Plasmidique vinculine-GFP a été un cadeau de Kenneth Yamada, National Institutes of Health, Institut national de recherche dentaire et craniofaciale, Bethesda, Maryland, et l&#39;actine-MRFP plasmide a été un cadeau de Michael Davidson, Florida State University, Tallahassee, en Floride. Ce travail a été soutenu par la NSF Career Award # 0747334 et AHA-National SDG # 0835205N d&#39;Andreea Trache.

1. Présentation du système de microscope intégré Le système de microscope qui est utilisé pour ces études a été décrite en détail 1. En bref, une inversé Olympus IX-81 microscope TIRF attachement (Olympus, Center Valley, Pennsylvanie) est combiné avec la CSU-22 Yokogawa numérisation tête (Yokogawa Electric Inc, Japon). Un microscope à force atomique Bioscope SZ (Veeco Instruments Inc, Santa Barbara, Californie) est monté sur le dessus du microscope optique inversé, et l&#39;ensemble du système est placé sur le dessus d&#39;une table de qualité optique de recherche (Newport, Irvine, CA). Le montage confocale composé à partir du scanner tourne-disque, roue à filtres externes et sa caméra EMCCD (Roper Scientific-Photometrics, Tucson, AZ) sont solidaires d&#39;une plaque de base en aluminium placée sur un coussin de silicium nécessaire à l&#39;amortissement des vibrations. Le scanner se connecte confocale avec le microscope grâce à une bride avec un collier qui peut être foulé découplé du microscope lors des mesures AFM, telle qu&#39;aucune vibration du scanner tourne-disque aura une incidence sur les mesures AFM. Un laser argon-krypton (Spectra Physics, Mountain View, CA) est utilisé comme source d&#39;excitation pour les deux FRBR et les modes d&#39;imagerie confocale, et peut être couplé alternativement dans deux fibres optiques qui offrent le laser à l&#39;attachement FRBR ou la tête de balayage, respectivement . Deux ordinateurs de contrôle du système: un logiciel de contrôle Slidebook l&#39;imagerie optique et un logiciel de contrôle Nanoscope l&#39;AFM. 2. Culture de cellules Les cellules vivantes exprimant la GFP des constructions ciblées contre des protéines spécifiques doivent être placés dans un plat en verre à fond la culture de cellules 24 heures avant l&#39;expérience de stimulation mécanique. Le jour de l&#39;expérience de remplacer le milieu de culture cellulaire avec du phénol rouge sans moyen. La boîte de culture cellulaire est monté sur la scène AFM avec un collier magnétique. 3. AFM Astuce Préparation et Microscope Set-up La pointe AFM personnalisé avec un micron 2 biotinilés billes de verre seront lavées cinq fois avec du DPBS et ensuite incubées 5 min avec l&#39;avidine (1 mg / ml), lavé à nouveau avec du DPBS cinq fois, puis incubées 5 min à 1 mg / mL biotinilés fibronectine. La pointe sera lavé cinq fois plus à la fin 2, monté sur le scanner AFM, puis la jupe de protection en silicone sera placé autour du support. Régler le microscope pour visionner des vidéos d&#39;aligner la pointe AFM dans le centre du champ de vue. Aligner le levier optique en plaçant le faisceau laser à la fin du cantilever, puis modifier l&#39;objectif d&#39;un objectif à fort grossissement approprié pour l&#39;imagerie cellulaire unique. La constante du ressort de la pointe doit être déterminé avant de commencer l&#39;expérience. Ce paramètre sera mesuré en utilisant la méthode de réglage thermique disponible dans le logiciel Nanoscope. Commutateur de caméra vidéo pour caméra EMCCD pour permettre l&#39;imagerie de fluorescence. Ensuite, choisissez une cellule et amener la pointe AFM en contact avec cette cellule spécifique. Après 20 min de temps d&#39;attente, ce qui est nécessaire pour une forte adhérence focale pour former au point de l&#39;AFM de contact avec la surface des cellules, la procédure de stimulation mécanique va commencer. 