Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Matteo Fossati; Nica Borgese; Sara Francesca Colombo; Maura Francolini

doi:10.3791/50985

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

JoVE Journal
Biology

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

JoVE Journal Biology

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Full Text

13,780 Views

16:43 min

February 18, 2014

DOI: 10.3791/50985-v

Matteo Fossati^1,2,3, Nica Borgese^2,3,4, Sara Francesca Colombo^2,3, Maura Francolini^1,2

¹Fondazione Filarete, ²Department of Biotechnology and Translational Medicine,University of Milan, ³Neuroscience Institute,National Research Council (CNR), ⁴Department of Health Science,"Magna Graecia" University of Catanzaro

Overview

This study investigates the distribution and mobility of transfected fluorescent proteins in the endoplasmic reticulum (ER) using live cell confocal imaging. Additionally, it examines the impact of these proteins on the ultrastructural architecture of the ER.

Key Study Components

Area of Science

Cell Biology
Fluorescence Imaging
Subcellular Structures

Background

The endoplasmic reticulum (ER) plays a crucial role in protein and lipid synthesis.
Fluorescent proteins are commonly used to study cellular processes.
Understanding protein mobility in the ER can reveal insights into cellular function.
Ultrastructural analysis provides detailed information about cellular architecture.

Purpose of Study

To investigate the distribution and movement of fluorescent proteins in the ER.
To assess the effects of protein expression on ER structure.
To utilize advanced imaging techniques for real-time analysis.

Methods Used

Transient transfection of cultured cells with GFP-tagged ER resident proteins.
Live cell confocal imaging to assess fluorescence recovery after photobleaching (FRAP).
Transmission electron microscopy for ultrastructural analysis.
Data collection on the organization and structure of the ER.

Main Results

Expression of GFP-tagged proteins significantly alters ER organization.
Formation of aggregates within the ER was observed post-transfection.
Fluorescence recovery and loss provided insights into protein mobility.
Results contribute to understanding basic cell biology processes.

Conclusions

The study demonstrates the impact of fluorescent protein expression on ER architecture.
Advanced imaging techniques can elucidate cellular dynamics.
Findings may inform future research on protein and lipid trafficking.

Frequently Asked Questions

What is the significance of studying the endoplasmic reticulum?

The ER is essential for protein and lipid synthesis, and understanding its dynamics can reveal important cellular processes.

How does fluorescence recovery after photobleaching work?

FRAP involves bleaching a specific area of a cell and monitoring the recovery of fluorescence to study protein mobility.

What techniques were used in this study?

The study utilized live cell confocal imaging and transmission electron microscopy for analysis.

What were the main findings regarding ER structure?

The expression of GFP-tagged proteins led to significant alterations in the organization of the ER, including aggregate formation.

Why is it important to understand protein mobility?

Understanding protein mobility is crucial for insights into cellular functions such as trafficking and signaling.

What implications do these findings have for cell biology?

The findings provide a deeper understanding of the dynamics of cellular compartments and their roles in cellular processes.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The overall goal of the following experiment is to investigate how transfected fluorescent proteins are distributed and move in the endoplasmic reticulum and to assess the effect of expression on the ultra structural architecture. To do this, cultured cells are transiently transfected with GFP tagged tail anchored ER resident proteins. Following transfection live cell confocal imaging is performed to assess fluorescence recovery after photobleaching or frap and fluorescence loss.

In photobleaching or flip, ultra structural analysis is performed using transmission electron microscopy. The resulting data demonstrate that the expression of GFP tagged B five construct dramatically alters the organization and structure of the endoplasmic reticulum inducing the formation of aggregates. The method we demonstrate here can provide insight into basic cell biology processes, such as protein and lipid mobility and trafficking.

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