Ligand Coupling to the AAV Capsid for Cell-Specific Gene Transfer.
Abstract Cell entry of AAV vectors is initiated by contacting the cell surface attachment receptor. This process can be rationally engineered through mutating the contact residues on the AAV capsid and covalently coupling targeting ligands to the capsid surface that exhibit high affinity for a cell surface protein of choice. This way, selective gene delivery to target-receptor positive cell types has been achieved. Two methods for coupling targeting ligands to the AAV capsid can be distinguished. Genetic coupling is achieved through expressing fusion proteins composed of the capsid protein VP2 and the targeting li...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Reul J, Muik A, Buchholz CJ Tags: Methods Mol Biol Source Type: research

Quantitative and Digital Droplet-Based AAV Genome Titration.
Abstract The adeno-associated viral vector (AAV) platform has developed into a primary modality for efficient in vivo, and in more limited settings, in vitro or ex vivo gene transfer. Its applications range from a tool for experimental purposes to preclinical and clinical gene therapy. The ability to accurately and reproducibly quantify vector concentration is critical for any of these applications. While several quantification assays are available, here we outline a detailed protocol for the quantification of DNase-I protected vector genomes reliant on the polymerase chain reaction (PCR) as a measure of the activ...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Sanmiguel J, Gao G, Vandenberghe LH Tags: Methods Mol Biol Source Type: research

Single-Stranded DNA Virus Sequencing (SSV-Seq) for Characterization of Residual DNA and AAV Vector Genomes.
Abstract With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV ...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Lecomte E, Leger A, Penaud-Budloo M, Ayuso E Tags: Methods Mol Biol Source Type: research

In Situ Hybridization for Detection of AAV-Mediated Gene Expression.
Abstract Techniques to localize vector transgenes in cells and tissues are essential in order to fully characterize gene therapy outcomes. In situ hybridization (ISH) uses synthesized complementary RNA or DNA nucleotide probes to localize and detect sequences of interest in fixed cells, tissue sections, or whole tissue mounts. Variations in techniques include adding labels to probes, such as fluorophores, which can allow for the simultaneous visualization of multiple targets. Here we provide the steps necessary to: (1) label probes for colorimetric visualization and (2) perform ISH on OCT cryo-preserved fixed froz...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Hunter JE, Gurda BL, Yoon SY, Castle MJ, Wolfe JH Tags: Methods Mol Biol Source Type: research

Use of AAV Vectors for CRISPR-Mediated In Vivo Genome Editing in the Retina.
Abstract Degenerative retinal diseases such as retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA) may lead to blindness without effective treatment. With the rapid advancement of the CRISPR/Cas9 genome editing technology, in vivo application of CRISPR/Cas9 holds immense potential for treatment of these diseases. Adeno-associated virus (AAV) vectors are an ideal gene transfer tool for delivery of CRISPR components to the retina. Here, we describe a protocol for utilizing an AAV-based CRISPR/Cas9 system for in vivo genome editing in the retina. PMID: 30783971 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Yu W, Wu Z Tags: Methods Mol Biol Source Type: research

Intraspinal and Intracortical Delivery of AAV Vectors for Intersectional Circuit Tracing in Non-transgenic Species.
Abstract The mapping of long-range axonal projections is a rapidly growing endeavor in the field of neuroscience. Recent advances in the development of adeno-associated viral vectors that can achieve efficient retrograde transport now enable the characterization and manipulation of specific neuronal subpopulations using Cre-dependent, intersectional approaches. Importantly, these approaches can be applied to non-transgenic animals, making it possible to carry out detailed anatomical studies across a variety of species including nonhuman primates. In this chapter, we demonstrate the utility of such intersectional s...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Conner JM, Bain GL, Dulin JN Tags: Methods Mol Biol Source Type: research

