Latency Locus Complements MicroRNA 155 Deficiency In Vivo

MicroRNA-155 (miR-155) is expressed in many cancers. It also executes evolutionary conserved functions in normal B cell development. We show that the Kaposi''s sarcoma-associated herpesvirus (KSHV) latency locus, which contains an ortholog of miR-155, miR-K12-11, complements B cell deficiencies in miR-155 knockout mice. Germinal center (GC) formation was rescued in spleen, lymph node, and Peyer''s patches. Immunoglobulin levels were restored. This demonstrates that KSHV can complement the normal, physiological function of miR-155.     

In Vivo and In Vitro Morphological Analysis of Melanocytes Homozygous for the misp Allele at the Murine Microphthalmia Locus

The mi^8p allele (microphthalmia-spotted), a mutant allele at the murine microphthalmia (mi) locus, when homozygous, results in a normal phenotype in which there is no apparent alteration in pelage pigmentation or ocular development. However, when heterozygous with other mi locus alleles, specifically Mi^wh (microphthalmia-white) the mi^8p allele exerts an affect on the phenotype. We examined the ultrastructure of melanocytes in the anagen hair bulb and the choroid plus the retinal pigmented epithelium of C57BL/6J-mi^8p/mi^8p mice, C57BL/6J-Mi^wh/Mi^wh mice, C57BL/6J-Mi^wh/mi^8p mice, and C57BL/6J-⁺/⁺ control mice. Melanocytes of the mi^8p/mi^8p mice appeared normal in situ. However, melanocyte cultures derived from neonatal skins of mi^8p/mi^8p mice exhibited small primary colonies that did not dramatically expand in size. Occasionally, abnormalities in the structure of the Golgi apparatus were observed in primary cultures of mi^8p/mi^8p melanocytes. These results demonstrate that while the mi^8p allele has no obvious effect on the phenotype of the mouse, it does dramatically suppress the survival of melanocytes in normal culture conditions.     

Genome-wide Chromatin Interactions of the Nanog Locus in Pluripotency,Differentiation, and Reprogramming

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In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers

Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.A combination of genetically encoded fluorescent probes, advances in light microscopy, and interdisciplinary approaches has revolutionized our understanding of organelle transport. Organelle movement in highly vacuolated leaf epidermal cells appears erratic, with individual organelles undergoing a range of movements within a relatively short time frame: they stop-go, change direction (trajectory), and move at varying speeds. The use of pharmacological inhibitors indicated a role for actin, and therefore myosins, in this process; however, myosin-organelle specificity is poorly characterized (Madison and Nebenführ, 2013; Tamura et al., 2013; Buchnik et al., 2015). Therefore, we are still at a relatively rudimentary stage in the understanding of the molecular and physical control, and interaction, of organelles in plant cells compared with that known in other model systems (Hammer and Sellers, 2012; Prinz, 2014). However, it is clear that organelle movement plays important roles in physological processes in plants; reduced movement effects growth and development, and movement is correlated with responses to extracellular stresses such as pathogens and heavy metals (for refs., see Sparkes, 2011; Madison and Nebenführ, 2013; Buchnik et al., 2015). Organelle interactions in other systems have important roles in calcium and lipid exchange, setting a precedent for physiologically important roles in plants (Prinz, 2014). However, characterization of the molecular factors required to physically tether organelles, as opposed to those that function in the exchange of molecules at the interaction site, is challenging. Monitoring organelle interactions in highly vacuolated plant epidermal cells is further complicated by the constraints imposed by the large central vacuole. Static snapshots provided through electron microscopy of highly vacuolated cells, where the vacuole can effectively push organelles together, giving the impression of direct interaction between organelles, is not a suitable method to determine dynamic interactions. Other techniques, such as the laser-induced shockwave by explosion method used by Oikawa et al. (2015), works globally without directly manipulating the individual organelle. Here, using optical tweezers with submicron precision, we provide a means to assess and quantify the dynamic interaction between peroxisomes and chloroplasts in vivo in leaf epidermal cells.Peroxisomes are responsible for several biochemical reactions, including the glyoxylate cycle and β-oxidation, which provides an energy source for germination in oilseeds. They also produce and scavenge free radicals, synthesize jasmonic acid and indole-3-acetic acid, and are required for photorespiration (for refs., see Hu et al., 2012). The photorespiratory pathway spans peroxisomes, chloroplasts, and mitochondria, where phosphoglycolate produced in the chloroplast is converted back to 3-phosphoglycerate. It has been suggested that functional connectivity between these organelles accounts for the close association observed in ultrastructural micrographs (Frederick and Newcomb, 1969). Several Arabidopsis (Arabidopsis thaliana) pex10 (peroxisomal membrane protein) mutants show altered chloroplast-peroxisome juxtaposition with a defect in photorespiration, while others do not (Schumann et al., 2007; Prestele et al., 2010). Both CLUMPED CHLOROPLASTS1 (CLMP1) and CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1) encode for proteins that localize to the chloroplast, with CHUP1 playing a role in chloroplast-actin formation (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008; Yang et al., 2011). While CHUP1 and CLMP1 affect chloroplast positioning, they have differential effects on peroxisome and mitochondrial location; clmp1 causes chloroplast clustering without affecting mitochondria or peroxisome location (Yang et al., 2011), whereas chup1 was reported to affect peroxisome location (Oikawa et al., 2003). In vitro analysis through density centrifugation highlighted chloroplast sedimentation with peroxisomes under certain conditions (Schnarrenberger and Burkhard, 1977), although this does not necessarily reflect the organelle interaction in live cells. Peroxisome proteomics studies have been hampered by difficulties in isolating pure peroxisomal fractions (Bussell et al., 2013). This could be indicative of interaction, where associated membranes are isolated together, or sticky nonspecific contaminating chloroplast membranes. The work by Oikawa et al. (2015) provides insight into the physiological processes controlling peroxisome-chloroplast interaction (photosynthesis dependent), but they did not determine the effective baseline force required to move peroxisomes that were not next to chloroplasts under control or altered environmental conditions. Comparisons between the relative forces required to move peroxisomes next to chloroplasts versus those that are not next to chloroplasts are critical in understanding and probing the physical interaction between the two organelles, the hypothesis being that tethering would increase the force required to move peroxisomes compared with organelles that are not tethered. Since peroxisomes have diverse biochemical roles that affect a wide range of physiological processes throughout the plant life cycle (Hu et al., 2012), an understanding of if and how peroxisomes may interact with other subcellular structures is likely to be an important consideration for efficient peroxisome function.Peroxisomes are highly pleomorphic, dynamic organelles bounded by a single membrane (Hu et al., 2012), whose movement is driven by acto-myosin-dependent processes (Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002; Avisar et al., 2008; Sparkes et al., 2008). Tubular emanations termed peroxules (Scott et al., 2007) can extend from the main peroxisome body, yet it is unclear what function they may play. Formation is quite frequent in hypocotyl cells (Cutler et al., 2000; Mano et al., 2002; Sinclair et al., 2009), can occur around chloroplasts in cotyledonary leaf pavement cells (Sinclair et al., 2009), and is not always from the trailing edge of the peroxisome (Sinclair et al., 2009). Exogenous addition of hydroxyl reactive oxygen species (ROS), or exposure to UV light, induces peroxule formation (Sinclair et al., 2009). It has been suggested that they represent an increased surface area for increased biochemical function or might represent a morphological precursor for peroxisome division (Jedd and Chua, 2002). Based on subcellular coalignment, a retro-flow model for the potential exchange of luminal content between the endoplasmic reticulum (ER) and peroxisome through the peroxule has been suggested (Sinclair et al., 2009; Barton et al., 2013). However, these studies, as with many others, interpret the close association between organelles to indicate physical connectivity between organelles, whereas, in fact, in highly vacuolated leaf epidermal cells, organelles can be closely packed within the cytoplasm due to mere spatial constrictions generated through the large central vacuole. This is further complicated by the highly motile, and seemingly stochastic, nature of acto-myosin-driven organelle movement, resulting in frequent apparent organelle collisions that may not reflect a functional requirement for organelle interaction.Optical trapping provides a highly specific and sensitive means to measure physical connectivity between organelles. By focusing an infrared beam, it allows the user to trap objects that have a significantly different refractive index from the surrounding medium. Upon trapping, the user can then move the trapped object relative to its original position to gain an understanding of whether the movement affects the position and motion of other structures (such as other organelles) that may be physically attached to the trapped organelle. For example, unlike the ER, Golgi bodies are amenable to trapping. By trapping and micromanipulating (i.e. precisely moving) the Golgi, a physical association between the ER and the Golgi was determined in a qualitative manner (Sparkes et al., 2009b). Here, we have developed a system to generate quantitative measures for organelle interaction by standardizing and automating how far we move the trapped organelle (which we call the translation step) at a defined speed and assessing how trapping efficiency alters in response to the power of the laser trap itself. By using these parameters, we can then model the forces imparted on the organelle, providing further insight into the tethering processes.Our results indicate that peroxisomes are amenable to being trapped, that they physically interact with chloroplasts in leaf epidermal cells, and, surprisingly, that peroxisomes are also tethered to other unknown structures within the cell. This approach highlights that organelle interactions within plant cells are not random but regulated through tethering. In addition, we provide a novel role for peroxules and a simple biophysical model to describe peroxisome motion during the trapping process.     

Identification of New Interactions between Endolysosomal Tethering Factors

Proper functioning of the precisely controlled endolysosomal system is essential for maintaining the homeostasis of the entire cell. Tethering factors play pivotal roles in mediating the fusion of different transport vesicles, such as endosomes or autophagosomes with each other or with lysosomes. In this work, we uncover several new interactions between the endolysosomal tethering factors Rabenosyn-5 (Rbsn) and the HOPS and CORVET complexes. We find that Rbsn binds to the HOPS/CORVET complexes mainly via their shared subunit Vps18 and we mapped this interaction to the 773–854 region of Vps18. Based on genetic rescue experiments, the binding between Rbsn and Vps18 is required for endosomal transport and is dispensable for autophagy. Moreover, Vps18 seems to be important for β1 integrin recycling by binding to Rbsn and its known partner Vps45.     

