Extended and dynamic linker histone-DNA Interactions control chromatosome compaction.

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as approximately ±140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosome's entry and exit. These interactions are dynamic, exhibit rapid binding and dissociation, are sensitive to phosphorylation of a specific residue, and are crucial to determining the symmetry of the chromatosome's core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

Curvature sensing amphipathic helix in the C-terminus of RTNLB13 is conserved in all endoplasmic reticulum shaping reticulons in Arabidopsis thaliana.

The reticulon family of integral membrane proteins are conserved across all eukaryotes and typically localize to the endoplasmic reticulum (ER), where they are involved in generating highly-curved tubules. We recently demonstrated that Reticulon-like protein B13 (RTNLB13) from Arabidopsis thaliana contains a curvature-responsive amphipathic helix (APH) important for the proteins' ability to induce curvature in the ER membrane, but incapable of generating curvature by itself. We suggested it acts as a feedback element, only folding/binding once a sufficient degree of curvature has been achieved, and stabilizes curvature without disrupting the bilayer. However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons-to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. thaliana isoforms. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities.

Gene Regulation and Cellular Metabolism: An Essential Partnership.

It is recognized that cell metabolism is tightly connected to other cellular processes such as regulation of gene expression. Metabolic pathways not only provide the precursor molecules necessary for gene expression, but they also provide ATP, the primary fuel driving gene expression. However, metabolic conditions are highly variable since nutrient uptake is not a uniform process. Thus, cells must continually calibrate gene expression to their changing metabolite and energy budgets. This review discusses recent advances in understanding the molecular and biophysical mechanisms that connect metabolism and gene regulation as cells navigate their growth, proliferation, and differentiation. Particular focus is given to these mechanisms in the context of organismal development.

Biophysical ambiguities prevent accurate genetic prediction.

A goal of biology is to predict how mutations combine to alter phenotypes, fitness and disease. It is often assumed that mutations combine additively or with interactions that can be predicted. Here, we show using simulations that, even for the simple example of the lambda phage transcription factor CI repressing a gene, this assumption is incorrect and that perfect measurements of the effects of mutations on a trait and mechanistic understanding can be insufficient to predict what happens when two mutations are combined. This apparent paradox arises because mutations can have different biophysical effects to cause the same change in a phenotype and the outcome in a double mutant depends upon what these hidden biophysical changes actually are. Pleiotropy and non-monotonic functions further confound prediction of how mutations interact. Accurate prediction of phenotypes and disease will sometimes not be possible unless these biophysical ambiguities can be resolved using additional measurements.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation.

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 µM. As the KD of CBMCipA binding to cellulose is below 1 µM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.

Establishment and Evaluation of an In Vitro System for Biophysical Stimulation of Human Osteoblasts.

While several studies investigated the effects of mechanical or electrical stimulation on osseointegration and bone fracture healing, little is known about the molecular and cellular impact of combined biophysical stimulation on peri-implant osseointegration. Therefore, we established an in vitro system, capable of applying shear stress and electric fields simultaneously. Capacitively coupled electric fields were used for electrical stimulation, while roughened Ti6Al4V bodies conducted harmonically oscillating micromotions on collagen scaffolds seeded with human osteoblasts. Different variations of single and combined stimulation were applied for three days, while samples loaded with Ti6Al4V bodies and untreated samples served as control. Metabolic activity, expression of osteogenic markers and bone remodeling markers were investigated. While combined stimulation showed no substantial benefit compared to sole mechanical stimulation, we observed that 25 µm micromotions applied by roughened Ti6Al4V bodies led to a significant increase in gene expression of osteocalcin and tissue inhibitor of metalloprotease 1. Additionally, we found an increase in metabolic activity and expression of bone remodeling markers with reduced procollagen type 1 synthesis after 100 mVRMS electrical stimulation. We were able to trigger specific cellular behaviors using different biophysical stimuli. In future studies, different variations of electrical stimulation will be combined with interfacial micromotions.

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta.

The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and experimental force-length curves, thin filament lengths were estimated as ~1.5 µm and thick filament lengths were measured as ~2.5 µm. This structural information may help provide an interpretive framework for future studies using this muscle system.

Conscious uncoupling of riboswitch functions.