Peripheral AAV Injection for Retrograde Transduction of Dorsal Root and Trigeminal Ganglia.
Abstract Adeno-associated Virus (AAV) vectors are useful vehicles for delivering transgenes to a number of different tissues and organs in vivo. To date, most of these applications deliver the vectors to their target by either infusion into the bloodstream or direct injection into the target tissue. Recently there has been progress in delivering AAV vectors to neurons of the peripheral nervous system (PNS) following application of vectors to the peripheral epithelium, such as the skin or eye. This delivery only requires treatment of the epithelium to access the underlying nerve termini, and following treatment the...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Bloom DC, Watson ZL, Neumann DM Tags: Methods Mol Biol Source Type: research

SubILM Injection of AAV for Gene Delivery to the Retina.
Abstract Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retin...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Gamlin PD, Alexander JJ, Boye SL, Witherspoon CD, Boye SE Tags: Methods Mol Biol Source Type: research

AAV-Mediated Gene Delivery to Taste Cells of the Tongue.
Abstract Taste sensation is initiated in sensory cells within the taste buds (taste cells), in which the cooperation of many signaling molecules leads to the coding and transmission of information on the quality and intensity of taste to the afferent gustatory nerves. Here, we describe our method for inducing foreign gene expression in taste cells of fungiform papillae in a living mouse using a recombinant adeno-associated virus (AAV) vector, enabling us to study and control the function of a gene product in vivo. Among the serotypes tested to date, only AAV-DJ, a synthetic serotype, can transduce taste cells in v...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Taruno A, Kashio M Tags: Methods Mol Biol Source Type: research

AAV-Mediated Gene Delivery to the Lung.
Abstract Adeno-associated virus (AAV) vectors are an efficient method of gene delivery to various tissues including the lung. Mouse models are often used as a preliminary preclinical model in order to advance AAV lung gene therapy vectors. In this chapter we describe an AAV purification protocol using heparin affinity chromatography as well as an intranasal and intratracheal method of delivering AAV vectors to the lungs of mice. PMID: 30783985 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: van Lieshout LP, Domm JM, Wootton SK Tags: Methods Mol Biol Source Type: research

Simplified Purification of AAV and Delivery to the Pancreas by Intraductal Administration.
Abstract Genetic manipulation is a very powerful tool for studying diabetes, pancreatitis, and pancreatic cancer. Here we discuss the use of an adeno-associated virus (AAV) vector to modify gene expression, such as to introduce a green fluorescence protein (GFP) in wild-type mice, cre recombinase in loxP mice, or to inactivate a gene with shRNA. The use of viruses for genetic modification allows for time-specific genetic changes which have advantages over time-consuming and often complex cross-breeding strategies. Here we provide a detailed approach for this process from viral production and purification through p...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Guo P, Wiersch J, Xiao X, Gittes G Tags: Methods Mol Biol Source Type: research

MicroRNA-5195-3p enhances the chemosensitivity of triple-negative breast cancer to paclitaxel by downregulating EIF4A2.
Conclusion: These data demonstrate that miR-5195-3p might be a potential therapeutic target to reverse chemoresistance in TNBC through its targeting of EIF4A2. PMID: 31308851 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Liu M, Gong C, Xu R, Chen Y, Wang X Tags: Cell Mol Biol Lett Source Type: research

Testing the Ability of Compounds to Induce Swarming.
Abstract One of the most distinctive features of Proteus mirabilis is its ability to undergo differentiation from short, rod-shaped vegetative cells with peritrichous flagella to massively elongated swarm cells that express hundreds to thousands of flagella. The unique bull's-eye pattern that forms from cycles of active swarming and consolidation back to the vegetative state has long been a distinguishing characteristic of this species. Many factors involved in regulation of flagellar synthesis and swarm cell differentiation have been characterized, but the exact conditions sensed by P. mirabilis that send a signa...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Armbruster CE Tags: Methods Mol Biol Source Type: research

