Name: imaging molecular adhesion in cell rolling by adhesion footprint assay
Uploaded: 2021-09-27T15:00:00
Description: Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Este trabalho foi apoiado pela Fundação de Inovação do Canadá (CFI 35492), Conselho de Ciências Naturais e Engenharia do Canadá Discovery Grant (RGPIN-2017-04407), New Frontiers in Research Fund (NFRFE-2018-00969), Michael Smith Foundation for Health Research (SCH-2020-0559) e a University of British Columbia Emin Fund.

1. Rotulagem e hibridização de oligonucleotídeos
<ol><li>Redução de ligações de dissulfeto de proteína G<ol>
<li>Dissolva 10 mg de Proteína G (ProtG) em 1 mL de água ultrauso. NOTA: A proteína G aqui é modificada com um único resíduo de cisteína no C-terminus e uma tag N-terminus poly-histidine.</li>
		<li>Troca de buffer ≥20 μL de ProtG (10 mg/mL) em 1x PBS (pH 7.2) com uma coluna P6.</li>
		<li>Meça a concentração de proteína após a troca de tampão. NOTA: Concentração ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+interpretation+of+cell+rolling+velocity+distribution.">Quantitative interpretation of cell rolling velocity distribution.</a> Biophysical Journal. 120 (12), 2511-2520 (2021).</li><li>Li, I. T. S., Ha, T., Chemla, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cell+surface+adhesion+by+rotation+tracking+and+adhesion+footprinting.">Mapping cell surface adhesion by rotation tracking and adhesion footprinting.</a> Scientific Reports. 7 (1), 44502 (2017).</li><li>Yasunaga, A. B., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+fast+molecular+adhesion+by+fluorescence+footprinting.">Quantification of fast molecular adhesion by fluorescence footprinting.</a> Science Advances. 7 (34), (2021).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nature Methods. 8 (12), 1027-1040 (2011).</li><li>Zhu, M., Lerum, M. Z., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+prepare+reproducible,+homogeneous,+and+hydrolytically+stable+aminosilane-derived+layers+on+silica.">How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica.</a> Langmuir. 28 (1), 416-423 (2012).</li><li>Chandradoss, S. D., Haagsma, A. C., Lee, Y. K., Hwang, J. H., Nam, J. M., Joo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments: JoVE. (86), e50549 (2014).</li><li>Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., Jungmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+with+DNA-PAINT.">Super-resolution microscopy with DNA-PAINT.</a> Nature Protocols. 12 (6), 1198-1228 (2017).</li><li>Chalfoun, J., 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=MIST:+Accurate+and+scalable+microscopy+image+stitching+tool+with+stage+modeling+and+error+minimization.">MIST: Accurate and scalable microscopy image stitching tool with stage modeling and error minimization.</a> Scientific Reports. 7 (1), 4988 (2017).</li><li>Wang, X., 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=Constructing+modular+and+universal+single+molecule+tension+sensor+using+protein+G+to+study+mechano-sensitive+receptors.">Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors.</a> Scientific Reports. 6, 1-10 (2016).</li><li>Crockett-Torabi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectins+and+mechanisms+of+signal+transduction.">Selectins and mechanisms of signal transduction.</a> Journal of Leukocyte Biology. 63 (1), 1-14 (1998).</li><li>Ye, Z., 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+P-selectin+and+PSGL-1+axis+accelerates+atherosclerosis+via+activation+of+dendritic+cells+by+the+TLR4+signaling+pathway.">The P-selectin and PSGL-1 axis accelerates atherosclerosis via activation of dendritic cells by the TLR4 signaling pathway.</a> Cell Death and Disease. 10 (7), 507 (2019).</li><li>Saha, K., Bender, F., Gizeli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+IgG+binding+to+proteins+G+and+A:+Nonequilibrium+kinetic+and+binding+constant+determination+with+the+acoustic+waveguide+device.">Comparative study of IgG binding to proteins G and A: Nonequilibrium kinetic and binding constant determination with the acoustic waveguide device.</a> Analytical Chemistry. 75 (4), 835-842 (2003).</li><li>Murad, Y., Li, I. 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O ensaio de pegada de adesão permite a visualização dos eventos de adesão molecular entre PSGL-1 e P-selectin durante a adesão ao rolamento celular. Este processo é iniciado por captura mediada por P-selectin seguido de rolagem sob estresse fluido de tesoura. Problemas potenciais durante o experimento geralmente envolvem más rolamentos celulares ou rastros fluorescentes ausentes mesmo quando as células rolam bem. Esses problemas muitas vezes são resultantes de controles de qualidade nas etapas críticas do proto...

