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 FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

Detta arbete stöddes av Thomas F. och Kate Miller Jeffress Memoria Trust (till DEC) och NIH bevilja R35GM119617 (till december). Konfokalmikroskop avbildning utfördes vid VCU Nanomaterial Karakterisering Kärna (NCC) Facility.

1. Erhåll Nesprin-2G Sensor-DNA och annan plasmid-DNA <ol><li> Erhålla Nesprin-2G TS (spänningssensor), Nesprin-2G HL (huvudlös) kontroll, mTFP1, venus, och TSmod från en kommersiell källa. Propagera alla DNA-plasmider och rena dem med användning av standard E. coli-stammar, såsom DH5-α, såsom beskrivits tidigare 12, 13. </li></ol> 2. transfektera celler med Nesprin-2G och Annan Plasmid-DNA <ol><li> Växa NIH 3T3-fi...

<ol>
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Ett förfarande och en demonstration av levande cell imaging av mekanisk spänning tvärs Nesprin-2G, ett protein i det nukleära LINC-komplexet, skisserades ovan. Före detta arbete, olika tekniker, såsom mikropipett aspiration, magnetisk-vulst-cytometri, och mikroskopisk laser-ablation, använts för att applicera belastning på cellkärnan och för att mäta dess bulkmaterialegenskaper 16, 17, 18. Emellertid, t...

Kraft-känslig, har genetiskt kodade FRET sensorer nyligen dykt upp som ett viktigt verktyg för att mäta dragbaserade krafter i levande celler, som ger insikt i hur mekaniska krafter appliceras tvärs proteiner 1, 2, 3, 4. Med dessa verktyg, forskare kan icke-invasivt bild intracellulära krafter i levande celler med hjälp av konventionella fluorescerande mikroskop. Dessa sensorer består av ett FRET-par (donator- och acceptor fluorescerande proteiner, oftast en blå donator och gul acceptor) åtskilda av en elastisk peptid 3. I motsats till C- eller N-terminal taggning är denna sensor införd i ett inre ställe i ett protein för att mäta den mekaniska kraften som överförs över proteinet, beter sig som en molekylär töjningsgivare. Ökad mekanisk spänning över sensorn resulterar i ett ökat avstånd mellan FRET-pluft, vilket resulterar i minskade FRET 3. Som en följd av detta FRET omvänt relaterad till dragkraft. Dessa fluorescerande baserade sensorer har utvecklats för fokala vidhäftningsproteiner (vinkulin 3 och talin 4), cytoskelettproteiner (α-actinin 5), och cell-cell-junction-proteiner (E-cadherin 6, 7, VE-cadherin 8, och PECAM 8). Den mest använda och väl känne elastisk linker i dessa biosensorer är känd som TSmod och består av en repetitiv sekvens av 40 aminosyror, (GPGGA) 8, som var härledda från spindelsilkesprotein flagelliform. TSmod har visats uppträda som en linjär elastisk nano våren, med FRET känslighet för 1 till 5 pN av dragkraft 3. Olika längder av flagelliform kan användas för att förändra den dynamiska range av TSmod FRET-force känslighet 9. Förutom flagelliform upprepar spektrin 5 och villin headpiece peptid (känd som HP35) 4 har använts som de elastiska peptiderna mellan FRET-par i liknande kraft biosensorer 4. Slutligen en färsk rapport visade att TSmod också kan användas för att upptäcka tryckkrafter 10. Vi utvecklade nyligen en kraftsensor för linkern av nukleo-cytoskelettet (LINC) komplex protein Nesprin2G med hjälp TSmod införd i en tidigare utvecklad trunkerad Nesprin2G protein känt som mini-Nesprin2G (figur 2C), som uppför sig på liknande sätt som endogen Nesprin-2G 11. LINC-komplexet innehåller multipla proteiner som leder från utsidan till insidan av kärnan, förbinder den cytoplasmatiska cytoskelettet till den nukleära lamina. Nesprin-2G är ett strukturellt protein som binder till bådeaktin cytoskelettet i cytoplasman och SUN proteiner i perinukleära utrymmet. Med vår biosensor, kunde vi visa att Nesprin-2G är föremål för aktomyosin beroende spänningar i NIH3T3 fibroblaster 2. Detta var första gången som kraft direkt mätt över ett protein i kärn LINC komplexa, och det kommer sannolikt att bli ett viktigt verktyg för att förstå vilken roll kraft kärnan i mechanobiology. Protokollet nedan ger en detaljerad metod för hur man använder Nesprin-2G kraftsensor, innefattande expression av Nesprin spänningssensorn (Nesprin-TS) i däggdjursceller, samt insamling och analys av FRET bilder av celler som uttrycker Nesprin- TS. Användning av ett inverterat konfokalt mikroskop utrustat med en spektral detektor, FRET en beskrivning av hur man mäter sensibiliserade emission med användning av spektral unmixing och ratiometrisk FRET avbildning tillhandahålls. Utgången ratiometrisk bilder kan användas för att göra relativa quantitative kraft jämförelser. Även om detta protokoll fokuserar på uttrycket av Nesprin-TS i fibroblaster, är det lätt att anpassa till andra däggdjursceller, inklusive både cellinjer och primära celler. Vidare kan detta protokoll eftersom det gäller bildförvärvande och FRET-analys lätt anpassas till andra FRET-baserade kraft biosensorer som har utvecklats för andra proteiner.

