They greatly increase the surface area for enhanced nutrient absorption.
Contrastingly, dynamic protrusions are primarily involved in cell migration. The majority of these structures use actin polymerization as the driving force for membrane deformation. Additionally, actin binds myosin motor proteins to form contractile bundles. The myosin motors pull on the actin filaments, thus rearranging them during migration. Based on their appearance, actin-dependent protrusions are classified as lamellipodia, pseudopodia, filopodia, and invadopodia.
Many migrating cells like keratinocytes or fibroblasts exhibit lamellipodia, which are thin, sheet-like projections at the leading edge of these cells. They are involved in the rapid migration of cells to sites of injury for repair and regeneration. Lamellipodia&#160; can extend bundled actin filaments to form finger-like projections called filopodia. For example,&#160; neurons form these outgrowths to sense environmental signals and rewire the nervous system after trauma.
Other membrane protrusions include the podosomes&#160; and invadopodia. While podosomes are transient peg-like structures developed by migrating cells like macrophages, invadopodia are projections produced by metastatic cells on the extracellular matrix. Additionally, invadopodia secrete proteolytic enzymes, degrading the extracellular matrix to break tissue architecture and metastasize into adjacent tissues. Lastly, blebs are spherical membrane projections formed due to internal hydrostatic pressure. They do not depend on the actin cytoskeleton for their enlargement. Fibroblasts and immune cells exhibit bleb formation for migration and other cellular processes such as apoptosis.

Tipos de Protrusões de Membranas

The protrusion of the cell surface is an initial step for several cellular processes, including cell migration, phagocytosis, and neurite outgrowth. These ...

Education

JoVE Core

Biologia Celular

Unidade 1

Polarização e Migração Celular

TERMOS E CONCEITOS CHAVE

Mechanism of Lamellipodia Formation

Cells migrating in response to external stimuli form lamellipodia, which are thin membrane protrusions supported by a mesh of linked, branched, or unbranched actin filaments. These actin filaments interact with myosin motor proteins, creating the dynamic actomyosin complex within the cytoskeleton. Contractility, or the ability to generate contractile stress, is inherent to the actomyosin complex. It helps cells detect the stiffness of the surrounding ECM and exert contractile force for migration.
At the leading edge, the binding of an activated Rac GTPase to the WAVE complex initiates lamellipodia formation. Subsequently, the WAVE complex changes conformation to prime actin nucleators like the Arp2/3 complex for polymerization and branching of filaments. Additionally, members of the formin family localize at the sites of lamellipodia formation, elongating the unbranched filaments. The force generated by continuous actin polymerization pushes the membrane outwards and forms the membrane protrusion. The actin mesh at the leading edge contributes to the formation of focal adhesion points on the ECM, preventing the retraction of lamellipodia.
After lamellipodia formation, the dissociation of filaments begins at the rear end of the dynamic actin meshwork. The actin monomers steadily detach from the minus ends and attach to the plus ends of filaments in the actin network, resulting in treadmilling. This cyclic assembly and disassembly causes&#160; the forward movement of the entire actin filament network even though the position of individual filaments remains stationary to the substratum.
Behind the dynamic lamellipodial meshwork lies a stable network of bundled linear actin filaments called the lamella. This actin network supports the lamellipodium by developing mature and strong adhesion sites, which resist cell compression during migration. Additionally, stable actin filaments of the lamella associate with myosin proteins and use their inherent contractibility to propel the cell forward.

Mecanismo de Formação de Lamelipódio

Cells migrating in response to external stimuli form lamellipodia, which are thin membrane protrusions supported by a mesh of linked, branched, or ...

Mechanism of Filopodia Formation

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication between two cells. For example, the formation of filopodial bridges between two adjacent endothelial or epithelial cells is important for the subsequent establishment of adherens junctions. In addition, filopodia can also help the cells to reach and internalize distant targets, such as pathogens.
Filopodia Formation and Disassembly
Actin nucleation, elongation, and bundling are critical for filopodia formation and function. The filopodia core comprises 12-20 actin filaments spaced 12 nm apart. Bundling of these filaments by fascin is crucial to give filopodia the rigidity to maintain its elongated shape.
The disassembly of actin filaments during the retraction phase involves periodic helical and rotational motion of the actin shaft and is regulated by several factors. For instance, capping proteins promote filopodial retraction by shielding the polymerizing ends of the filaments from further elongation. RhoA kinase activity also regulates actin polymerization within filopodia.

Mecanismo de Formação do Filopódio

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, ...