A cascata de aderência rolando descreve como as células circulantes se amarram e rolam ao longo da parede do vaso sanguíneo1. O rolamento passivo é mediado principalmente por selectins, uma grande classe de moléculas de adesão celular (CAMs)1. Sob o fluxo de cisalhamento de sangue, leucócitos expressando ligante de glicoproteína P-selectin -1 (PSGL-1) formam ligações altamente transitórias com p-selectina, que podem ser expressas na superfície de células endoteliais inflamadas. Esse processo é fundamental para que os leucócitos migrem para um local de inflamação2. Além disso, o PSGL-1 também é um receptor mecanosensível capaz de desencadear o estágio subsequente de adesão firme da cascata de adesão rolando após seu engajamento com P-selectin3.
Mutações genéticas que afetam a função CAM podem afetar severamente o sistema imunológico, como na rara doença da deficiência de adesão leucócito (LAD), onde o mau funcionamento das moléculas de adesão que mediam o rolamento leva a indivíduos severamente imunocomprometidos4,5,6. Além disso, as células tumorais circulantes têm sido mostradas para migrar após um processo de rolagem semelhante, levando à metástase7,8. No entanto, como o rolamento celular é rápido e dinâmico, os métodos de mecanobiologia experimental convencionais são inadequados para estudar interações moleculares durante o rolamento celular. Embora métodos de manipulação de células únicas e moléculas únicas, como microscopia de força atômica e pinça óptica, foram capazes de estudar interações moleculares, como a interação dependente da força de P-selectin com o PSGL-1 no nível de molécula única9, eles são inadequados para investigar eventos de adesão ao vivo durante o rolamento celular. Além disso, a interação caracterizada in vitro não pode responder diretamente à pergunta sobre a adesão molecular in vivo. Por exemplo, que faixa de tensão molecular é biologicamente relevante quando as células estão funcionando em seu ambiente nativo? Métodos computacionais, como simulação de dinâmica adesiva10 ou modelo simples de estado estável11, capturaram certos detalhes moleculares e como influenciam o comportamento de rolamento, mas são altamente dependentes da precisão dos parâmetros e suposições de modelagem. Outras técnicas como microscopia de força de tração podem detectar forças durante a migração celular, mas não fornecem resolução espacial suficiente ou informações quantitativas sobre tensão molecular. Nenhuma dessas técnicas pode fornecer observações experimentais diretas da dinâmica temporal, distribuição espacial e heterogeneidade de magnitude das forças moleculares, que se relacionam diretamente com a função celular e o comportamento em seu ambiente nativo.
Portanto, implementar um sensor de força molecular capaz de medir com precisão interações seletivas mediadas é crucial para melhorar nossa compreensão da adesão de rolamento. Aqui, descrevemos o protocolo para o ensaio de pegada de adesão12 onde as contas revestidas psgl-1 são enroladas em uma superfície apresentando amarras de medidor de tensão funcionalizadas p-selectin (TGTs)13. Estes TGTs são sensores de força irreversíveis baseados em DNA que resultam em um histórico permanente de eventos de ruptura na forma de leitura de fluorescência. Isso é conseguido através da ruptura do TGT (dsDNA) e, em seguida, posterior rotulagem do TGT rompido (ssDNA) com uma cadeia complementar fluorescentemente rotulada. Uma grande vantagem deste sistema é sua compatibilidade com imagens limitadas à difração e super-resolução. O fio complementar fluorescente ou rotulado pode ser permanentemente vinculado (>12 bp) para imagens limitadas à difração ou vinculado transitoriamente (7-9 bp) para imagens de super-resolução através de TINTA DE DNA. Este é um sistema ideal para estudar a adesão de rolamento, pois os TGTs são rompidos durante o rolamento ativo, mas a leitura da fluorescência é analisada após a rolagem. Os dois métodos de imagem também proporcionam ao usuário mais liberdade para investigar a adesão em rolamento. Normalmente, a imagem limitada à difração é útil para extrair força de ruptura molecular através da intensidade da fluorescência13, enquanto a imagem de super-resolução permite a análise quantitativa da densidade do receptor. Com a capacidade de investigar essas propriedades de adesão de rolamento, essa abordagem fornece uma plataforma promissora para entender o mecanismo de regulação da força sobre a adesão molecular das células rolantes sob fluxo de cisalhamento.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-channel drill guide</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>4-holes slide</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>BDH1101-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic spacer</td>
			<td>Custom made</td>
			<td></td>
			<td>Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder.</td>
		</tr>
		<tr>
			<td>Aluminium chip holder</td>
			<td>Custom made</td>
			<td></td>
			<td>Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread.</td>
		</tr>
		<tr>
			<td>Aminosilane</td>
			<td>AlfaAesar</td>
			<td>L14043</td>
			<td>CAS 1760-24-3</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution</td>
			<td>Cytiva HyClone</td>
