Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Summary

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis.

Abstract

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

Introduction

We describe a Total Internal Reflection Fluorescence-Number and Brightness (TIRF-N&B) imaging approach for determining the average oligomeric state of receptor molecules at the plasma membrane of live cells, aiming at linking the receptor assembly dynamics to the biological function of the proteins (Figure 1).

Upon extracellular ligand binding, receptors initiate the intracellular signal transduction depending on their conformation, oligomerization, potential co-receptors and membrane composition. Despite the importance and ubiquity of receptor oligomerization, recognized as a key event in cellular signaling

Protocol

1. Sample Preparation

1. Day 1. Seed HeLa cells in complete medium at a concentration of 100,000-200,000 cell/mL in glass-bottom dishes. Seed 6-8 replicate dishes.
   NOTE: In this example, the medium is supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), 1 mM sodium pyruvate, 100 U/100 µg penicillin/streptomycin. Several replicate dishes are prepared.
2. Day 2-3. When cells are at sub-confluency, transfect half of the dishes with the protein plasmid and the second half with reference plasmids (monomer and dimer), in serum-free medium.
   NOTE: Transfection is made in serum free medium supplem....

Results

The results for two representative HeLa-mEGFP-FGFR1 cells seeded in the same culture dish are shown in Figure 5 and Supplemental Table 1. The two cells were captured at time 0 min (Figure 5A, top) and 7 min (Figure 5A, bottom) after addition of the FGF2 ligand.

Figure 5.......

Discussion

N&B requires several precautions in the choice of the cell model and labelling strategy. It can be applied only to live cells that remain stably adhered during the image capture time. Extra fluctuations due to the whole cell rigid displacement might be handled with appropriate image restoration approaches³⁸. However, generally when a cell moves, the cell membrane also deforms, and structure deformation, producing large extra variance, introduces serious limitation to the analysis of membrane p.......

Materials

Name	Company	Catalog Number	Comments
3-Colour Fast TIRF Leica AM TIRF MC inverted microscope, with smi-automatic TIRF alignment. The microscope is equipped with a diode 488 nm laser, a 100x1.46 oil TIRF objective, Ex/Em Bandpass filters at 490/20 and 525/50, temperature/CO2 incubator and Andor DU 8285 VP EMCCD camera. The microscope is operated by Leica LIF software.	Leica Microsystems, Wetzlar, Germany
Albumin from Bovine Serum 98% minimun	Sigma-Aldrich, St. Louis, MI, USA	A7906-100G
DMEM without Phenol Red with 25 mM HEPES	GIBCO Thermo Fisher Scientific,Waltham, MA, USA	21063029	Used serum free for microscopy
DMEM high-glucose GlutaMAX I	GIBCO Thermo Fisher Scientific,Waltham, MA, USA	10566-016	Used for complete medium
Dulbecco's Phosphate Buffered Saline 10x (PBS)	Biowest, Nuaillé, France	X0515-500
Emission splitting system Photometrics DV2	TeledynePhotometrics, Tucson, AZ, USA
Fetal Bovine Serum, qualified, Brazil	GIBCO Thermo Fisher Scientific,Waltham, MA, USA	10270106	10% inactivated supplement for complete medium
Glass bottom 35-mm sterile 1.5 dishes	MatTek, Ashland, MA, USA	P35G-0.170-14-C	uncoated, glass thickness 0.17 microns
GraphPad Prism	GraphPad Software Inc., San Diego, CA, USA
Human cervical carcinoma (HeLa), serum-free animal component (AC) cells	Millipore-Sigma ECACC, Darmstadt, Germany	CB_08011102
iXonEM+ 897 EMCCD (back-illuminated) ANDOR camera controlled by ANDOR Solis software	Oxford Instruments, Andor TM Technology, Abingdon-on-Thames, UK		This camera, installed in an additional port of the microscope, is used for acquiring the N&B time series
Matlab Executable N&B routine	Unit of Microscopy and Dynamic Imaging, CNIC, Madrid, Spain		download at https://www.cnic.es/en/investigacion/2/1187/tecnologia
MatLab v.2018b	The MathWorks, Inc. Natick, MA, USA		download at https://www.mathworks.com/products/matlab.html
Penicillin:Streptomycin for tissue culture 100x	Biowhittaker Inc. Walkersville, MD, USA	LONZA 17-602E	supplement for medium at Penicillin/Streptomycin 100U/100µg.
pN1-mEGFP-FGFR1 expression vector	Unit of Gynecological Oncology Research, European Institute of Oncology IRCCS, Milan, Italy		Zamai et al., 2019
pN1-N-Gly-mEGFP-GPI expression vector	Unit of Microscopy and Dynamic Imaging, CNIC, Madrid, Spain		Hellriegel et al., 2011
pN1-N-Gly-mEGFP-mEGFP-GPI expression vector	Unit of Microscopy and Dynamic Imaging, CNIC, Madrid, Spain		Hellriegel et al., 2011
Recombinant FGF2	PeproTech EC, Ltd., London, UK		Ligand solution: 20ng/mL of FGF2 in PBS supplemented with 0.01%BSA.
Sodium pyruvate GIBCO	ThermoFisher Scientific	11360070	1mM supplement for medium
TransIt-LT1 Transfection Reagent	MirusBio LLC, Madison, WI, USA	MIR 2300
Trypsin-EDTA (0.25%), phenol red	GIBCO Thermo Fisher Scientific,Waltham, MA, USA	25200056
Type F Immersion liquid 10 mL	Leica Microsystems, Wetzlar, Germany	11513 859
UltraPure BSA (50 mg/mL)	ThermoFisher Scientific	AM2618	0.1% supplement for medium without phenol red used for transfections