This work was supported by a National Science Foundation CAREER Award (NSF-CMMI-14-54257) as well as grants from the American Heart Association (16GRNT30930019) and National Institutes of Health (R01GM121739-01) awarded to Dr. Brenton Hoffman and a National Science Foundation Graduate Research Fellowship awarded to Katheryn Rothenberg.&#160;The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF or NIH.

1. Generate Samples for Imaging
<ol>
	<li>Stably express tension sensor construct in desired cell type.
	<ol>
		<li>Clone tension sensor construct into pRRL vector or other viral expression plasmid. 
		NOTE: Several different molecular cloning tools are available to achieve this step including the use of restriction enzymes, overlap extension, and Gibson Assembly35. The pRRL vector is used in lenti viral transduction and enables a substantial degree of protein production through the use of ...

The FRET-FRAP method allows for direct measurement of force-sensitive protein dynamics, a property that has been difficult to directly probe inside living cells. The sensitivity of protein dynamics to molecular load is critical to the protein&#39;s function as a force transmitter or transducer. Loading is required for the transmission of both internally-generated and externally-applied forces, called mechanotransmission, and for the conversion of those forces into biochemically-detectable signals, called mechanotransduct...

The extracellular environment is a rich source of both biochemical and physical cues that dictate cell behavior. In particular, the physical nature of the microenvironment can mediate key cellular functions, including cell growth, migration, and differentiation1,2,3,4. Dysregulation of the mechanics of the microenvironment is a critical component to many diseases that do not yet have adequate treatments, such as cancer5, atherosclerosis6, and fibrosis7. A complete understanding of how cells convert physical stimuli into biochemically-detectable signals, a process termed mechanotransduction, requires the elucidation of the molecular mechanisms mediating force transmission, both into and out of the cells and within multiple subcellular structures.
Inside subcellular structures, proteins are constantly turning over; binding and unbinding based on the strength of their interactions with binding partners8. For forces to be successfully transmitted across a physical distance, there must be an unbroken chain of protein-protein interactions, meaning that a protein&#39;s turnover must be slow enough to sustain and transmit force to its binding partner9. While protein-protein interactions generally consist of several non-covalent bonds between the protein domains, the interaction is often conceptualized as a bound state that can transition to an unbound state under different conditions10,11. For a given protein-protein interaction, it is possible that force can have no effect on the lifetime of the interaction, known as an &#34;ideal bond&#34;, reduce the lifetime of the interaction, known as a &#34;slip bond&#34;, or increase the lifetime of the interaction, known as a &#34;catch bond&#34;10. Thus, there is an intricate relationship between protein load and protein dynamics, which we refer to as force-sensitive dynamics.
Towards understanding the effect of load on bond dynamics, a number of highly informative experiments have been performed on the single-molecule level. Using isolated proteins, or fragments of proteins and manipulation techniques such as magnetic tweezers, optical tweezers, and atomic force microscopy, these studies have demonstrated force-sensitive protein-protein interactions for several relevant proteins11,12. Both integrins13 and cadherins14, which are transmembrane proteins important for forming cell-matrix and cell-cell interactions, respectively, have demonstrated alterations in dynamics due to load. Within the cell, vinculin is recruited to both talin15 and &#945;-catenin16 in a force-dependent manner and can form a catch bond with actin17, indicating a crucial role for vinculin at both focal adhesions (FAs) and adherens junctions (AJs) under load. Single-molecule studies allow for the isolation of specific protein-protein interactions and yield unambiguous results, but they do not account for the complexity of the cellular environment.
Landmark experiments demonstrated that several subcellular structures, including FAs and AJs, are mechanosensitive, and exhibit enhanced assembly in response to internally-generated or externally-applied loads18,19,20,21,22. Additionally, several theoretical models have suggested that mechanosensitive assembly could be driven by force-sensitive protein dynamics23,24,25. To examine these force-sensitive dynamics within living cells, a few indirect approaches have been taken. FRAP and related techniques provide a relatively simple methodology for measuring protein dynamics in cells26,27,28,29. However, the measurement of protein load has been more limited. A typical approach is to compare protein dynamics in cells with and without the exposure to a cytoskeletal inhibitor used to reduce overall cell contractility8,30,31. Conceptually, this is a comparison between a high load and low load state. However, there is no quantification of the load across the protein in either state, and there may be unintended biochemical effects of the inhibitor, such the loss of key binding sites along an F-actin filament. Another approach, specific to FAs, has been to measure total force exertion on the substrate by the FA using traction force microscopy to approximate molecular load and examine the relationship with the dynamics of a single protein within the FA32. While this approach allows for the quantification of total force, it does not provide molecularly specific information. FAs are made up of over 200 different proteins, many of which can bear load33. Thus, measuring the total force output of an FA potentially obscures the possibility of multiple force transmission pathways and does not reliably provide a measure of load on a specific protein.
Unlike previous approaches in mechanobiology, the advent of FRET-based tension sensors allows direct measurement of loads experienced by specific proteins inside living cells34,35,36. Here, we present a protocol that combines FRET-based tension sensors with FRAP-based measure of protein dynamics. We refer to this technique as FRET-FRAP. This approach enables the simultaneous measurement of protein load and protein dynamics, thus allowing the assessment of the force-sensitive protein dynamics in living cells (Figure 1). Already, the FRET-FRAP technique has been applied to the study of the force-sensitive dynamics of the mechanical linker protein vinculin37. Tension sensors have been developed for numerous proteins that are relevant in a variety of subcellular structures. For example, sensors have been developed for vinculin34 and talin38,39 in FAs, cadherins and catenins in AJs40,41,42, nesprin in the nuclear LINC complex43, &#945;-actinin44 and filamin36 in the cytoskeleton, and MUC-1 in the glycocalyx45, among others46. Similarly, FRAP is a commonly used technique has been used on mechanosensitive proteins within the focal adhesions8,31, adherens junctions47, actin cortex26, and nucleus48. Moving forward, the FRET-FRAP technique should be broadly applicable to any of these existing sensors or newly developed sensors, allowing for the measurements of force-sensitive dynamics in a wide variety of subcellular structures and contexts. Towards this end, we provide a detailed, generalized protocol for implementing the FRET-FRAP technique applicable in these different systems. Hopefully, this will enable a wide variety of experiments elucidating the roles of various mechanosensitive proteins in regulating force transmission and in mediating cell behavior.

