Gli autori sono grati ad Anna Labernadie, Guillaume Charrière e Patrick Delobelle per il loro contributo iniziale di questo lavoro e di Matthieu Sanchez e Françoise Viala per il loro aiuto con riprese video e montaggio. Quest'opera è stata sostenuta dall'Agence Nationale de la Recherche (ANR14-CE11-0020-02), la Fondation pour la Recherche Médicale (FRM DEQ2016 0334894), INSERM piano cancro, Fondation Tolosa e Human Frontier Science Program (RGP0035/2016).

1. preparazione di griglie rivestite con Formvar
<ol><li>Pulire le griglie di microscopia elettronica con acetone puro e farle asciugare su carta da filtro. Quindi pulire un vetrino da microscopio con etanolo puro, pulire con una carta per lenti e rimuovere la polvere con un soffietto.</li>
	<li>Posto un vetrino pulito etanolo verticalmente nell'imbuto di un dispositivo di fusione pellicola contenente una soluzione di Formvar nella parte inferiore. Coprire la parte superiore dell'imbuto.</li>
	<li>Con la lampadina atomizzatore pompa 100 mL di soluzione di Formvar (0,5%, dicloruro di etilene) fino a quando il livello raggiunge due terzi della diapositiva.</li>
	<li>Mantenere il vetrino nella soluzione per 1 min.</li>
	<li>Aprire la valvola del dispositivo per drenare la soluzione di Formvar in un flusso costante. Una portata di 10 a 15 mL/s produrrà un film Formvar di 30-80 nm. Lo spessore del film è determinato dalla concentrazione della soluzione Formvar e per il tasso di drenaggio. Nota: Il tasso di drenaggio può essere controllata scaricando la pressione tramite la valvola aria aprendo lentamente la valvola. Il più veloce il drenaggio, il più spesso il film. In pratica, perché è difficile da predire lo spessore del film dalla portata Formvar, prepariamo diversi lotti di Formvar film e controllare il loro spessore di AFM (vedi passo 2).</li>
	<li>Rimuovere il vetrino dalla camera e asciugarlo delicatamente su carta da filtro per rimuovere qualsiasi liquido Formvar.</li>
	<li>Tagliare un rettangolo di 20 x 50 mm dal film Formvar con una lama di rasoio.</li>
	<li>Riempire un bicchiere con acqua distillata e immergersi lentamente la diapositiva verticalmente in acqua per galleggiare fuori la pellicola: il film deve galleggiare sulla superficie dell'acqua.</li>
	<li>Luogo asciutto pulito acetone griglie sulla pellicola, splendente verso l'alto.</li>
	<li>Posizionare un coprioggetto in vetro tondo (Ø 12 mm) su alcune delle griglie. Questo vetrino coprioggetti servirà a misurare lo spessore del film (vedi passo 2).</li>
	<li>Coprire un vetrino da microscopio con un adesivo bianco ridimensionato per adattarlo. Immergere la diapositiva verticalmente al bordo del film Formvar galleggiante fino a quando l'intero film aderisce alla diapositiva. Rimuovere l'acqua in eccesso dal vetrino con carta da filtro e lasciarlo asciugare a temperatura ambiente per una notte.</li>
</ol>2. misurazione dello spessore del Film
<ol><li>Tagliare il Formvar intorno il coprioggetto con pinzette per staccarlo dalla diapositiva.</li>
	<li>Segnare la posizione della griglia sotto il vetrino coprioggetto con una penna.</li>
	<li>Tagliare il Formvar intorno alla griglia contrassegnata per staccarla dal vetrino coprioggetti. Pertanto non vi sarà un buco nello strato Formvar rimanente, che verranno permetterà di misurare lo spessore del film. Coprire il vetrino coprioggetto con PBS e collocarla su un vetrino sul microscopio.</li>
	<li>Montare un cantilever di nitruro di silicio sul mattone di vetro AFM, assicurandosi che la striscia dorata non è coperto dalla punta della molla. Nota: La sensibilità e la costante elastica del cantilever dovrebbe avere precedentemente stati calibrati in PBS.</li>
	<li>Montare il blocco di vetro sul modulo del AFM e quindi posizionare il modulo sul microscopio.</li>
	<li>Collocare il vetrino coprioggetto rivestite con Formvar sulla camera di osservazione e coprirlo con 500 µ l di PBS.</li>
	<li>Il bordo del foro nel foglio Formvar utilizzando AFM in modalità di contatto e un punto di regolazione forza di 0,5 nN la scansione.</li>
	<li>Utilizzare la superficie del vetrino coprioggetti di vetro come il riferimento per misure di altezza e valutare lo spessore di Formvar come l'altezza della sezione trasversale (eseguire almeno 10 misurazioni per ogni batch di Formvar).</li>
</ol>3. cellule su griglie di semina
<ol><li>Sotto una cappa sterile, mettere una striscia (12 x 3 mm) del nastro bi adesivo su un vetrino coprioggetti.</li>
	<li>Tagliare una griglia Formvar-rivestito con pinzette e metterlo a testa in giù sulla striscia adesiva (solo il bordo della griglia deve essere attaccata al nastro). Il film di Formvar deve affrontare il vetrino coprioggetti.</li>
	<li>Mettere il dispositivo in una cultura ben.</li>
	<li>Mettere una goccia di 10 µ l di RPMI senza FCS contenente 104 macrofagi differenziati da monociti umani16. Nota: Assicurarsi di non toccare la griglia con la punta della pipetta per evitare di danneggiare il rivestimento di Formvar.</li>
	<li>Incubare per 30 min (37 ° C, 5% CO2) che le cellule aderiscono.</li>
	<li>Riempire il pozzo con 2 mL di RPMI contenente 10% FCS.</li>
	<li>Incubare il dispositivo per 2 ore a 37 ° C e 5% di CO2 di lasciare le cellule aderiscono prima osservazione AFM.</li>
</ol>4. topografia misure delle deformazioni indotte da Podosome
<ol><li>Attaccare due strisce di raddoppiato-nastro biadesivo su un fondo di vetro capsula di Petri. Nota: La distanza tra le due strisce dovrebbe essere inferiore al diametro di griglia.</li>
	<li>Staccare con cura la griglia placcata con macrofagi dal nastro adesivo.</li>
	<li>Capovolgere la griglia al fine di avere le cellule rivolto verso il vetro e la griglia tra le due strisce adesive di bastone.</li>
	<li>Correzione della griglia con due nuove strisce di adesivo raddoppiato: la griglia sarà così essere intramezzata fra due strisce adesive, lasciando l'area centrale della griglia accessibile a punta dell'AFM. Nota: Assicurarsi che la griglia è ben fissata e non attorcigliata; altrimenti il cantilever AFM potrebbe non essere in grado di avvicinarsi alla superficie di Formvar.</li>
	<li>Riempire la capsula di Petri con 2 mL di terreno di coltura pre-riscaldata 37 ° C (RPMI con 10% FCS) completati con 10 mM HEPES (pH 7.4).</li>
	<li>Posizionare la piastra di coltura su un riscaldatore di piatto di 37 ° C.</li>
	<li>Installare il riscaldatore di piatto sul palco AFM.</li>
	<li>Lasciare che il sistema si stabilizza a 37 ° C.</li>
	<li>In modalità a contatto con una forza di 0,5 nN, fare una prima immagine globale per individuare podosomes e poi la scansione la topografia di Formvar a 3 Hz con un circa 20 nm-grande pixel. Nota: Evitare l'analisi vicino ai bordi della griglia.</li>
</ol>

