Name: the cryoapex method for electron microscopy analysis of membrane protein localization within ultrastructurally-preserved cells
Uploaded: 2020-02-27T13:00:00
Description: Watch this Scientific Journal Video about The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells at JoVE.com

The protocol described here stems from a publication by Sengupta et al., Journal of Cell Science, 132 (6), jcs222315 (2019)48. This work is supported by grants R01GM10092 (to S.M.) and AI081077 (R.V.S.) from the National Institutes of Health, CTSI-106564 (to S.M.) from Indiana Clinical and Translational Sciences Institute, and PI4D-209263 (to S.M.) from the Purdue University Institute for Inflammation, Immunology, and Infectious Disease.

1. Cell Culture and Transfection
<ol>
	<li>Seed HEK-293T cells on a 60 mm diameter or larger tissue culture dish and grow to 60%&#8211;90% confluence in a cell culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>Transfect cells with APEX2-tagged mammalian expression plasmids using transfection reagent (see Table of Materials) according to the manufacturer&#39;s directions.</li>
	<li>At 12&#8211;15 h post-transfection, wash cells once with phosphate buffered saline (PBS). Remove cells from ...

The cryoAPEX protocol presented here provides a robust method to characterize the localization of membrane proteins within the cellular environment. Not only does the use of a genetically encoded APEX2 tag provide precise localization of a protein of interest, but the use of cryofixation and low-temperature dehydration provides excellent preservation and staining of the surrounding cellular ultrastructure. Combined, these approaches are a powerful tool for localizing a protein with high precision within its subcellular c...

Biological studies often include questions of resolving subcellular protein localization within cells and organelles. Immunofluorescence microscopy provides a useful low-resolution view of protein localization, and recent advances in super-resolution imaging are pushing the bounds of resolution for fluorescently tagged proteins1,2,3. However, electron microscopy (EM) remains the gold standard for imaging high-resolution cellular ultrastructure, though the labeling of proteins is a challenge.
Historically, several EM methods have been used to approach questions of ultrastructural protein localization. One of the most commonly utilized methods is immunoelectron microscopy (IEM), where antigen-specific primary antibodies are used to detect the protein of interest. EM signal is generated by the application of secondary antibodies conjugated with electron-dense particles, most commonly colloidal gold4,5. Alternately, antibodies conjugated with enzymes such as horse radish peroxidase (HRP) can be used to produce an electron-dense precipitate6,7,8. Two main approaches exist for IEM, termed pre-embedding and post-embedding labeling. In pre-embedding IEM, antibodies are introduced directly into cells, which necessitates light fixation and permeabilization of the cells9,10,11. Both steps can damage ultrastructure12,13. Development of significantly smaller antibodies consisting of an antibody Fab fragment conjugated with 1.4 nm nanogold allows very gentle permeabilization conditions to be used; however, nanogold is too small for direct visualization under TEM and requires additional enhancement steps to become visible14,15,16. In post-embedding IEM, antibodies are applied on thin sections of cells which have been fully processed by fixation, dehydration, and embedding in resin17. While this approach avoids the permeabilization step, preserving the epitope of interest throughout sample preparation is challenging18,19,20. The Tokuyasu method of light fixation followed by freezing, cryo-sectioning, and antibody detection provides improved epitope preservation21,22. However, the technical requirements of cryo-ultramicrotomy, as well as the sub-optimal contrast achieved in the cell, are disadvantages23.
The use of genetically encoded tags eliminates many of the difficulties of IEM related to detection of the protein of interest. A variety of tags are available, including HRP, ferritin, ReAsH, miniSOG, and metallothionein24,25,26,27,28,29,30,31,32. Each of these has advantages over previous methods, but each also has drawbacks preventing widespread use. These drawbacks range from inactivity of HRP in the cytosol to the large size of the ferritin tag, light sensitivity of ReAsH, and small size and lack of compatibility with cellular staining of metallothionein. Recently, a protein derived from ascorbate peroxidase has been engineered as an EM tag, named APEX233,34. As a peroxidase, APEX2 can catalyze the oxidation of 3,3&#39; diaminobenzidine (DAB), producing a precipitate that reacts with osmium tetroxide to provide local EM contrast with minimal diffusion from the protein of interest (less than 25 nm)33,35. Unlike traditional HRP-based methods, APEX2 is extremely stable and remains active in all cellular compartments33. Samples can be processed for TEM using traditional EM sample staining and methods that allow good visualization of the surrounding structures33,34,36. Because of its small size, stability, and versatility, APEX2 has emerged as an EM tag with great potential.
Many of the approaches discussed above either cannot be or have not yet been combined with the current state of the art in ultrastructural preservation, cryofixation and low-temperature freeze-substitution. Thus, they suffer from a lack of membrane preservation and/or cell staining to determine accurate protein localization. This necessarily limits the resolution and interpretation of the data that can be obtained. Cryofixation by high pressure freezing (HPF) involves rapid freezing of samples in liquid nitrogen at a high pressure (~2,100 bar), which causes vitrification rather than crystallization of aqueous samples, thus preserving cells in a near-native state37,38,39. HPF is followed by freeze substitution (FS), a low temperature (-90 &#176;C) dehydration in acetone combined with incubation with typical EM stains such as osmium tetroxide and uranyl acetate. HPF and FS together provide a distinct advantage over traditional chemical fixation (a longer process which can lead to artefacts) and alcohol dehydration at room temperature or on ice (which can lead to extraction of lipids and sugars), and thus are desirable to combine with the best EM tags for protein detection.
One reason that HPF/FS has not been combined with APEX2 labeling is that light chemical fixation is a prerequisite for the peroxidase reaction, limiting the diffusion of the DAB reaction product. In APEX2 studies thus far, fixation and peroxidase reaction are followed by traditional EM methods for staining and alcohol dehydration33,36. However, it has been shown that following chemical fixation with HPF/FS provides a distinct advantage in preservation over traditional chemical fixation and alcohol dehydration alone40. The loss of ultrastructural integrity seen in traditional TEM samples appears less connected to fixation than to dehydration, which is typically done using alcohol at room temperature or on ice, and can lead to extraction of lipids and sugars40,41. To develop the cryoAPEX method, we hypothesized that chemical fixation and peroxidase reaction, followed by HPF and FS, would produce an optimal result in terms of ultrastructural preservation.
Here we present the cryoAPEX protocol, which combines APEX2 tagging with cryofixation and freeze substitution methods (Figure 1). This straightforward protocol consists of transfection of an APEX2-tagged protein of interest, chemical fixation of cells, and the peroxidase reaction. HPF and FS are then performed followed by typical resin embedding and thin sectioning. TEM imaging reveals excellent preservation of ultrastructure using this method. Additionally, high-resolution subcellular localization and spatial distribution of an endoplasmic reticulum (ER) lumenal protein were observed. This method is widely useful for detection of membrane protein localization within cells for electron microscopy analysis. In our hands, the method has worked successfully for a variety of cell lines grown in tissue culture, including HEK-293T (human embryonic kidney), HeLa (human cervical cancer), Cos7 (African green monkey kidney fibroblast), and BHK (baby hamster kidney). Detailed instructions are described below using HEK-293T cells.

