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

Moreno Zamai; Antonio Trullo; Elvira Arza; Ugo Cavallaro; Valeria  R. Caiolfa

doi:10.3791/60398

Dinâmica de oligomerização dos receptores de superfície celular em células vivas pela microscopia...

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

Dinâmica de oligomerização dos receptores de superfície celular em células vivas pela microscopia de fluorescência de reflexão interna total combinada com análise de número e brilho

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10:43 min

November 6, 2019

DOI: 10.3791/60398-v

Moreno Zamai¹, Antonio Trullo², Elvira Arza¹, Ugo Cavallaro³, Valeria R. Caiolfa^1,2

¹Unit of Microscopy and Dynamic Imaging,Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, ²Centro di Imaging Sperimentale,Ospedale San Raffaele, Milan, Italy, ³Unit of Gynecological Oncology Research,European Institute of Oncology IRCCS, Milan, Italy

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Overview

This study presents an innovative imaging technique to assess the oligomeric state of mEGFP-tagged receptor oligomers on the plasma membrane of living cells, particularly in response to ligand binding. Using Total Internal Reflection Fluorescence (TIRF) microscopy alongside Number and Brightness (N&B) analysis, the researchers can explore receptor dynamics in real time.

Key Study Components

Research Area

Cell biology
Microscopy
Molecular biology

Background

Receptor clustering is critical for signaling pathways.
Measuring receptor clustering has been challenging due to limited methods.
TIRF microscopy provides fast imaging capabilities near the cell surface.

Methods Used

Total Internal Reflection Fluorescence (TIRF) microscopy
HeLa cells
Number and Brightness (N&B) analysis

Main Results

The protocol successfully tracks diffusion of FGFR1-mEGFP molecules.
Average oligomeric states were determined post-ligand binding.
This method facilitates real-time observation of receptor dynamics.

Conclusions

The study offers a reliable approach to connect spatial and temporal organization of receptors.
This method has broad applications for studying protein clustering and signaling in live cells.

Frequently Asked Questions

What is TIRF microscopy?

TIRF microscopy is a technique that allows imaging of fluorescent molecules at or near the surface of cells.

Why is measuring receptor clustering important?

Receptor clustering is essential for effective signal transduction in cellular signaling pathways.

What is Number and Brightness (N&B) analysis?

N&B analysis determines the fluorescence intensity of molecules to infer their oligomeric state.

Can this method be used for proteins inside the cell?

No, this method is designed for proteins on the cell membrane; other microscopy techniques are needed for intracellular proteins.

What type of cells were used in the study?

HeLa cells were used for the experiments.

Is prior experience with fluorescence methods necessary?

Basic knowledge of fluorescence fluctuation methods is required to apply this technique.

What temperature is required for the experiment?

The experiments are conducted at 37 degrees Celsius.

Descrevemos uma abordagem de imagem para a determinação do estado oligomérico médio de oligomeros de receptores marcados por mEGFP induzidos pela ligação de ligand na membrana plasmática das células vivas. O protocolo é baseado na microscopia de Fluorescência Total de Reflexão Interna (TIRF) combinada com análise de Número e Brilho (N&B).

O agrupamento de receptores é onipresente e muitas vezes necessário para a sinalização de uma ativação em cascata. No entanto, poucos métodos estão disponíveis para medir o grau de agrupamento. Usamos fluorescência de reflexão interna total, microscopia DE TIRF, para acompanhar a difusão de moléculas FGFR1-mEGFP na membrana plasmática das células vivas.

Em seguida, aplicamos brilho numérico, a análise de N&B, para descrever a dinâmica do agrupamento estimulado do receptor. O TIRF é ideal para imagens temporais rápidas de eventos moleculares que ocorrem perto da superfície celular, enquanto n&B determina o estado oligomerico médio de moléculas fluorescentes com base em onde a difusão entra e sai do volume de iluminação do microscópio. Na verdade, esta é uma ferramenta para conectar a organização temporal espacial dos receptores na superfície celular onde eles estão sinalizando.

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