A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Summary

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.

Abstract

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 Förster 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.

Introduction

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 proteins^1,2,3,4. 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 (α-actinin⁵), and cell-cell junction proteins (E-Cadherin^6,7, 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.

Protocol

1. Obtain Nesprin-2G Sensor DNA and Other Plasmid DNA

1. 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-α, as described previously^12,13.

2. Transfect Cells with Nesprin-2G and Other Plasmid DNA

1. Grow NIH 3T3 fibroblasts cells to 70-90% confluence in a 6-well cell culture dish in a standard cell culture incubator with temperature (37 °C) and CO₂ (5%) regulation. For the cell growth medium, use Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum.
2. In a cell culture hood, remove the medium and rinse each well with approximately 1 mL of a reduced-serum cell medium. Add 800 µL of reduced-serum cell medium to each well and place the 6-well chamber in the incubator.
3. Pipette 700 µL of reduced-serum cell medium into a 1.5 mL tube with 35 µL of lipid carrier solution to form the "lipomix". Mix by pipetting. Label the tube with an "L."
4. Gather six 1.5 mL tubes and label them 1 through 6. Pipette 100 µL of reduced-serum cell medium into each tube.
   1. Using the plasmid DNA concentration from step 1, pipette 2 µg of Nesprin 2G-TS into tubes 1 and 2. Pipette 2 µg of Nesprin-HL into tubes 3 and 4. Pipette 1 µg of mTFP into tube 5. Pipette 1 µg of mVenus into tube 6. Do not re-use pipette tips when pipetting different types of DNA.
5. Pipette 100 µL of the lipomix from the "L" tube into each labeled tube (1-6) and mix by repeated pipetting. Use a clean pipette for each tube. Incubate for 10-20 min.
6. Add 200 µL from each labeled tube to a well in the 6-well chamber with 70-90% confluent cells. Label the top of each well with the corresponding DNA added. Place the 6-well chamber in an incubator for 4-6 h.
7. Aspirate the medium and add 1-2 mL of reduced-serum cell medium to rinse. Aspirate the reduced-serum cell medium, add 2 mL of trypsin to each well, and place the 6-well dish in the incubator (5-15 min).
8. While the cells detach in the incubator, coat 6 glass-bottom viewing dishes with a layer of fibronectin at a concentration of 20 µg/mL dissolved in phosphate-buffered saline (PBS). Allow the dishes to coat the surface in the cell culture hood (approximately 20 min).
9. Neutralize the trypsin by adding 2 mL of DMEM once the cells are detached.
10. Transfer the contents of each well to a labeled 15-mL centrifuge tube and spin down at 90 x g for 5 min in a swinging rotor centrifuge. Aspirate the supernatant and re-suspend each cell pellet in 1,000 µL of DMEM by pipette mixing.
11. Aspirate the fibronectin mixture from the glass dishes and pipette 1,000 µL of each cell suspension onto the glass dishes.
12. After the cells settle to the bottom of the glass dishes (~15 min), add another 1 mL of DMEM + 10% FBS + 1% Pen-Strep to each well and place in the cell incubator. Allow the cells to attach and express sensor for at least 18-24 h.
    NOTE: Cells are transfected using commercial cationic lipid transfection reagents (see the Table of Materials). Alternatively, stable cell lines can be selected by using a plasmid with a gene conferring resistance to a toxin (the pcDNA plasmids for Nesprin-TS and -HL are available on a DNA repository website (see the Table of Materials) provide cells expressing geneticin resistance). Additionally, viral infection methods (lentivirus, retrovirus, or adenovirus) can be utilized to express the sensor in cells that are hard to transfect.
13. In addition to Nesprin-TS, transfect additional cells with the Nesprin HL zero-force control, as described in steps 2.1-2.12; TSmod can also be used as a zero-force control and should exhibit similar FRET to Nesprin-HL.
14. Transfect cells with mTFP1 and venus to generate spectral fingerprints (see step 4).
    NOTE: mTFP1 and venus typically express at higher levels than Nesprin-TS, and as such, lower amounts of DNA may need to be transfected to achieve similar expression levels.

