Pigment epithelium-derived factor (PEDF) is a secreted serpin that exhibits a variety of interesting biological activities. The multifunctional PEDF has neurotrophic and antiangiogenic properties, and acts in retinal differentiation, survival, and maintenance. It is also antitumorigenic and antimetastatic, and has stem cell self-renewal properties. It is widely distributed in the human body and exists in abundance in the eye as a soluble extracellular glycoprotein. Its levels are altered in diseases characterized by retinopathies and angiogenesis. Its mechanisms of neuroprotection and angiogenesis are associated with receptor interactions at cell-surface interfaces and changes in protein expression. This serpin lacks demonstrable serine protease inhibitory activity, but has binding affinity to extracellular matrix components and cell-surface receptors. Here we describe purification protocols, methods to quantify PEDF, and determine interactions with specific molecules, as well as neurotrophic and angiogenesis assays for this multifunctional protein.
In eukaryotes, ribosome biogenesis involves the nucleolar transcription and processing of pre-ribosomal RNA molecules (pre-rRNA) in a complex pathway requiring the participation of myriad protein and ribonucleoprotein factors. Through efforts aimed at categorizing and characterizing these factors, at least 20 RNA helicases have been shown to interact with or participate in the activities of the major ribosome biogenesis complexes. Unfortunately, little is known about the enzymatic properties of most of these helicases, and less is known about their roles in ribosome biogenesis and pre-rRNA maturation. This chapter presents approaches for characterizing RNA helicases involved in ribosome biogenesis. Included are methods for depletion of specific protein targets, with standard protocols for assaying the typical ribosome biogenesis defects that may result. Procedures and rationales for mutagenic studies of target proteins are discussed, as well as several approaches for identifying protein–protein interactions in order to determine functional context and potential cofactors of RNA helicases.
Aquaporin-4 (AQP4) is a water channel expressed in astrocytes throughout the central nervous system, as well as in epithelial cells in various peripheral organs. AQP4 is involved in brain water balance, neuroexcitation, astrocyte migration, and neuroinflammation and is the target of pathogenic autoantibodies in neuromyelitis optica. Two AQP4 isoforms produced by alternative splicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular aggregates called orthogonal arrays of particles (OAPs). OAPs have been studied morphologically, by freeze-fracture electron microscopy, and biochemically, by native gel electrophoresis. We have applied single-molecule and high-resolution fluorescence microscopy methods to visualize AQP4 and OAPs in live cells. Quantum dot single particle tracking of fluorescently labeled AQP4 has quantified AQP4 diffusion in membranes, and has elucidated the molecular determinants and regulation of OAP formation. The composition, structure, and kinetics of OAPs containing fluorescent protein-AQP4 chimeras have been studied utilizing total internal reflection fluorescence microscopy, single-molecule photobleaching, and super-resolution imaging methods. The biophysical data afforded by live-cell imaging of AQP4 and OAPs has provided new insights in the roles of AQP4 in organ physiology and neurological disease.
The biogenesis of microRNAs (miRNAs) in plants is similar to that in animals, however, the processing of plant miRNAs consists of an additional step, the methylation of the miRNAs on the 3′ terminal nucleotides. The enzyme that methylates Arabidopsis miRNAs is encoded by a gene named HEN1, which has been shown genetically to be required for miRNA biogenesis in vivo. Small interfering RNAs (siRNAs) are also methylated in vivo in a HEN1-dependent manner. Our biochemical studies demonstrated that HEN1 is a methyltransferase acting on both miRNAs and siRNAs in vitro. HEN1 recognizes 21 to 24 nt small RNA duplexes, which are the products of Dicer-like enzymes, and transfers a methyl group from S-adenosylmethionine (SAM) to the 2′ OH of the last nucleotides of the small RNA duplexes. Here we describe methods to characterize the biochemical activities of the HEN1 protein both in vitro and in vivo, and methods to analyze the methylation status of small RNAs in vivo.
