Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Summary

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

Abstract

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).

Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.

A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Introduction

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research^1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution³; however, the precise identification and localization of protein molecules remain challenging.

The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-a....

Protocol

All the supplies used in this experiment are listed in the Table of Materials. The step-by-step workflow of the current protocol is shown in Figure 1.

1. Cell culture on sapphire discs

1. Transfer 3 mm x 0.16 mm sapphire discs to a 2 mL centrifuge tube containing 1 mL of ethanol, and sonicate in an ultrasonic cleaner for 10 min.
2. Burn each sapphire disc in an alcohol lamp flame until there are no visible deposits on the surface.
   NOTE: Through the above method, sapphire discs can be recycled many times.
3. Label one side of each sapphire disc with....

Results

The ANSM-based AuNP synthesis technique is an extremely useful tool for labeling and detecting MT-tagged proteins with TEM²⁶. To validate its robustness in mammalian cells, three stable cell lines expressing EGFP-MTn-KDEL, Ost4-EGFP-MTn, or Mito-acGFP-MTn in Hela cells were generated. KDEL is a canonical C-terminal endoplasmic reticulum (ER) retention/retrieval sequence, which maintains the fusion protein EGFP-MTn-KDEL within the ER lumen or the perinuclear space of the nuclear envelope (NE). Ost4.......

Discussion

The study presents here a robust clonable EM labeling technology for the single-molecule visualization of protein molecules within the cellular environment with ultrastructural resolution. The AuNPs directly synthesized on genetically encoded cysteine-rich tags provide unambiguous and precise localization of the target proteins. High-pressure freezing and freeze-substitution technique excellently preserve the ultrastructure of biological samples. Taken together, the clonable electron microscopy labeling technology presen.......

Materials

Name	Company	Catalog Number	Comments
0.025 mm/0.275 mm Aluminum carrier	Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend
0.2 M HEPES buffer			Dissolve HEPES (0.2 M) in 980 mL of ddH2O, then add 10 mL of 100 mM MgCl2 and 10 mL of 100 mM CaCl2 (final concentration 1 mM), respectively, adjust pH to 5.5
1.5 mL MaxyClear snaplock microtube	Axygen Scientific	MCT150C
2 mL polypropylene screw cap microtubes	Biologix	81-0204
200 mesh hexagonal copper grid	Tedpella inc	G200HEX
2-mercaptoethanol	Amresco	0482-250ML
35 mm cell culture dishes	Corning	430165
50 mL polypropylene centrifuge tubes	Corning	430928
Acetone	Beiijng Tong Guang Fine Chemicals Company	31025
Automated freeze substitution machine	Leica	AFS2
Customized 3.05 mm x 0.66 mm specimen holders for HPF	Beijing Wulundes Biotech Ltd.
D-penicillamine	TCI	P0147
Dulbecco’s modified Eagle medium	GBICO	C11965500BT
Fetal bovine serum	GBICO	10099-141C
Flat bottom embedding capsule	Tedpella inc
Foam cryobox
Formvar 15/95 resin	Electron Microscopy Sciences	15800
HAuCl4	Sigma	4022-1G
HEPES	sigma	H3375-500G
HPF machine	Wohlwend	HPF compact01
Methonal	Beiijng Tong Guang Fine Chemicals Company	12397
NaBH4	Sigma	480886-25G
OsO4	Electron Microscopy Sciences	19110
PBS-A buffer			Dissolve NaH2PO4 (1.125 mM), Na2HPO4 (3.867 mM), NaCl (100 mM) in 1 L of ddH2O, adjust to pH 7.4
Qualitative filter paper (medium speed)	Beyotime Biotechnology	FFT08
Sapphire discs	Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend
Solvent resistant pen	Electron Microscopy Sciences	62053-B
SPI-Pon 812 resin	SPI Inc	02659-AB
Transmission electron microscopy	FEI	Tecnai G2 spirit
Trypsin-EDTA	GBICO	C25200-056
Tweezers	Dumont
Ultramictotome	Leica	FC7
Uranyl acetate	Electron Microscopy Sciences	22400