3. Verify Transfection Efficiency

1. Roughly 18-24 h 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 (usually 5-30%).
   1. Use a fluorescent microscope equipped with an excitation frequency near 462 nm (mTFP1) or 525 nm (venus) and an emission filter centered near 492 nm (mTFP1) or 525 nm (venus).
      NOTE: Alternatively, GFP filter sets will capture a combination of mTFP1 and venus emissions and will allow the confirmation of the transfection efficiency.
2. Image live cells within 48 h after transfection.
   1. Alternatively, fix cells in 4% paraformaldehyde (in PBS with calcium and magnesium) for 5 min, store in PBS, and view after 48 h⁸.
      NOTE: Beyond 48 h, the signal quality and strength decays. Fixing the cells preserves their state, including the FRET being expressed⁸; however, cells should only be imaged in PBS, as mounting medium may affect FRET¹⁴. Fixed cells can only be compared to other fixed cells, as there may be a change in expression.
      Caution: Paraformaldehyde is toxic. Wear appropriate personal protective equipment (PPE).

4. Capture Spectral Fingerprints of mTFP1 and Venus Fluorophores for Spectral Unmixing

1. In a cell culture hood, replace the cell medium with imaging medium (HEPES-buffered) supplemented with 10% fetal bovine serum.
2. Place the viewing dishes in a temperature-controlled (37 °C) confocal microscope stage.
3. Place the glass viewing dish with mTFP1-transfected cells over the oil objective at 60X magnification with a numerical aperture of 1.4.
   NOTE: The oil is used on a laser scanning microscope on the 60X objective to closely resemble the refractive index of the glass substrate. The oil is placed on top of the objective lens and comes in direct contact with the glass coverslip.
4. Locate mTFP1 expressing cells with a 458 nm excitation source and an emission bandpass filter centered at 500 nm.
5. With a fluorescent cell in the field of view, select the spectral detection mode ("Lambda Mode" in the software used here) and capture the spectral image (Figure 3-1); include all frequencies beyond 458 nm using 10 nm increments (Figure 3-3). Select a bright fluorescent region (ROI) on the cell (20-pixel radius).
   NOTE: The spectral shape of the mTFP1-expressing ROI should remain relatively constant across the cell. If the shape varies considerably, re-adjust the laser and gain settings to improve the signal-to-noise ratio.
6. Add the fluorescent ROI mean, normalized intensity to the spectral database by clicking "save spectra to database."
7. Optimize the laser power and gain such that a good signal-to-noise ratio is achieved. Settings will vary for different equipment. Using non-fluorescent, untransfected cells as a background reference, increase the gain and power until the cells are bright, but not beyond the dynamic range of the detector (saturation point). Background cells should not have detectable fluorescence after averaging.
8. Repeat the process for the venus-transfected cells with the following exceptions:
   1. Ensure that the excitation frequency is at 515 nm and that the bandpass filter is centered near 530 nm when locating venus-expressing cells.
   2. In "Spectral Mode," use an excitation frequency of 515 nm instead of 458 nm.

5. Capture Unmixed Images

1. Switch the capturing mode to "Spectral Unmixing."
2. Add the spectral fingerprints of venus and mTFP1 into the unmixing channels (Figure 3, Arrow-2).
3. Set the excitation laser back to a 458 nm argon source.
4. Place the Nesprin-TS viewing dish above the 60X oil objective.
5. After focusing on a fluorescent cell with sufficiently bright expression, adjust the gain and laser power to optimize the signal-to-noise ratio.
   NOTE: Since the FRET efficiency of Nesprin-TS is near 20%, the unmixed venus image should be substantially dimmer than the unmixed mTFP1 image. Once an acceptable power and gain setting have been determined iteratively, these parameters must remain constant for all images captured.
6. Capture a minimum of 15-20 images of Nesprin-TS cells with relatively similar brightness and avoid excessive pixel saturation (all saturated pixels are removed during image processing).
7. Repeat the image capturing process with Nesprin-HL cells using identical imaging parameters.

6. Image Processing and Ratio Image Analysis

1. Using open-source ImageJ (FIJI) software (http://fiji.sc/), open native format images using BioFormats Reader.
2. Pre-process the images and compute the ratio images using previously established protocols¹⁵.

Results

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 µg/m...

Discussion

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 properties^16,17,18. However, until our recent work, no stud...

Materials

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