4. Stimulation mécanique des cellules vivantes FRBR 3-4 imagerie sera la méthode de choix pour la visualisation et la quantification des adhérences focales, et le filage du disque imagerie confocale 5 sera utilisé pour visualiser le cytosquelette d&#39;actine dans tout le corps cellulaire. L&#39;AFM 6 sera utilisé en imagerie mode de contact sous fluide. Une image TIRF ou confocale de la cellule va être acquis avant de commencer la procédure de stimulation mécanique. La stimulation mécanique sera induite par la sonde AFM fonctionnalisées qui sera déplacé vers le haut par étapes discrètes tous les 3-5 min sur une période de temps (1-1,5 heure). La cellule réactive-réponse sera enregistré en continu dans les logiciels Nanoscope comme une série d&#39;images de hauteur, chaque ligne de l&#39;image représentant une unité de temps de 1 sec. Alors que l&#39;acquisition des données AFM, l&#39;opérateur surveille en tout temps, le z-position de la pointe AFM pour déterminer le moment approprié pour l&#39;attraction suivante. Après chaque stimulation mécanique, il est possible d&#39;acquérir une image TIRF ou confocale de la cellule dans Slidebook logiciels, tout en continuant à acquérir des données de l&#39;AFM. Les images optiques peuvent être traitées hors ligne pour créer des films qui montrent cytosquelette d&#39;actine et la réorganisation focale adhérences. En outre, chaque image peut être traitée afin de déterminer quantitativement surface cellulaire, la zone de protéines spécifiques, et d&#39;analyser la morphologie cellulaire en général. A la fin de l&#39;expérience de la pointe AFM est retirée de la cellule et la sensibilité pointe AFM seront mesurés dans le logiciel Nanoscope. L&#39;expérience est très sensible au bruit! Toute parler ou de microscope toucher pendant l&#39;expérience devrait être évitée. Afin de minimiser le bruit dans les mesures AFM, l&#39;confocale à disque rotatif doit être découplée de la loupe. Le temps d&#39;attente pour former une forte adhésion focale entre la surface des cellules et la pointe de l&#39;AFM peut varier en fonction du type cellulaire utilisé et le revêtement de matrice à la pointe. 5. Les résultats représentatifs <img src="/files/ftp_upload/2072/2072fig1.jpg" alt="Figure 1" /> Figure 1. Idem cellules musculaires lisses vasculaires (CMLV) transfectées avec l&#39;actine et RDRF vinculine-GFP a été imagée par: (A) l&#39;AFM à la surface cellulaire apicale; (B) la FRBR à la surface des cellules basales et (C) tourne-disque microscopie confocale partout dans le corps cellulaire. L&#39;image confocale est montré que la projection maximale d&#39;une pile en 3-D. Bonne chevauchements entre l&#39;AFM et de la FRBR ou le filage des images de disque confocale sont présentés dans (D) et (E), respectivement. Taille de l&#39;image est de 64 x 50 microns. <img src="/files/ftp_upload/2072/2072fig2.jpg" alt="Figure 2" /> Figure 2. Un CMLV transfectées avec l&#39;actine-RDRF était mécaniquement stimulées par l&#39;AFM et imagée par disque rotatif microscopie confocale Les flèches indiquent que les fibres d&#39;actine.: (A) polymériser / épaissir, (b) dépolymériser / disparaître, ou (c) bundle ensemble en raison à la restructuration de l&#39;actine. Blanc lignes pointillées représentent la pointe de l&#39;AFM-dessus de la cellule. Taille de l&#39;image 91 x 91 microns.

