Questo lavoro è stato supportato da NIH R01 EB014869.

1. preparare le celle per l'Imaging
Nota: La sonda di forza diretta (DFP) può essere utilizzata per qualsiasi tipo di cellula aderente. Qui, fibroblasti NIH 3T3 del mouse sono usati come la linea cellulare modello per questo protocollo.
<ol><li>Cellule di cultura NIH 3T3 del fibroblasto dell'Aquila per volta di Dulbecco Medium (DMEM) completati con 10% siero bovino di donatore e 1% penicillina-streptomicina su un fondo di 35 mm vetro piatto fino al confluency di desiderata. Mantenere le cellule a 37 ° C e 5% CO2.<ol>
<li>Assicurarsi di ricoprire tutti i piatti di fondo di vetro 35mm con 5 µ g/mL di fibronectina (o proteina simile ECM), prima della semina di cellule NIH 3T3 per l'imaging. Nota: Le cellule devono essere pienamente diffuso e aderente sul piatto per l'esperimento. Non ci sono vincoli in termini di confluenza per utilizzare il metodo DFP.</li>
	</ol>
</li>
	<li>Immediatamente prima dell'esperimento, lavare le cellule due volte con PBS, seguita da un lavaggio singolo con il mezzo di crescita completa.</li>
	<li>Aggiungere 3 mL di terreno di crescita completa il piatto di fondo di vetro.</li>
</ol>2. microscopia e acquisizione di immagini
Nota: Un invertito fluorescenza Microscopio (o equivalente) con micromanipolatore installato per il braccio di lato, secondo le raccomandazioni del produttore. Il microscopio deve anche essere dotato di una camera climatica per mantenere la temperatura a 37 ° C e il livello di CO2 al 5%. È necessario anche un micromanipolatore e microinjector collegato al microscopio. Un olio immersione 40x / 1.3 NA o 60x / 1,49 NA (o obiettivi equivalenti) sono raccomandati per gli esperimenti. Il microscopio deve essere montato su un tavolo di isolamento delle vibrazioni.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> Figura 1 . Microscopio di messa a fuoco e di deformazione nucleare A. massima deformazione nucleare e rilassamento della deformazione nucleare. Prima di calcolare la deformazione massima nucleare, i bordi posteriori delle forme nucleare erano in primo luogo ha coinciso per correggere per la traduzione del nucleo deformato. La forma del nucleo al momento del distacco di punta della micropipetta è stata sovrapposta la forma nucleare iniziale prima di tirare. La differenza nell'area tra le due forme è stata misurata come Δun1. La massima deformazione nucleare è stata definita come ΔA1 diviso per l'area nucleare originale. Allo stesso modo, un secondo parametro, ΔA2, può essere definito mediante la sovrapposizione della forma nucleare finale allo stato stazionario dopo distacco micropipetta della forma nucleare originale. B. lo stato attivo della cella al piano A e quindi spostare il piano focale fino a piano B per trovare la punta di una micropipetta. Durante la formazione immagine, la micropipetta è stato tradotto a destra (direzione della freccia arancione). Questa figura è stata modificata da Neelam et al. 15. <a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol><li>Accendere il microinjector secondo il protocollo del produttore.</li>
	<li>Con un contagocce di olio di immersione, applicare una sola goccia di olio per immersione in cima la lente dell'obiettivo.</li>
	<li>Fissare saldamente il piatto nel titolare del piatto e caricare il titolare di piatto sul palco. Nota: Le celle devono essere mantenute a 37 ° C e 5% di CO2 in tutto l'esperimento.</li>
	<li>Regolare l'altezza dell'obiettivo di portare le cellule a fuoco (aereo A, Figura 1B).</li>
	<li>Spostare il tavolino del microscopio per trovare una cella di interesse.</li>
	<li>Ruotare il joystick sul micromanipolatore per spostare il supporto di dispensare alla posizione superiore. Caricare la micropipetta di punta di diametro 0,5 µm al supporto per dispensare.<ol>
<li>Per evitare l'adesione delle cellule per la micropipetta, pre-trattare la punta della micropipetta con 0,3 mg/mL di soluzione di PLL-g-PEG per 1 h a temperatura ambiente. Test per adesione toccando la micropipetta al nucleo senza alcuna pressione di aspirazione, e quindi tradurre la micropipetta dal nucleo. L'assenza di adesione possa essere individuata da una completa mancanza di deformazione nucleare e traduzione. Nota: Si prega di seguire suggerimenti di fabbricazione per aprire il pacchetto.</li>
	</ol>
</li>
	<li>Sollevare il piano focale obiettivo sopra A aereo e parte superiore della cella per piano B regolando il controllo fine (Figura 1B, vedere il punto 2.4).</li>
	<li>Impostare il micromanipolatore controllo grossolana . Portare lentamente la micropipetta giù per piano B guardando per la silhouette della micropipetta, fino a quando la micropipetta viene completamente messa a fuoco.</li>
	<li>Una volta che la punta della micropipetta è a fuoco, impostare il micromanipolatore controllo bene .</li>
	<li>Abbassare l'obiettivo per il piano equatoriale della cellula (aereo A, Figura 1B) e abbassare la micropipetta a circa 15 µm sopra piano A (Figura 1B, micropipetta tratteggiata).</li>
	<li>Impostare la pressione di compensazione (Pc) su microinjector alla pressione desiderata; attendere alcuni secondi per la pressione si stabilizzi. Nota: Il set point di pressione ottimale dipende dal tipo di cellula sia gli obiettivi specifici dell'esperimento. Per la maggior parte dei casi, 300 hPa sarebbe un buon punto di partenza.</li>
	<li>Garantire che la micropipetta non sia intasata utilizzando l'impostazione pulite sul pannello micromanipolatore e controllo per assicurarsi che le bolle d'aria emergere dalla punta della micropipetta.</li>
	<li>Inserire la punta nella cella abbassando gradualmente la micropipetta fino a quando la punta è leggermente toccando la superficie nucleare. Nota: Quando si abbassa la micropipetta, la silhouette della punta della micropipetta diventerà chiara come essa viene messa a fuoco. Prima la micropipetta tocca il nucleo, sollevare l'obiettivo messa a fuoco e allineare la punta della micropipetta con nucleo (stessa coordinata x-y, z-piano superiore). Tornare il fuoco al piano equatoriale del nucleo (aereo A, Figura 1B) e abbassare gradualmente la punta della micropipetta.</li>
	<li>Creare una guarnizione tra la punta della micropipetta e la membrana nucleare scollegando il tubo di alimentazione di pressione dal sistema di microiniezione, aprendo così l'estremità del tubo micropipetta all'atmosfera. Questo passaggio crea una pressione negativa uguale a Pc sulla superficie nucleare.</li>
	<li>Acquisire immagini con il software di raccolta immagini di microscopi. Impostare un'acquisizione avi (video) o nd-acquisizione (immagini) del software di raccolta di immagine. Nota: Per qualsiasi imaging l'acquisizione di software, è istituito da formazione immagine video in tempo reale o acquisizioni di time-lapse immagine con un intervallo di tempo breve.</li>
	<li>Attiva/disattiva per le fluorescenti corrispondente canale di imaging (cioè, GFP, RFP, ecc.) e iniziare la formazione immagine.</li>
	<li>Tradurre la punta della micropipetta lontano dal corpo della cella (a destra, Figura 1B) finché il nucleo non si stacca dalla micropipetta. Nota: Tirare la punta lungo la direzione x positiva (a destra nel campo visivo). Il tasso di trazione può essere programmato e controllato da computer o il joystick può essere spostato manualmente. Non abbiamo trovato alcuna correlazione tra la trazione tasso e deformazione nucleare15 suggerendo una risposta prevalentemente elastica per forzare.</li>
</ol>3. analisi dei dati
<ol><li>Eseguire l'analisi di immagine con qualsiasi software di elaborazione delle immagini base disponibile. L'entità della deformazione nucleare può essere quantificata dal ceppo di lunghezza (Ɛ) o il ceppo di zona (Figura 1A). Quantificare il ceppo di lunghezza utilizzando l'equazione 1, dove L e L0 rappresentano le lunghezze del nucleo a deformazione massima e la posizione iniziale, rispettivamente. <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(Equazione 1)</li>
</ol>