Siderophore Detection Using Chrome Azurol S and Cross-Feeding Assays.
Abstract More than 500 siderophores that bind ferric iron have been characterized and grouped by type based on their chemical structure. The chrome azurol S (CAS) assay is a universal colorimetric method that detects siderophores independent of their structure. In this assay, siderophores scavenge iron from an Fe-CAS-hexadecyltrimethylammonium bromide complex, and subsequent release of the CAS dye results in a color change from blue to orange. Solution-based experiments with CAS result in a quantitative measure of siderophore production, while an observable color change on CAS agar plates can be performed for qual...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Himpsl SD, Mobley HLT Tags: Methods Mol Biol Source Type: research

Detection of Neutrophil Extracellular Traps in Urine.
Abstract Neutrophils are important mediators of the antimicrobial defense during urinary tract infections (UTIs). When activated at the site of infection, these innate immune cells phagocytose and neutralize an invading pathogen. Another neutrophil defense strategy is the release of effectors, such as antimicrobial peptides and proteins stored in neutrophil granules and reactive oxygen species. Their release can be facilitated by cellular signals that trigger chromatic decondensation and the disruption of nuclear membranes, followed by granule and plasma membrane disintegration, DNA release into the extracellular ...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Yu Y, Kwon K, Pieper R Tags: Methods Mol Biol Source Type: research

Using Proteomics to Identify Inflammation During Urinary Tract Infection.
Abstract Urinary tract infections (UTIs) are one of the most common bacterial infections. Conventional approaches to diagnose these infections rely on microbial urine culture, urine sediment microscopy and basic molecular urinalysis tests, in combination with assessments of patient symptoms that are indicative of UTI. The last decade has seen a more widespread clinical use of standardized MALDI-TOF methods to identify UTI-causing microbial agents. Shotgun proteomics methods to determine the extent of inflammation and types of immune cell effectors in urine have not become part of routine clinical tests. However, s...
Source: Mol Biol Cell - July 18, 2019 Category: Molecular Biology Authors: Yu Y, Pieper R Tags: Methods Mol Biol Source Type: research

Identification of Human Cutaneous Squamous Cell Carcinoma Cancer Stem Cells.
Abstract Epithelia are under constant threat from environmental carcinogens and none more so than squamous epithelia, which form the outermost linings of our bodies. Hence malignancies of squamous epithelia are collectively the most common cancer type and with the highest mortality, despite a constant cell turnover and only relatively rare long-lived adult tissue stem cells. Genetic analysis from SCC whole genome sequencing reveals commonality in mutated genes, despite various etiological factors. Most SCC types have been shown to exhibit hierarchical growth, in which a high frequency of cancer stem cells is assoc...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Olivero C, Morgan H, Patel GK Tags: Methods Mol Biol Source Type: research

Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.
Abstract The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Morgan H, Olivero C, Patel GK Tags: Methods Mol Biol Source Type: research

Isolation of Normal Fibroblasts and Their Cancer-Associated Counterparts (CAFs) for Biomedical Research.
, Smetana K Abstract Cancer-associated fibroblasts (CAFs) represent a crucial component of cancer microenvironment. CAFs significantly influence biological properties of various types of cancer in terms of local aggressiveness, recurrence, and metastatic behaviour. This chapter summarizes a simple protocol for isolation of normal fibroblasts and their cancer-associated counterparts from normal human skin and mucosa, respectively, as well as from samples of human tumours such as basal/squamous carcinoma, melanoma, and breast cancer, and employment of this procedure for other types of cancer is possible. Isolated fi...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Dvořánková B, Lacina L, Smetana K Tags: Methods Mol Biol Source Type: research

Isolation and Cultivation of Skin-Derived Precursors.
Abstract Skin-derived precursors (SKPs) have been shown recently to be capable of inducing hair genesis and hair follicle regeneration. Here, we describe a protocol for SKP isolation and culture based on our experience and previous publications. PMID: 29736805 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Wang X, Dong S, Wu Y Tags: Methods Mol Biol Source Type: research