Riboswitches alter gene expression in response to ligand binding, coupling sensing and regulatory functions to help bacteria respond to their environment. The structural determinants of ligand binding in the prequeuosine (7-aminomethyl-7-deazaguanine, preQ1) bacterial riboswitches have been studied, but the functional consequences of structural perturbations are less known. A new article combining biophysical and cell-based readouts of 15 mutants of the preQ1-II riboswitch from Lactobacillus rhamnosus demonstrates that ligand binding does not ensure successful gene regulation, providing new insights into these shapeshifting sequences.

Comparison of Rosetta flexible-backbone computational protein design methods on binding interactions.

Computational design of binding sites in proteins remains difficult, in part due to limitations in our current ability to sample backbone conformations that enable precise and accurate geometric positioning of side chains during sequence design. Here we present a benchmark framework for comparison between flexible-backbone design methods applied to binding interactions. We quantify the ability of different flexible backbone design methods in the widely used protein design software Rosetta to recapitulate observed protein sequence profiles assumed to represent functional protein/protein and protein/small molecule binding interactions. The CoupledMoves method, which combines backbone flexibility and sequence exploration into a single acceptance step during the sampling trajectory, better recapitulates observed sequence profiles than the BackrubEnsemble and FastDesign methods, which separate backbone flexibility and sequence design into separate acceptance steps during the sampling trajectory. Flexible-backbone design with the CoupledMoves method is a powerful strategy for reducing sequence space to generate targeted libraries for experimental screening and selection.

Skeletal development in the sea urchin relies upon protein families that contain intrinsic disorder, aggregation-prone, and conserved globular interactive domains.

The formation of the sea urchin spicule skeleton requires the participation of hydrogel-forming protein families that regulate mineral nucleation and nanoparticle assembly processes that give rise to the spicule. However, the structure and molecular behavior of these proteins is not well established, and thus our ability to understand this process is hampered. We embarked on a study of sea urchin spicule proteins using a combination of biophysical and bioinformatics techniques. Our biophysical findings indicate that recombinant variants of the two most studied spicule matrix proteins, SpSM50 and SpSM30B/C (S. purpuratus) have a conformational landscape that include a C-terminal random coil/intrinsically disordered MAPQG sequence coupled to a conserved, folded N-terminal C-type lectin-like (CTLL) domain, with SpSM50 > SpSM30B/C with regard to intrinsic disorder. Both proteins possess solvent-accessible unfolded MAQPG sequence regions where Asn, Gln, and Arg residues may be accessible for protein hydrogel interactions with water molecules. Our bioinformatics study included seven other spicule matrix proteins where we note similarities between these proteins and rare, unusual proteins that possess folded and unfolded traits. Moreover, spicule matrix proteins possess three types of sequences: intrinsically disordered, amyloid-like, and folded protein-protein interactive. Collectively these reactive domains would be capable of driving protein assembly and hydrogel formation. Interestingly, three types of global conformations are predicted for the nine member protein set, wherein we note variations in the arrangement of intrinsically disordered and interactive globular domains. These variations may reflect species-specific requirements for spiculogenesis. We conclude that the molecular landscape of spicule matrix protein families enables them to function as hydrogelators, nucleators, and assemblers of mineral nanoparticles.

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi.

BACKGROUND: VhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose. METHODS: VhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water. RESULTS: The role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H2O and D2O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H2O as solvent by D2O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp136 with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp136 was replaced by Arg, or 5-fold and 3-fold for Trp136 replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel. CONCLUSIONS: Hydrogen-bonds contribute to sugar permeation.

Phloem function and development-biophysics meets genetics.

Evolution of the vascular tissues allowed plants to efficiently settle land, occupy new ecological niches, and thereby crucially shape earth's biosphere. Of the two conducting cell types in the plant vasculature, the tubular network of phloem sieve elements transports phloem sap from source to sink organs. Recent years have witnessed the identification of ever more regulators of sieve element differentiation, as well as a more detailed understanding of phloem physiology and function. From molecular regulators of the commitment to sieve element fate, to enzymatic executors of the differentiation process, the toolbox to investigate sieve element formation has been greatly enlarged. To connect the various players in different genetic layers, and thus to ultimately attain a comprehensive description and understanding of sieve element development at the molecular level, appears to be within reach.

Increased functional coupling of 5-HT1A autoreceptors to GIRK channels in Tph2-/- mice.