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Thermo Fisher</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>60x Objective NA1.35</td>
			<td>Olympus</td>
			<td>UPLSAPO 60XO</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Stage</td>
			<td>Prior Scientific</td>
			<td>H117EIX3</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Dichroic Mirror</td>
			<td>Chroma Technology Corp</td>
			<td></td>
			<td>T450/514rpc</td>
		</tr>
		<tr>
			<td>Custom mTFP1 Emission Filter</td>
			<td>Chroma Technology Corp</td>
			<td></td>
			<td>ET485/20m</td>
		</tr>
		<tr>
			<td>Custom mTFP1 Excitation Filter</td>
			<td>Chroma Technology Corp</td>
			<td></td>
			<td>ET450/30x</td>
		</tr>
		<tr>
			<td>Custom Venus Excitation Filter</td>
			<td>Chroma Technology Corp</td>
			<td></td>
			<td>ET514/10x</td>
		</tr>
		<tr>
			<td>DMEM-gfp Live Cell Visualization Medium</td>
			<td>Sapphire</td>
			<td>MC102</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td>with L-glutamine and sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin, Human</td>
			<td>Corning</td>
			<td>47743-654</td>
			<td></td>
		</tr>
		<tr>
			<td>FRAPPA Calibration Slide</td>
			<td>Andor</td>
			<td></td>
			<td>provided along with FRAPPA unit</td>
		</tr>
		<tr>
			<td>FRAPPA System with 515 nm Laser</td>
			<td>Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottomed Fluoro Dishes</td>
			<td>World Precision Instruments</td>
			<td>FD35</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293-T Cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide, Polybrene</td>
			<td>Sigma Aldrich</td>
			<td>H9268-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>High-glucose Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma Aldrich</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescent Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>JMP Pro Software</td>
			<td>SAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda 10-3 Motorized Filter Wheels</td>
			<td>Sutter Instruments</td>
			<td>LB10-NW</td>
			<td></td>
		</tr>
		<tr>
			<td>LambdaLS Arc Lamp with 300W Ozone-Free Xenon Bulb</td>
			<td>Sutter Instruments</td>
			<td>LS/OF30</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB Software</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph for Olympus</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Humidification System</td>
			<td>Bioptechs</td>
			<td>130708</td>
			<td></td>
		</tr>
		<tr>
			<td>MoFlo Astrios EQ Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective Heater Medium</td>
			<td>Bioptechs</td>
			<td>150819-13</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>Thermo Fisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G Envelope Plasmid</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>pRRL Vector</td>
			<td></td>
			<td></td>
			<td>gift from Dr. Kam Leong (Columbia University)</td>
		</tr>
		<tr>
			<td>psPax2 Packaging Plasmid</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS ORCA-Flash4.0 V2 Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-22CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend XT/XF Centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75004505</td>
			<td></td>
		</tr>
		<tr>
			<td>Stable Z Stage Warmer</td>
			<td>Bioptechs</td>
			<td>403-1926</td>
			<td></td>
		</tr>
		<tr>
			<td>Venus Emission Filter</td>
			<td>Semrock</td>
			<td>FF01-571/72</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

