Name: method to visualize and analyze membrane interacting proteins by transmission electron microscopy
Uploaded: 2017-03-05T14:00:00
Description: Watch this Scientific Journal Video about Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy at JoVE.com

The authors thank the Swedish Research Council, Stockholm County Council, and KI funds for their support. The expression and purification of MSP was performed at the Karolinska Institutet/SciLifeLab Protein Science Core Facility (http://PSF.ki.se). The authors would also like to thank Dr. Pasi Purhonen and Dr. Mathilda Sj&#246;berg for sharing their technical expertise and for their timely assistance.

1. Preparation of Nanodiscs
<ol>
	<li>Expression and purification of the membrane scaffolding protein8,35
	<ol>
		<li>Express the His-tagged MSP1E3D1 in the E. coli BL21 (DE3) T1R pRARE2 strain in flasks. Prepare a 50-mL overnight starter culture with LB medium supplemented with 50 &#181;g/mL Kanamycin at 37 &#176;C. Dilute the overnight starter culture in 2 L of terrific broth medium supplemented with 50 &#181;g/mL kanamycin.</li>
		<li>Grow the cells...

The method can be separated into three parts: the reconstitution of empty nanodiscs, the preparation of protein-nanodisc complexes, and the negative staining for the TEM of these complexes. Each part will be addressed separately regarding limitations of the technique, critical steps, and useful modifications.
Reconstitution of empty nanodiscs. Critical steps and limitations in the production and use of nanodiscs.
For the preparation of the empty nanodiscs, it is essent...