Amniotic Membrane Seeded Fetal Fibroblasts as Skin Substitute for Wound Regeneration.
Abstract Various natural and synthetic biomaterials have been applied as skin substitutes for regenerating damaged skin. Here, we describe a straightforward method for fabrication of a tissue-engineered skin substitute by seeding human fetal fibroblasts on acellular human amniotic membrane (HAM). Fetal fibroblasts are achieved from the skin of normal and non-macerated fetus of 11-14 weeks old after spontaneous pregnancy termination. Acellular HAM is obtained by separation of the outer membrane of the chorion and removing its epithelial cells. PMID: 29736806 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Taghiabadi E, Beiki B, Aghdami N, Bajouri A Tags: Methods Mol Biol Source Type: research

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.
el NO Abstract Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and techno...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Auvré F, Coutier J, Martin MT, Fortunel NO Tags: Methods Mol Biol Source Type: research

Isolation of Cancer Stem Cells from Squamous Cell Carcinoma.
Abstract Different cancer stem cell (CSC) populations can be found in many types of cancer, including squamous cell carcinoma (SSC). Diverse reports showed that CSC play a crucial role in the relapse of different types of cancer. CSC sustains tumor growth due to their capacity to self-renew and their potential to initiate secondary tumors with metastatic cancer features. Therefore, the development of methods for the isolation of CSC is a key step to explore the mechanisms underlying CSC maintenance. In this chapter, we provide a method for isolating CSC from cutaneous SSC using immunofluorescence labeling to allow...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Fontenete S, Perez-Moreno M Tags: Methods Mol Biol Source Type: research

Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells.
Abstract The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells. PMID: 29896657 [PubMed - indexed for MEDL...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Badarinath K, Dutta A, Hegde A, Pincha N, Gund R, Jamora C Tags: Methods Mol Biol Source Type: research

Whole-Mount Immunofluorescent Staining Coupled to Multicolor Lineage Tracing Model for Analyzing the Spatiotemporal Organization of Epidermal Stem Cells.
Abstract An overall three-dimensional picture of the distribution of epidermal cells at a given time point is of importance to better characterize epidermal progenitors. We introduce a whole-mount immunofluorescent staining coupled to a multicolor lineage tracing model for analyzing the spatiotemporal organization of epidermal stem cells. Laser scanning confocal microscopy with multiple labelling allows for robust imaging of dorsal skin with excellent resolution. PMID: 29896659 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Roy E, Khosrotehrani K Tags: Methods Mol Biol Source Type: research

Human Hair Follicle Associated-Pluripotent (hHAP) Stem Cells Differentiate to Cardiac Muscle Cells.
Abstract Human hair follicle-associated pluripotent (hHAP) stem cells were isolated from the upper parts of human hair follicles. hHAP stem cells were suspended in DMEM containing 10% fetal bovine serum (FBS) where they differentiated to cardiac muscle cells as well as neurons, glial cells, keratinocytes, and smooth muscle cells. The methods of this chapter are appropriate for use of human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve, and spinal cord regeneration in the clinic. PMID: 29992514 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Hoffman RM Tags: Methods Mol Biol Source Type: research

Melanoblasts as Multipotent Cells in Murine Skin.
Abstract Melanoblasts (MBs) are melanocyte precursors that are derived from neural crest cells (NCCs). We recently demonstrated the multipotency of MBs; they differentiate not only into pigmented melanocytes but also other NCC derivatives. We herein describe methods for the isolation of MBs from mouse skin by flow cytometry. Methods to culture isolated MBs that retain their multipotency and isolation methods for MBs using gene-modified mouse are also described. PMID: 30006864 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Motohashi T, Kunisada T Tags: Methods Mol Biol Source Type: research

High-Grade Purification of Third-Generation HIV-Based Lentiviral Vectors by Anion Exchange Chromatography for Experimental Gene and Stem Cell Therapy Applications.
Abstract Lentiviral vectors (LVs) have been increasingly used in clinical gene therapy applications particularly due to their efficient gene transfer ability, lack of interference from preexisting viral immunity, and long-term gene expression they provide. Purity of LVs is essential in in vivo applications, for a high therapeutic benefit with minimum toxicity. Accordingly, laboratory scale production of LVs frequently involves transient cotransfection of 293T cells with packaging and transfer plasmids in the presence of CaPO4. After clearance of the cellular debris by low-speed centrifugation and filtration, lenti...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Olgun HB, Tasyurek HM, Sanlioglu AD, Sanlioglu S Tags: Methods Mol Biol Source Type: research