Firing activity of serotonergic neurons is under regulatory control by somatodendritic 5-HT1A autoreceptors (5-HT1AARs). Enhanced 5-HT1AAR functioning may cause decreased serotonergic signaling in brain and has thereby been implicated in the etiology of mood and anxiety disorders. Tryptophan hydroxylase-2 knockout (Tph2-/-) mice exhibit sensitization of 5-HT1A agonist-induced inhibition of serotonergic neuron firing and thus represents a unique animal model of enhanced 5-HT1AAR functioning. To elucidate the mechanisms underlying 5-HT1AAR supersensitivity in Tph2-/- mice, we characterized the activation of G protein-coupled inwardly-rectifying potassium (GIRK) conductance by the 5-HT1A receptor agonist 5-carboxamidotryptamine using whole-cell recordings from serotonergic neurons in dorsal raphe nucleus. Tph2-/- mice exhibited a mean twofold leftward shift of the agonist concentration-response curve (p < 0.001) whereas the maximal response, proportional to the 5-HT1AAR number, was not different (p = 0.42) compared to Tph2+/- and Tph2+/+ littermates. No differences were found in the basal inwardly-rectifying potassium conductance, determined in the absence of agonist, (p = 0.80) nor in total GIRK conductance activated by intracellular application of GTP-γ-S (p = 0.69). These findings indicate increased functional coupling of 5-HT1AARs to GIRK channels in Tph2-/- mice without a concomitant increase in 5-HT1AARs and/or GIRK channel density. In addition, no changes were found in α1-adrenergic facilitation of firing (p = 0.72) indicating lack of adaptive changes Tph2-/- mice. 5-HT1AAR supersensitivity may represents a previously unrecognized cause of serotonergic system hypofunction and associated disorders and provides a possible explanation for conflicting results on the correlation between 5-HT1AAR density and depression in clinical imaging studies.

Human ATG3 binding to lipid bilayers: role of lipid geometry, and electric charge.

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic α-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.

Substrate stiffness regulates arterial-venous differentiation of endothelial progenitor cells via the Ras/Mek pathway.

Cells sense and respond to the biophysical properties of their surrounding environment by interacting with the extracellular matrix (ECM). Therefore, the optimization of these cell-matrix interactions is critical in tissue engineering. The vascular system is adapted to specific functions in diverse tissues and organs. Appropriate arterial-venous differentiation is vital for the establishment of functional vasculature in angiogenesis. Here, we have developed a polydimethylsiloxane (PDMS)-based substrate capable of simulating the physiologically relevant stiffness of both venous (7kPa) and arterial (128kPa) tissues. This substrate was utilized to investigate the effects of changes in substrate stiffness on the differentiation of endothelial progenitor cells (EPCs). As EPCs derived from mouse bone marrow were cultured on substrates of increasing stiffness, the mRNA and protein levels of the specific arterial endothelial cell marker ephrinB2 were found to increase, while the expression of the venous marker EphB4 decreased. Further experiments were performed to identify the mechanotransduction pathway involved in this process. The results indicated that substrate stiffness regulates the arterial and venous differentiation of EPCs via the Ras/Mek pathway. This work shows that modification of substrate stiffness may represent a method for regulating arterial-venous differentiation for the fulfilment of diverse functions of the vasculature.

Effects of LRRK2 Inhibitors on Nigrostriatal Dopaminergic Neurotransmission.

INTRODUCTION: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent cause of familial and sporadic Parkinson's disease (PD). Because most pathogenic LRRK2 mutations result in enhanced kinase activity, it suggests that LRRK2 inhibitors may serve as a potential treatment for PD. To evaluate whether LRRK2 inhibitors are effective therapies for PD, it is crucial to know whether LRRK2 inhibitors will affect dopaminergic (DAergic) neurotransmission. However, to date, there is no study to investigate the impact of LRRK2 inhibitors on DAergic neurotransmission. AIMS: To address this gap in knowledge, we examined the effects of three types of LRRK2 inhibitors (LRRK2-IN-1, GSK2578215A, and GNE-7915) on dopamine (DA) release in the dorsal striatum using fast-scan cyclic voltammetry and DA neuron firing in the substantia nigra pars compacta (SNpc) using patch clamp in mouse brain slices. RESULTS: We found that LRRK2-IN-1 at a concentration higher than 1 µM causes off-target effects and decreases DA release, whereas GSK2578215A and GNE-7915 do not. All three inhibitors at 1 µM have no effect on DA release and DA neuron firing rate. We have further assessed the effects of the inhibitors in two preclinical LRRK2 mouse models (i.e., BAC transgenic hG2019S and hR1441G) and demonstrated that GNE-7915 enhances DA release and synaptic vesicle mobilization/recycling. CONCLUSION: GNE-7915 can be validated for further therapeutic development for PD.

