Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Summary

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Abstract

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Introduction

Cells employ many mechanisms to respond to stress. Some responses occur at the post-transcriptional level and involve regulating mRNA translation and/or stability^1,2. Stress-modulated mRNA translational arrest and degradation are associated with the formation of specific nonmembranous cellular foci, the most well characterized being Stress Granules (SGs)³. SGs are cytoplasmic foci that concentrate nontranslating mRNPs in translationally-arrested cells responding to stress (e.g., oxidation, heat shock, nutrient starvation, and viral infection)⁴. In additi....

Protocol

1. Cell Preparation

1. Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in Figure 1A; this can be done using a sterile Pasteur pipette attached to a vacuum to pick up each coverslip. Gently tap the coverslip on the side of the well to release the suction, allowing the coverslip to fall into the well.
2. Plate 1 x 10⁵ U-2 OS (U2OS) osteosarcoma cells per well at a final volume of 500 µL of medium. Move the plate side to side and up and down several times to ensure that the cells are evenly distributed.
3. Gently press the coverslips down with a clean P1000 tip to ensure that the coverslip is ....

Results

Stress-induced foci are not necessarily SGs. SGs are classified as cytoplasmic foci that contain mRNAs, translation initiation factors, and RNA-binding proteins and are in equilibrium with active translation. The above protocol can be used as a template to characterize whether a given stress induces bona fide SGs.

SGs colocalize with other known SG markers, including both proteins and mRNA. SA and VRB induce cytoplasmic.......

Discussion

As evidenced by immunohistological studies in multiple diseases, chronic stress leads to the formation of different intracellular foci. For example, most neurodegenerative diseases are characterized by intracellular aggregates of insoluble proteins. The presence of SG-associated proteins in such aggregates is often the basis for concluding that such foci are SGs. A similar conclusion is also drawn when SG marker-positive cytoplasmic foci are observed in cells treated with new stress stimuli. This protocol provides a simp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
U-2 OS	ATCC	ATCC®HTB-96™	Commonly written "U2OS" Culture at 37 °C under 5% CO2 in 10% FBS/DMEM
Dulbecco's Modified Eagle's Medium (DMEM)	Corning	10-013-CV	Contains 4.5 g/L glucose, pyruvate, and phenol red Supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin Prewarm prior drug(s) dilution
Fetal Bovine Serum (FBS)	Sigma	F2442-500ml	Used to supplement media
HEPES (1 M)	Thermo Fisher Scientific	15630-080	Used to supplement media
penicilline streptomycine	Sigma	P0781-100ml	Used to supplement media
24-well plate	Costar	3524
lab tissues (kimwipes)	KIMTECH SCIENCE	34120
Coverglass for Growth Cover Glasses	Fisher Scientific	12-545-82	Autoclaved before use Keep sterile in the tissue culture hood
Sodium (meta)arsenite ≥90% (SA)	Sigma	S7400-100G	Dissolve in water to 1 M concentration, then dilute to 100 µM as a working stock
Vinorelbine (VRB)	TSZ CHEM	RS055	Dissolve in water to 10 mM
Puromycin	Sigma	P9620	Used to assess translation level (ribopuromycylation) -Used to assess SG connection with active translation Dilute in water to 10 mg/mL
Emetine dihydrochloride hydrate	Sigma	E2375	Used in combination with puromycin to assess general translation level Used to assess SG connection with active translation -Dilute in water to 10 mg/mL
Cycloheximide (CHX)	Sigma	C4859	Used to assess SG connection with active translation Dilute in water to 10 mg/mL
Phosphate Buffered Saline (PBS)	Lonza / VWR	95042-486	Wash buffer for immunofluorescence
Paraformaldehyde reagent grade, crystalline (PFA)	Sigma	P6148-500G	Make a 4% solution in hot PBS in fume hood, stir until dissolved Aliquots can be stored at -20 °C for several months Hazardous, use ventilation Discard in special waste
Methanol, ACS	BDH / VWR	BDH1135-4LP	Prechill to -20 °C before use Discard in special waste
Normal Horse Serum (NHS)	Thermo Fisher Scientific	31874	Dilute to 5% in PBS Add sodium azide for storage at 4 °C Blocking solution for immunofluorescence
Ethanol, Pure, 200 Proof (100%),USP, KOPTEC	Decon Labs / VWR	89125-188	Dilute to 70% with water
Fisherbrand Superfrost Plus Microscope Slides	Fisherbrand	12-550-15
mouse anti G3BP1 antibody (TT-Y)	Santa Cruz	sc-81940	Store at 4 °C 1/100 dilution
rabbit anti eIF4G antibody (H-300)	Santa Cruz	sc-11373	Store at 4 °C 1/250 dilution
goat anti eIF3η (N-20) antibody	Santa Cruz	sc-16377	eIF3η is also known as eIF3b Store at 4 °C 1/250 dilution
mouse anti puromycin 12D10 antibody	Millipore	MABE343	Store at 4 °C 1/1,000 dilution
Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L)	Jackson Immunoresearch	715-225-150	Reconstitute in water per manufacturer’s instructions then store at 4 °C 1/250 dilution
Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson Immunoresearch	711-165-152	Reconstitute in water as per manufacturer’s instructions then store at 4°C 1/2,500 dilution
Cy5 AffiniPure Donkey Anti-Goat IgG (H+L)	Jackson Immunoresearch	705-175-147	Reconstitute in water as per manufacturer’s instructions then store at 4°C 1/250 dilution
Hoechst 33258 solution	Sigma	94403-1ML	Incubate with secondary antibodies Stock solution 0.5 mg/mL in dH20, protect from light -Store at 4 °C
Cy3 Streptavidin	Jackson Immunoresearch	016-160-084	Reconstitute in water per manufacturer’s instructions, then store at 4 °C 1/250 dilution
oligo-(dT)40x probe biotinilated	Integrated DNA Technologies (IDT)	/	Reconstitute in water to 100 ng/mL, aliquot and store at -20 °C Dilute in hybridation buffer 1/50 prior to use Custom order
hybridation box	/	/	Make a moisture chamber with any storing slide box by adding humidified paper on the bottom - prewarm the box prior use
20x SSC buffer	Thermo fisher Ambion	AM9763	Dilute to 2x SSC using RNase Free water (DEPC treated) Store at RT Wash buffer for FISH
PerfectHybPlus Hybridization Buffer	Sigma	H7033-50ml	Block and probe incubation buffer for FISH
slide mounting media	home made	/	Mix 5 g of “cold-soluble” poly(vinyl alcohol) in 20 mL of PBS Mix by sonication, followed by stirring O/N at RT Add 5 mL of glycerol and 0.2 mL of 20 % sodium azide, and stir for 16 h at RT Centrifugation at 20,000 g for 20 min, discard large pellet Aliquot viscous liquid, long-term storage at -20 °C, 1 week at 4 °C
Parafilm "M"	Sigma	P7793-1EA
Poly(vinyl alcohol)	Sigma	P-8136-250G	Reagent to make vinol
Glycerol	Sigma	G5516-100ML	Reagent to make vinol
sodium azide	Fisher Scientific	S2002-5G	Preservative agent for blocking solution and vinol Make a 20% dilution in water