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 understanding of how cells convert physical stimuli into biochemically-detectable signals, a process termed mechanotransduction, requires the elucidation of the molecular mechanisms mediating force transmission, both into and out of the cells and within multiple subcellular structures.

Inside subcellular structures, proteins are constantly turning over; binding and unbinding based on the strength of their interactions with binding partners⁸. For forces to be successfully transmitted across a physical distance, there must be an unbroken chain of protein-protein interactions, meaning that a protein's turnover must be slow enough to sustain and transmit force to its binding partner⁹. While protein-protein interactions generally consist of several non-covalent bonds between the protein domains, the interaction is often conceptualized as a bound state that can transition to an unbound state under different conditions^10,11. For a given protein-protein interaction, it is possible that force can have no effect on the lifetime of the interaction, known as an "ideal bond", reduce the lifetime of the interaction, known as a "slip bond", or increase the lifetime of the interaction, known as a "catch bond"¹⁰. Thus, there is an intricate relationship between protein load and protein dynamics, which we refer to as force-sensitive dynamics.

Towards understanding the effect of load on bond dynamics, a number of highly informative experiments have been performed on the single-molecule level. Using isolated proteins, or fragments of proteins and manipulation techniques such as magnetic tweezers, optical tweezers, and atomic force microscopy, these studies have demonstrated force-sensitive protein-protein interactions for several relevant proteins^11,12. Both integrins¹³ and cadherins¹⁴, which are transmembrane proteins important for forming cell-matrix and cell-cell interactions, respectively, have demonstrated alterations in dynamics due to load. Within the cell, vinculin is recruited to both talin¹⁵ and α-catenin¹⁶ in a force-dependent manner and can form a catch bond with actin¹⁷, indicating a crucial role for vinculin at both focal adhesions (FAs) and adherens junctions (AJs) under load. Single-molecule studies allow for the isolation of specific protein-protein interactions and yield unambiguous results, but they do not account for the complexity of the cellular environment.

Landmark experiments demonstrated that several subcellular structures, including FAs and AJs, are mechanosensitive, and exhibit enhanced assembly in response to internally-generated or externally-applied loads^{18,19,20,21,22}. Additionally, several theoretical models have suggested that mechanosensitive assembly could be driven by force-sensitive protein dynamics^23,24,25. To examine these force-sensitive dynamics within living cells, a few indirect approaches have been taken. FRAP and related techniques provide a relatively simple methodology for measuring protein dynamics in cells^26,27,28,29. However, the measurement of protein load has been more limited. A typical approach is to compare protein dynamics in cells with and without the exposure to a cytoskeletal inhibitor used to reduce overall cell contractility^8,30,31. Conceptually, this is a comparison between a high load and low load state. However, there is no quantification of the load across the protein in either state, and there may be unintended biochemical effects of the inhibitor, such the loss of key binding sites along an F-actin filament. Another approach, specific to FAs, has been to measure total force exertion on the substrate by the FA using traction force microscopy to approximate molecular load and examine the relationship with the dynamics of a single protein within the FA³². While this approach allows for the quantification of total force, it does not provide molecularly specific information. FAs are made up of over 200 different proteins, many of which can bear load³³. Thus, measuring the total force output of an FA potentially obscures the possibility of multiple force transmission pathways and does not reliably provide a measure of load on a specific protein.

Unlike previous approaches in mechanobiology, the advent of FRET-based tension sensors allows direct measurement of loads experienced by specific proteins inside living cells^34,35,36. Here, we present a protocol that combines FRET-based tension sensors with FRAP-based measure of protein dynamics. We refer to this technique as FRET-FRAP. This approach enables the simultaneous measurement of protein load and protein dynamics, thus allowing the assessment of the force-sensitive protein dynamics in living cells (Figure 1). Already, the FRET-FRAP technique has been applied to the study of the force-sensitive dynamics of the mechanical linker protein vinculin³⁷. Tension sensors have been developed for numerous proteins that are relevant in a variety of subcellular structures. For example, sensors have been developed for vinculin³⁴ and talin^38,39 in FAs, cadherins and catenins in AJs^40,41,42, nesprin in the nuclear LINC complex⁴³, α-actinin⁴⁴ and filamin³⁶ in the cytoskeleton, and MUC-1 in the glycocalyx⁴⁵, among others⁴⁶. Similarly, FRAP is a commonly used technique has been used on mechanosensitive proteins within the focal adhesions^8,31, adherens junctions⁴⁷, actin cortex²⁶, and nucleus⁴⁸. Moving forward, the FRET-FRAP technique should be broadly applicable to any of these existing sensors or newly developed sensors, allowing for the measurements of force-sensitive dynamics in a wide variety of subcellular structures and contexts. Towards this end, we provide a detailed, generalized protocol for implementing the FRET-FRAP technique applicable in these different systems. Hopefully, this will enable a wide variety of experiments elucidating the roles of various mechanosensitive proteins in regulating force transmission and in mediating cell behavior.

Protocol

1. Generate Samples for Imaging Stably express tension sensor construct in desired cell type. 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 …

Representative 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 sensors34,46. 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

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