			<td>SV3007901</td>
			<td>Pen/Strep/Fungiezone</td>
		</tr>
		<tr>
			<td>Beads, ProtG coated polystyrene</td>
			<td>Spherotech</td>
			<td>PGP-60-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>VWR</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer, DNA PAINT</td>
			<td></td>
			<td></td>
			<td>0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Buffer, T50M5</td>
			<td></td>
			<td></td>
			<td>10mM Tris, 50 mM NaCl, 5 mM MgCl2</td>
		</tr>
		<tr>
			<td>Buffer, Rolling</td>
			<td></td>
			<td></td>
			<td>HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA</td>
		</tr>
		<tr>
			<td>Buffer, Wash</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR</td>
			<td>BDH9224</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>VWR</td>
			<td>10062-868</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>VWR</td>
			<td>BDH3072-2.5LG</td>
			<td>95-98%</td>
		</tr>
		<tr>
			<td>Coverslip holding tweezers</td>
			<td>Techni-Tool</td>
			<td>758TW150</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond-coated drill bits</td>
			<td>Abrasive technology</td>
			<td>C5250510</td>
			<td>0.75 mm diamond drill</td>
		</tr>
		<tr>
			<td>DNA, amine-ssDNA (top strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGGAGGG /3AmMO/</td>
		</tr>
		<tr>
			<td>DNA, biotin-ssDNA (bottom strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG</td>
		</tr>
		<tr>
			<td>DNA, imager strand for DNA-PAINT</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>GAGGGAAA TT/3Cy3Sp/</td>
		</tr>
		<tr>
			<td>DNA, imager strand for permanent labelling</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGG /3Cy3Sp/</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>237</td>
			<td>3/4 inch width, permanent double-sided tape</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermofisher</td>
			<td>15575020</td>
			<td>0.5 M EDTA, pH 8.0</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Gorilla</td>
			<td>42001</td>
			<td>5 minute curing time</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Avantor</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GelGreen</td>
			<td>Biotium</td>
			<td>41005</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>VWR</td>
			<td>BDH3094-2.5LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Microscope slide</td>
			<td>VWR</td>
			<td>48300-026</td>
			<td>75 mm x 25 mm x 1 mm</td>
		</tr>
		<tr>
			<td>Glass, Staining jar</td>
			<td>VWR</td>
			<td>74830-150</td>
			<td>Wheaton Staining Jar (900620)</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution (HBSS)</td>
			<td>Lonza</td>
			<td>04-315Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber slide support</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>VWR</td>
			<td>BDH7690-1</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Inlets/outlets</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm&#160; polyethylene tubing (Tygon, I.D. 1/32&#8221; O.D. 3/32&#8221;) into each hollow setscrew</td>
		</tr>
		<tr>
			<td>Iscove Modified Dulbecco Media (IMDM)</td>
			<td>Lonza</td>
			<td>12-722F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>BDH9244</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Ni-NTA beads</td>
			<td>Invitrogen</td>
			<td>10103D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mailer tubes</td>
			<td>EMS</td>
			<td>EMS71406-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin P-6 Gel Columns</td>
			<td>Biorad</td>
			<td>7326200</td>
			<td>In SSC Buffer</td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>Laysan Bio</td>
			<td>MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-biotin</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>VWR</td>
			<td>470302-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein, Protein G</td>
			<td>Abcam</td>
			<td>ab155724</td>
			<td>N-terminal His-Tag and C-terminal cysteine</td>
		</tr>
		<tr>
			<td>Protein, P-selectin-Fc</td>
			<td>R&#38;D System</td>
			<td>137-PS</td>
			<td>Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF</td>
		</tr>
		<tr>
			<td>Protein, Streptavidin</td>
			<td>Cedarlane</td>
			<td>CL1005-01-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Syringe</td>
			<td>Harvard Apparatus</td>
			<td>704801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ward&#8217;s Science</td>
			<td>470302-444</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>97061-274</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermofisher</td>
			<td>22322</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>81520</td>
			<td>Syringes with PTFE luer lock, 5 mL</td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>BD</td>
			<td>305115</td>
			<td>Precision Glide 26 G, 5/8 Inch Length</td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>BDH4502-500GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, Adaptor</td>
			<td>Tygon</td>
			<td>ABW00001</td>
			<td>Formulation 3350, I.D. 1/32&#8221;; O.D. 3/32&#8221;</td>
		</tr>
		<tr>
			<td>Tubing, Polyethylene</td>
			<td>BD Intramedic</td>
			<td>427406</td>
			<td>Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>93773</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging molecular adhesion in cell rolling by adhesion footprint assay

