Assistant Managing Editor
Anca Flavia Savulescu
  • Honorary Research Affiliate, University of Cape Town
Research fields
  • Biochemistry, Cell Biology, Molecular Biology, Systems Biology
Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection
Authors:  Lukas Ded and Jean-Ju Chung, date: 10/20/2021, view: 2284, Q&A: 0

Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation; instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the utero-tubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37°C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time- and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating (Ded et al., 2020). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm.

Electron Tomography to Study the Three-dimensional Structure of the Reovirus Egress Pathway in Mammalian Cells

Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway.

Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles
Authors:  Surya P. Aryal, Xu Fu, Abdullah A. Masud, Khaga R. Neupane and Christopher I. Richards, date: 05/20/2021, view: 3576, Q&A: 0

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with isolating single proteins within a biological system. Here we provide a detailed protocol to conduct ex vivo single molecule imaging where single transmembrane proteins are isolated by rapidly extracting nanovesicles containing receptors of interest from different regions of the brain and subjecting them to single molecule study by using total internal reflection fluorescence (TIRF) microscopy. This protocol discusses the isolation and separation of brain region specific nanovesicles as well as a detailed method to perform TIRF microscopy with those nanovesicles at the single molecule level. This technique can be applied to study trafficking and stoichiometry of various transmembrane proteins from the central nervous system. This approach can be applied to a wide range of animals that are genetically modified to express a membrane protein-fluorescent protein fusion with a wide range of potential applications in many aspects of neurobiology.


Graphic abstract:



EX vivo single molecue imaging of membrane receptors


Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method
Authors:  Zhou Han, Anne Christiansen, Meena and Gene Liau, date: 02/05/2021, view: 3127, Q&A: 0

Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA (enzyme-linked immunosorbent assay) and uses electrochemiluminescence to produce measurable signals. Two different antibodies are used in this assay to capture and detect NaV1.1 respectively in brain tissue lysate. The specificity of the antibodies is confirmed by Scn1a gene knock-out tissue. The calibration curve standards used in this assay were generated with mouse liver lysate spiked with mouse brain lysate, instead of using a recombinant protein. We showed that this method was qualified and used for quantification of NaV1.1 in mouse brain tissues with specificity, accuracy and precision.

Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 2973, Q&A: 0
Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and resources used for protein purification and further studies. This protocol describes the use of the Thermofluor assay, in combination with a custom 24-condition screen, to identify buffer conditions that increase protein thermostability, using the conserved herpesviral protein UL37 as an example. Detailed instructions on screen conditions, running the Thermofluor MATLAB script, and analyzing the data are provided. In comparison to circular dichroism (CD), the buffer screen in combination with Thermofluor assay provides a faster and more informative method to analyze protein thermostability.
A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol
Author:  Chiara Sinigaglia, date: 09/05/2019, view: 5327, Q&A: 0
In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence–the hybridization step, followed by detection of annealed sequences by means of fluorescent or colorimetric reactions. Successful hybridization requires permeabilization of tissues, followed by denaturation of nucleic acids strands, which is usually carried out in a formamide-based buffer and at high temperatures. Such reaction conditions, besides posing a health hazard (both concerning manipulation and waste disposal), can be excessively harsh for the delicate tissues of some species or developmental stages. We detail here an alternative method for in situ hybridization, where the toxic formamide is replaced with a urea solution. This substitution improved both tissues preservation and signal-to-noise detection, in several animal species. The protocol described here, originally developed for the hydrozoan jellyfish Clytia hemisphaerica, provides guidelines for adapting formamide-based traditional protocols to the urea variant. Urea-based protocols have already been successfully applied to diverse invertebrate and vertebrate species, showing the ease of such a modification, and providing the scientific community with a promising, safer and versatile tool.
Immunoprecipitation and Sequencing of Acetylated RNA
Authors:  Daniel Arango, David Sturgill and Shalini Oberdoerffer, date: 06/20/2019, view: 6183, Q&A: 0
Generation of the epitranscriptome through chemical modifications of protein-coding messenger RNAs (mRNAs) has emerged as a new mechanism of post-transcriptional gene regulation. While most mRNA modifications are methylation events, a single acetylated ribonucleoside has been described in eukaryotes, occurring at the N4-position of cytidine (N4-acetylcytidine or ac4C). Using a combination of antibody-based enrichment of acetylated regions and deep sequencing, we recently reported ac4C as a novel mRNA modification that is catalyzed by the N-acetyltransferase enzyme NAT10. In this protocol, we describe in detail the procedures to identify acetylated mRNA regions transcriptome-wide using acetylated RNA immunoprecipitation and sequencing (acRIP-seq).
Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation
Authors:  Christopher Fields, Peike Sheng, Bret Miller, Tianqi Wei and Mingyi Xie, date: 04/20/2019, view: 9925, Q&A: 1
Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we found that IR-labeled probes can be used to multiplex or detect different species of RNA at the same time. Here we describe three methods for generating an IR-labeled probe as well as how to perform irNorthern blot. In conclusion, our irNorthern protocol offers a convenient method for RNA detection.
Preparation of RNA 3’ End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae
Authors:  Manfred Schmid, Agnieszka Tudek and Torben Heick Jensen, date: 03/20/2019, view: 4351, Q&A: 0
Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have been developed to measure transcription and RNA decay rates. However, to our knowledge, none of those techniques can simultaneously interrogate transcription and RNA decay. The presented protocol allows this and provides a simple approach to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. It is based on brief metabolic labeling of RNA and subsequent concurrent sequencing of polyA+ and polyA- RNA 3’ ends. The protocol was developed in S. cerevisiae and should be broadly applicable.
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