<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.

Enligt det protokoll som ovan, var plasmid-DNA förvärv från DNA förvaret och transformerades in i E. coli-celler. E. coli som uttrycker sensor DNA valdes från LB / ampicillinplattor och amplifieras i ett flytande LB-buljong. Efter amplifiering av de vektorer, var DNA-plasmider renas till TRIS-EDTA-buffert med användning av en standard, kommersiellt tillgänglig DNA-isoleringskit. Användning av en spektrofotometer, renades DNA kvantifierades i en standardkoncentra...

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;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.

Ett protokoll för användning av Förster resonansenergiöverföring (FRET) -force Biosensorer för att mäta mekaniska krafter över 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. ...

The overall goal of this procedure is to determine forces on the linker of the nucleoskeleton cytoskeleton, or LINC complex, through FRET microscopy in living cells via a biosensor designed for the LINC complex protein, nesprin-2G. This method can help answer keys questions in the field of mechanotransduction, such as the forces placed on proteins in the nucleus of living cells, and how these forces change under variant conditions. The main advantage of this technique is its ability to measure very small levels of force on proteins and living cells.Generally, individuals new to this method will struggle with capturing images. Optimizing the microscope laser power and gain to receive a good signal between all experimental groups is difficult. To begin, grow NIH 3T3 fiber blast cells to between 70%and 90%confluence in a six-well cell culture dish in a standard cell culture incubator with temperature and CO2 regulation.In a cell culture hood, remove the medium and rinse each well with approximately 1 milliliter of a reduced serum cell medium. Add 800 microliters of reduced serum cell medium to each well, and place the six-well chamber in the incubator. Then, pipette 700 microliters of reduced serum cell medium into a 1.5 milliliter tube labeled L with 35 microliters of lipid carrier solution to form the lipo mix.Mix the suspension by pipetting. Next, pipette 100 microliters of the reduced serum cell medium into each of six 1.5 milliliter tubes labeled one through six. Pipette the plasma DNA for nesprin tension sensor, nesprin headless zero force control, mTFP1, and venus into tubes one through six, as described in the text protocol.Do not reuse pipette tips when pipetting different types of DNA. Then, pipette 100 microliters of the lipo mix from the L tube into each labeled tube, and mix by repeated pipetting, using a clean pipette for each tube. Incubate the suspension for 10 to 20 minutes.Next, add 200 microliters from each labeled tube to a well of the prepared six-well chamber containing cells. Label the top of each well with the corresponding DNA added. Then, place the six-well chamber in an incubator for four to six hours.Following incubation, aspirate the medium, and add one to two milliliters of reduced serum cell medium to rinse. Aspirate the reduced serum cell medium, and add two milliliters of trypsin to each well before placing the six-well dish in the incubator for five to 15 minutes. While the cells detach in the incubator, coat six glass-bottom viewing dishes with a layer of fibronectin at a concentration of 20 micrograms per milliliter dissolved in phosphate-buffered saline.Allow the dishes to coat the surface in the cell culture hood. Neutralize the trypsin by adding two milliliters of DMEM once the cells are detached. Then, transfer the contents of each well to a labeled 15 milliliter centrifuge tube and spin down at 90 times g for five minutes in a swinging rotor centrifuge.Aspirate the supernatant and re-suspend each cell pellet in 1, 000 microliters of DMEM using a pipette. Aspirate the fibronectin mixture from the glass dishes and pipette 1, 000 microliters of each cell suspension onto the dishes. After the cells settle to the bottom of the glass dishes, add another one milliliter of media to each well.Allow the cells to attach and express sensor in the cell incubator