A adesão de rolamento, facilitada por interações seletivas mediadas, é uma motilidade altamente dinâmica e passiva no recrutamento de leucócitos para o local da inflamação. Este fenômeno ocorre em venules pós-capilares, onde o fluxo sanguíneo empurra leucócitos em um movimento de rolamento sobre as células endoteliais. O rolamento estável requer um delicado equilíbrio entre a formação de vínculos de adesão e sua dissociação mecanicamente conduzida, permitindo que a célula permaneça presa à superfície enquanto rola na direção do fluxo. Ao contrário de outros processos de adesão que ocorrem em ambientes relativamente estáticos, a adesão é altamente dinâmica à medida que as células rolante viajam sobre milhares de mícrons a dezenas de mícrons por segundo. Consequentemente, métodos convencionais de mecanobiologia, como microscopia de força de tração, são inadequados para medir os eventos de adesão individual e as forças moleculares associadas devido à curta escala de tempo e alta sensibilidade necessária. Aqui, descrevemos nossa mais recente implementação do ensaio de pegada de adesão para imagem das interações P-selectin: PSGL-1 na adesão de rolamento no nível molecular. Este método utiliza amarras irreversíveis de medidor de tensão baseada em DNA para produzir uma história permanente de eventos de adesão molecular na forma de trilhas de fluorescência. Essas faixas podem ser imagens de duas maneiras: (1) costurando milhares de imagens limitadas à difração para produzir um grande campo de visão, permitindo a extração de pegada de adesão de cada célula rolante sobre milhares de mícrons de comprimento, (2) realizando DNA-PAINT para reconstruir imagens de super-resolução das faixas de fluorescência dentro de um pequeno campo de visão. Neste estudo, o ensaio de pegada de adesão foi utilizado para estudar células HL-60 rolando em diferentes estresses de tesoura. Ao fazê-lo, fomos capazes de imaginar a distribuição espacial da interação P-selectin: PSGL-1 e obter insights sobre suas forças moleculares através da intensidade da fluorescência. Assim, este método fornece as bases para a investigação quantitativa das diversas interações célula-superfície envolvidas na adesão de rolamento a nível molecular.

