A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Summary

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Abstract

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.

In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

Introduction

Many biological processes occur at the sites of cell-cell interactions, e.g., cell-cell adhesion^1,2,3, cell-cell fusion⁴ and cellular recognition⁵. Such events are particularly important during the development of multicellular organisms and for cell-cell communication, e.g., during immune responses. These processes are typically mediated by proteins that are localized at the surface, i.e., at the plasma membrane (PM) of neighboring cells and undergo specific interactions at the cell-cell contact that a....

Protocol

1. Sample Preparation: Cell-Cell Mixing Assay

NOTE: The following protocol describes the mixing procedure for adherent cells. It may be modified for cells cultured in suspension.

1. Seed an appropriate number of cells on a 6-well plate, e.g., 800,000 HEK 293T cells (counted with a Neubauer counting chamber), a day before transfection. The number can be modified depending on the time between seeding and transfection and adjusted for other cell types. To perform a basic experiment (i.e., proteins of interest and negative control), prepare at least 4 wells. Culture cells at 37 °C, 5% CO₂ in Dulbecco's Modi....

Results

A first test for the protein-protein interaction assay, i.e., mixing of cells expressing spectrally distinct fluorescent proteins followed by sFCCS/ccN&B measurements (Figure 1), should be performed on proteins that are not expected to interact at the cell-cell contact (i.e., a negative control). Therefore, HEK 293T cells expressing myristoylated-palmitoylated-mEYFP (myr-palm-mEYFP) or -mCardinal were mixed and sFCCS was performed across.......

Discussion

The experimental procedure described here allows the investigation of protein-protein trans interactions at cell-cell contacts, employing fluorescence fluctuation spectroscopy techniques, namely sFCCS and ccN&B. These methods involve a statistical analysis of fluorescence fluctuations emitted by two spectrally separated FPs fused to the protein(s) of interest at a contact of two neighboring cells, each expressing one or the other fusion protein. The presence of trans complexes is quantified by probi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM growth medium	PAN-Biotech	P04-01548
DPBS w/o: Ca2+ and Mg2+	PAN-Biotech	P04-36500
DPBS w: Ca2+ and Mg2+	PAN-Biotech	P04-35500
Trypsin EDTA	PAN-Biotech	P10-023100
TurboFect Transfection Reagent	Thermo Fisher Scientific	R0531
HEK 293T cells	DSMZ	ACC 635
Alexa Fluor 488 NHS Ester	Thermo Fisher Scientific	A20000
Rhodamine B	Sigma-Alderich	83689-1G
Plasmid DNA	Addgene	NA	See reference 12 (Dunsing et. al., MBoC 2017),for a detailed description of all plasmids
6-well plate	Starlab	CC7672-7506
35-mm glass bottom dishes	CellVis	D35-14-1.5-N
Zeiss LSM780 confocal	Carl Zeiss	NA
MATLAB software package	MathWorks	2015b
Neubauer cell counting chamber	Marienfeld	640110