Cancer Cell Migration through Invadopodia

Invadosome is a broad category of cell surface structures with proteolytic activity that&#160; degrades the extracellular matrix (ECM). Invadosomes are present in normal cell types, including macrophages, endothelial cells, and neurons, as well as tumor cells. Although the macrophage podosomes and tumor cell invadopodia are classified as invadosomes, they have different structures, molecular pathways, and functions. Podosomes are short structures that last for a few minutes. However, invadopodia can last for hours and are many microns in length.
In tumors, invadopodia are essential for cell intravasation and extravasation through blood vessels. They are similar to lamellipodia and filopodia, with a filamentous actin core shaped by actin nucleators and regulatory factors. The lifecycle of an invadopodium is initiated by complex processes involving various signaling cascades that remodel the cytoskeleton and cell membrane. The actin cytoskeleton is reorganized and assembled into new filaments and branches, which requires the activity of actin nucleators such as formins or the ARP2/3 complex.
The Arp2/3 complex requires nucleation-promoting factors (NPF) for optimal activity as it is slow to spontaneously initiate new actin branches. Cortactin, an NPF and scaffold protein, recruits ARP2/3 to filaments, allowing branching of the actin network. Additionally, cortactin can stabilize the newly generated branches. The formin proteins induce the elongation of unbranched actin filaments, while filament bundling is coordinated by fascin. Together these events promote actin filament polymerization and maturation of the invadopodium.&#160;
After the invadopodium stabilizes, kinesins use the neighboring microtubules as tracks to transport vesicles with proteases from the Golgi network. These vesicles release enzymes such as&#160; matrix metalloproteases, cathepsins, and serine proteases from the membrane of the invadopodia to degrade the surrounding ECM. Lastly, the actin core is disassembled to retract the invadopodium. Several proteins are implicated in the retraction pathway, but it is unclear how these proteins interact during the final stage of the invadopodium life cycle.&#160;

Migração de Células Cancerosas Através de Invadopódios

Invadosome is a broad category of cell surface structures with proteolytic activity that&#160; degrades the extracellular matrix (ECM). Invadosomes are ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms throughout their life. When cells are not able to migrate properly to their ordained locations, various disorders may occur. For example, disruption in cell migration causes chronic inflammatory diseases such as arthritis.

<h4>General Mechanism</h4>

Generally, cellular migration begins when a cell, such as a fibroblast, responds to an external-polarizing-chemical signal. As a result, one end extends itself as a protrusion called the leading edge, which attaches itself to substrates via secreted adhesive compounds, in its microenvironment. The trailing edge&mdash;the area that serves as the back of the cell&mdash;also adheres to substrates to anchor the cell. After adhesion, the cell is propelled towards its destination by a sequence of contractions that are generated by cytoskeletal motility structures. Then, the adhesive attachment at the trailing edge gets released. These steps are repeated cyclically until the fibroblast reaches its destination.

<h4>Polarization</h4>

There is a diversity in the different types of signaling molecules that initiate cell migration. They illicit two types of responses: chemokinetic and chemotactic. Chemokinesis refers to movement that occurs when signaling molecules either symmetrically or asymmetrically stimulate cell migration without dictating the directionality of the resultant movement. Chemotaxis refers to a movement where a gradient of soluble (chemotactic) or substrate-bound (haptotactic) signaling molecules dictates the directionality of cellular movement.

Membrane receptors such as G-protein coupled receptors (GPCR) and receptor tyrosine kinase receptors (RTK) detect external signaling molecules and cause an accumulation of phosphatidylinositol (3,4,5) triphosphate (PIP3) at the leading edge. The accumulation of PIP3 then leads to the activation of Rho-family Ras-like small proteins called Rac, Cdc42, and Rho. Rac and/or Cdc42 cause cytoskeletal changes such as actin polymerization at the leading edge while Rho causes actin-myosin contractions at the trailing edge. As a result of actin polymerization, protrusions are generated at the leading edge.

<h4>Types of Protrusions</h4>

Actin serves as a physical scaffold for protrusions. Consequently, the shape of protrusion structures varies depending on how actin is assembled. Two commonly studied types of protrusions are lamellipodia and filopodia. Lamellipodia are broad, sheet-like protrusions that contain a branched network of thin, short actin filaments. When lamellipodia lift away from the substrate and move backward, a notably distinct ruffling movement occurs. Lamellipodia protrusions can be found in cells like fibroblasts, immune cells, and neurons. Filopodia are thin-finger-like protrusions that emanate from cell membranes. They are often observed in cells, such as neurons, working in tandem with lamellipodia during migration.