O protocolo acima descreve o procedimento experimental do ensaio de pegada de adesão. O fluxo de trabalho do experimento geral é ilustrado na Figura 1, desde a montagem da câmara de fluxo (Figura 1A) até a funcionalização da superfície (Figura 1B) e etapas de experimento e imagem (Figura 1C).
A Figura 2 é um resultado representativo para ...

Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Este protocolo apresenta os procedimentos experimentais para realizar o ensaio da pegada de adesão para a imagem dos eventos de adesão durante a adesão rápida ao rolamento celular.

A adesão molecular de imagem em rolamento celular por ensaio de pegada de adesão

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Este protocolo ajuda a estabelecer a conexão entre a força molecular e o comportamento de rolagem celular, além de permitir mapear espacialmente e quantificar a adesão ao rolamento no nível molecular. Esta técnica permite estudar eventos de adesão individual durante o rolamento celular real, permitindo medir diretamente a força de adesão molecular relevante fisiológica. A preparação da superfície usando o tempo de concentração e incubação adequado em cada etapa é crucial para alcançar a funcionalidade de superfície de alta qualidade.A qualidade das bioconjugações e as condições adequadas também são cruciais. A demonstração visual é fundamental para ajudar os pesquisadores a replicar esse protocolo. Em particular, as etapas como a montagem da câmara de fluxo e imagens pós-processamento se beneficiarão muito de uma demonstração visual.Comece espalhando uma pequena quantidade de epóxi em ambos os lados da fita de dois lados com uma lâmina de barbear. Usando um laser, corte a fita revestida de epóxi para criar quatro canais. Crie o chip de fluxo sanduitando a fita epóxi entre um slide de quatro buracos e uma mancha de cobertura PEG.Usando uma ponta de pipeta, aplique pressão suave ao longo do comprimento dos canais para criar uma boa vedação e, em seguida, cure o epóxi por um mínimo de uma hora. Alinhe o chip para que a abertura de cada canal esteja posicionada nos centros do adaptador. Em seguida, coloque dois espaçadores acrílicos transparentes em cima do chip.Aplique pressão firme no meio do bloco e parafuso nas extremidades de cada espaçador. Enrosque as entradas nos orifícios roscados do outro lado do suporte e monitore a condição de vedação através do bloco acrílico transparente. Flua 200 microliters de tampão de lavagem na câmara para verificar se há vazamento.Se as bolhas se formarem no canal, empurre agressivamente mais 200 microliters de tampão de lavagem para remover as bolhas. Adicione 40 microliters de BSA à câmara de fluxo para evitar a ligação inespecífica e incubar por 10 minutos na câmara de umidade. Após a incubação, adicione 40 microliters na Tween 20 à câmara de fluxo e incuba novamente por 10 minutos para reduzir ainda mais a ligação inespecífica.Em seguida, lave o canal com 200 microliters de tampão de lavagem para remover todos os agentes de passivação. Para a funcionalização da superfície da câmara, adicione 40 microliters de Streptavidin à câmara de fluxo e incuba por 20 minutos, depois lave a câmara com 200 microliters de tampão de lavagem. Agora, adicione 40 microliters de proteína hibridizada G-TGT à câmara de fluxo e incubar por 20 minutos.Depois de lavar com tampão de lavagem, adicione 40 microliters de proteína G-TGT top strand e incuba por 20 minutos para completar qualquer fio inferior TGT não higiido na superfície e depois lave com tampão de lavagem. Por fim, adicione 40 microliters de P-selectin-Fc à câmara de fluxo e incubar por 60 minutos antes de lavar com tampão de lavagem. Encha uma seringa de vidro de cinco mililitros com o tampão de rolamento e toque nas laterais da seringa para desalojar e empurrar as bolhas para fora enquanto flutuam em direção à ponta.Depois de inserir uma agulha estéril em um pedaço de tubo de polietileno de 200 milímetros, conecte a agulha à seringa de vidro. Fixar a seringa na bomba de seringa e inclinar a bomba de seringa de tal forma que o lado do êmbolo seja elevado para evitar que bolhas de ar entrem no canal. Insira a extremidade do tubo na entrada da câmara de fluxo.Insira uma extremidade de outra peça de 200 milímetros da tubulação de polietileno na tomada e submerse a outra extremidade em um béquer de resíduos. Pegue de um a dois mililitros da amostra de suspensão celular e centrífuga para pelotar as células. Remova o meio e levemente resuspenque as células em 500 microliters do buffer de rolamento.Desconecte cuidadosamente o tubo das entradas e a saída e pipeta 40 microliters da suspensão celular na câmara de fluxo. Reconecte a