Genome Sequencing and Analysis Methods in Chronic Lymphocytic Leukemia.
pez-Otín C Abstract The genomic sequencing of chronic lymphocytic leukemia (CLL) samples has provided exciting new venues for the understanding and treatment of this prevalent disease. This feat is possible thanks to high-throughput sequencing methods, such as Illumina sequencing. The interpretation of these data sources requires not only appropriate software and hardware, but also understanding the biology and technology behind the sequencing process. Here, we provide a primer to understand each step in the analysis of point mutations from whole-genome or whole-exome sequencing experiments of tumor and nor...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Quesada V, Araujo-Voces M, Pérez-Silva JG, Velasco G, López-Otín C Tags: Methods Mol Biol Source Type: research

Characterization of Somatically-Acquired Copy Number Alterations in Chronic Lymphocytic Leukaemia Using Shallow Whole Genome Sequencing.
Abstract Shallow whole genome sequencing (sWGS) is a simple, robust, and cost-effective technique recently optimized for the identification of copy number aberrations (CNAs) in tumor samples. This multiplexed methodology sequences 50 bp from one end of the DNA molecule, generating ˜0.1× coverage, and utilizes the observed sequence depth across the genome to infer copy number. It is amenable to low quantities of input DNA, sequencing costs are modest, processing is compatible with low-output instruments, and downstream analysis is simplified by the use of freely available bioinformatics tools and a...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Parker H, Carr L, Syeda S, Bryant D, Strefford JC Tags: Methods Mol Biol Source Type: research

Quantification of Alpha-Synuclein in Biological Fluids by Electrochemiluminescence-Based Detection.
Abstract Several potential marker candidates for Parkinson's disease (PD) in cerebrospinal fluid (CSF) have been identified. These include α-synuclein, a major constituent of the intracellular aggregates Lewy bodies, a neuropathological hallmark of PD, and others. The extracellular presence of α-synuclein in blood, CSF, saliva, and conditioned media motivated the hypothesis that the quantification of CSF α-synuclein is a biomarker for α-synuclein-related disorders. We here describe the development of an electrochemiluminescence-based assay by conversion of an established ELISA for quantific...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Kruse N, Mollenhauer B Tags: Methods Mol Biol Source Type: research

Studying α-Synuclein Conformation by Intact-Cell Cross-Linking.
Studying α-Synuclein Conformation by Intact-Cell Cross-Linking. Methods Mol Biol. 2019;1948:77-91 Authors: Imberdis T, Fanning S, Newman A, Ramalingam N, Dettmer U Abstract β-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SD...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Imberdis T, Fanning S, Newman A, Ramalingam N, Dettmer U Tags: Methods Mol Biol Source Type: research

C. elegans as a Model for Synucleinopathies and Other Neurodegenerative Diseases: Tools and Techniques.
Abstract Caenorhabditis elegans is widely used to investigate biological processes related to health and disease. Multiple C. elegans models for human neurodegenerative diseases do exist, including those expressing human α-synuclein. Even though these models do not feature all pathological and molecular hallmarks of the disease they mimic, they allow for the identification and dissection of molecular pathways that are involved. In line with this, genetic screens have yielded multiple modifiers of proteotoxicity in C. elegans models for neurodegenerative diseases. Here, we describe a set of common screening a...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Koopman M, Seinstra RI, Nollen EAA Tags: Methods Mol Biol Source Type: research