In medical research, much attention is focused on membrane proteins, either intrinsic or extrinsic, involved in a variety of lipid interactions. Working with lipid-interacting proteins includes either selecting a substitute to the lipids, such as detergents, amphipols1, or small proteins2, or finding a membrane substitute that keeps the protein soluble and active. Lipoic membrane substitutes include liposomes and nanodiscs (ND)3,4.
Nanodiscs are near-native membrane platforms developed by engineering the protein part, ApoA-1, of the high-density lipoprotein (HDL) naturally occurring in blood. ApoA-1 is a 243 residue-long chain of short amphipathic &#945;-helices and has a lipid-free soluble conformation. In vitro when in the presence of lipids, two copies of the protein ApoA-1 spontaneously rearrange to encircle the hydrophobic acyl chain portion of a lipid bilayer patch5. Engineered versions of ApoA-1 are generally called membrane scaffolding proteins (MSP), and an increasing number are commercially available as plasmids or as purified proteins. Repetitions or deletions of the &#945;-helices in ApoA-1 result in longer6 or shorter7 membrane scaffolding proteins. This in turn makes it possible to form discs around 6 nm7 to 17 nm8 in diameter. There are different types of applications for the nanodiscs3,9. The most commonly used application is to provide a near-native membrane environment for the stabilization of an integral membrane protein8, reviewed previously3,9. A less-explored use is to provide a nanoscale membrane surface for the study of peripheral membrane proteins10,11,12,13,14,15,16,17. Section 1 of the protocol below visualizes the procedure for making nanodiscs composed of phospholipids and membrane scaffolding protein.
Sample preparation is a bottleneck in most methods. Method-specific samples may add particular information, but they also make comparisons of results difficult. Therefore, it is simpler when samples are multimodal and can be used directly in several different methods. One advantage with the use of nanodiscs is the small size of the nanodisc in comparison to liposomes (e.g., the samples can be directly used for both TEM and non-denaturing gel electrophoresis, as in the present protocol).
Vesicles and liposomes have long been used to understand the function of membrane-interacting proteins. For structural studies and visualization, an example of the structural determination of a transmembrane protein in liposomes is available18. However, no high-resolution 3D structure of a monotopic membrane protein embedded on a liposome membrane has been published yet, as far as we know. Gold nanoparticles or antibodies can be used to visualize proteins binding to liposomes or vesicles using TEM19. Even though these probes are very specific, they might interfere with membrane-binding proteins by veiling the membrane binding site or by masking areas of interest with the flexible parts. Gold-labeled or antibody-complexed proteins could probably be analyzed on a gel, but this would increase the cost of the experiment.
Though liposomes are an excellent platform, one cannot be certain that the population has a particular ratio of protein per liposome, a feature that can be explored by the use of nanodiscs20. In a liposome, cofactors and substrates can be trapped in the soluble interior. Substances that are membrane-soluble will share the same fate for both types of membrane mimetics. Nevertheless, as the bilayer area is smaller in nanodiscs, a smaller amount of substance is required to saturate the nanodisc membranes.
Understanding protein function through the determination of the atomic structure has been essential for many fields of research. Methods for protein structure determination include X-ray21; nuclear magnetic resonance (NMR)22,23; and transmission electron microscopy (TEM)24 at cryogenic temperatures, cryoEM. The resolution by cryoEM has lately been greatly improved, mainly due to the use of direct electron detectors25,26. The macromolecules are imaged in thin, vitreous ice27 in a near-native state. However, due to the low contrast of biological molecules, they become hard to detect in the size range of 100 - 200 kDa. For suitably sized samples, data collection can be made and the method of single particle reconstruction can be applied to obtain a structure28.
However, the determination of protein structure by TEM is a multistep process. It usually starts with the evaluation of sample monodispersity by negative-stain TEM29 using salts of heavy metals like phosphotungsten (PT)30 or uranium31. Reconstruction of a low-resolution model of the negatively stained macromolecule is usually made and may yield important information on the molecular structure29. In parallel, data collection using cryoEM may start. Care should be taken when evaluating negative-stain TEM data to avoid the misinterpretation of artefact formation. One particular artefact is the effect of the PT stain on phospholipids and liposomes32, resulting in the formation of long rods resembling stacks of coins viewed from the side33. Such &#34;rouleau&#34; or &#34;stacks&#34; (hereafter denoted as &#34;stacks&#34;) were observed early on for HDL34, and later also for nanodiscs35.
The stacking and reshaping of membranes may occur for many reasons. For example, it can be induced by co-factors like copper, shown by TEM imaging in an ammonium molybdate stain36. A fraction of the membrane lipids in liposomes contained an iminodiacetic acid head group mimicking metal complexation by EDTA, thus stacking liposomes after the addition of copper ions36. Stacking could also be due to a protein-protein interaction by a protein in or on the lipid bilayers (the stain used is not mentioned)37. The stack formation of phospholipids by PT was observed early on; however, later work has focused on removing or abolishing this artifact formation38.