<ol>
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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINC+complexes+form+by+binding+of+three+KASH+peptides+to+domain+interfaces+of+trimeric+SUN+proteins.">LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins.</a> Cell. 149 (5), 1035-1047 (2012).</li><li>Tapley, E. C., Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+the+nucleus+to+the+cytoskeleton+by+SUN-KASH+bridges+across+the+nuclear+envelope.">Connecting the nucleus to the cytoskeleton by SUN-KASH bridges across the nuclear envelope.</a> Current Opinion in Cell Biology. 25 (1), 57-62 (2013).</li><li>Arsenovic, P. T., 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=Nesprin-2G,+a+Component+of+the+Nuclear+LINC+Complex,+Is+Subject+to+Myosin-Dependent+Tension.">Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.</a> Biophysical Journal. 110 (1), 34-43 (2016).</li><li>Rowat, A. C., Lammerding, J., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+cell+nucleus+and+the+effect+of+emerin+deficiency.">Mechanical properties of the cell nucleus and the effect of emerin deficiency.</a> Biophysical Journal. 91 (12), 4649-4664 (2006).</li><li>Rowat, A. C., Foster, L. J., Nielsen, M. M., Weiss, M., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+elastic+properties+of+the+nuclear+envelope.">Characterization of the elastic properties of the nuclear envelope.</a> Journal of the Royal Society Interface. 2 (2), 63-69 (2005).</li><li>Pagliara, S., 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=Auxetic+nuclei+in+embryonic+stem+cells+exiting+pluripotency.">Auxetic nuclei in embryonic stem cells exiting pluripotency.</a> Nature Materials. 13 (6), 638-644 (2014).</li><li>Liu, H., 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=In+situ+mechanical+characterization+of+the+cell+nucleus+by+atomic+force+microscopy.">In situ mechanical characterization of the cell nucleus by atomic force microscopy.</a> ACS Nanotechnology. 8 (4), 3821-3828 (2014).</li><li>Krause, M., Te Riet, J., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+compressibility+of+tumor+cell+nuclei+by+combined+atomic+force-confocal+microscopy.">Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy.</a> Physical Biology. 10 (6), 065002 (2013).</li><li>Neelam, S., 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=Direct+force+probe+reveals+the+mechanics+of+nuclear+homeostasis+in+the+mammalian+cell.">Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 5720-5725 (2015).</li><li>Pajerowski, J. 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Gli autori non hanno nulla a rivelare.

