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Past Issue in 2020
Volume: 10, Issue: 18
Biochemistry
A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.
Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag
Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification methods. This novel and efficient conjugation method can be used to prepare HaloTag-barcoded proteins using a click chemistry-based labeling technique. Here, we describe the preparation of protein-DNA complexes and detection of protein-protein interactions which can be used in a HaloTag protein barcode assay to detect an antibody. The protocol includes procedures for preparing the ligand-oligonucleotide complex, plasmid DNA preparation for protein expression, and preparation of the protein-oligonucleotide complex. The described click reaction-based protocols simplify the conventional amine-ester reaction methods which require additional steps for chromatography purification.
Cell Biology
Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells
As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial cells). The scheme provides the readers with clear steps so that this method could be applied to detect the acetylation level of various proteins.
Quantification of the Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays
G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. Thus, the quantity of receptor expression at the cell surface is a crucial factor regulating the functionality of the receptors. Over the past decades, many methods have been developed to measure the cell surface expression of GPCRs. Here, we describe an intact live-cell radioligand binding assay to quantify the surface expression of GPCRs at the endogenous levels or after overexpression. In this assay, cell cultures will be incubated with specific cell-nonpermeable radioligands which selectively and stoichiometrically bind to individual GPCRs and the receptor numbers at the cell surface are quantified by the radioactivity of receptor-bound ligands. This method is highly specific for measuring the functional GPCRs at the surface of intact live cells and is particularly useful for endogenous, low-abundant GPCRs.
A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms
This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.
Developmental Biology
Production and Bioassay of a Diffusible Factor that Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus
The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.
Immunology
Site-specific Labeling of B Cell Receptor and Soluble Immunoglobulin
B lymphocyte activation is regulated by its membrane-bound B cell receptors (BCRs) upon recognizing diverse antigens. It is hypothesized that antigen binding would trigger conformational changes within BCRs, followed by a series of downstream signaling activation. To measure the BCR conformational changes in live cells, a fluorescent site-specific labeling technique is preferred. Genetically encoded fluorescent tags visualize the location of the target proteins. However, these fluorescent proteins are large (~30 kDa) and would potentially perturb the conformation of BCRs. Here, we describe the general procedures of utilizing short tag-based site-specific labeling methodologies combining with fluorescence resonance energy transfer (FRET) assay to monitor the conformational changes within BCR extracellular domains upon antigen engagement.
Surgical Induction of Endometriosis in Female Mice
Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking the endometriotic lesions observed in women with endometriosis. Therefore, this mouse model provides a valuable tool to study the pathophysiology of endometriosis and the efficacy of potential treatments.
Immunohistochemistry of Kidney a-SMA, Collagen 1, and Collagen 3, in A Novel Mouse Model of Reno-cardiac Syndrome
Cardiorenal syndrome defines a synergistic pathology of the heart and kidneys where failure of one organ causes failure in the other. The incidence of cardiovascular mortality caused by this syndrome, is 20 fold higher in the end stage renal disease (ESRD) population compared to the population as a whole thus necessitating the need for improved therapeutic strategies to combat reno-cardiac pathologies. Murine in vivo models play a major role in such research permitting precise genetic modification thus reducing miscellany, however presently there is no steadfast model of reno-cardiac syndrome in the most common genetically modified mouse strain, the C57BL/6 mouse. In this study we have modified an established model of chronic renal disease using adenine diet and extended the associated pathology achieving chronic renal failure and consequent reno-cardiac syndrome in the C57BL/6 mouse. Eight week-old male C57BL/6 mice were acclimatized for 7 days before administration of a 0.15% adenine diet or control diet for 20 weeks after which the experiment was terminated and blood, urine and organs were collected and analyzed biochemically and by immunohistochemistry. Administration of 0.15% adenine diet caused progressive renal failure resulting in a reno-cardiac syndrome confirmed by a significantly increased heart to body weight ratio (P P P We present a novel regimen of adenine diet which induces both chronic kidney disease and reno-cardiac syndrome in the C57BL/6 mouse strain. The non-surgical nature of this model makes it highly reproducible compared to other models currently available.
Microbiology
Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.
Evaluation of Viable Cells in Pseudomonas aeruginosa Biofilms by Colony Count and Live/Dead Staining
Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or antibiotics, but also, according to their high resistance to standard antimicrobial approaches, alternative strategies employing photocatalysis or control of biofilm formation by modifying surfaces, have been proposed. Colony forming units (cfu) counting and live/dead staining, two classic techniques used for biofilm quantification, are detailed in this work. Both methods assess cell viability, a key factor to analyze the microbial susceptibility to given treatment, then, they represent a good approach for evaluation of an antibiofilm strategy.