Recombinant Expression and Purification of N-Acetylated Alpha-Synuclein.
Abstract The majority of proteins in eukaryotic cells are subject to amino-terminal (Nt) acetylation. Recombinant protein expressed using prokaryotic expression systems such as E. coli would not normally be Nt-acetylated as these cells lack the appropriate N-α-terminal acetylation complex. Here we describe a simple protocol that allows the recombinant expression and purification of Nt-acetylated alpha-synuclein (aS) from E. coli. PMID: 30771174 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Eastwood TA, Mulvihill DP Tags: Methods Mol Biol Source Type: research

Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes.
Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes. Methods Mol Biol. 2019;1948:123-143 Authors: Rovere M Abstract α-Synuclein's physiology and pathology have been linked by numerous reports to its ability to bind and remodel membranes, especially at synaptic terminals. It is therefore critical for researchers investigating the determinants of these interactions to rely on methods capable of providing an accurate and complete physicochemical snapshot of the binding events. Circular dichroism (CD) and isothermal titration calorimet...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Rovere M Tags: Methods Mol Biol Source Type: research

In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.
Abstract In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Sanz R, Ovando-Roche P, Udeshi ND, Carr SA, Chung CY, Khurana V Tags: Methods Mol Biol Source Type: research

Expression and Purification of Untagged α-Synuclein.
Expression and Purification of Untagged α-Synuclein. Methods Mol Biol. 2019;1948:261-269 Authors: Powers AE, Patel DS Abstract α-Synuclein (αS) is an abundant neuronal protein which has been implicated, among others, in the pathogenesis of neurodegenerative diseases like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In fact, αS is the major constituent of Lewy bodies, the primarily proteinaceous inclusions found in the nervous tissue of PD and DLB patients. While its physiological role is unclear, it is believed to be involved in the regulation of synaptic vesic...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Powers AE, Patel DS Tags: Methods Mol Biol Source Type: research

Generation and Analysis of Viral Vector-Mediated Rodent Models for Parkinson's Disease.
Abstract The use of viral vector-mediated α-synuclein-overexpressing rodent models for Parkinson's disease has become increasingly accepted to study associated pathology and to use as a platform for therapeutic efficacy testing. We here describe methods to generate such models and how to analyze them by behavioral assessments, histological investigations, and examination of nigrostriatal dopaminergic function. PMID: 30771185 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Karikari AA, Koprich JB, Ip CW Tags: Methods Mol Biol Source Type: research

Analysis of Phosphatidylinositol Transfer at ER-PM Junctions in Receptor-Stimulated Live Cells.
Abstract Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM junctions, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to mon...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Chang CL, Liou J Tags: Methods Mol Biol Source Type: research

Monitoring Non-vesicular Transport of Phosphatidylserine and Phosphatidylinositol 4-Phosphate in Intact Cells by BRET Analysis.
Abstract Non-vesicular lipid transport via lipid transfer proteins (LTPs) at membrane contact sites (MCSs) is critical for the maintenance of the lipid composition of biological membranes. The ability to measure lipid transfer activity of diverse LTPs in live cells without interrupting the fine structural organization is essential to better understand the contribution of non-vesicular lipid transport to membrane organization. Here, we describe a semiquantitative method to analyze phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine (PS) changes at the plasma membrane (PM) as they relate to LTP functions....
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Sohn M, Toth DJ, Balla T Tags: Methods Mol Biol Source Type: research

Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells.
, Várnai P Abstract There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that refle...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Tóth JT, Gulyás G, Hunyady L, Várnai P Tags: Methods Mol Biol Source Type: research

Imaging Lipid Metabolism at the Golgi Complex.
Abstract The development of fluorescence-based molecular imaging has revolutionized cell biology allowing the visualization of specific biomolecules at the microscopic and, more recently, at the nanoscopic scale while in their relevant biological contexts. Nonetheless, despite the imaging toolkit for biologists interested in exploring the subcellular localization and dynamics of proteins and nucleic acids has expanded exponentially in the last decades, the means to visualize and track lipids in cells did not develop to the same extent until recently. Here we described some basic fluorescence-based techniques that ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Capasso S, D'Angelo G Tags: Methods Mol Biol Source Type: research