FRET-FRAP is made up of the combination of two fluorescent techniques, FRET and FRAP. As we focused on the effects of protein load, we used FRET-based tension sensors34,46. These sensors are often based on a tension sensing module consisting of two fluorescent proteins, such as mTFP1 and VenusA206K, connected by a flagelliform linker (Figure 1A). When the module is placed between the head and tail dom...

Watch this Scientific Journal Video about Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques at JoVE.com

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

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.

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

This method can answer key questions in the field of mechanobiology, such as how forces are transmitted and sensed within cells as well as between cells and their environment. The main advantage of this technique is it allows access to the molecular scale information regarding how proteins respond to mechanical forces inside the native context of the living cell. Though this method has been applied specifically to studying the focal adhesion protein vinculin, it can also be applied to other mechanically-sensitive proteins, such as talin, cadherins or nesprin.Visual demonstration of this method is critical, as proper imaging setup is difficult but necessary for the success of the analysis. Begin this procedure with stable expression of the tension sensor construct in the desired cell type, as detailed in the text protocol. To prepare substrates for cell seeding, acquire four 35 millimeter glass-bottomed dishes.Working in a cell culture hood, in a 15 milliliter conical tube, make four milliliters of 10 micrograms per milliliter fibronectin in sterile phosphate-buffered saline solution, or PBS. Gently invert the tube once to mix and let the solution sit for five minutes in the cell culture hood. Then pipette one milliliter of fibronectin solution onto each glass-bottomed dish.Leave the fibronectin solution on the dishes for one hour at room temperature or overnight at four degrees Celsius. Then aspirate the fibronectin solution and rinse once with PBS. Leave one milliliter of PBS on the dishes until the cells are ready for seeding.After counting the cells, seed 30, 000 cells onto each fibronectin-coated glass dish with the appropriate complete media for a final volume of 1.5 milliliters. Allow the cells to spread for four hours following seeding. At two hours of spreading, aspirate the growth media and rinse once with imaging media.Leave 1.5 milliliters of the imaging media. Warm up the microscope as described in the text protocol before beginning calibration. To calibrate the FRAP laser, first open the laser configuration window.Set Illumination setting to the appropriate FRAP Illumination settings for laser exposure to the sample. Then set Illumination setting to the illumination settings appropriate for imaging only the acceptor fluorophore. Select the objective to calibrate under Coordinate system setting.Uncheck Manually click calibration points and check Display images during calibration. Set the Dwell time to 10, 000 microseconds and the Number of pulses to 100. Now place the calibration slide, made of ethidium bromide sealed between a glass slide and a cover slip, into the stage adapter with the cover slip side down.Use the acceptor illumination settings to focus on the surface of the slide, identifiable as the focal plane with the brightest signal. Small defects in the coating will be visible to aid in focusing. Move the slide to an area with uniform fluorescence across the imaging plane.Click on Create Setting for the first calibration or Update Setting for subsequent calibrations. The software will initialize the calibration process, automatically bleaching and detecting the position of the bleached point. Ensure a successful calibration by assessing the final image, which will be a three-by-three grid of bleached points that should be evenly distributed and in focus.Save the calibration image for future reference. Remove the calibration slide and safely store. Calibration should be performed before beginning each experiment but does not need to be performed between samples.Prepare the