Quantitative Immunofluorescence to Measure Global Localized Translation

Summary

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Abstract

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

Introduction

The regulation of translation by different cellular functions has prompted many research teams to develop new tools and methods to determine the subcellular localization of mRNA translation and regulated protein synthesis^1,2,3,4. These recent technological advances allow for an improved understanding of the mechanisms involving translation upregulation or the repression of specific mRNAs during biological processes, such as neuronal development, drug response, and metastasis^5,6,....

Protocol

1. Determination of Puromycilation Conditions

NOTE: This technique describes the method used to assess localized translation during the MRC-5 cell adhesion process⁵. As puromycilation can be done in any cell, it is important to optimize the puromycilation conditions for the specific cell lines to be used, because the treatment conditions are not identical for each cell line in terms of the puromycin concentration and the desired incubation time. To show how these conditions are defined, three example cell lines (i.e., HeLa, MRC-5, and Huh-7) were treated with increasing concentrations of puromycin for differen....

Results

To accurately observe translation events using puromycin incorporation, it is critical to determine the optimal conditions for each cell line because each shows different puromycin incorporation kinetics (Figure 1)^9,11,12,18. Hence, to validate puromycin incorporation, it is necessary to treat the desired cell line with a standardiz.......

Discussion

Recent technological advances have allowed for a better understanding of the mechanisms involved in translational upregulation or the repression of specific mRNAs in biological processes, such as neuronal development, drug response, and metastasis. The cost-effective methodology described here allows translation events to be visualized in cells to study how RNA-binding proteins regulate metastatic processes, such as cellular adhesion, migration, and invasion.

Although numerous methods to asses.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	wisent	319-005-CL
Trypsine	wisent	325-043 EL
FBS	Thermo Fisher Scientific	12483020
Puroycin antibody 12D10	EMD millipore	MABE343	western blot dilution 1:25,000 Immunofluoresence dilution 1:10,000
Anti-mouse IgG, HRP-linked Antibody	cell signaling technology	7076	western blot dilution 1:8,000
Western Lightning Plus-ECL	Perkin Elmer	NEL104001EA
Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate)	cell signaling technology	4408	immunofluoresecence dilution 1:400
CF568 Phalloidin	biotium	00044	immunofluoresecence dilution 1:400
Cyclohexmide	Sigma	C1988-1G	50µg/ml final concentration
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306	final concentration 1µg/ml
Puromycin	bio-Basic	PJ593	2.5µg/ml to 10µg/ml
Ibidi µ-Dish 35 mm, high, ibiTreat	Ibidi	81156
MRC-5 cells	ATCC	CCL-171
HeLa cells	ATCC	CCL-2
Huh-7 cells	from Dr. Mazroui (Université Laval)
Fv1000	olympus		confocal imaging system
Fiji software			http://fiji.sc
PBS (Phosphate BuffeRed Saline)	bio-Basic	PD8117
Formaldehyde 37% Solution	bio-Basic	C5300-1
Triton X-100	bio-Basic	TB0198
BSA	Fisher Bioreagents	BP9702-100
Tween20	Fisher Bioreagents	BP337-500