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

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomer...

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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

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomerizasyon Dinamiği Sayı ve Parlaklık Analizi ile Birlikte

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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.

Canlı hücrelerin plazma zarında ligand bağlanması ile indüklenen mEGFP etiketli-reseptör oligomerlerinin ortalama oligomerik durumunun belirlenmesi için bir görüntüleme yaklaşımını tanımladık. Protokol, Sayı ve Parlaklık (N&B) analizi ile birlikte Toplam İç Yansıma Floresan (TIRF) mikroskobuna dayanmaktadır.

Reseptör kümeleme her yerde ve genellikle bir basamaklı aktivasyon sinyal için gereklidir. Ancak, kümeleme derecesini ölçmek için birkaç yöntem kullanılabilir. Canlı hücrelerin plazma zarındaki FGFR1-mEGFP moleküllerinin difüzyonunu takip etmek için total internal resmefleks, TIRF mikroskopisi kullanıyoruz.

Daha sonra uyarılmış reseptör kümeleme dinamiklerini tanımlamak için sayı parlaklığı, N & B analizi uygulayın. TIRF hücre yüzeyine yakın meydana moleküler olayların hızlı zamansal görüntüleme için idealdir, N & B mikroskop aydınlatma hacmi içinde ve dışında difüzyon nerede dayalı floresan moleküllerin ortalama oligomerik durumunu belirler. Gerçekten de, bu sinyal hücre yüzeyinde ki reseptörlerin mekansal zamansal organizasyon bağlamak için bir araçtır.

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Biyoloji Sayı 153 Reseptör kümeleri N&B Sayı ve Parlaklık moment analizi protein oligomerleri hücre zarı TIRF mikroskobu