Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Published: January 05, 2024

doi:

¹Division of Chemistry and Chemical Engineering,California Institute of Technology, ²Division of Biology and Biological Engineering,California Institute of Technology

Summary

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

Abstract

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Introduction

A growing body of work suggests that biomolecular condensates play an important role in cellular organization and numerous cellular functions, e.g., transcriptional regulation¹^,²^,³^,⁴^,⁵, DNA damage repair⁶^,⁷^,⁸, chromatin organization⁹^,¹⁰^,¹¹^,¹², X-chromosome inactivation¹³^,¹⁴^,¹⁵, and intracellular signaling¹⁶^,¹⁷^,¹⁸. In addition, the dysregulation of biomolecular condensates is implicated in many diseases, including cancers¹⁹^,²⁰^,²¹ and neurodegenerative disorders²²^,²³^,²⁴^,²⁵^,²⁶. Condensate formation is often driven by transient, selective, and multivalent protein-protein, protein-nucleic acid, or nucleic acid-nucleic acid interactions²⁷. Under certain conditions, these interactions can lead to liquid-liquid phase separation (LLPS), a density transition that locally enriches specific biomolecules in membraneless droplets. Such multivalent interactions are often mediated by the intrinsically disordered regions (IDRs) of proteins¹^,²⁸^,²⁹. Biophysical characterization of these interactions at the molecular level is critical to our understanding of numerous healthy and aberrant cellular functions, given the pervasiveness of condensates across them. Although techniques based on confocal fluorescence microscopy, e.g., fluorescence recovery after photobleaching (FRAP)³⁰^,³¹^,³², have been widely used to qualitatively show that the molecular exchanges between condensates and the surrounding cellular environment are dynamic, quantifying the interaction dynamics of specific biomolecules within condensates is generally not possible using conventional confocal microscopy or single-molecule microscopy without specialized data analysis methods. The single-particle tracking (SPT) technique described in this protocol is based on live-cell single-molecule microscopy³³ and provides a uniquely powerful tool to quantify the interaction dynamics between specific proteins within condensates. The readout of SPT for such measurement is the mean residence time of a protein of interest in the condensates.

The protocol can be broken down into two parts – data acquisition and data analysis. The first step of imaging data acquisition is to express in cells a protein of interest that is fused to a HaloTag³⁴. This enables labeling of the protein of interest with two fluorophores, where a majority of the protein molecules are to be labeled with a non-photoactivatable fluorophore (e.g., JFX549 Halo ligand³⁵) and a small fraction of them are to be labeled with a spectrally distinct, photoactivatable fluorophore (e.g., PA-JF646 Halo ligand³⁶). This allows for the simultaneous acquisition of all condensate locations in the cell and the acquisition of single-molecule movies of the protein of interest binding and unbinding to the condensates. Meanwhile, the same type of cells are modified to stably express Halo-tagged H2B, a histone that is largely immobile on chromatin. The cells are then stained with the PA-JF646 Halo ligand to enable single-molecule imaging of H2B. As will be discussed in detail below, this experiment accounts for the contribution of photobleaching to enable precise quantification of the interaction dynamics of the protein of interest. Cells for imaging experiments must then be cultured on clean coverslips, stained with HaloTag ligand(s), and assembled into a live-cell imaging chamber. From there, the sample is imaged under highly inclined and laminated optical sheet (HILO) illumination on a total internal reflection fluorescence (TIRF) microscope capable of two-channel imaging and single-molecule detection. The emission is then split onto two cameras, one tracking condensate positions and one tracking single molecules. Acquisition is performed with a long integration time (on the order of hundreds of ms) to blur out freely-diffusing proteins and only capture proteins that are less mobile due to binding to stable structures in the cell³⁷.

The first step of data analysis is using an established single-particle tracking (SPT) algorithm³⁸^,³⁹ to localize individual protein molecules in each frame of the movie and assemble the localizations into a trajectory for each molecule over its detectable lifetime. The trajectories are then sorted into those representing molecules inside and those representing molecules outside the condensates by comparing the localizations of the molecules throughout their trajectories to the localizations of all the condensates at the corresponding times¹.

Next, a survival curve (1 – CDF) is generated using the lengths of all the in-condensate trajectories. The apparent mean residence time of the molecules is then extracted by fitting the survival curve to the following two-component exponential model of protein binding,

with A as the fraction of molecules non-specifically bound and with k_obs,ns and k_obs,s as the observed dissociation rates of the non-specifically bound and specifically bound molecules, respectively. Only k_obs,s is considered from here onward. The dynamics of both protein dissociation, k_true,s, and photobleaching of the fluorophore, k_pb, contribute to k_obs,s as

;

thus, to isolate the effects of protein dissociation, the specific dissociation rate of H2B-Halo in the cell line mentioned prior is measured.

H2B is a protein that is stably integrated into chromatin and that experiences minimal dissociation in the time scale of a single-molecule movie acquisition³⁷. Its specific dissociation rate is then equal to the photobleaching rate of the PA-JF646 Halo ligand, or

The mean in-condensate residence time of the protein of interest, , is then

Representative results from Irgen-Gioro et al.⁴⁰ are shown, where this protocol was applied to demonstrate that fusing an oligomerization domain to IDR results in longer residence times of the IDR in its condensates. This result suggests that the added oligomerization domain stabilizes the homotypic interactions of the IDR that drives LLPS. In principle, the same method with slightly modified protocols can be applied to characterize the homotypic or heterotypic interactions of any protein that participates in the formation of any types of condensates.

