Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Summary

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

Abstract

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and γ-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Introduction

A change of protein subcellular localization is a common mechanism in response to internal or external signals such as heat stress, starvation, oxidative stress, apoptosis, protein phosphorylation, and others. For example, heat stress induces a FOXO member DAF-16 nuclear translocation^1,2, and the pro-apoptotic BCL-2 protein BID translocates to the mitochondria upon receiving death signaling^3,4. Various techniques are available to detect these changes. A combination of western blotting and biochemically isolating subcellular structures (e.g., m....

Protocol

1. Solutions

1. NGM plates
   1. In a 2 L Erlenmeyer flask, add 3 g of NaCl, 17 g of agar, 2.5 g of Peptone, and 975 mL of dH₂O. Cover the mouth of the flask with aluminum foil. Autoclave the flask for 50 min. Cool it for 20–30 min.
   2. Then add sterilized solutions: 1 mL of 1 M CaCl₂, 1 mL of 5 mg/mL cholesterol in ethanol, 1 mL of 1 M MgSO₄, and 25 mL of 1 M KPO₄ (pH 6.0) buffer. Swirl the solutions to mix them well.
   3. Using a liquid dispenser, dispense the NGM solution to 60 mm Petri plates. Fill the plates 2/3 full of agar. Store them in an air-tight container at 4 &#....

Results

Chloride intracellular channel proteins (CLIC) are multifunctional proteins that are highly conserved across species¹². Much research shows that CLICs regulate cellular stress, autophagy, apoptosis, carcinogenesis, angiogenesis, and the macrophage innate immune response in the mammalian system^{13,14,15,16,17}

Discussion

This article presented a fast and efficient method to verify protein subcellular changes from the cytoplasm to the nucleus. The protein expression was shown by a GFP fusion, while the nucleus structure was verified by DAPI staining (Figure 3). Since immunostaining C. elegans proteins is challenging, most C. elegans protein subcellular localizations are characterized by tagging them with marker proteins such as GFP, Laz, mCherry, and others^18,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DAPI	Biotium	40043	both Hoechst 33258 and Hoechst 33342 also works well
OP50	CGC	OP50	https://cgc.umn.edu/
Caenorhabditis Genetics Center (CGC)			https://cgc.umn.edu/
Kim Wipe	Kimberly-Clark Corporation		soft tissue
Zeiss  AX10	Zeiss		fluorescence microscope
Axiovision Rel 4.8	Zeiss		microscope software
AxioCam MR Rev3	Zeiss		digital camera
Incubator
Micro centrifuge
Transparent nail polish gel
60 mm petri dishes
Glass slides
Glass coverslips
1-20 µL pipettor
20-200 µL pipettor
200-1000 µL pipettor
1-200 µL pipet tips
200-1000 µL pipet tips
1.5 mL microcentrifuge tubes
Platinum wire worm pick