The nematode Caenorhabditis elegans is an excellent model system in which to study long-distance cell migration in vivo. This chapter describes methods used to study a subset of migratory cells in the hermaphrodite nematode, the distal tip cells. These methods take advantage of the organism’s transparent body and the expression of green fluorescent protein to observe cell migration and behavior. Additionally, the availability of nematode mutants and gene knockdown techniques that affect cell migration allow the analysis and comparison of wild-type and aberrant migratory paths. Methods for nematode growth and maintenance, strain acquisition, observation and live imaging, gene knockdown, and analysis of cell migration defects are covered.

Summary

The balance between self-renewal and differentiation must be tightly regulated in somatic stem cells to ensure proper tissue generation and to prevent tumor-like overgrowth. A Drosophila larval brain lobe consists of the central brain and the optic lobe, and possesses three well-defined neural stem cell lineages that generate differentiated cells in a highly reproducible pattern. Unambiguous identification of various cell types in these stem cell lineages is pivotal for studying the regulation of neural stem cells and progenitor cells at a single-cell resolution. This chapter will describe the methodology for collection and processing of larval brains for examination by fluorescence confocal microscopy.

Molecular details and temporal organization of the early (pre-integration) phase of HIV life cycle remain among the least investigated and most controversial problems in the biology of HIV. To perform reverse transcription and intracellular transport of the viral genetic material, HIV forms multimolecular complexes termed reverse transcription complexes (RTCs). Analysis of the kinetic of reverse transcription and nuclear import of RTCs, as well as assessment of the changes in their protein content in the course of reverse transcription and nuclear translocation is a necessary step in understanding mechanisms of cytoplasmic maturation and nuclear import of HIV-1 RTCs. Here, we review methods allowing to quantitatively assess the dynamics of the maturation of HIV-1 RTCs and transformations of RTC protein composition associated with nuclear import of the complexes.

Animal models of fetal alcohol spectrum disorder (FASD) have been instrumental in isolating alcohol as a teratogen and demonstrating behavioral and neural effects. There are a number of different models for rodents with various strengths and weaknesses. A three-trimester model of FASD is described here; the model uses intragastric intubation of both pregnant dams and pups to mimic alcohol exposure across all three trimesters in humans. The model does not use expensive equipment and is relatively easy to accomplish. The model allows excellent control of alcohol dose and uses an oral route of administration. There are no undernutrition effects with the doses used here. A drawback of the model is the stress of the intubation procedures and ways in which to minimize this stress are discussed. In addition, a method to measure blood alcohol levels is described.

Following AAV-based gene transfer, the occurrence of adaptive immune responses specific to the vector or the transgene product is a major roadblock to successful clinical translation. These responses include antibodies against the AAV capsid, which can be neutralizing and therefore prevent the ability to repeatedly administer the vector, and CD8+ cytotoxic T lymphocytes, which can eliminate transduced cells. In addition, humans may have both humoral and cellular pre-existing immunity, as a result from natural infection with parent virus or related serotypes. The need for assays to detect and measure these anti-capsid immune responses in humans and in experimental animals is profound. Here, ELISPOT, immunocapture (ELISA), and neutralization assays are explained and provided in detail. Furthermore, such techniques can readily be adapted to monitor and quantify immune responses against therapeutic transgene products encoded by the vector genome.

Summary

The proteasome is an ATP-dependent molecular machine that degrades proteins through the concerted activity of dozens of subunits. It is the yin to the ribosome’s yang, and together these entities mold the protein landscape of the cell. Native gels are generally superior to conventional and affinity purifications for the analytical resolution proteasomal variants, and have thus become a staple of proteasome work. Here we describe the technique of using native gels to observe proteasomes in complex with ubiquitin conjugates. We discuss the consequences of ubiquitin conjugate length and concentration on the migration of these complexes, the use of this mobility shift to evaluate the relative affinity of mutant proteasomes for ubiquitin conjugates, and the effects of deubiquitinating enzymes and competing ubiquitin binding proteins on the interactions of ubiquitin conjugates with the proteasome.

During mitosis, the Golgi membranes in mammalian cells undergo a continuous disassembly process and generate mitotic fragments that are distributed into the daughter cells and reassembled into new Golgi after mitosis. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins that are associated with interphase and mitotic Golgi membranes using quantitative proteomics that combines the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and LC-MALDI-MS/MS. A total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories: Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, and secretory and cytoskeletal proteins. More importantly, the combination of the quantitative proteomic approach with Western blot analysis allowed us to unveil 86 proteins with significant changes in abundance under the mitotic condition compared to the interphase condition. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that controls the Golgi disassembly and reassembly processes in the cell cycle.

DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations.

Activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and intracellular levels of ODC protein are controlled very tightly. Numerous studies have described ODC regulation at the levels of transcription, translation and protein degradation in normal cells, and dysregulation of these processes in response to oncogenic stimuli. Although post-transcriptional regulation of ODC has been well-documented, the RNA binding proteins (RBPs) that interact with ODC mRNA and control synthesis of the ODC protein have not been defined. Using Ras-transformed rat intestinal epithelial cells (Ras12V cells) as a model, we have begun identifying the RBPs that associate with the ODC transcript. Binding of RBPs could potentially regulate ODC synthesis by either changing mRNA stability or rate of mRNA translation. Techniques for measuring RBP binding and translation initiation are described here. Targeting control of ODC translation or mRNA decay could be a valuable method of limiting polyamine accumulation and subsequent tumor development in a variety of cancers.

This chapter describes the methods to form and optimize samples of protein–DNA complexes that are suitable for detailed structure and dynamics studies by NMR spectroscopy.

Summary

Fluorescence-activated cell sorting (FACS) permits specific biologic parameters of cellular populations to be quantified in a high throughput fashion based on their unique fluorescent properties. Relative quantitation of mitochondrial-localized dyes in human cells using FACS analysis allows sensitive analysis of a variety of mitochondrial parameters including mitochondrial content, mitochondrial membrane potential, and matrix oxidant burden. Here, we describe protocols that utilize FACS analysis of human lymphoblastoid cell lines (LCL) for relative quantitation of mitochondrial-localized fluorescent dye intensity. The specific dyes described include MitoTracker Green FM to assess mitochondrial content, tetramethylrhodamine ethyl ester (TMRE) to assess mitochondrial membrane potential, and MitoSOX Red to assess mitochondrial matrix oxidant burden. Representative results of FACS-based mitochondrial analyses demonstrate the variability of these three basic mitochondrial parameters in LCLs from healthy individuals, as well as the sensitivity of applying FACS analysis of LCLs to study the effects of pharmacologic induction and scavenging of oxidant stress.

Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of post-translational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS), with its qualitative and quantitative capabilities, is ideally suited to the task. MS provides fast, sensitive, accurate, and high-throughput peptidomic analyses of neuropeptides without requiring prior knowledge of the peptide sequences. Aided by liquid chromatography (LC) separations and bioinformatics, MS is quickly becoming a leading technique in neuropeptidomics. This chapter describes several LC-MS analytical methods to identify, characterize and quantify neuropeptides, while emphasizing the sample preparation steps so integral to experimental success.

In genetic association studies, it is necessary to correct for population structure to avoid inference bias. During the past decade, prevailing corrections often only involved adjustments of global ancestry differences between sampled individuals. Nevertheless, population structure may vary across local genomic regions due to the variability of local ancestries associated with natural selection, migration, or random genetic drift. Adjusting for global ancestry alone may be inadequate when local population structure is an important confounding factor. In contrast, adjusting for local ancestry can more effectively prevent false-positives due to local population structure. To more accurately locate disease genes, we recommend adjusting for local ancestries by interrogating local structure. In practice, locus-specific ancestries are usually unknown and cannot be accurately inferred when ancestral population information is not available. For such scenarios, we propose employing local principal components (PC) to represent local ancestries and adjusting for local PCs when testing for genotype–phenotype association. With an acceptable computation burden, the proposed algorithm successfully eliminates the known spurious association between SNPs in the LCT gene and height due to the population structure in European Americans.

This chapter describes the use of glutathione S-transferase (GST) gene fusion proteins as a method for inducible, high-level protein expression and purification from bacterial cell lysates. The protein is expressed in a pGEX vector, with the GST moiety located at the N-terminus followed by the target protein. The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies. Additionally, the GST fusion protein can be affinity purified facilely without denaturation or use of mild detergents. The fusion protein is captured by immobilized glutathione and impurities are washed away. The fusion protein then is eluted under mild, non-denaturing conditions using reduced glutathione. If desired, the removal of the GST affinity tag is accomplished by using a site-specific protease recognition sequence located between the GST moiety and the target protein. Purified proteins have been used successfully in immunological studies, structure determinations, vaccine production, protein-protein, and protein-DNA interaction studies and other biochemical analysis.

Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes.

Experimental autoimmune encephalomyelitis (EAE) and Theiler’s Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question being asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed. In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro proliferation, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assessment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.

Reconstituted high density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoproteinA-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10-11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptors function.

Summary

The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components and preserving DNA structures. Here we describe a series of assays used to study the replication checkpoint. These assays allow us to investigate the specific functions of proteins involved in the replication checkpoint in fission yeast.