L'integrazione meccanica del nucleo con il citoscheletro di misura è una sfida per i metodi più attuali, ad esempio micropipetta aspirazione16, perché richiedono entrambi nuclei isolati (dove il nucleo è disaccoppiato dal citoscheletro) o nuclei in cellule sospese (dove le forze extracellulari, quali le forze di trazione, sono assenti). Vigore è stato applicato al nucleo applicando deformazione biassiale a aderente celle a una membrana17,18...

Patologie come il cancro coinvolgono alterazioni nucleare forma e struttura1,2, che sono generalmente accompagnati da un 'addolcimento' del nucleo3,4. Nucleare resistenza alla deformazione meccanica è stata caratterizzata generalmente applicando una forza di nuclei isolati5.
Il nucleo in cellule è molecolarmente collegato al citoscheletro dal Linker di Nucleoskeleton e citoscheletro (LINC) complesso6,7,8,9. Di conseguenza, il nucleo è integrato meccanicamente con il citoscheletro e, attraverso le adesioni cellula-substrato, la matrice extracellulare. Meccanicamente sondando il nucleo all'interno di cellule aderenti può fornire la comprensione in questa integrazione meccanica. Metodi per manipolare i nuclei in cellule viventi includono micropipetta aspirazione10,11e a forza atomica microscopia12,13,14. Recentemente abbiamo descritto una sonda di forza diretta (DFP) che si applica forze meccaniche direttamente sul nucleo in un vivente cellulari aderenti15.
Qui, descriviamo la procedura per l'utilizzo di un sistema di microiniezione che è comunemente disponibile in strutture di microscopia di applicare una forza meccanica di nano-scala nota, direttamente al nucleo in una cellula aderente. Un femtotip (punta della micropipetta diametro di 0,5 µm) è montato e collegato al sistema di microiniezione di un tubo. La punta, posizionata ad un angolo di 45° rispetto alla superficie della piastra di coltura, si abbassa fino a adiacente alla superficie nucleare. Il tubo viene quindi disconnesso e aperto all'atmosfera, che crea una pressione negativa di aspirazione sulla superficie nucleare e sigilla la punta della micropipetta contro la superficie nucleare. Attraverso la traduzione della punta della micropipetta, il nucleo è deformato e alla fine (a seconda della grandezza della forza applicata), staccato dalla micropipetta. Questo distacco si verifica quando le forze antagoniste (resiste), esercitate dal nucleo e cella, uguale la forza di aspirazione applicata dalla micropipetta. Analisi può essere effettuata misurando lo spostamento del nucleo, il ceppo di lunghezza (equazione 1), o il ceppo di zona (Figura 1A).

