Name: a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex
Uploaded: 2017-04-11T12:30:00
Description: Watch this Scientific Journal Video about A Protocol for Using FÃƒÂ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

This work was supported by the Thomas F. and Kate Miller Jeffress Memoria Trust (to DEC) and NIH grant R35GM119617 (to DEC). The confocal microscope imaging was performed at the VCU Nanomaterials Characterization Core (NCC) Facility.

1. Obtain Nesprin-2G Sensor DNA and Other Plasmid DNA
<ol>
	<li>Obtain Nesprin-2G TS (tension sensor), Nesprin-2G HL (headless) control, mTFP1, venus, and TSmod from a commercial source. Propagate all the DNA plasmids and purify them using standard E. coli strains, such as DH5-&#945;, as described previously12,13.</li>
</ol>
2. Transfect Cells with Nesprin-2G and Other Plasmid DNA
<ol>
	<li>Grow NIH 3T3 fibroblasts cells to 70...

<ol>
	<li>Conway, D. E., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+cellular+mechanotransduction+in+atherosclerosis.">Flow-dependent cellular mechanotransduction in atherosclerosis.</a> J Cell Sci. 126, 5101-5109 (2013).</li><li>Arsenovic, P. T., Ramachandran, I., 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> Biophys J. 110 (1), 34-43 (2016).</li><li>Grashoff, C., Hoffman, B. D., 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=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>Austen, K., Ringer, P., 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=Extracellular+rigidity+sensing+by+talin+isoform-specific+mechanical+linkages.">Extracellular rigidity sensing by talin isoform-specific mechanical linkages.</a> Nat Cell Bio. 17 (12), 1597-1606 (2015).</li><li>Meng, F., Sachs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dynamic+cytoplasmic+forces+with+a+compliance-matched+FRET+sensor.">Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor.</a> J Cell Sci. 124, 261-269 (2011).</li><li>Borghi, N., Sorokina, M., 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=E-cadherin+is+under+constitutive+actomyosin-generated+tension+that+is+increased+at+cell-cell+contacts+upon+externally+applied+stretch.">E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (31), 12568-12573 (2012).</li><li>Cai, D., Chen, S. -. C., 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=Mechanical+Feedback+through+E-Cadherin+Promotes+Direction+Sensing+during+Collective+Cell+Migration.">Mechanical Feedback through E-Cadherin Promotes Direction Sensing during Collective Cell Migration.</a> Cell. 157 (5), 1146-1159 (2014).</li><li>Conway, D. E., Breckenridge, M. T., Hinde, E., Gratton, E., Chen, C. S., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+Shear+Stress+on+Endothelial+Cells+Modulates+Mechanical+Tension+across+VE-Cadherin+and+PECAM-1.">Fluid Shear Stress on Endothelial Cells Modulates Mechanical Tension across VE-Cadherin and PECAM-1.</a> Curr Bio CB. 23 (11), 1024-1030 (2013).</li><li>Brenner, M. D., Zhou, R., 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=Spider+Silk+Peptide+Is+a+Compact,+Linear+Nanospring+Ideal+for+Intracellular+Tension+Sensing.">Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.</a> Nano Lett. 16 (3), 2096-2102 (2016).</li><li>Rothenberg, K. E., Neibart, S. S., LaCroix, A. S., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Cell+Geometry+Affects+the+Spatial+Distribution+of+Load+Across+Vinculin.">Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin.</a> Cell Mol Bioeng. 8 (3), 364-382 (2015).</li><li>Ostlund, C., Folker, E. S., Choi, J. C., Gomes, E. R., Gundersen, G. G., Worman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+molecular+interactions+of+linker+of+nucleoskeleton+and+cytoskeleton+(LINC)+complex+proteins.">Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.</a> J Cell Sci. 122, 4099-4108 (2009).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J Vis Exp. (6), e253 (2007).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> J Vis Exp. (6), e247 (2007).</li><li>Rodighiero, S., Bazzini, C., 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=Fixation,+mounting+and+sealing+with+nail+polish+of+cell+specimens+lead+to+incorrect+FRET+measurements+using+acceptor+photobleaching.">Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.</a> Cell. Physiol. Biochem. 21 (5-6), 489-498 (2008).</li><li>Kardash, E., Bandemer, J., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+activity+in+live+embryos+using+fluorescence+resonance+energy+transfer+biosensors.">Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors.</a> Nat Protoc. 6 (12), 1835-1846 (2011).</li><li>Vaziri, A., Mofrad, M. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+deformation+of+the+nucleus+in+micropipette+aspiration+experiment.">Mechanics and deformation of the nucleus in micropipette aspiration experiment.</a> J Biomech. 40 (9), 2053-2062 (2007).</li><li>Wang, N., Naruse, K., 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=Mechanical+behavior+in+living+cells+consistent+with+the+tensegrity+model.">Mechanical behavior in living cells consistent with the tensegrity model.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7765-7770 (2001).</li><li>Nagayama, K., Yahiro, Y., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+stabilize+the+position+of+intranuclear+DNA+through+mechanical+connection+with+the+nucleus+in+vascular+smooth+muscle+cells.">Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells.</a> FEBS letters. 585 (24), 3992-3997 (2011).</li><li>Luxton, G. W. G., Gomes, E. R., Folker, E. S., Vintinner, E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+arrays+of+nuclear+envelope+proteins+harness+retrograde+actin+flow+for+nuclear+movement.">Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement.</a> Science. 329 (5994), 956-959 (2010).</li><li>Chen, Y., Mauldin, J. P., Day, R. N., Periasamy, A. <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+spectral+FRET+imaging+microscopy+for+monitoring+nuclear+protein+interactions.">Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions.</a> J Microsc. 228, 139-152 (2007).</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>LaCroix, A. S., Rothenberg, K. E., Berginski, M. E., Urs, A. N., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction,+imaging,+and+analysis+of+FRET-based+tension+sensors+in+living+cells.">Construction, imaging, and analysis of FRET-based tension sensors in living cells.</a> Methods Cell Biol. , (2015).</li></ol>