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Conflitti di interesse non dichiarati.

Proprietà del materiale
La scelta del materiale per la membrana deformabile, nel nostro caso Formvar, deve soddisfare alcuni requisiti. Il materiale deve essere trasparente alla luce visibile e presentano fluorescenza auto limitato per consentire osservazioni in campo chiaro e microscopia a fluorescenza. La rugosità del film sottile deve essere ben di sotto di 10 nm per evitare qualsiasi effetto topografica su adesione delle cellule e per consentire la chiara osservazione de...

Cellule animali interagiscono fisicamente con la matrice e le altre cellule che costituiscono il loro ambiente1. Ciò è necessario per loro di migrare, interiorizzare corpi, acquisire informazioni esterne o differenziare. In tali processi, la cella deve generare forze meccaniche e, come numerosi studi hanno dimostrato negli ultimi anni, la capacità di una cellula di generare forze e sonda ambiente influenza il comportamento biologico, dirigendo per esempio proliferazione o differenziazione2,3. A sua volta, la misurazione delle forze cellulare è un aiuto importante per studiare la regolazione della forza di generazione e capire la sua implicazione nella cella comportamento e tessuto destino4,5.
Ultimi anni hanno visto lo sviluppo di numerose tecniche per misurare le forze che una cella può esercitare sul suo ambiente6. La maggior parte di questi è stati strumentale nel rivelare che la trazione delle forze che cellule esercitano come si tirano su sonde mobili o un substrato deformabile. Tuttavia, le forze meccaniche coinvolte in protrusione nell'ambiente extracellulare soffrono di una mancanza di tecniche di misurazione e sono fino ad oggi non è ben caratterizzato.
Per ovviare a questa limitazione, presentiamo un metodo per misurare forze esercitate ortogonalmente al substrato. Essa consiste nella placcatura cellule viventi in un sottile foglio elastico che può deformare in direzione ortogonale, che permette di misurare la deformazione di substrato dalle cellule e dedurre le forze coinvolte. Topografia di substrato è misurata con risoluzione nanometrica mediante microscopia a forza atomica e la valutazione delle forze di deformazione si basa sulla conoscenza della geometria delle strutture cellulari protrusiva7,8, 9.
Qui, descriviamo l'installazione e la sua applicazione per misurare le forze generate dal podosomes, strutture di adesione protrusiva formate dai macrofagi per la loro migrazione mesenchimale in ambienti tridimensionali10,11, 12,13,14,15,16,17. Noi crediamo che questa tecnica avanzerà la comprensione della creazione delle forze e la sua regolamentazione in molti processi cellulari che coinvolgono la protrusione.