Protocol

1. Labeling of proteins in cells Express the protein of interest fused to HaloTag in the desired cell line. Stably express Halo-tagged H2B in the same type of cells as in 1.1 using transposons or viral transduction. 2. Preparation of coverslips Before using coverslips for cell culture, clean coverslips to remove autofluorescent contaminants. Mount 25 mm diameter, #1.5 coverslips on a ceramic staining rack an…

Representative Results

Here, we present representative results from Irgen-Gioro et al.40, where we used this SPT protocol to compare the interaction dynamics of two proteins in their respective self-assembled LLPS condensates. TAF15 (TATA-box binding protein associated factor 15) contains an IDR that can undergo LLPS upon overexpression in human cells. We hypothesized that fusing TAF15(IDR) to FTH1 (ferritin heavy chain 1), which forms a 24-subunit oligomer, would lead to more stable homotypic protein-protein interactio…

Discussion

The protocol as presented here is designed for systems like those investigated in Irgen-Gioro et al.⁴⁰. Depending on the application, some components of the protocol can be modified, e.g., the method for generating fluorescently labeled cell lines, the fluorescent labeling system, and the style of coverslip used. Halo-tagging of a protein in a cell can be done using two strategies, depending on which is more suitable for a given experiment. 1) Exogenous expression: fusing the protein of interest t…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1745301 (S.Y.), Pew-Stewart Scholar Award (S.C.), Searle Scholar Award (S.C.), the Shurl and Kay Curci Foundation Research Grant (S.C.), Merkin Innovation Seed Grant (S.C.), the Mallinckrodt Research Grant (S.C.), and the Margaret E. Early Medical Research Trust 2024 Grant (S.C.). S.C. is also supported by the NIH/NCI under Award Number P30CA016042.

Materials

0.1 µm TetraSpeck microsphere	Invitrogen	T7279	Single-molecule imaging
25 mm Diameter, #1.5 Coverslips	Marienfeld Superior	111650	Preparation of coverslips
593/40 nm bandpass filter	Semrock	FF01-593/40-25	Single-molecule imaging
676/37 nm bandpass filter	Semrock	FF01-676/37-25	Single-molecule imaging
6-Well TC Plate	Genesee	25-105MP	Preparation of cells for microscopy
Cell Line: U-2 OS	ATCC	HTB-96	Labeling of proteins in cells
ConvertASCII_SlowTracking_css3 .m			Analysis of single-molecule imaging data: Available in Chong et al., 2018
Coverglass Staining Rack	Thomas	24957	Preparation of coverslips
Deuterated Janelia Fluor 549 (JFX549)	Janelia Research Campus		Preparation of cells for microscopy
DMEM, Low Glucose	Gibco	10-567-022	Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep
Eclipse Ti2-E Inverted Microscope	Nikon		Single-molecule imaging
Ethanol 200 Proof	Lab Alley	EAP200-1GAL	Preparation of coverslips
evalSPT			Analysis of single-molecule imaging data: Available in Drosopoulos et al., 2020
Fetal Bovine Serum	Cytiva	SH30396.03	Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep
Fiji			Analysis of single-molecule imaging data
Ikon Ultra CCD Camera	Andor	X-13723	Single-molecule imaging
Longpass dichroic beamsplitter	Semrock	Di02-R635-25×36	Single-molecule imaging: Red/Far Red beamsplitter
LUN-F Laser Unit	Nikon		Single-molecule imaging: 405/488/561/640
MatTek glass-bottom dish	MatTek	P35G-1.5-20-C	Preparation of cells for microscopy: 35 mm, #1.5 coverslip dish for cell culture.
NIS-Elements	Nikon		Single-molecule imaging: Microscope acquisition software
nucleus and cluster mask_v2.txt			Analysis of single-molecule imaging data: Available in Chong et al., 2018
Penicillin-Streptomycin	Gibco	15-140-122	Labeling of proteins in cells: Growth media used: DMEM with 5% fetal bovine serum, 1% penstrep
Phosphate Buffered Saline	Thermo Fisher Scientific	18912014	Labeling of proteins in cells
Photoactivatable Janelia Fluor 646 (PA-JF646)	Janelia Research Campus		Preparation of cells for microscopy
PLOT_ResidenceHist_css.m			Analysis of single-molecule imaging data: Available in Chong et al., 2018
Potassium Hydroxide	Mallinckrodt Chemicals	6984-06	Preparation of coverslips
pretracking_comb.txt			Analysis of single-molecule imaging data: Available in Chong et al., 2018
SLIMfast			Analysis of single-molecule imaging data: Available in Teves et al., 2016
Stage-top incubation system	Tokai Hit		Single-molecule imaging: For live-cell imaging
TwinCam dual emission image splitter	Cairn Research		Single-molecule imaging
Ultrasonic Cleaner	Branson	5800	Preparation of coverslips

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Yoshida, S. R., Chong, S. Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates. J. Vis. Exp. (203), e66169, doi:10.3791/66169 (2024).