Name: quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy
Uploaded: 2022-02-07T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

This work was supported by the Swedish Olympic Committee.

The present protocol using human biopsied skeletal muscle samples was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20170198). Muscle biopsies were obtained through an incision in the skin from the vastus lateralis muscle using a Bergstr&#246;m needle with suction after local anesthesia was given subcutaneously (1-3 mL of Lidocaine 2% per incision). If isolated whole rat muscles were used, the animals were sacrificed by cervical dislocation before the muscle biopsies were ...

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The critical step of the method is the use of reduced osmium by potassium ferrocyanide during post-fixation. The selectivity of this modified fixative for glycogen detection cannot be fully explained by chemistry, but also includes experimental findings demonstrating no detection of such particles in tissues known to be free of glycogen or in the extracellular space11.
Critical parameters are the precision of the estimates and the fiber-to-fiber variation. By following ...

Glycogen particles are composed of branched polymers of glucose and various associated proteins1 and constitute an important fuel during high metabolic demands2. Although not widely recognized, glycogen particles also constitute a local fuel, where some subcellular processes preferentially utilize glycogen despite the availability of other and more long-lasting fuels as plasma glucose and fatty acids3,4.
The importance of storing glycogen as a subcellular specific localized fuel has been discussed in several reviews5,6 mainly based on some of the earliest documentations of the subcellular distribution of glycogen by transmission electron microscopy (TEM)7,8. The first studies used different protocols to increase the contrast of glycogen from histochemical staining techniques to negative and positive stainings9,10. An important methodological development was the refined post-fixation protocol with the potassium ferrocyanide-reduced osmium11,12,13,14, which significantly improved the contrast of glycogen particles. This refined protocol was not used in some of the pioneering work on exercise-induced glycogen depletion15 but was re-introduced by Graham and colleagues16,17.
Based on the 2-dimensional images, the subcellular distribution of glycogen is most often described as glycogen particles located in three pools: subsarcolemmal (just beneath the surface membrane), intermyofibrillar (between the myofibrils), or intramyofibrillar (within the myofibrils). However, glycogen particles could also be described as associated with, for example, sarcoplasmic reticulum7 or nuclei18. In addition to the subcellular distribution, the advantage of TEM-estimated glycogen content is also that quantification can be conducted at the single fiber level. This allows investigation of fiber-to-fiber variability and correlative analyses with fiber types and cellular components as mitochondria and lipid droplets.
Here, the protocol for the TEM-estimated fiber type-specific volumetric content of the three common subcellular pools of glycogen (subsarcolemmal, intermyofibrillar, and intramyofibrillar) in skeletal muscle fibers is described. The method has been applied to skeletal muscles from humans19, rats20, and mice21; as well as birds and fish22; and cardiomyocytes from rats23.

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			<td>Embedding 812 resin medium kit</td>
			<td>Taab</td>
			<td>T031</td>
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			<td>Glutaraldehyde solution 25%</td>
			<td>Merck</td>
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			<td>Osmium tetroxide 4% solution</td>
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			<td>Philips CM 100 Transmission EM</td>
			<td>Philips</td>
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			<td>Potassium hexacyanoferrate (II) trihydrate</td>
			<td>Sigma-Aldrich</td>
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			<td>Sodium cacodylatbuffer 0,2 M ph 7.4</td>
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			<td>Leica</td>
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			<td>Polysciences</td>
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quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

Using this protocol, glycogen particles appear black and distinct (Figures 1 and Figure 2). The normal values of glycogen are depicted in Figure 3. These data are based on a total of 362 fibers from 41 healthy young men as collected in different previous studies19,24,29,30,31</...

Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

The measure of glycogen content in single cells and subcellular organizations is important because in most cell types, single cells adapt differently to changing physiological conditions and diseases. So, the main advantage of this technique is the very high resolution in the transmission electron microscope. This improves the possibility to have localized subcellular structures and also in this content in this technique here, it's important that the glycogen staining is antibody free.So, we don't have any specificity to worry about. In order to get a better contrast on glycogen, just use potassium ferrocyanide 1.5%instead of potassium ferrocyanide. Prepare 1.6 milliliters of the primary fixative solution by adding 2.5%glutaraldehyde in 0.1 molar sodium cacodylate buffer in a two milliliter micro-centrification tube.Store it at five degrees Celsius for a maximum of 14 days. Isolate a small specimen from the muscle biopsy or whole muscle which has a maximum diameter of one millimeter in any direction and is longer longitudinally than cross-sectionally. Place the specimen in the tube containing the cold primary fixation solution.Store it at five degrees Celsius for 24 hours. Then wash the specimen four times in 0.1 molar sodium cacodylate buffer. Using transfer pipettes, remove the used buffer from the tube leaving the specimen untouched and add the fresh buffer.Post-fix the specimen with 1%osmium tetroxide and 1.5%potassium ferrocyanide in 0.1 molar sodium cacodylate buffer for 120 minutes at four degrees Celsius. Rinse the specimen twice in double distilled water at room temperature. Dehydrate by submerging in a graded series of ethanol at room temperature.Infiltrate the specimen with propylene oxide and epossidic resin graded mixtures at room temperature using the mentioned volume ratios. The following day, embed specimens in 100%fresh epossidic resin in molds and polymerize at 60 degrees Celsius for 48 hours. Mount the block of a specimen on the ultramicrotome holder.Trim the block on the surface with a razor blade to reach the tissue level. Mount a diamond knife in front of the sample and align the sample surface parallel to the knife. Produce a semi-thin section of one micrometer thickness with the diamond knife to check the orientation of the sample.Stain the semi-thin section with toluidine blue for observation with light microscopy. Then trim the block further to reduce the area of interest to get proper ultrathin sections. Cut 60 to 70 nanometer thickness ultrathin sections with a second diamond knife.Collect one to two sections on one whole copper grids using a perfect loop. For contrasting the sections, immerse the grids in uranyl acetate solution for 20 minutes and wash the grids in double distilled water and then immerse the grids in lead citrate for 15 minutes and re-wash the grids in double distilled water. Turn on the transmission electron microscope computer and image recording software, record digital images with a digital Slow Scan CCD Camera and the associated imaging software.After inserting the grid with multiple sections in the microscope stage, screen the grid initially at low magnification to choose the best quality sections and determine the direction of the muscle fibers. Next focus, the image at magnification above 30, 000 with the beam centered on a peripheral fiber in the section and record images with one second exposure time at the desired magnification. Ensure that the images are distributed across the length and width of the fiber in a randomized bot systematic order to obtain unbiased results and repeat the imaging until six to 10 fibers are imaged.Import images to image J by clicking on file followed by open. Set global scale to match the original size of the image by clicking on analyze and then set scale. To zoom in 100%click on image, go to zoom and click on in.Measure the thickness of one Z-disk per image of the myofibrillar space using the straight line tool from the tools menu and calculate the average Z-disk thickness of each of the six to 10 fibers. Use the segmented line tool to measure the length of the outermost myofibril visible just below the subsarcolemmal region. To insert a grid, click on analyze, select tools and then select grid.Now set the area per point at 32, 400 square nanometers. Count the number of hits within the available length in the 12 subsarcolemmal images where a cross hits the subsarcolemmal glycogen. Insert a grid by clicking on analyze, then select tools, followed by grid and set area per point at 160, 000 square nanometers.Count the number of hits in the 12th myofibrillar images where a cross hits the intramyofibrillar space. Then again, to insert a grid, click on analyze, select tools and then select grid. Now set the area per point at 3, 600 square nanometers.Count the number of hits in the 12th myofibrillar images where a cross hits the intramyofibrillar glycogen. Insert a grid by clicking on analyze, then select tools, followed by grid and set area per point at 32, 400 square nanometers. Count the number of hits in the 12th myofibrillar images where a cross hits the intermyofibrillar glycogen.Using the 32, 400 square nanometers grid to randomly choose the glycogen particles, start with the upper left square and move left if necessary. Using straight line tool, measure the diameter of five randomly chosen glycogen particles of each pool for each of the 12 images to obtain an average of 60 particles per pool per fiber. This figure shows the normal values of the three glycogen pools.It can be observed that intermyofibrillar glycogen values are distributed close to normal, whereas both intramyofibrillar and subsarcolemmal glycogen show skewed distribution where fibers sometimes have an excessive amount of glycogen. Amongst all the steps it's very important to use potassium ferrocyanide. The analysis of glycogen can be combined with analysis of mitochondrion and lipid droplets which can improve our understanding of how other key metabolic components correlate with glycogen and muscle health.

quantification-subcellular-glycogen-distribution-skeletal-muscle

Research

JoVE Journal

Biochemistry

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.

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 ...

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.

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 ...

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 ...

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 ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached to each other by &#945;-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, &#946;-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.

Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis FACE Method

In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.

Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by &#945;-1,4 glucoside bonds. The latter are attached ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...