This chapter describes two types of FRET-based fluorescence assays that can be used to identify and analyze compounds that inhibit the helicase encoded by the hepatitis C virus (HCV). Both assays use a fluorescently labeled DNA or RNA oligonucleotide to monitor helicase-catalyzed strand separation, and they differ from other real-time helicase assays in that they do not require the presence of other nucleic acids to trap the reaction products. The first assay is a molecular beacon-based helicase assay (MBHA) that monitors helicase-catalyzed displacement of a hairpin-forming oligonucleotide with a fluorescent moiety on one end and a quencher on the other. DNA-based MBHAs have been used extensively for high-throughput screening (HTS), but RNA-based MBHAs are typically less useful because of poor signal to background ratios. In the second assay discussed, the fluorophore and quencher are split between two hairpin-forming oligonucleotides annealed in tandem to a third oligonucleotide. This split beacon helicase assay can be used for HTS with either DNA or RNA oligonucleotides. These assays should be useful to the many labs searching for HCV helicase inhibitors in order to develop new HCV therapies that are still desperately needed.
Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins.
Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss our adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells.
Primary neuron; High-throughput microscopy; High-content screen; Neurodegeneration
Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation.
We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.
Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein (Rho-GAP), was one of the first Rho-linked MR gene identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evidence for the requirement of OPHN1 in dendritic spine morphogenesis and synaptic function of CA1 pyramidal neurons. Organotypic hippocampal brain slice cultures are commonly used as a model system to investigate the morphology and synaptic function of neurons, mainly because they allow for the long-term examination of neurons in a preparation where the gross cellular architecture of the hippocampus is retained. In addition, the maintenance of the tri-synaptic circuitry in hippocampal slices enables the study of synaptic connections. Today, a multitude of gene transfer methods for postmitotic neurons in brain slices are available to easily manipulate and scrutinize the involvement of signaling molecules, such as Rho GTPases, in specific cellular processes in this system. This chapter covers techniques detailing the preparation and culturing of organotypic hippocampal brain slices, as well as the production and injection of lentivirus into brain slices.
One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo.
The life and operation of cells involve many physiological processes that take place over fast timescales of milliseconds to minutes. Genetically-encoded technologies for driving or suppressing specific fast physiological processes in intact cells, perhaps embedded within intact tissues in living organisms, are critical for the ability to understand how these physiological processes contribute to emergent cellular and organismal functions and behaviors. Such “synthetic physiology” tools are often incredibly complex molecular machines, in part because they must operate at high speeds, without causing side effects. We here explore how synthetic physiology molecules can be identified and deployed in cells, and how the physiology of these molecules in cellular contexts can be assessed and optimized. For concreteness, we discuss these methods in the context of the “optogenetic” light-gated ion channels and pumps that we have developed over the past few years as synthetic physiology tools, and widely disseminated for use in neuroscience for probing the role of specific brain cell types in neural computations, behaviors, and pathologies. We anticipate that some of the insights revealed here may be of general value for the field of synthetic physiology, as they raise issues that will be of importance for the development and use of high-performance, high-speed, side-effect free physiological control tools, in heterologous expression systems.
Enveloped virus particles select their lipid-protein components and egress by budding from the host cell membranes. The matrix protein of many enveloped viruses has been proposed as a crucial element for viral budding; however, molecular mechanisms behind membrane budding by the matrix protein are yet to be unraveled. Here we describe a set of in vitro functional reconstitution assays that allows quantitative evaluation of both, membrane binding and creation of membrane curvature by the matrix protein isolated from Newcastle Disease Virus. Individual budding events orchestrated by the matrix protein can be resolved in real time. The assays may be applied for direct reconstitution of the on-membrane action of cellular proteins involved in membrane curvature induction upon binding in vivo.