Advanced In Vitro Assay System to Measure Phosphatidylserine and Phosphatidylethanolamine Transport at ER/Mitochondria Interface.
Abstract A number of previous studies have shown that phospholipid molecules come and go between the endoplasmic reticulum (ER) and mitochondrial membranes while the molecular basis of non-vesicular phospholipid transport is still not understood well. In this chapter, we describe an optimized method that uses membrane fractions isolated from yeast cells to directly analyze phospholipid transport between the ER and mitochondria. With this assay, we are able to assess not only the ER-to-mitochondria but also mitochondria-to-ER transports at the same time. We believe that this assay system can accelerate the research...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Tamura Y, Kojima R, Endo T Tags: Methods Mol Biol Source Type: research

Measurement of Lipid Transport in Mitochondria by the MTL Complex.
Abstract Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This li...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Jouhet J, Gros V, Michaud M Tags: Methods Mol Biol Source Type: research

Bi- and Trifunctional Lipids for Visualization of Sphingolipid Dynamics within the Cell.
r D Abstract Investigations into lipid localization and transport are often hampered by a lack of methods and tools to faithfully visualize lipids in the context of living cells, since fluorescent modifications drastically change lipid properties. Here, we describe the use of bifunctional as well as trifunctional sphingosine to reveal its subcellular localization via crosslinking, fixation, and specific staining by click reaction with a fluorophore. Additionally, these probes allow investigations into lipid metabolism as revealed by thin-layer chromatography. PMID: 30790251 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Höglinger D Tags: Methods Mol Biol Source Type: research

Measurement of Intracellular Sterol Transport in Yeast.
Abstract Intracellular sterol transport occurs largely by non-vesicular mechanisms in which sterol transport proteins extract sterol from one membrane and transfer it to another across the cytoplasm. Here we describe a suite of complementary assays to measure intracellular sterol transport in the model eukaryote Saccharomyces cerevisiae, as well as to quantify protein-mediated sterol transport between populations of vesicles in vitro. The in vivo assays can be adapted to study sterol transport in other cell types. PMID: 30790253 [PubMed - indexed for MEDLINE] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Chauhan N, Jentsch JA, Menon AK Tags: Methods Mol Biol Source Type: research

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.
Abstract Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at th...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Wilhelm LP, Voilquin L, Kobayashi T, Tomasetto C, Alpy F Tags: Methods Mol Biol Source Type: research

Monitoring and Modulating Intracellular Cholesterol Trafficking Using ALOD4, a Cholesterol-Binding Protein.
Abstract Mammalian cells carefully control their cholesterol levels by employing multiple feedback mechanisms to regulate synthesis of cholesterol and uptake of cholesterol from circulating lipoproteins. Most of a cell's cholesterol (~80% of total) is in the plasma membrane (PM), but the protein machinery that regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell's cholesterol. How does the ER communicate with PM to monitor cholesterol levels in that membrane? Here, we describe a tool, ALOD4, that helps us answer this questi...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Endapally S, Infante RE, Radhakrishnan A Tags: Methods Mol Biol Source Type: research

Measurement of Lysophospholipid Transport Across the Membrane Using Escherichia coli Spheroplasts.
Abstract In the inner membrane of Gram-negative bacteria lysophospholipid transporter (LplT) and the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase (Aas) form a glycerophospholipid remodeling system, which is capable of facilitating rapid retrograde translocation of lyso forms of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin across the cytoplasmic membrane. This coupled remodeling enzyme tandem provides an effective method for the measurement of substrate specificity of the lipid regeneration and lysophospholipid transport per se across t...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Lin Y, Zheng L, Bogdanov M Tags: Methods Mol Biol Source Type: research

In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.
Abstract The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial lipid-binding Protein (SMP) domain, a lipid-harboring module through which they mediate lipid transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and lipid transport functions of E-Syt1. Its membrane tethering activity is monitored through ...
Source: Mol Biol Cell - July 16, 2019 Category: Molecular Biology Authors: Bian X, De Camilli P Tags: Methods Mol Biol Source Type: research