for at least 18 to 24 hours. To verify the transfection efficiency, use a fluorescent microscope equipped with an excitation frequency near 462 nanometers for mTFP1, or 525 nanometers for venus. Use an emission filter centered near 492 nanometers for mTFP1, or 525 nanometers for venus.Roughly 18 to 24 hours after completing the transfection, use an inverted fluorescent microscope to examine the efficiency of transfection by comparing the number of fluorescent cells to the total number of cells in view. If live cells cannot be imaged within 48 hours after transfection, fix the cells in 4%paraformaldehyde for five minutes. Allow cells to express sensor for 24 to 48 hours before fixation.Store the cells in PBS and view after 48 hours. In a cell culture hood, replace the cell medium with imaging medium supplemented with 10%fetal bovine serum. Place the viewing dishes in a temperature controlled confocal microscope stage.Place the glass viewing dish with mTFP1 transfected cells over the oil objective at 60X magnification with a numerical aperture of 1.4. Locate mTFP1-expressing cells in widefield fluorescent mode. With a fluorescent cell in the field of view, select the spectral detection mode and capture the spectral image.Include all frequencies beyond 458 nanometers using 10 nanometer increments. Select a bright fluorescent region on the cell. Add the fluorescent ROI mean as the normalized intensity to the spectral database by clicking Save Spectra to Database.Optimize the laser power and gain such that a good signal to noise ratio is achieved. Settings will vary for different equipment. Repeat the process for venus-expressing cells and remain in spectral mode.Use an excitation frequency of 488 nanometers instead of 458 nanometers. Adjust the power settings as needed, but do not change the emission frequencies. Select a bright fluorescent ROI of around a 20-pixel radius.Add the fluorescent ROI mean as the normalized intensity to the spectral database, by clicking Save Spectra to Database. To capture the unmixed images, first switch the capturing mode to spectral unmixing. Add the spectral fingerprints of venus and mTFP1 to the unmixing channels.Set the excitation laser back to a 458 nanometer argon source. Place the nesprin tension sensor viewing dish above the 60X oil objective. After focusing on a fluorescent cell with sufficiently bright expression, adjust the gain and laser power to optimize the signal to noise ratio.Capture a minimum of 15 to 20 images of nesprin tension sensor cells with relatively similar brightness and avoid excessive pixel saturation. Repeat the image capturing process with nesprin headless cells using identical imaging parameters. A representative 10X magnified image of 3T3 fibroblasts transfected with nesprin tension sensor using a lipid mediated carrier is shown here.Typically, only a fraction of cells are transfected in a given field of view. Shown here is a representative pair of two 3T3 nuclei expressing nesprin-TS in the spectrally unmixed venus and mTFP channels. A merged color image of the two channels is shown in the far right panel.Here is a representative sampling of nesprin-TS masked nuclei from stable expressing MDCK cells on patterned and unpatterned substrates. Ratio images are defined as the ratio of the venus channel divided by the mTFP channel and can be used to make relative quantitative force comparisons. Once mastered, this technique can be done in about eight hours if it's performed properly.While attempting this procedure, it's important to remember to carefully label DNA tubes during the transfection, and keep the imaging parameters constant while capturing spectrally result images. After watching this video, you should have a good understanding of how to transfect cells with the nesprin tension sensor and capture spectrally resolved images to computer FRET index that correlates with force on the tension sensor. Though in this case, a FRET sensor is used for nesprin, it can also be applied to other proteins in the cell where measuring force is desired.

Research

JoVE Journal

Bioengineering

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