<ol>
	<li>Trache, A., Lim, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microscopy+for+real-time+imaging+of+mechanotransduction+studies+in+live+cells.">Integrated microscopy for real-time imaging of mechanotransduction studies in live cells.</a> J Biomed Opt. 14, 034024-03 (2009).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy.">Atomic Force Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.17 (2008).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J Biomed Opt. 6, 6-13 (2001).</li><li>Trache, A., Meininger, G. A., Coico, R., Kowalik, T., Quarles, J. M., Stevenson, B., Taylor, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Current Protocols in Microbiology. , 2.1-2.22 (2008).</li><li>Inoue, S., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-view+high-speed+confocal+scanner:+the+CSU-10.">Direct-view high-speed confocal scanner: the CSU-10.</a> Methods Cell Biol. 70, 87-127 (2002).</li><li>Binnig, G., Quate, C. F., Gerber, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Phys Rev Lett. 56, 930-933 (1986).</li><li>Stamenovi&#263;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+mechanics+in+airway+smooth+muscle+cells.">Cytoskeletal mechanics in airway smooth muscle cells.</a> Respir Physiol Neurobiol. 163, 25-32 (2008).</li><li>Gunst, S. J., Tang, D. D., Opazo Saez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+remodeling+of+the+airway+smooth+muscle+cell:+a+mechanism+for+adaptation+to+mechanical+forces+in+the+lung.">Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung.</a> Respir Physiol Neurobiol. 137, 151-168 (2003).</li><li>Worth, N. F., Rolfe, B. E., Song, J., Campbell, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+cell+phenotypic+modulation+in+culture+is+associated+with+reorganization+of+contractile+and+cytoskeletal+proteins.">Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganization of contractile and cytoskeletal proteins.</a> Cell Motility and the Cytoskeleton. 49, 130-145 (2001).</li><li>Romer, H. R., Birukov, K. G., Garcia, J. G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+paradigm+for+a+signaling+nexus.">Focal Adhesions: paradigm for a signaling nexus.</a> Circulation Research. 98, 606-616 (2006).</li><li>Opazo Saez, A., Zhang, W., Wu, Y., Turner, C. E., Tang, D. D., Gunst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+development+during+contractile+stimulation+of+smooth+muscle+requires+recruitment+of+paxillin+and+vinculin+to+the+membrane.">Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane.</a> Am J Physiol Cell Physiol. 286, C433-C447 (2004).</li><li>Alenghat, F. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction:+all+signals+point+to+cytoskeleton,+matrix+and+integrins.">Mechanotransduction: all signals point to cytoskeleton, matrix and integrins.</a> Sciences STKE. 119, pe6-pe6 (2002).</li><li>Zamir, E., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+complexity+and+dynamics+of+cell-matrix+adhesions.">Molecular complexity and dynamics of cell-matrix adhesions.</a> J Cell Sci. 114, 3583-3590 (2001).</li><li>Geiger, B., Bershdsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+mechanosensory+function+of+focal+contacts.">Assembly and mechanosensory function of focal contacts.</a> Curr Opin Cell Biol. 13, 584-592 (2001).</li><li>Sastry, S. K., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Adhesions:+a+nexus+for+intracellular+signaling+and+cytoskeletal+dynamics.">Focal Adhesions: a nexus for intracellular signaling and cytoskeletal dynamics.</a> Exp Cell Res. 261, 25-36 (2000).</li><li>Orr, A. W., Helmke, B. P., Blackman, B. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mechanotransduction.">Mechanisms of mechanotransduction.</a> Dev Cell. 10, 11-20 (2006).</li></ol>

