Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Summary

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

Abstract

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Introduction

Stress granules (SGs) are non-membranous cytoplasmic foci formed as a cellular protective response to environmental stress, such as elevated temperature, oxidative stress, hypoxia, osmotic shock, UV irradiation, glucose deprivation, or viral infection¹. They can be induced chemically by treatment with compounds like sodium arsenite, which triggers oxidative stress. SGs accumulate stalled translation arrested messenger ribonucleoprotein (mRNPs) complexes², sequestering mRNAs from the translational machinery, and their assembly can be triggered by the phosphorylation of eIF2α (eukaryotic initiation factor 2 α). SGs are dynamic structures ....

Protocol

All the animal procedures in this protocol are in strict adherence with the guidelines of the European Community Council Directive of 24 November 1986 (86-609/EEC). House the mice in group, allowing food and water ad libitum. Maintain them in a controlled environment (22 ±1 °C, 55 ±5% humidity) with a 12 hr:12 hr light:dark cycle (light on at 7:00 am).

1. Culture of Murine Primary Cells: Mouse Embryonic Fibroblasts (MEFs)

1. Autoclave thin forceps, as well as curved forceps and dissecting scissors. Store in a sterile container. Use sterile D-PBS (Dulbecco's Phosphate-Buffered Saline). Prepare complete ME....

Results

Stress granule formation is important in the response of cells to stress, permitting a cellular adaptation with stalled translation, until the stress has cleared, associated to prevention of apoptosis. This assay permits to study the SGs in primary cells, by following the localization of key SGs protein components (Figure 1). G3BP, a key factor of SGs assembly, is present rather diffusively in the cytoplasm of cultured MEFs or neurons (Figure 2A a and c).......

Discussion

The most critical steps in the protocol concern the transfection and more particularly the time lapse acquisitions, which have to be carefully monitored in order to lower down the cytotoxicity.

Culture of primary cells is not a difficult part, as long as sterile conditions are maintained and caution is taken in order to prevent damage during the dissection and cell dissociation steps. MEFs can be kept frozen at early passages. Neuron transfection with calcium phosphate has been shown to work w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Gibco	21331	Prewarm at 37 °C
Sodium pyruvate	Gibco	11360
Nonessential amino acids	Gibco	11140
L-Glutamine	Gibco	25030
Trypsin	Gibco	15096
Glass bottom B-35	Greiner	627860/627861	Treated or not (treated: increases attachment of adherent cells)
Poly-L-lysine	Sigma-Aldrich	P2636
DMEM	Gibco	31966	Prewarm at 37 °C
HeBS	Sigma-Aldrich	51558
Neurobasal	Gibco	21103	Prewarm at 37 °C
2-mercaptoethanol	Gibco	31350
Forceps	Biotek	DU-110-A	Very thin, tips 0.1 mm (useful to remove meninges)
Curved forceps	Biotek	P-110-BUF	Very thin, tips 0.1 mm
Small scissors	Biotek	CM-85-BS	Can be useful to remove hippocampi
Polyplus transfection JetPEI reagent	Ozyme	101-10	MEFs transfection, follow the forward protocol
Inverted laser scanning confocal microscope	Leica	SP5