Obesity is reaching epidemic proportions so there is growing interest in the mechanisms that regulates adipose tissue development and function. Although murine adipose cell lines are useful for many mechanistic studies, primary human adipose stromal cells (ASCs), which can be isolated from distinct adipose depots and cultured in vitro, have clear translational relevance. We describe the methods to isolate, culture, and differentiate human ASCs to adipocytes that respond to physiologically relevant hormones, such as insulin and β-adrenergic agonists. We also describe methods for assaying hormonal effects on glucose transport and lipolysis.
Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We therefore developed a genetically-encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.
ATP; ADP; metabolism; energy; fluorescence; genetically-encoded; sensor; microscopy; imaging; ratiometric
The majority of existing antibacterial and anticancer drugs are natural products or their derivatives. However, the characterization and engineering of these compounds are often hampered by limited ability to manipulate the corresponding biosynthetic pathways. Recently, we developed a genomics-driven, synthetic biology-based method, DNA assembler, for discovery, characterization, and engineering of natural product biosynthetic pathways (Shao et al., 2011). By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in individual hosts in a single-step manner. In this chapter, we describe the general guidelines for construct design. By using two distinct biosynthetic pathways, we demonstrate that DNA assembler can perform multiple tasks, including heterologous expression, introduction of single or multiple point mutations, scar-less gene deletion, generation of product derivatives and creation of artificial gene clusters. As such, this method offers unprecedented flexibility and versatility in pathway manipulations.
synthetic biology; natural product biosynthesis; heterologous expression; spectinabilin; aureothin; genetic manipulation of gene clusters
Cilia are prevalent biological structures that are important for cell signaling and for generating fluid flow (or motility). Cilia are found throughout biology from single-celled organisms to vertebrates, and many model systems have been employed for their analysis. Here, we describe the use of Xenopus larval skin as a system for the study of ciliogenesis and ciliary function. In particular, we describe basic molecular and embryological manipulations and imaging techniques that have proven particularly useful for understanding the polarized beating of cilia and the generation of directed fluid flow (Werner & Mitchell, 2012). However, these same tools have the potential to benefit a large number of cilia-related biological questions.
Hydrogen peroxide (H2O2) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H2O2 within neuronal and non-neuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H2O2 in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H2O2 production. A promising means of regulating endogenous H2O2 is through the expression of peroxide-producing enzyme D-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H2O2 production by applying the substrate D-alanine or permeable analogs of D-alanine. In this chapter, we describe the use of DAAO to produce H2O2 in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.
Chaperone-mediated autophagy (CMA) is a selective type of autophagy responsible for the lysosomal degradation of soluble cytosolic proteins. In contrast to other forms of autophagy where cargo is sequestered and delivered to lysosomes through membrane fusion/excision, CMA substrates reach the lysosomal lumen after direct translocation across the lysosomal membrane. CMA is part of the cellular quality control systems and as such, essential for the cellular response to stress. CMA activity decreases with age, likely contributing to the accumulation of altered proteins characteristic in tissues from old organisms. Furthermore, impairment of CMA underlies the pathogenesis of certain human pathologies such as neurodegenerative disorders. These findings have drawn renewed attention to CMA and a growing interest in the measurement of changes in CMA activity under different physiological and pathological conditions. In this chapter we review the different experimental approaches utilized to assess CMA activity both in cells in culture and in different organs from animals.
G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells.
Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.
CRISPR; Disease model; sgRNA design; Amphibian mutagenesis; Targeted mutagenesis; Loss-of-function; Off-target effects; Genome engineering
The endo-lysosomal system and autophagy are essential components of macromolecular turnover in eukaryotic cells. The low-abundance signaling lipid PI(3,5)P2 is a key regulator of this pathway. Analysis of mouse models with defects in PI(3,5)P2 biosynthesis have revealed the unique dependence of the mammalian nervous system on this signaling pathway. This insight led to the discovery of the molecular basis for several human neurological disorders, including Charcot-Marie-Tooth Disease and Yunis-Varon Syndrome. Spontaneous mutants, conditional knockouts, transgenic lines and gene-trap alleles of Fig4, Vac14 and Pikfyve (Fab1) in the mouse have provided novel information regarding the role of PI(3,5)P2
in vivo. This review summarizes what has been learned from mouse models and highlights the utility of manipulating complex signaling pathways in vivo.
FIG4; VAC14; FAB1; PIKFYVE; lysosome; autophagy; transgenic; conditional; neurological mutant
White adipose tissue (WAT) is a dynamic and modifiable tissue that develops late during gestation in humans and through early postnatal development in rodents. WAT is unique in that it can account for as little as 3% of total body weight in elite athletes or as much as 70% in the morbidly obese. With the development of obesity, WAT undergoes a process of tissue remodeling in which adipocytes increase in both number (hyperplasia) and size (hypertrophy). Metabolic derangements associated with obesity, including type 2 diabetes, occur when WAT growth through hyperplasia and hypertrophy cannot keep pace with the energy storage needs associated with chronic energy excess. Accordingly, hypertrophic adipocytes become overburdened with lipids, resulting in changes in the secreted hormonal milieu. Lipids that cannot be stored in the engorged adipocytes become ectopically deposited in organs such as the liver, muscle, and pancreas. WAT remodeling therefore coincides with obesity and secondary metabolic diseases. Obesity, however, is not unique in causing WAT remodeling: changes in adiposity also occur with aging, calorie restriction, cancers, and diseases such as HIV infection. In this chapter, we describe a semiautomated method of quantitatively analyzing the histomorphometry of WAT using common laboratory equipment. With this technique, the frequency distribution of adipocyte sizes across the tissue depot and the number of total adipocytes per depot can be estimated by counting as few as 100 adipocytes per animal. In doing so, the method described herein is a useful tool for accurately quantifying WAT development, growth, and remodeling.
Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.
Adiponectin is a circulating bioactive hormone secreted by adipocytes as oligomers ranging in size from 90 kDa trimers and 180 kDa hexamers to larger high molecular weight oligomers that may reach 18- or 36-mers in size. While total circulating adiponectin levels correlate well with metabolic health, it is the relative distribution of adiponectin complexes that is most clinically relevant to glucose sensitivity and inflammation. High molecular weight adiponectin best mirrors insulin sensitivity, while trimeric adiponectin dominates with insulin resistance and adipose tissue inflammation. Experimental animal and in vitro models have also linked the relative fraction of high molecular weight adiponectin to its positive effects. Quantitating adiponectin size distribution thus provides a window into metabolic health and can serve as a surrogate marker for adipose tissue fitness.
Here, we present a detailed protocol for isolating and quantitating adiponectin complexes in serum or plasma that has been extensively utilized for both human clinical samples and numerous animal models under various experimental conditions. Examples are presented of different adiponectin distributions and tips are provided for optimization using available equipment. Comparison of this rigorous approach to other available methods is also discussed. In total, this summary is a blueprint for the expanded quantitation and study of adiponectin complexes.
Many proteins and protein complexes perform sophisticated, regulated functions in vivo. Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause-effect relationships, for example between a protein and its phosphorylating kinase. Here, we describe a protocol to purify kinetochores, the protein complexes that attach chromosomes to microtubules during mitosis, and quantitatively assay their microtubule binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of microtubules to kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of kinetochore-microtubule attachments, which is believed to be critical for correcting erroneous kinetochore-spindle attachments and thereby avoiding chromosome mis-segregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.
Transfer RNAs (tRNAs) are cellular courier molecules that decipher the genetic code in messenger RNAs and enable the transfer of appropriate esterified amino acids to the growing peptide chain. The preparation of biophysical quantities of homogeneous aminoacylated tRNAs has remained a significant technical challenge. This is primarily due to the difficulty in removing contaminating non-aminoacylated tRNAs that are have very similar properties overall, as well as the hydrolytic instability of the aminoacyl linkage. We describe a flexible, scalable method to prepare homogeneous aminoacylated tRNAs that is also broadly compatible with mutant, misacylated or otherwise aberrant tRNAs and other RNAs. This method combines ribozyme-mediated aminoacylation with reversible N-pentenoylation of the esterified amino acid, which not only protects against spontaneous deacylation but also provides a hydrophobic purification handle. This protocol makes it straightforward to produce biophysical quantities of natural and unnatural aminoacylated tRNAs, and has proven essential for mechanistic investigations of the T-box riboswitches.
The nematode Caenorhabditis. elegans has served as a fruitful setting for cell death research for over three decades. A conserved pathway of four genes, egl-1/BH3-only, ced-9/Bcl-2, ced-4/Apaf-1, and ced-3/caspase, coordinates most developmental cell deaths in C. elegans. However, other cell death forms, programmed and pathological, have also been described in this animal. Some of these share morphological and/or molecular similarities with the canonical apoptotic pathway, while others do not. Indeed, recent studies suggest the existence of an entirely novel mode of programmed developmental cell destruction that may also be conserved beyond nematodes. Here we review evidence for these noncanonical pathways. We propose that different cell death modalities can function as backup mechanisms for apoptosis, or as tailor-made programs that allow specific dying cells to be efficiently cleared from the animal.
cell death; apoptosis; necrosis; linker; elegans; morphology
Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second messenger binding, and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule “fragments” that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.
White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several non-adipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, non-adipocyte cell populations have critical roles in WAT maintenance, growth and function.
As mature adipocytes are terminally-differentiated post-mitotic cells, differentiation of adipocyte precursors is required for hyperplastic WAT growth during development and in obesity. In this chapter, we present methods to separate adipocyte precursor cells from other non-adipocyte cell populations within WAT for analysis by flow cytometry or purification by fluorescence-activated cell sorting (FACS). Additionally, we provide methods to study the adipogenic capacity of purified adipocyte precursor cells ex vivo.
Adipocyte precursor; adipogenesis; stromal vascular; preadipocytes; stem cell; progenitor; adipose tissue
An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.
For more than a decade, mitochondria-targeted nucleases have been used to promote double-strand breaks in the mitochondrial genome. This was done in mitochondrial DNA (mtDNA) homoplasmic systems, where all mtDNA molecules can be affected, to create models of mitochondrial deficiencies. Alternatively, they were also used in a heteroplasmic model, where only a subset of the mtDNA molecules were substrates for cleavage. The latter approach showed that mitochondrial-targeted nucleases can reduce mtDNA haplotype loads in affected tissues, with clear implications for the treatment of patients with mitochondrial diseases. In the last few years, designer nucleases, such as ZFN and TALEN, have been adapted to cleave mtDNA, greatly expanding the potential therapeutic use. This chapter describes the techniques and approaches used to test these designer enzymes.
Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through non-shivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including brightfield colorimetric imaging of paraffin sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.
adipose; whole mount; confocal; frozen; paraffin; cell profiler; lineage tracing