Determination of Intracellular Ca2+ Concentration in the Human Pathogens Trypanosomatids Leishmania mexicana and Trypanosoma cruzi by the Use of the Fluorescent Ca2+ Indicator Fura-2
Ca2+ is an essential signaling messenger in all eukariotic cells, playing a pivotal role in many cellular functions as cell growth control (differentiation, fertilization and apoptosis), secretion, gene expression, enzyme regulation, among many others. This basic premise includes trypanosomatids as Trypanosoma cruzi and various species of Leishmania, the causative agents of Chagas disease and leishmaniasis respectively, where intracellular Ca2+ concentration ([Ca2+]i) has been demonstrated to be finely regulated. Nevertheless [Ca2+]i has been difficult to measure because of its very low cytoplasmic concentration (typically around 50-100 nM), when compared to the large concentration in the outside milieu (around 2 mM in blood). The development of intracellular fluorescent Ca2+-sensitive indicators has been of paramount importance to achieve this goal. The success was based on the synthesis of acetoximethylated derivative precursors, which allow the fluorescent molecules typically composed of many hydrophilic carboxyl groups responsible for its high affinity Ca2+-binding (and therefore very hydrophilic), to easily cross the plasma membrane. Once in the cell interior, unspecific esterases split the hydrophobic moiety from the fluorescent backbone structure, releasing the carboxyl groups, transforming it in turn to the acid form of the molecule, which remain trapped in the cytoplasm and regain its ability to fluoresce in a Ca2+-dependent manner. Among them, Fura-2 is by far the most used, because it is a ratiometric (two different wavelength excitation and one emission) Ca2+ indicator with a Ca2+ affinity compatible with the [Ca2+]i. This protocol essentially consists in loading exponential phase parasites with Fura-2 and recording changes in [Ca2+]i by mean of a double wavelength spectrofluorometer. This technique allows the acquisition of valuable information about [Ca2+]i changes in real time, as a consequence of diverse stimuli or changes in conditions, as addition of drugs or different natural modulators.
Neuroscience
Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue
Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms.
Long-term Devocalization of Zebra Finches
Songbirds, such as the zebra finch, are a popular animal model for studying the neural basis of vocal and complex skill learning. Adult male zebra finches produce courtship song toward females (referred to as ‘directed song’) and recording and analyzing sounds of directed song along with underlying neural activity is important for investigating behavioral and neural mechanisms of song production and learning. However, recording of directed song is easily contaminated by calls that are often as loud as directed songs and frequently produced by a female bird is presented in the same sound-recording chamber to elicit directed song. We developed a new surgical procedure to relatively easily and almost completely devocalize female zebra finches semi-permanently, without affecting other behaviors. This procedure enables researchers to record directed songs with almost no contamination by female calls. The procedure can also be used to devocalize male birds as well and, thus, has great potential for a variety of experimental purposes, such as long-term elimination of auditory feedback during singing in male birds.
In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD
One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction in α-syn fibrillation in vitro upon posttranslational modification (PTM) by the Fic (Filamentation induced by cAMP) family adenylyltransferase HYPE/FICD (Huntingtin yeast-interacting protein E/FICD). Specifically, HYPE utilizes ATP to covalently decorate key threonine residues in α-syn’s N-terminal and NAC (non-amyloid-β component) regions with AMP (adenosine monophosphate), in a PTM termed AMPylation or adenylylation. Status quo in vitro AMPylation reactions of HYPE substrates, such as α-syn, use a variety of ATP analogs, including radiolabeled α-32P-ATP or α-33P-ATP, fluorescent ATP analogs, biotinylated-ATP analogs (N6-[6-hexamethyl]-ATP-Biotin), as well as click-chemistry-based alkyl-ATP methods for gel-based detection of AMPylation. Current literature describing a step-by-step protocol of HYPE-mediated AMPylation relies on an α-33P-ATP nucleotide instead of the more commonly available α-32P-ATP. Though effective, this former procedure requires a lengthy and hazardous DMSO-PPO (dimethyl sulfoxide-polyphenyloxazole) precipitation. Thus, we provide a streamlined alternative to the α-33P-ATP-based method, which obviates the DMSO-PPO precipitation step. Described here is a detailed procedure for HYPE mediated AMPylation of α-syn using α-32P-ATP as a nucleotide source. Moreover, our use of a reusable Phosphor screen for AMPylation detection, in lieu of the standard, single-use autoradiography film, provides a faster, more sensitive and cost-effective alternative.
Plant Science
Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae
Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro enzymatic assay of magnesium chelatase (Wang et al., 2020). Currently, the crystallization studies of the BCM1 are underway. This protocol could be adapted to purify BCM1-like transmembrane proteins from eukaryotic organisms for enzymatic and structural studies.
Stem Cell
In vitro Differentiation of Human iPSC-derived Cardiovascular Progenitor Cells (iPSC-CVPCs)
Induced pluripotent stem cell derived cardiovascular progenitor cells (iPSC-CVPCs) provide an unprecedented platform for examining the molecular underpinnings of cardiac development and disease etiology, but also have great potential to play pivotal roles in the future of regenerative medicine and pharmacogenomic studies. Biobanks like iPSCORE ( Stacey et al., 2013; Panopoulos et al., 2017), which contain iPSCs generated from hundreds of genetically and ethnically diverse individuals, are an invaluable resource for conducting these studies. Here, we present an optimized, cost-effective and highly standardized protocol for large-scale derivation of human iPSC-CVPCs using small molecules and purification using metabolic selection. We have successfully applied this protocol to derive iPSC-CVPCs from 154 different iPSCORE iPSC lines obtaining large quantities of highly pure cardiac cells. An important component of our protocol is Cell confluency estimates (ccEstimate), an automated methodology for estimating the time when an iPSC monolayer will reach 80% confluency, which is optimal for initiating iPSC-CVPC derivation, and enables the protocol to be readily used across iPSC lines with different growth rates. Moreover, we showed that cellular heterogeneity across iPSC-CVPCs is due to varying proportions of two distinct cardiac cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs), both of which have been shown to have a critical function in heart regeneration. This protocol eliminates the need of iPSC line-to-line optimization and can be easily adapted and scaled to high-throughput studies or to generate large quantities of cells suitable for regenerative medicine applications.
Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells
Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.