<table><tbody><tr><td>FluoroDish</td><td>WPI</td><td>FD35</td><td></td></tr><tr><td>SYTO 59</td><td>ThermoFisher Scientific</td><td>S11341</td><td></td></tr><tr><td>Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>InjectMan NI2</td><td>Eppendorf</td><td>NA</td><td>discontinued, current equivalent model: InjectMan 4</td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>NA</td><td>Current model FemtoJet 4i</td></tr><tr><td>Plan Fluor oil immersion 40x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Apo TIRF oil immersion 60x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Donor Bovine Serum (DBS)</td><td>ThermoFisher Scientific</td><td>16030074</td><td>NIH 3T3 serum</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s (DMEM)</td><td>Mediatech cellgro</td><td>MT10013CVRF</td><td>NIH 3T3 medium</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Mediatech</td><td>MT30004CIRF</td><td>NIH 3T3 medium supplement</td></tr><tr><td>Immersion Oil Type LDF Non-Fluorescing</td><td>Nikon</td><td>77007</td><td>Immersion oil for objective lens&amp;nbsp;</td></tr></tbody></table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

Le proprietà meccaniche del nucleo determinano la sua risposta a forze meccaniche generate nelle cellule. Poiché il nucleo è molecolarmente continuo con il citoscheletro, metodi sono necessari per sondare il suo comportamento meccanico in cellule aderenti. Qui, discutiamo la sonda di forza diretta (DFP) come strumento per applicare la forza direttamente al nucleo in una cellula aderente vivente. Noi attribuiamo una micropipetta stretta per la superficie nucleare con aspirazione. La micropipetta è traslata lontano il nucleo, che fa sì che il nucleo di deformare e tradurre. Quando la forza di ripristino è uguale alla forza di aspirazione, il nucleo si stacca e rilassa elasticamente. Poiché la pressione di aspirazione è noto con precisione, la forza sulla superficie nucleare è noto. Questo metodo ha rivelato che le forze su scala nanometrica sono sufficienti per deformare e tradurre il nucleo in cellule aderenti e identificati gli elementi del citoscheletro che consentono il nucleo resistere alle forze. La DFP può essere utilizzata per sezionare i contributi di componenti cellulari e nucleare nucleare proprietà meccaniche in cellule viventi.

La Figura 2A Mostra la forzatura di un nucleo di fibroblasto NIH 3T3 del mouse. Come la punta di una micropipetta è tradotto a destra, il nucleo si deforma e alla fine si stacca dalla punta della micropipetta. Il ceppo di lunghezza del nucleo è visto ad aumentare con l'aumento della forza di aspirazione (Figura 2B). Il bordo anteriore del nucleo (micropipetta tirando bordo) forma una protrusione nucleare ed il bordo d'uscita è...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

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

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

In questo protocollo, descriviamo un metodo micropipetta per applicare direttamente una forza controllata al nucleo in una cellula vivente. Questo test permette di interrogatorio di proprietà meccaniche nucleari nella cellula viva, aderente.

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

a-direct-force-probe-for-measuring-mechanical-integration-between

Research

JoVE Journal

Biochemistry

15.1K Views.  University of Florida. In questo protocollo, descriviamo un metodo micropipetta per applicare direttamente una forza controllata al nucleo in una cellula vivente. Questo test permette di interrogatorio di proprietà meccaniche nucleari nella cellula viva, aderente.

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

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 ...

Ricerca

Bioingegneria

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Un protocollo per l&#39;utilizzo di trasferimento di energia per risonanza (FRET) biosensori -force per misurare le forze meccaniche in tutto il complesso LINC nucleare

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

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 ...

Biologia dello sviluppo

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Misurazione delle dinamiche di sensibili alla forza della proteina in cellule viventi, utilizzando una combinazione di tecniche di fluorescenza

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Saggi biofisici per sondare le proprietà meccaniche del nucleo della cellula Interphase: Strain supporto Applicazione e manipolazione microago

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biologia

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

Sonde di tensione del DNA per mappare le forze transitorie del recettore di Piconewton da parte delle cellule immunitarie

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

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

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

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

Microscopia 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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Isolamento di Myofibrils da biopsie muscolari scheletriche e determinazione della funzione Contractile con un trasduttore della forza di risoluzione Nano-Newton

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Misurazione diretta delle forze all'interno di fasci di microtubuli attivi ricostituiti

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

Combinazione di attuatore di forza magnetica 3D e imaging a fluorescenza multifunzionale per studiare la meccanobiologia del nucleo

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...