tubulação como descrito anteriormente, garantindo que as bolhas não sejam introduzidas no canal de fluxo. Inicie o experimento de rolagem celular iniciando a bomba de seringa nas taxas de fluxo desejadas.Observe o rolamento de células usando um microscópio de campo escuro com um objetivo de 10X. Uma vez que o experimento seja concluído, remova as células do canal infundindo o tampão de rolamento a 100 mililitros por hora até que a superfície esteja livre de células. Para a imagem das faixas locais por DNA-PAINT, adicione 40 microliters de fio imager DNA-PAINT preparado em buffer DNA-PAINT ao canal.Realize microscopia de fluorescência de reflexão interna total utilizando as condições mencionadas no manuscrito do texto. Localize e torne as imagens de super resolução. Para fotografar as faixas longas por rotulagem permanente, adicione o fio imager permanente e incubar por 120 segundos no buffer T50M5.Em seguida, lave o canal infundindo 200 microliters de tampão de lavagem. Grave uma imagem com o laser de excitação desligado para obter ruído de câmera de fundo. Imagem uma grande área em um padrão de grade pela microscopia TIRF.Programe o microscópio para digitalizar a área de 400 por 50 imagens e divida os dados brutos em arquivos TIP individuais cada um contendo um máximo de 10.000 imagens usando o programa ImageJ. Achate todas as imagens usando o perfil de iluminação e use o plugin MIST para costurar as imagens. O resultado para a caracterização da bioconjugação da proteína G ssDNA mostrou que quase uma é uma razão de proteína G para ssDNA onde a proteína G Maleimide ssDNA e espectro tampão de eluição imidazol da base ortogonal para a montagem do espectro do produto de bioconjugação.Native PAGE foi usado para confirmar a bioconjugação mostrando as bandas verdes brilhantes coincidindo com a banda de proteína monomérica G indicando conjugação bem sucedida e bom rendimento. O perfil de iluminação TIRF introduzido a partir de uma fibra de modo único é geralmente mais brilhante no meio do campo de visão e dimmer ao redor das bordas. Para compensar a iluminação desigual e achatar as imagens para análise quantitativa, o perfil de iluminação foi determinado pela média de milhares de quadros individuais.As imagens achatadas foram produzidas subtraindo o ruído da câmera tanto de perfis brutos quanto de iluminação e, em seguida, normalizando pelo perfil de iluminação. A costura bruta mostrou padrões periódicos claros correspondentes às imagens não corrigidas, enquanto o mesmo campo de visão costurado a partir de imagens achatadas produziu um fundo plano. Um perfil de fluxo de rampa foi usado para determinar a amplitude de estresse da cisalhamento que resultou em rolagem celular e fluxo de fluorescência sob as quais uma pegada típica de adesão unicelular poderia ser vista.Resultados subótimos e ótimos de trilhas fluorescentes com contraste insuficiente, densidade excessiva da faixa, densidade e contraste da pista ideais e difusão limitada e imagem DNA-PAINT são mostrados. O tempo de incubação superior a 60 minutos garante a funcionalização da superfície e a construção adequada da câmara impede a passagem de bolhas que danificam a superfície funcionalizada. Este procedimento é aplicável para análise quantitativa da força molecular envolvida na adesão ao rolamento e permite que os pesquisadores entendam o comportamento de rolagem dos diferentes tipos celulares.Essa técnica permite que os pesquisadores explorem novas questões sobre eventos de adesão rápida que levem a um avanço da pesquisa de molécula única e mecanobiologia.

imaging-molecular-adhesion-in-cell-rolling-by-adhesion-footprint-assay

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Ensaio para a Adesão e Invasão Agar em S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Biologia

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Dissecção do Organizador e Explantes Pólo animal a partir de embriões Xenopus laevis e Montagem de um ensaio de Adesão Celular

Title Cell Encapsulation by Droplets

Encapsulation título celular por Droplets

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

Um Ensaio Comportamental para Medida de capacidade de resposta a alterações em Zebrafish intensidades de luz

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Adesão e agregação plaquetária sob fluxo usando células de fluxo microfluídicos

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Ativando Autophagy pelo exercício aeróbio em ratos

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analisando celular superfície de adesão remodelação em resposta à tensão mecânica usando pérolas magnéticas

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...