microscopy setup for live cell imaging, preferably with a heated stage and objective, as well a carbon dioxide control. Allow to equilibrate for 20 minutes. Now place one of the generated samples of cells expressing the tension sensor into the microscope holder for imaging.Allow the cells to equilibrate for 10 minutes. To ensure the health of the imaged cells, maintain the temperature at 37 degrees Celsius in the imaging chamber. Use a peristaltic pump to pass humidified 5%carbon dioxide over the samples at 15 milliliters per minute to maintain the pH.Open the Multi-Dimensional Acquisition, or MDA, tool and set it up with FRET imaging parameters, including the different filter sets. Save this MDA to the Experimental Folder with the name of MDA_FRET_date. Set up another MDA with the FRAP imaging parameters, including the different filter sets, the Timelapse settings and the Journal to pulse the laser after pre-bleach acquisition.Save this MDA to the Experimental Folder with the name of MDA_FRAP_date. In the toolbar at the top of the screen, select Journal, Start Recording. Open the MDA window, load the MDA_FRET_date state and press Acquire.Then load the MDA_FRAP_date state and press Acquire. At the end of the acquisition, in the toolbar at the top of the screen, select Journal, Stop Recording. Save this Journal to the Experimental Folder with the name of FRETFRAP_date and add it to a toolbar for easy access.Close the MDA window. Navigate the sample using the image acquisition under Acquire, Acquire with minimal exposure time and neutral density filter to identify the cells of interest. Set continuous autofocus by navigating to Devices, Focus.Manually focus on the sample until reaching the correct imaging plane. Click Set Continuous Focus and wait for the system to adjust. Once the system adjusts, click Start Continuous Focusing.Then find a cell expressing the tension sensor with clear localization to a structure of interest and snap an image. Draw a rectangular region of interest, or ROI, to highlight where to bleach. Store this ROI location.Now initialize the FRETFRAP_date journal, which will begin the acquisition of FRET images, followed by the initialization of the FRAP timelapse. Repeat these steps until 10 to 15 image sets are acquired. Then analyze the FRET-FRAP data as detailed in the text protocol.Shown here are representative results for FRET imaging of the vinculin tension sensor, following segmentation and masking to isolate focal adhesions. Spacial variation of vinculin load can be seen across the cell. FRAP imaging and analysis can be affected by focal adhesion stability.A focal adhesion that is translocating rapidly during the imaging period can be difficult to accurately track. Often this is indicated by a FRAP curve that contains discontinuities and in uninterpretable. For a wild-type vinculin, there is an inverse correlation between protein load and turnover rate, indicating that vinculin is stabilized by force.Introducing a mutation in vinculin to effect its binding to talin results in a reversal of the correlation, indicating that vinculin that is unable to bind talin is destabilized by force. While attempting this procedure, it's important to remember to prepare samples properly, ensure that cells are expressing the sensor at endogenous levels and choose imaging parameters carefully. Following this procedure, other methods like immunofluorescence, measuring cellular scale dynamics or challenging cells with various inhibitors can be performed in order to answer additional questions like how the protein of interest interacts with other subcellular components to coordinate response to force.

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8.1K visualizações.  Duke University. Esse método pode responder a perguntas-chave no campo da mecanobiologia, como como as forças são transmitidas e sentidas dentro das células, bem como entre as células e seu ambiente. A principal vantagem dessa técnica é que permite o acesso às informações em escala molecular sobre como as proteínas respondem às forças mecânicas dentro do contexto nativo da célula viva. Embora este método tenha sido aplicado especificamente ao estudo da proteína de adesão focal vinculina, ele...