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	<tbody>
		<tr>
			<td>3,3&#39;-Diaminobenzidine tetrahydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5637-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone (Glass Distilled)</td>
			<td>Electron Microscopy Sciences</td>
			<td>10016</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers; Plastic, Disposable 120 cc</td>
			<td>Electron Microscopy Sciences</td>
			<td>60952</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Storage Vials, 2 mL</td>
			<td>VWR</td>
			<td>82050-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM Fluka, single component A, M epoxy resin</td>
			<td>Sigma-Aldrich</td>
			<td>44611-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM Fluka, single component B, hardener 964</td>
			<td>Sigma-Aldrich</td>
			<td>44612-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM Fluka, single component C, accelerator 960 (DY 060)</td>
			<td>Sigma-Aldrich</td>
			<td>44613-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM Fluka,single component D</td>
			<td>Sigma-Aldrich</td>
			<td>44614-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold, standard flat, 14 mm x 5 mm x 6 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70901</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold, standard flat, 14 mm x 5 mm x 4 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum; Nu-Serum IV Growth Medium Supplement</td>
			<td>Corning</td>
			<td>355104</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Knife Boats, 6.4 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>71008</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Knifemaker</td>
			<td>Leica Microsystems</td>
			<td>EM KMR3</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 10% Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>16120</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293 Cells</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>High Pressure Freezer with Rapid Transfer System</td>
			<td>Leica Microsystems</td>
			<td>EM PACT2</td>
			<td>Archived Product Replaced by Leica EM ICE</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30% Solution</td>
			<td>Fisher Scientific</td>
			<td>50-266-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane carrier for EM PACT2, 1.5 mm x 0.1 mm</td>
			<td>Mager Scientific</td>
			<td>16707898</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide, crystalline</td>
			<td>Electron Microscopy Sciences</td>
			<td>19110</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 20X, Ultra Pure Grade</td>
			<td>VWR</td>
			<td>97062-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Capsules for AFS/AFS2, 5 mm x 15 mm</td>
			<td>Mager Scientific</td>
			<td>16702738</td>
			<td></td>
		</tr>
		<tr>
			<td>Slot grids, 2 x 1 mm copper with Formvar support film</td>
			<td>Electron Microscopy Sciences</td>
			<td>FF2010-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate Buffer, 0.2 M, pH 7.4</td>
			<td>Electron Microscopy Sciences</td>
			<td>102090-962</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, Pellet 500 G (ACS)</td>
			<td>Avantor Macron Fine Chemicals</td>
			<td>7708-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic Acid</td>
			<td>Electron Microscopy Sciences</td>
			<td>21710</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dishes, Polystyrene, Sterile, Corning, 100 mm</td>
			<td>VWR</td>
			<td>25382-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Glass Knife Strips</td>
			<td>Electron Microscopy Sciences</td>
			<td>71012</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems</td>
			<td>EM UC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate Dihydrate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

the cryoapex method for electron microscopy analysis of membrane protein localization within ultrastructurally-preserved cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

In order to compare the ultrastructural preservation using the cryoAPEX method with traditional fixation and dehydration, we prepared samples in which an endoplasmic reticulum membrane (ERM; ER membrane) peptide was tagged with APEX2 and transfected into HEK-293T cells. ERM-APEX2 localizes to the cytoplasmic face of the ER and remodels the ER structure into morphologically distinct structures known as organized smooth ER (OSER)34,42,4...

Watch this Scientific Journal Video about The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells at JoVE.com

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

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