Stressful learning paradigm precludes manifestation of cognitive ability in sphingomyelin synthase-2 knockout mice.

Sphingomyelin synthases (SMSs) are enzymes converting ceramide to sphingomyelin. The behavioral phenotype attributed to their disruption has not been well described. We examined learning ability and hippocampal synaptic plasticity in mice deficient in SMS2 (SMS2 KO). In context-dependent fear learning and novel object recognition test, no difference in learning ability was detected in SMS2 KO and wild-type (WT) mice. By contrast, achievement of the Morris water maze (MWM) test was deteriorated in SMS2 KO mice. In the hippocampal CA1, while the basic synaptic transmission was normal, both short- and long-term synaptic plasticity was moderately suppressed. We interpret that the MWM test taking place in wet environment may represent learning paradigm under more stressful condition than those performed in dry conditions, and that the learning ability of SMS2 KO mice failed to manifest itself fully in stressful situations. In agreement, forced swimming induced depression-like behavior more easily in SMS2 KO mice. Mass spectrometry suggested a slightly altered species distribution of ceramide in the hippocampus of SMS2 KO mice. These findings support the proposal that altered synthesis of ceramide, which is the substrate of SMS2 and therefore expected to be modified in SMS2 KO mice, is associated with depression-like tendency in animal models and depressive disorder in humans.

Dopamine D1 receptor modulation of calcium channel currents in horizontal cells of mouse retina.

Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 µM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 µM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 µM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 µM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gßγ-blocking peptide suggested involvement of Gßγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gßγ proteins in horizontal cells.

Cannabinoid Control of Learning and Memory through HCN Channels.

The mechanisms underlying the effects of cannabinoids on cognitive processes are not understood. Here we show that cannabinoid type-1 receptors (CB1Rs) control hippocampal synaptic plasticity and spatial memory through the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that underlie the h-current (Ih), a key regulator of dendritic excitability. The CB1R-HCN pathway, involving c-Jun-N-terminal kinases (JNKs), nitric oxide synthase, and intracellular cGMP, exerts a tonic enhancement of Ih selectively in pyramidal cells located in the superficial portion of the CA1 pyramidal cell layer, whereas it is absent from deep-layer cells. Activation of the CB1R-HCN pathway impairs dendritic integration of excitatory inputs, long-term potentiation (LTP), and spatial memory formation. Strikingly, pharmacological inhibition of Ih or genetic deletion of HCN1 abolishes CB1R-induced deficits in LTP and memory. These results demonstrate that the CB1R-Ih pathway in the hippocampus is obligatory for the action of cannabinoids on LTP and spatial memory formation.

Voltage-gated potassium channels involved in regulation of physiological function in MrgprA3-specific itch neurons.

Itch is described as an unpleasant or irritating skin sensation that elicits the desire or reflex to scratch. MrgprA3, one of members of the Mrgprs family, is specifically expressed in a subpopulation of dorsal root ganglion (DRG) in the peripheral nervous system (PNS). These MrgprA3-expressing DRG neurons have been identified as itch-specific neurons. They can be activated by the compound, chloroquine, which is used as a drug to treat malaria. In the present study, we labeled these itch-specific neurons using the method of molecular genetic markers, and then studied their electrophysiological properties. We also recorded the cutaneous MrgprA3(-) neurons retrogradely labeled by Dil dye (MrgprA3(-)-Dil). We first found that MrgprA3(+) neurons have a lower excitability than MrgprA3(-) neurons (MrgprA3(-)-non-Dil and MrgprA3(-)-Dil). The number of action potential (AP) was reduced more obviously in MrgprA3(+) neurons than that of in MrgprA3(-) neurons. In most cases, MrgprA3(+) neurons only generated single AP; however, in MrgprA3(-) neurons, the same stimulation could induce multiple AP firing due to the greater voltage-gated potassium (Kv) current existence in MrgprA3(+) than in MrgprA3(-) neurons. Thus, Kv current plays an important role in the regulation of excitability in itch-specific neurons.