Extracting the Stepping Dynamics of Molecular Motors in Living Cells from Trajectories of Single Particles

Molecular motors are responsible of transporting a wide variety of cargos in the cytoplasm. Current efforts are oriented to characterize the biophysical properties of motors in cells with the aim of elucidating the mechanisms of these nanomachines in the complex cellular environment. In this study, we present an algorithm designed to extract motor step sizes and dwell times between steps from trajectories of motors or cargoes driven by motors in cells. The algorithm is based on finding patterns in the trajectory compatible with the behavior expected for a motor step, i.e., a region of confined motion followed by a jump in the position to another region of confined motion with similar characteristics to the previous one. We show that this algorithm allows the analysis of 2D trajectories even if they present complex motion patterns such as active transport interspersed with diffusion and does not require the assumption of a given step size or dwell period. The confidence on the step detection can be easily obtained and allows the evaluation of the confidence of the dwell and step size distributions. To illustrate the possible applications of this algorithm, we analyzed trajectories of myosin-V driven organelles in living cells.     

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing¹ as well as in pathological processes such as neuropathic pain². Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Extracting Diffusive States of Rho GTPase in Live Cells: Towards In Vivo Biochemistry

Resolving distinct biochemical interaction states when analyzing the trajectories of diffusing proteins in live cells on an individual basis remains challenging because of the limited statistics provided by the relatively short trajectories available experimentally. Here, we introduce a novel, machine-learning based classification methodology, which we call perturbation expectation-maximization (pEM), that simultaneously analyzes a population of protein trajectories to uncover the system of diffusive behaviors which collectively result from distinct biochemical interactions. We validate the performance of pEM in silico and demonstrate that pEM is capable of uncovering the proper number of underlying diffusive states with an accurate characterization of their diffusion properties. We then apply pEM to experimental protein trajectories of Rho GTPases, an integral regulator of cytoskeletal dynamics and cellular homeostasis, in vivo via single particle tracking photo-activated localization microcopy. Remarkably, pEM uncovers 6 distinct diffusive states conserved across various Rho GTPase family members. The variability across family members in the propensities for each diffusive state reveals non-redundant roles in the activation states of RhoA and RhoC. In a resting cell, our results support a model where RhoA is constantly cycling between activation states, with an imbalance of rates favoring an inactive state. RhoC, on the other hand, remains predominantly inactive.     

Global Quantitative Modeling of Chromatin Factor Interactions

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions.     

In Vivo Protein Interactions within the Escherichia coli DNA Polymerase III Core

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ ( subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutations do not affect the subunit-α subunit and subunit-θ subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the protein, which leads to altered interactions within the Pol III core.     

In Vivo Analysis of Voltage-Dependent Calcium Channels

The molecular cloning of calcium channel subunits has identified an unexpectedly large number of genes and splicing variants, many of whichhave complex expression patterns: a central problem of calcium channel biology is to understand the functional significance of this genetic complexity. The genetic analysis of voltage-dependent calcium channels (VDCCs) provides an approach to defining channel function that is complimentary to pharmacological, electrophysiological, and other molecular methods. By discovering or creating alleles of VDCC genes, one can gain an understanding of the VDCC function at the whole animal level. Of particular interest are mutations in the alpha1 genes that encode the pore forming subunits, as they define the specific channel subtypes. In fact, a variety of calcium channelopathies and targeted mutations have been described for these genes in the last 6 years. The mutant alleles described below illustrate how phenotype analysis of these alleles has uncovered very specific functional roles that can be localized to specific synapses or cells.     

Targeted In Vivo Inhibition of Specific Protein–Protein Interactions Using Recombinant Antibodies

With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated “silencing” represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein–protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.     

Quantitative Analysis of Single Particle Trajectories: Mean Maximal Excursion Method

An increasing number of experimental studies employ single particle tracking to probe the physical environment in complex systems. We here propose and discuss what we believe are new methods to analyze the time series of the particle traces, in particular, for subdiffusion phenomena. We discuss the statistical properties of mean maximal excursions (MMEs), i.e., the maximal distance covered by a test particle up to time t. Compared to traditional methods focusing on the mean-squared displacement we show that the MME analysis performs better in the determination of the anomalous diffusion exponent. We also demonstrate that combination of regular moments with moments of the MME method provides additional criteria to determine the exact physical nature of the underlying stochastic subdiffusion processes. We put the methods to test using experimental data as well as simulated time series from different models for normal and anomalous dynamics such as diffusion on fractals, continuous time random walks, and fractional Brownian motion.