Here, we propose a method to take advantage of the NaPT-induced nanodisc stacking for the study of membrane-binding proteins by TEM. In short, protein binding on the nanodiscs would prevent the nanodiscs from stacking. Though the reasons for the stacking are not clear, it was proposed39 that there is an electrostatic interaction between the phospholipids and the phosphoryl group of PT, causing the discs to stick to each other (Figure 1A). The hypothesis behind our protocol is that when a protein binds to a nanodisc, most of the phospholipid surface is not available for the interaction with the PT due to steric hindrance by the protein. This would prevent stack formation (Figure 1B). Two conclusions can be drawn. First, the prevention of stacking means that the protein of interest has bound to the membrane. Secondly, the protein-ND complex can be treated with standard single-particle processing methods24,40 to get a rough morphology of the complex. Furthermore, analyses by methods like non-denaturing gel electrophoresis or dynamic light scattering can be performed.
To demonstrate this hypothesis, we used the membrane-binding protein 5-lipoxygenase (5LO), which is involved in many inflammatory diseases41,42. This 78-kDa protein requires calcium ions to bind to its membrane43. Though this membrane association has been studied extensively using liposomes44,45,46 and membrane fractions47, these cannot be used for TEM analysis and structure determination.
The preparation of nanodiscs starts by mixing MSP with lipid resuspended in the detergent sodium cholate. After incubation on ice for 1 h, the detergent is slowly removed from the reconstitution mixture using an adsorbent resin. This kind of material is frequently made of polystyrene shaped into small beads. They are relatively hydrophobic and have a strong preference for binding detergent compared to lipids48. After removing the hydrophobic beads and performing clarification using centrifugation, the nanodiscs are purified by size exclusion chromatography (SEC). The purified nanodiscs are mixed with a monotopic membrane protein (and possible cofactors) in an equimolar ratio (or several ratios for a titration) and are left to react (15 min). Analysis by TEM is carried out by applying &#181;L-amounts of sample onto glow-discharged, carbon-coated grids and then by performing negative staining with NaPT. The same sample from when the aliquots were applied to the TEM grids can be used for analysis by non-denaturing or SDS PAGE gel-electrophoresis, as well as by various kinds of activity measurements, with no major changes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Transmission electron microscope: JEOL2100F</td>
			<td>JEOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Tiez Video and Imaging Processing System GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glow discharger</td>
			<td>Baltec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM grid: 400 mesh</td>
			<td>TAAB</td>
			<td>GM016/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Size exclusion chromatography: Agilent SEC-5</td>
			<td>Agilent Technologies</td>
			<td>5190-2526</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 HR 10/300</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5172-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid:MSP1E3D1</td>
			<td>Addgene</td>
			<td>20066</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria: BL21DE3</td>
			<td>NEB</td>
			<td>C2527H</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria: BL21 (DE3) T1R pRARE2</td>
			<td>Protein Science Facility, KI, Solna</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Matrix: ATP agarose</td>
			<td>Sigma Aldrich</td>
			<td>A2767</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Matrix: HisTrap HP-5 ml</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-5247-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid:POPC</td>
			<td>Avanti polar lipids</td>
			<td>850457C</td>
			<td>25 mg/ml in chloroform</td>
		</tr>
		<tr>
			<td>Hydrophobic beads: Bio-Beads, SM-2 Resin</td>
			<td>Bio-Rad</td>
			<td>1523920</td>
			<td></td>
		</tr>
		<tr>
			<td>13 mm syringe filter: 0.2 &#956;m</td>
			<td>Pall life sciences</td>
			<td>PN 4554T</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain: Sodium phosphotungstate tribasic hydrate</td>
			<td>Sigma Aldrich</td>
			<td>31648</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma Aldrich</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma Aldrich</td>
			<td>646547</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent: Sodium cholate hydrate</td>
			<td>Sigma Aldrich</td>
			<td>C6445-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cholate</td>
			<td>500 mM Sodium cholate</td>
			<td></td>
			<td>Resuspend in miliQ water and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Lipid Stock</td>
			<td>50 mM POPC, 100 mM sodium cholate, 20 mM Tris-HCl pH 7.5, 100 mM NaCl</td>
			<td></td>
			<td>Store at 4&#176;C for a week or 
			Store -80&#176;C for a month, after purging the solution with nitrogen</td>
		</tr>
		<tr>
			<td>MSP standard buffer</td>
			<td>20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mMEDTA</td>
			<td></td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis Anode Buffer</td>
			<td>50 mM Bis Tris 50 mM Tricine, pH 6.8</td>
			<td>BN2001</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis Cathode Buffer</td>
			<td>50 mM Bis Tris 50 mM Tricine, pH 6.8 0.002% Coomassie G-250</td>
			<td>BN2002</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Non-Denaturaing Electrophoresis 4X Sample loading Buffer</td>
			<td>50 mM BisTrispH 7.2, 6N HCl, 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S</td>
			<td>BN2003</td>
			<td>Purchased from Thermofisher Scientific</td>
		</tr>
		<tr>
			<td>Denaturaing Electrophoresis Running Buffer</td>
			<td>25 mM Tris-HCl pH 6.8, 200 mM Glycine, 0.1 % (w/v) SDS</td>
			<td></td>
			<td>Inhouse receipe</td>
		</tr>
		<tr>
			<td>Denaturaing Electrophoresis 5X Sample loading Buffer</td>
			<td>0.05 % (w/v) Bromophenolblue, 0.2 M Tris-HCl pH 6.8, 20 % (v/v) glycerol, 10% (w/v) SDS,10 mM 2-mercaptoethanol</td>
			<td></td>
			<td>Inhouse receipe</td>
		</tr>
		<tr>
			<td>Terrific broth</td>
			<td>Tryptone - 12.0g 
			Yeast Extract - 24.0g 
			100 mL 0.17M KH2PO4 and 0.72M K2HPO4 
			Glycerol - 4 mL</td>
			<td></td>
			<td>Tryptone, yeast extract and glycerol were prepared to 900 ml and autoclaved seperately. KH2PO4 and K2HPO4 were prepared and autoclaved separately. Both were mixed before using the medium</td>
		</tr>
	</tbody>
</table>