Summary

Regulation of protein function via reversible phosphorylation is an essential component of cell signaling. Our ability to understand complex phosphorylation networks in the physiological context of a whole organism or tissue remains limited. This is largely due to the technical challenge of isolating serine/threonine phosphorylated peptides from a tissue sample. In the present study, we developed a phosphoproteomic strategy to purify and identify phosphopeptides from a tissue sample by employing protein gel filtration, protein SAX (strong anion exchange) and SCX (strong cation exchange) chromatography, peptide SCX chromatography and TiO2 affinity purification. By applying this strategy to the mass spectrometry-based analysis of rat liver homogenates, we were able to identify with high confidence and quantify over four thousand unique phosphopeptides. Finally, the reproducibility of our methodology was demonstrated by its application to analysis of the mammalian Target of Rapamycin (mTOR) signaling pathways in liver samples obtained from rats in which hepatic mTOR was activated by refeeding following a period of fasting.

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the three leading bacteria species associated with otitis media. Defining the molecular epidemiology of bacteria known to cause otitis media is of great importance, in both clinical and research settings. PFGE and MLST provide data for the characterization of isolates’ genetic relatedness, yet they differ in the types of studies for which they are most useful. Consequently, knowledge of both techniques is important for laboratories intending to study the molecular epidemiology of otitis media–associated bacterial pathogens.

Specificity within the pathways of ubiquitin conjugation are defined by protein-binding affinities among the components. Enzyme kinetics provides a facile high-resolution experimental approach for quantitating such protein-binding affinities and yields additional mechanistic insights into the transition state of the enzyme-catalyzed reaction. Most ubiquitin ligases form free polyubiquitin chains at a slow rate in the absence of their cognate target protein as a normal step in their overall catalytic cycle. Rates of polyubiquitin chain formation can, therefore, be used as a reporter function kinetically to characterize binding interactions within the ligation pathway. We describe experimental approaches for: (1) precisely quantitating functional E1 and E2 concentrations by their stoichiometric formation of 125I-ubiquitin thiolester; (2) semiquantitative screens to define the cognate E2(s) for ubiquitin ligases based on their ability to support polyubiquitin chain formation; (3) initial rate studies to quantify Km and kcat as a measure of the ability of specific E2-ubiquitin thiolester substrates to support ligase-catalyzed polyubiquitin chain formation; and (4) an isopeptidase T-based technique for distinguishing between free and conjugated polyubiquitin chains formed in the functional assays. These kinetic methods provide mechanistic insights that are otherwise inaccessible by other experimental approaches and yield a precision in characterizing protein interactions that exceeds that of other techniques.

Natural Regulatory T (Treg) cells are a subset of CD4+ T cells characterized by expression of the transcription factor Foxp3 and the ability to suppress immune responses. Treg cells develop in the thymus in response to highly specific interactions between the T cell receptor (TCR) and self-antigens. These processes can be recapitulated in antigen-specific systems using transgenic mice that co-express a TCR with its cognate peptide as a neo-self antigen. Here we describe a method for using such a system to establish a flow cytometric profile of phenotype markers expressed by developing and mature Treg cells in the thymus. Our approach is to compare antigen-specific thymocytes developing in the presence or absence of Treg cell-selecting ligands to identify phenotypic changes that characterize thymocytes undergoing selection into the Treg cell lineage.

Populations of ethnic mixtures can be useful in genetic studies. Admixture mapping, or mapping by admixture linkage disequilibrium (MALD), is specially developed for admixed populations and can supplement traditional genome-wide association analyses in the search for genetic variants underlying complex traits. Admixture mapping tests the association between a trait and locus-specific ancestries. The locus-specific ancestries are in linkage disequilibrium (LD) which is generated by the admixture process between genetically distinct ancestral populations. Because of highly correlated locus-specific ancestries, admixture mapping performs many fewer independent tests across the genome than current genome-wide association analysis. Therefore, admixture mapping can be more powerful because of the smaller penalty due to multiple tests. In this chapter, I introduce the theory behind admixture mapping and how we conduct the analysis in practice.

Polyamine transport plays an important role in the homeostatic regulation of the polyamine levels. In animals, dietary polyamines are absorbed efficiently in the intestinal tract. In the colon, luminal bacterial derived polyamines are important contributors to cellular polyamine contents. Polyamine transport involves unique uptake and export mechanisms. The amino acid transporter SLC3A2 acts as a polyamine exporter in colon cancer-derived cells. Polyamine uptake is mediated by caveolin-1 dependent endocytosis. The K-RAS oncogene signals increased polyamine uptake and decreased polyamine export. Here, we describe the methods of polyamine transport analysis in the colon and the small intestine using membrane vesicles, culture cells, and mouse models.

The mammalian Target of Rapamycin (mTOR) defines a crucial link between nutrient sensing and immune function. In CD4+ T cells, mTOR has been shown to play a critical role in regulating effector and regulatory T cell differentiation as well as the decision between full activation versus the induction of anergy. In this chapter, we describe how our group has employed the Cre-lox technology to genetically delete components of the mTOR signaling complex in T cells. This has enabled us to specifically interrogate mTOR function in T cells both in vitro and in vivo. We also describe techniques used to assay immune function and signaling in mTOR-deficient T cells at the single-cell level.