With the aid of chemoselective sensors, fluorescence microscopy has emerged as an indispensable tool to visualize the distribution and dynamics of various biologically important molecules in live specimens. Motivated by our interest in understanding the chemistry and biology of mobile zinc underlying its physiological and pathological roles, over the past decade our laboratory has developed an extensive library of zinc fluorescence probes. In this article, we provide essential information about our sensor toolbox in order to assist investigators interested to apply our constructs to study various aspects of mobile zinc biology. We illustrate their use with several examples of imaging both exogenous and endogenous mobile zinc in live cells and tissues using various versions of fluorescence microscopy, including confocal and two-photon microscopy.
imaging; fluorescence; sensor; probe; zinc; live cell; hippocampus; mossy fiber; confocal microscopy; two-photon microscopy
RNA localization, dynamics, and regulation are becoming increasingly important to our basic understanding of gene expression and RNA virus pathogenesis. An improved understanding of these processes will be necessary in order to identify new drug targets, as well as to create models of gene expression networks. Much of this new understanding will likely come from imaging studies of RNA, which can generate the spatiotemporal information necessary to characterize RNA within the cellular milieu. Ideally, this would be performed imaging native, nonengineered RNAs, but the approaches for performing these experiments are still evolving. In order for them to reach their potential, it is critical that they have characteristics that allow for the tracking of RNA throughout their life cycle. This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells.
The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction.
Constitutive activity of the extracellular calcium-sensing receptor (CaSR) has been studied in kindreds with the human disorder autosomal dominant hypocalcemia (ADH) and in an animal model called the Nuf mouse. These families generally showed reduced parathyroid hormone (PTH) secretion and excessive renal calcium (Ca2+) excretion. Soft tissues calcifications in the kidney and basal ganglia are frequent (10–50% of ADH cases), and there is a single report of skeletal abnormalities in a family resulting in short stature and premature osteoarthritis. In the latter, a causative mechanism could not be determined. The phenotype of the Nuf mouse is one of ectopic calcifications and cataracts in addition to biochemical abnormalities (low serum Ca2+ and high serum phosphate concentrations). To better understand the role of CaSRs in the control of osteoblastic function, we generated a transgenic mouse model with constitutively active CaSRs in mature osteoblasts. An analysis of the skeletal phenotype of that mouse indicates that strong signaling by CaSRs in this cell lineage induces alterations in the bone homeostasis reflected in mild osteopenia in male and female mice during growth and in adulthood. These studies indicate that this approach can be readily adapted to assess CaSR actions in other cell systems.
We compare the use of two-dimensional total internal reflection fluorescence microscopy with a rapid, simple-to-implement method for three-dimensional (3D) imaging using spinning-disk confocal microscopy suitable for reliable 3D tracking of clathrin-coated endocytic and endosomal carriers. These carriers contain about 20 EGFP (enhanced green fluorescent protein) equivalents of a chimeric fluorescent protein (either clathrin light chain or one of the clathrin adaptor subunits). Under tissue culture conditions, the clathrin-containing carriers correspond to a variable number of relatively sparse, diffraction-limited, fluorescent objects that can be identified with a spatial precision of ~30 nm or better and a temporal resolution of <1 s. The applicability of these approaches to mammalian cells in culture allows investigators detailed monitoring of the composition dynamics of the clathrin-containing carriers which can then be used to study in living cells the molecular mechanisms required for the formation and traffic of clathrin-coated pits and vesicles.
Protein phosphorylations, as well as phosphate metabolite binding, are well characterized posttranslational mechanisms that regulate enzyme activity in the cytosol, but remain poorly defined in mitochondria. Recently extensive matrix protein phosphorylation sites have been discovered but their functional significance is unclear. Herein we describe methods of using 32P labeling of intact mitochondria to determine the dynamic pools of protein phosphorylation as well as phosphate metabolite association. This screening approach may be useful in not only characterizing the dynamics of these pools, but also provide insight into which phosphorylation sites have a functional significance. Using the mitochondrial ATP synthetic capacity under appropriate conditions, inorganic 32P was added to energized mitochondria to generate high specific activity γ-P32-ATP in the matrix. In general, SDS denaturing and gel electrophoresis was used to primarily follow protein phosphorylation, whereas native gel techniques were used to observe weaker metabolite associations since the structure of mitochondrial complexes were minimally affected. The protein phosphorylation and metabolite association within the matrix was found to be extensive using these approaches. 32P labeling in 2D gels was detected in over 40 proteins, including most of the complexes of the cytochrome chain and proteins associated with intermediary metabolism, biosynthetic pathways, membrane transport, and reactive oxygen species metabolism. 32P pulse-chase experiments further revealed the overall dynamics of these processes that included phosphorylation site turnover as well as phosphate-protein pool size alterations. The high sensitivity of 32P resulted in many proteins being intensely labeled, but not identified due to the sensitivity limitations of mass spectrometry. These low concentration proteins may represent signaling proteins within the matrix. These results demonstrate that the mitochondrial matrix phosphoproteome is both extensive and dynamic. The use of this, in situ, labeling approach is extremely valuable in confirming protein phosphorylation sites as well as examining the dynamics of these processes under near physiological conditions.
Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells.
Discrete stochastic chemical kinetics describe the time evolution of a chemically reacting system by taking into account the fact that in reality chemical species are present with integer populations and exhibit some degree of randomness in their dynamical behavior. In recent years, with the development of new techniques to study biochemistry dynamics in a single cell, there are increasing studies using this approach to chemical kinetics in cellular systems, where the small copy number of some reactant species in the cell may lead to deviations from the predictions of the deterministic differential equations of classical chemical kinetics.
This chapter reviews the fundamental theory related to stochastic chemical kinetics and several simulation methods that are based on that theory. We focus on non-stiff biochemical systems and the two most important discrete stochastic simulation methods: Gillespie's Stochastic Simulation Algorithm (SSA) and the tau-leaping method. Different implementation strategies of these two methods are discussed. Then we recommend a relatively simple and efficient strategy that combines the strengths of the two methods: the hybrid SSA/tau-leaping method. The implementation details of the hybrid strategy are given here and a related software package is introduced. Finally, the hybrid method is applied to simple biochemical systems as a demonstration of its application.
The assembly and disassembly of chromatin impacts all DNA dependent processes in eukaryotes. These processes are intricately regulated through stepwise mechanisms, requiring multiple proteins, post-translational modifications and remodeling enzymes, as well as specific proteins to chaperone the highly basic and aggregation-prone histone proteins. The histone chaperones are acidic proteins that perform the latter function by maintaining the stability of the histones when they are not associated with DNA and guiding the deposition and removal of histones from DNA. Understanding the thermodynamics of these processes provides deeper insights into the mechanisms of chromatin assembly and disassembly. Here we describe complementary thermodynamic and biochemical approaches for analysis of the interactions of a major chaperone of the H3/H4 dimer, Anti-silencing function 1 (Asf1) with histones H3/H4 and DNA. Fluorescence quenching approaches are useful for measuring the binding affinity of Asf1 for histones H3/H4 under equilibrium conditions. Electrophoretic mobility shift analyses are useful for examining Asf1 mediated tetrasome (H3/H4-DNA) assembly and disassembly processes. These approaches potentially can be used more generally for the study of other histone chaperone-histone interactions and provide a means to dissect the role of post-translational modifications and other factors that participate in chromatin dynamics.
Anti-silencing function 1 (Asf1); histone H3/H4; tetrasome; binding affinity; fluorescence; electrophoretic mobility shift assay
Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.
The gene expression, signaling, and cellular dynamics driving mouse embryo development have emerged through embryology and genetic studies. However, since mouse development is a temporally regulated three-dimensional process, any insight needs to be placed in this context of real-time visualization. Live imaging using genetically encoded fluorescent protein reporters is pushing the envelope of our understanding by uncovering unprecedented insights into mouse development and leading to the formulation of quantitative accurate models.
This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.
A procedure for an in vitro signaling assay is described for the MAPK and NFκB pathways. The method uses a membrane-cleared lysate that contains all the soluble components required for activating these signaling cascades. The pathways can be activated by variety of molecules, including kinases, G-proteins, and E3 ligases. We demonstrate that YopJ inhibits downstream of all these activators. The in vitro signaling assay is ideal for initial biochemical studies on activators and inhibitors of the MAPK and NFκB pathways.