method to visualize and analyze membrane interacting proteins by transmission electron microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

The method we propose depends upon the preparation of nanodiscs to provide the membrane surface for monotopic membrane-protein binding. As there is no transmembrane protein embedded into the nanodisc lipid bilayer, the nanodiscs are here denoted as &#34;empty nanodiscs&#34; (Figure 2A). These have a calculated molecular weight of 256 kDa for a composition of two MSP1E3D1 scaffolding proteins and around 260 molecules of POPC8. Using this protein:lip...

Watch this Scientific Journal Video about Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy at JoVE.com

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

The overall goal of this procedure is to visualize the binding of an extrinsic membrane protein on a nanodisc membrane surface by negative stain transmission electron microscopy as a first step towards high resolution structure determination of the protein. This method can help answer key questions in several research fields as it concerns protein activities that occur in and on cellular membranes. The main advantage of this technique is the characteristic stacks of nanodisc forms if the protein cannot bind to the membranes.These stacks are clearly visible by transmission electron microscopy. The implication of this technique extends towards drug development as compounds can be easily screened for the ability to block or enable the membrane protein interactions. Though this method can provide insight into the optimal conditions for monotopic protein binding to your membrane, it also provides a low resolution structure of the protein nanodisc complex.To begin the procedure, express and purify membrane scaffolding protein such as MSP1E3D1. Next, using a glass syringe with a metal needle, dispense 305 microliters of 25 milligrams per milliliter of POPC in chloroform into a glass round-bottom flask. Evaporate the chloroform under a gentle stream of nitrogen gas in a fume hood.Dry the remaining lipid overnight in a vacuum desiccator. Then dissolve the dried lipid cake in 200 microliters of MSP standard buffer enriched with 100 millimolar sodium cholate as detergent. Vortex the mixture until transparent to obtain a suspension of 50 millimolar POPC in buffer.Next, wash five grams of hydrophobic beads with 30 milliliters of 100%methanol then with 40 milliliters of ultrapure water and finally with 10 milliliters of MSP standard buffer. Store the beads under 15 milliliters of standard buffer at four degrees Celsius. Then combine 190 microliters of a 0.124 millimolar solution of MSP1E3D1 and 61.5 microliters of a 50 millimolar suspension of POPC in buffer resulting in a one to 130 molar ratio of MSP to POPC and a sodium cholate concentration of 25 millimolar.The ideal ratio of the lipid per MSP varies with each choice of lipid type and MSP lens and must be optimized to obtain a homogenous preparation of nanodiscs. Incubate the mixture on wet ice for one hour. Then add the solution to a tube containing 0.5 grams of washed hydrophobic beans per milliliter of reconstitution mixture to initiate nanodisc self assembly.Incubate the mixture at four degrees Celsius for 16 hours at seven to eight rotations per minute. After incubation, allow the beads to settle by gravity. Remove and store the supernatant at four degrees Celsius until ready to perform size-exclusion chromatography.Prior to size-exclusion chromatography, centrifuge the mixture at 13, 000 g for 10 minutes at four degrees Celsius. Decant the supernatant and discard the pellet. Equilibrate a size-exclusion chromatography column with MSP standard buffer until the baseline at 280 nanometers is stable.Inject the supernatant onto the column and collect the product in 0.5 milliliter fractions. Measure the absorbance of the fractions at 280 nanometers with a UV vis spectrophotometer. Calculate the concentration of nanodiscs using the molar extinction coefficient for the chosen MSP.To perform non-denaturing gel electrophoresis, first mix 15 microliters of the sample with five microliters of the appropriate loading buffer. Fill the cathode tank with light cathode buffer and the anode tank with running buffer. Load the sample onto a four to 16%Bis-Tris gel and begin the run.Stain the gel per the gel manufacturer's instructions. To begin preparation of a nanodisc monotopic protein complex with five lipoxygenase as the protein, prepare a batch of MSP standard buffer enriched with 1.5 millimolar calcium chloride. Express and purify the 5LO protein.Immediately prepare a 100 microliter mixture of 0.8 micromolar 5LO and 0.8 micromolar nanodiscs in calcium-enriched MSP standard buffer. Our example of a monotopic proteinase five lipoxygenase depends on calcium amounts to bind to the membrane. 