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

Summary

Here, we present a protocol for the simultaneous use of Förster 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.

Abstract

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 Förster 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.

Introduction

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 differentiation^1,2,3,4. 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 unders....

Protocol

1. Generate Samples for Imaging

1. Stably express tension sensor construct in desired cell type.
   1. 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 human cytomegalovirus (CMV) promoter. Different vectors may be needed for a particular context. For instance, the CMV promoter is silenced in some....

Results

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 sensors^34,46. 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.......

Discussion

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'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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Thermo Fisher	25300062
16% Paraformaldehyde	Electron Microscopy Sciences	30525-89-4
60x Objective NA1.35	Olympus	UPLSAPO 60XO
Antibiotic-Antimycotic Solution (100x)	Gibco	15240-062
Automated Stage	Prior Scientific	H117EIX3
Custom Dichroic Mirror	Chroma Technology Corp		T450/514rpc
Custom mTFP1 Emission Filter	Chroma Technology Corp		ET485/20m
Custom mTFP1 Excitation Filter	Chroma Technology Corp		ET450/30x
Custom Venus Excitation Filter	Chroma Technology Corp		ET514/10x
DMEM-gfp Live Cell Visualization Medium	Sapphire	MC102
Dulbecco's Modified Eagle's Medium	Sigma Aldrich	D5796	with L-glutamine and sodium bicarbonate
Fetal Bovine Serum	HyClone	SH30396.03
Fibronectin, Human	Corning	47743-654
FRAPPA Calibration Slide	Andor		provided along with FRAPPA unit
FRAPPA System with 515 nm Laser	Andor
Glass-bottomed Fluoro Dishes	World Precision Instruments	FD35
HEK293-T Cells	ATCC	CRL-3216
Hexadimethrine Bromide, Polybrene	Sigma Aldrich	H9268-5G
High-glucose Dulbecco's Modified Eagle's Medium	Sigma Aldrich	D6429
Inverted Fluorescent Microscope	Olympus	IX83
JMP Pro Software	SAS
Lambda 10-3 Motorized Filter Wheels	Sutter Instruments	LB10-NW
LambdaLS Arc Lamp with 300W Ozone-Free Xenon Bulb	Sutter Instruments	LS/OF30
Lipofectamine 2000	Invitrogen	11668-027
MATLAB Software	Mathworks
MEM Non-Essential Amino Acids	Thermo Fisher	11140050
MetaMorph for Olympus	Olympus
Micro-Humidification System	Bioptechs	130708
MoFlo Astrios EQ Cell Sorter	Beckman Coulter	B25982
Objective Heater Medium	Bioptechs	150819-13
OptiMEM	Thermo Fisher	31985070
Phosphate Buffered Saline	Sigma Aldrich	D8537
pMD2.G Envelope Plasmid	Addgene	12259
pRRL Vector			gift from Dr. Kam Leong (Columbia University)
psPax2 Packaging Plasmid	Addgene	12260
sCMOS ORCA-Flash4.0 V2 Camera	Hamamatsu Photonics	C11440-22CU
Sorvall Legend XT/XF Centrifuge	Thermo Fisher	75004505
Stable Z Stage Warmer	Bioptechs	403-1926
Venus Emission Filter	Semrock	FF01-571/72