A method and demonstration of live cell imaging of mechanical tension across Nesprin-2G, a protein in the nuclear LINC complex, was outlined above. Prior to this work, various techniques, such as micropipette aspiration, magnetic-bead cytometry, and microscopic laser-ablation, have been used to apply strain on the cell nucleus and to measure its bulk material properties16,17,18. However, until our recent work, no stud...

Force-sensitive, genetically encoded FRET sensors have recently emerged as an important tool for measuring tensile-based forces in live cells, providing insight into how mechanical forces are applied across proteins1,2,3,4. With these tools, researchers can non-invasively image intracellular forces in living cells using conventional fluorescent microscopes. These sensors consist of a FRET-pair (donor and acceptor fluorescent proteins, most frequently a blue donor and yellow acceptor) separated by an elastic peptide3. In contrast to C- or N-terminal tagging, this sensor is inserted into an internal site of a protein to measure the mechanical force transmitted across the protein, behaving as a molecular strain gauge. Increased mechanical tension across the sensor results in an increased distance between the FRET-pair, resulting in decreased FRET3. As a result, the FRET is inversely related to tensile force.
These fluorescent-based sensors have been developed for focal adhesion proteins (vinculin3 and talin4), cytoskeletal proteins (&#945;-actinin5), and cell-cell junction proteins (E-Cadherin6,7, VE-Cadherin8, and PECAM8). The most frequently used and well-characterized elastic linker in these biosensors is known as TSmod and consists of a repetitive sequence of 40 amino acids, (GPGGA)8, which was derived from the spider silk protein flagelliform. TSmod has been shown to behave as a linear elastic nano-spring, with FRET responsiveness to 1 to 5 pN of tensile force3. Different lengths of flagelliform can be used to alter the dynamic range of TSmod FRET-force sensitivity9. In addition to flagelliform, spectrin repeats5 and villin headpiece peptide (known as HP35)4 have been used as the elastic peptides between FRET-pairs in similar force biosensors4. Finally, a recent report showed that TSmod can also be used to detect compressive forces10.
We recently developed a force sensor for the linker of the nucleo-cytoskeleton (LINC) complex protein Nesprin-2G by using TSmod inserted into a previously developed truncated Nesprin-2G protein known as mini-Nesprin2G (Figure 2C), which behaves similarly to endogenous Nesprin-2G11. The LINC complex contains multiple proteins that lead from the outside to the inside of the nucleus, linking the cytoplasmic cytoskeleton to the nuclear lamina. Nesprin-2G is a structural protein binding to both the actin cytoskeleton in the cytoplasm and to SUN proteins in the perinuclear space. Using our biosensor, we were able to show that Nesprin-2G is subject to actomyosin-dependent tension in NIH3T3 fibroblasts2. This was the first time that force was directly measured across a protein in the nuclear LINC complex, and it is likely to become an important tool to understand the role of force on the nucleus in mechanobiology.
The protocol below provides a detailed methodology of how to use the Nesprin-2G force sensor, including the expression of the Nesprin tension sensor (Nesprin-TS) in mammalian cells, as well as the acquisition and analysis of FRET images of cells expressing Nesprin-TS. Using an inverted confocal microscope equipped with a spectral detector, a description of how to measure sensitized emission FRET using spectral unmixing and ratiometric FRET imaging is provided. The output ratiometric images can be used to make relative quantitative force comparisons. While this protocol is focused on the expression of Nesprin-TS in fibroblasts, it is easily adaptable to other mammalian cells, including both cell lines and primary cells. Furthermore, this protocol as it relates to image acquisition and FRET analysis can readily be adapted to other FRET-based force biosensors that have been developed for other proteins.

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the protocol above, plasmid DNA was acquired from the DNA repository and transformed into E. coli cells. E. coli expressing the sensor DNA were selected from LB/Ampicillin plates and amplified in a liquid LB broth. Following the amplification of the vectors, DNA plasmids were purified into TRIS-EDTA buffer using a standard, commercially available DNA isolation kit. Using a spectrophotometer, purified DNA was quantified into a standard concentration of &#181;g/m...

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A Protocol for Using FÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

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.

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. ...

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Bioengineering

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