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. ....

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. F....

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/mL (

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

List of materials used in this article

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®ion=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