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

数と明るさの分析と組み合わせた全内部反射蛍光顕微鏡による生細胞における細胞表面受容体のオリゴマー...

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

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

数と明るさの分析と組み合わせた全内部反射蛍光顕微鏡による生細胞における細胞表面受容体のオリゴマー化ダイナミクス

Full Text

7,302 Views

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

Please note that some of the translations on this page are AI generated. Click here for the English version.

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.

生細胞の形質膜におけるリガンド結合によって誘導されるmEGFPタグ付き受容体オリゴマーの平均オリゴマー状態の測定のためのイメージングアプローチについて説明する。このプロトコルは、数と明るさ(N&B)分析と組み合わせた全内部反射蛍光(TIRF)顕微鏡に基づいています。

受容体クラスタリングは、遍在しており、カスケード活性化のシグナル伝達に必要な場合が多い。ただし、クラスタリングの程度を測定する方法はほとんどありません。我々は、全内部反射蛍光、TIRF顕微鏡を使用して、生細胞の原形質膜中のFGFR1-mEGFP分子の拡散に従う。

次に、N&B分析という数値輝度を適用して、刺激された受容体クラスタリングのダイナミクスを記述します。TIRFは、細胞表面付近で発生する分子事象の迅速な時間的イメージングに最適であり、N&Bは顕微鏡の照射量の拡散に基づいて蛍光分子の平均オリゴマー状態を決定します。実際、これは、シグナル伝達している細胞表面における受容体の空間的時間的組織を接続するツールである。

View the full transcript and gain access to thousands of scientific videos