<table><tbody><tr><td>200 mesh nickel grids</td><td>Electron Microscopy Sciences</td><td>G200-Ni</td><td></td></tr><tr><td>Filter paper</td><td>Sigma-Aldrich</td><td>1001-055</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>10235612</td><td></td></tr><tr><td>White stickers 26 x 70 mm</td><td>Avery</td><td>DP033-100</td><td></td></tr><tr><td>Film casting device with valve in its outlet</td><td>Electron Microscopy Sciences</td><td>71305-01</td><td></td></tr><tr><td>Razorblades</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>1.08543.0250</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>20066.321</td><td></td></tr><tr><td>Formvar 0.5% solution in ethylene dichloride</td><td>Electron Microscopy Sciences</td><td>15820</td><td></td></tr><tr><td>12 mm coverslips</td><td>VWR</td><td>631-0666</td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>JPK Instruments</td><td>NanoWizard III</td><td></td></tr><tr><td>Temperature-controlled sample holder&amp;nbsp;</td><td>JPK Instruments</td><td>BioCell</td><td></td></tr><tr><td>Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td><td>Veeco Instruments</td><td>MLCT-AUHW</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Double-sided adhesive tape</td><td>APLI AGIPA</td><td>118100</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>WPI</td><td>FD35-100</td><td></td></tr></tbody></table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

In numerosi contesti biologici, cellule animali hanno bisogno di interagire fisicamente con il loro ambiente attraverso lo sviluppo di forze meccaniche. Tra questi, le forze di trazione sono stati ben caratterizzate, ma c'è una mancanza di tecniche che permettono la misurazione delle forze protrusione esercitata dalle cellule ortogonalmente al loro substrato. Abbiamo progettato un setup sperimentale per misurare le forze di protrusione esercitate dalle cellule aderenti il loro substrato. Cellule piastrate su un foglio di Formvar compatibile con questo substrato di deformazione e la topografia risultante è mappata per microscopia a forza atomica (AFM) su scala nanometrica. I valori di forza vengono quindi estratti da un'analisi del profilo deformazione basato sulla geometria delle strutture cellulari protrusive. Quindi, le forze esercitate dalle singole unità sporgente di una cellula vivente possono essere misurate nel tempo. Questa tecnica consentirà lo studio della generazione di forza e la sua regolamentazione in molti processi cellulari che coinvolgono la protrusione. Qui, descriviamo la sua applicazione per misurare le forze protrusive generate da podosomes formata dai macrofagi umani.

Il protocollo di cui sopra viene descritto come preparare la messa a punto sperimentale di quantificare le forze di protrusione applicate dal macrofago podosomes su un substrato di Formvar. Questa operazione viene eseguita mediante AFM ed è illustrata nella Figura 1.
Quando si analizza un'immagine topografica di rigonfiamenti sotto podosomes utilizzando il software di elaborazione dati JPK, un poli...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

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

Qui, abbiamo dettaglio le tecniche sperimentali utilizzate per valutare le forze di protrusione che podosomes si applicano su una pellicola compatibile, dalla preparazione del film per l'analisi automatizzata delle immagini topografiche.

Microscopia a forza protrusione: Un metodo per quantificare le forze sviluppate dalla cella sporgenze

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

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Biology

5.5K Views.  IPBS, Université de Toulouse, CNRS, UPS. Qui, abbiamo dettaglio le tecniche sperimentali utilizzate per valutare le forze di protrusione che podosomes si applicano su una pellicola compatibile, dalla preparazione del film per l'analisi automatizzata delle immagini topografiche.

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

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

Una sonda di forza diretta per la misura di integrazione meccanica tra il nucleo e il citoscheletro

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

Ricerca

Biochimica

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Biomeccanica edonteliale perturbatrice tramite Connexin 43 Disgregazione strutturale

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescenza Biomembrane Forza Probe: quantificazione simultanea di Cinetica recettore-ligando e Binding-indotta intracellulare segnalazione su una singola cella

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Bioingegneria

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Tastatura meccanica delle cellule con pinzette ottiche senza tallone nell'embrione della drosofila

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Biologia dello sviluppo

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Misure di forza diretta della meccanica subcellulare nel confinamento mediante pinzette ottiche

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

Studio del citoscheletro di actina in vivo le cellule endoteliali Esprimendo GFP-Actina

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Biologia

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Fabbricazione e implementazione di una piattaforma di microscopia della forza di trazione senza riferimenti

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Microscopia a forza di trazione per studiare l'attivazione dei linfociti B

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Generazione di modelli per microscopia a trazione micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

Analisi della dinamica dell'actina, dell'accoppiamento della frizione e della forza di trazione per l'avanzamento del cono di crescita

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...