5LO is highly sensitive and must be used within a few hours of preparation.Incubate the mixture on ice for 10 minutes. Store the resulting nanodisc protein complex sample at four degrees Celsius for up to one month. To begin preparation for the TEM analysis, dissolve one gram of phosphotungstate sodium salt in 50 milliliters of ultrapure water at room temperature.Adjust the solution pH to 7.4 with a one molar solution of sodium hydroxide. Filter the phosphotungstate sodium solution through a 0.22 micrometer syringe filter and store the solution at room temperature. Next, glow discharge of 400 mesh carbon coated copper grid for 20 seconds at 30 milliamps to render the grid surface hydrophilic.Place between 2.5 and five microliters of the nanodisc monotopic protein complex sample on the grid and allow the sample to sit for 30 seconds. Then use filter paper to blot excess solution from the grid. Immediately apply a drop of phosphotungstate sodium solution and let the solution sit for 30 seconds.Blot the excess solution and allow the grid to air dry. Perform transmission electron microscopy with an accelerated voltage of 120 to 200 kilovolts. Exclude images showing long stacks from subsequent image processing.For the selected images, use standard processing methods to determine the class averages and to generate a low resolution 3D model of the nanodisc monotopic protein. Using this method, empty nanodiscs were prepared with a one to 130 ratio of membrane scaffolding proteins to lipids. Only one major peak was observed during size-exclusion chromatography and only one band was observed in blue native gel electrophoresis.When the empty nanodiscs are treated with a phosphotungstate sodium salt solution, stacking is induced. These long stacks can then be observed by TEM. This stacking was not disrupted by the inclusion of calcium ions in the nanodisc solution before application of the phosphotungstate sodium solution.When a monotopic protein such as five lipoxygenase is bound to the nanodisc surface, stacking is hindered by stearic obstruction by the protein. Both sides of the nanodisc are available for binding allowing formation of one to one and two to one 5LO nanodisc complexes. The sample of two to one 5LO nanodisc complexes exhibited less stacking than the one to one sample.5LO binding requires the presence of calcium ions. This was confirmed by observation of stacking in a mixture of 5LO and nanodiscs without calcium which indicated that 5LO had not bound to the membrane surfaces. Once the monotopic protein and the nanodiscs are prepared, evaluation of protein binding to your membrane can be done in an afternoon if it is performed properly.While attempting this procedure, it is important to estimate the area of the protein on the nanodisc to choose the correct MSP length. For well-matched sizes, maximally two extrinsic proteins may bind one on each side of the disc. It is also important that the sodium salt of phosophotungsten is used for the negative stand to ensure maximal stacking as binding is confirmed by comparison of the absence of stacks to the long stacks of a sample without a monotopic protein.Use of the nanodiscs instead of liposomes enables the use of dynamic light scattering, small angle x-ray scattering to answer additional questions about sample homogeneity size or molecular structure. The low resolution 3D structure of a monotopic membrane protein bound to a nanodisc could be an easily accessible first step in the path to high resolution structure of those bound proteins.

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A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Analysis of &amp;#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...