Adhérences Cytosquelette 7-10 et 11-16 focale sont des structures dynamiques qui s&#39;assemblent, se dispersent et retournent comme des cellules migrent ou répondre à une force mécanique 17. Ils ont des fonctions importantes dans la régulation de la croissance cellulaire, la survie et l&#39;expression des gènes. Connaissant leur distribution spécifique et de la dynamique permet de mieux comprendre comment les cellules communiquent avec la matrice et entre eux. <p class="jove_...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AFM tip</td>
<td>&nbsp;</td>
<td>Novascan Technologies</td>
<td>&nbsp;</td>
<td>2 micron Borosilicate bead, 0.01N/m, Biotin Surface</td>
</tr>
<tr>
<td>avidin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A9275</td>
<td>1mg/mL in DPBS</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>F4759</td>
<td>1 mg/mL</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21600-051</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa transfection kit for Smooth Muscle Cells</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPI-1004</td>
<td>&nbsp;</td>
<tr>
<td>Mattek glass bottom dishes</td>
<td>&nbsp;</td>
<td>MatTek Corporation</td>
<td>P60G-1.5-30-F</td>
<td>Sterile 60mm Falcon dishes with glass bottom coverslip no. 1.5</td>
</tr>		</tbody>
		</table>
		</div>

live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy

Pour comprendre le mécanisme par lequel les cellules vivantes sens des forces mécaniques, et comment ils réagissent et s&#39;adaptent à leur environnement, une nouvelle technologie capable de d&#39;étudier le comportement des cellules au niveau sub-cellulaire avec une résolution spatiale et temporelle élevée a été développé. Ainsi, un microscope à force atomique (AFM) a été intégré à la réflexion interne totale de fluorescence (FRBR) microscopie et rotation rapide du disque (DSE) microscopie confocale. Le système intégré est largement applicable dans un large éventail d&#39;études moléculaires dynamiques dans toute cellules adhérentes en direct, permettant d&#39;imagerie optique directe des réponses des cellules à la stimulation mécanique en temps réel. Réarrangement significatif des filaments d&#39;actine et adhésions focales a été montré en raison de la stimulation mécanique locale à la surface cellulaire apicale qui induit des changements dans la structure cellulaire dans le corps cellulaire. Ces techniques innovantes fournira de nouvelles informations pour comprendre la restructuration de cellules vivantes et dynamiques en réponse à une force mécanique. Un protocole détaillé et un ensemble de données représentatives que la réponse cellulaire spectacle à la stimulation mécanique sont présentées.

Watch this Scientific Journal Video about Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy at JoVE.com

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Ce document vise à instruire le lecteur dans le fonctionnement d&#39;un système intégré à force atomique, microscope optique d&#39;imagerie pour la stimulation mécanique des cellules vivantes en culture. Un protocole étape par étape est présentée. Un ensemble représentatif de données qui montre la réponse de cellules vivantes à la stimulation mécanique est présenté.

Réponse Live Cell à la stimulation mécanique étudiée par microscopie à force atomique et optique intégré

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

live-cell-response-to-mechanical-stimulation-studied-integrated

Research

JoVE Journal

Biology

11.2K vues.  Texas A&M Health Science Center. Ce document vise à instruire le lecteur dans le fonctionnement d'un système intégré à force atomique, microscope optique d'imagerie pour la stimulation mécanique des cellules vivantes en culture. Un protocole étape par étape est présentée. Un ensemble représentatif de données qui montre la réponse de cellules vivantes à la stimulation mécanique est présenté.

Vidéo: Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de phase et les interférences de contraste différentiel (DIC) Microscopie

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Recherche

Biologie

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Synapses présynaptique silencieux étudiée en microscopie optique

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Mécanique de caractérisation du tissu pulmonaire par microscopie à force atomique

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Préparation d’échantillons pour la microscopie à force atomique à virion unique et l’imagerie par fluorescence à super-résolution

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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.

Analyse de surface cellulaire Adhérence remodelage en réponse à la tension mécanique utilisant des billes magnétiques

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

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Mesure de l&#39;absorption de glucose et réponse à la stimulation de l&#39;insuline dans<em&gt; In Vitro</em&gt; Myotubes primaires humains cultivés

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Évaluation de Dictyostelium discoideum réponse à la Stimulation mécanique aiguë

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

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.

Microscopie à Force saillie : Une méthode pour quantifier les Forces développées par cellule saillies

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

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Caractérisation des propriétés mécaniques de la paroi cellulaire primaire dans les organes vivants de plantes à l’aide de la microscopie à force atomique

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...