Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level.

While the structure of a multitude of viral particles has been resolved to atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues are particle nucleation, particle growth, and the mode of genome compaction. These issues are difficult to address in bulk approaches and are effectively only accessible by the real-time tracking of assembly dynamics of individual particles. This we do here by studying the assembly into rod-shaped viruslike particles (VLPs) of artificial capsid polypeptides. Using fluorescence optical tweezers, we establish that small oligomers perform one-dimensional diffusion along the DNA. Larger oligomers are immobile and nucleate VLP growth. A multiplexed acoustic force spectroscopy approach reveals that DNA is compacted in regular steps, suggesting packaging via helical wrapping into a nucleocapsid. By reporting how real-time assembly tracking elucidates viral nucleation and growth principles, our work opens the door to a fundamental understanding of the complex assembly pathways of both VLPs and naturally evolved viruses.

Template-Free Self-Assembly of Artificial De Novo Viral Coat Proteins into Nanorods: Effects of Sequence, Concentration, and Temperature.

The self-assembly of protein polymers is a promising route to prepare sophisticated functional nanostructures. However, the interplay between protein self-assembly by itself and its co-assembly with a template is not well understood. Silk-based protein polymers that co-assemble with DNA to form rod-like artificial viruses are herein developed and the effects of silk block length, concentration, and temperature in the self-assembly of the proteins alone are characterized by using a combination of bulk dynamic light scattering (DLS) and single-molecule atomic force microscopy (AFM). Protein nanorods were slowly formed (up to hours) through the interaction of the silk-like blocks. The proteins present a silk-length dependent critical elongation concentration, and above it the amount and size of nanorods rapidly increase. Temperature-dependent light scattering data was adequately fitted into a cooperative model of nucleation-elongation. These results are also important to understand the self-assembly of designed viral coat proteins with DNA templates to form artificial virus-like particles and help us to define general guidelines to design proteins with the ability to precisely organize matter at the nanoscale.

Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4.

Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca(2+)-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4's recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.

Heterogeneous Diffusion of Alkanes in the Hierarchical Metal-Organic Framework NU-1000.

The metal-organic framework (MOF) NU-1000 is a hierarchical material that comprises both micropores and mesopores in its crystalline structure. Because the pore structure is perfectly defined, NU-1000 is an interesting material for improving our understanding of diffusion in hierarchically structured materials. Here, we present molecular dynamics simulations aimed at probing the transport properties of n-alkanes in NU-1000 and introduce methods from the microrheology literature for analyzing the mean-squared displacements and their spatial heterogeneity. Adsorption occurs initially in the smaller channels, and diffusion at low loading is limited by interaction between adsorbate and framework atoms. The larger channels provide a region of low density where molecules are able to diffuse at higher rates predominantly along the channel axes. The disparate size of the channels gives rise to heterogeneity in the diffusivity of the guest molecules, whereas the asymmetry of the channels leads to anisotropic diffusion. Together, the channels form a network of "highways" and "side streets" that provide enhanced diffusion in one dimension.

Spike-and-wave discharge mediated reduction in hippocampal HCN1 channel function associates with learning deficits in a genetic mouse model of epilepsy.

The GABAAγ2(R43Q) mouse is an established model of absence epilepsy displaying spontaneous spike-and-wave discharges (SWD) and associated behavioral arrest. Absence epilepsy typically results from cortico-thalamic networks. Nevertheless, there is increasing evidence for changes in hippocampal metabolism and electrical behavior, consistent with a link between absence seizures and hippocampus-related co-morbidities. Hyperpolarization-activated-cyclic-nucleotide-gated (HCN) channels are known to be transcriptionally regulated in a number of seizure models. Here we investigate the expression and function of these channels in the hippocampus of the genetic epilepsy model. A reduction in HCN1, but not HCN2 transcript, was observed in GABAAγ2(R43Q) mice relative to their littermate controls. In contrast, no change in HCN1 transcript was noted at an age prior to seizure expression or in a SWD-free model in which the R43Q mutation has been crossed into a seizure-resistant genetic background. Whole-cell recordings from CA1 pyramidal neurons confirm a reduction in Ih in the GABAAγ2(R43Q) mouse. Further, a left-shift in half-activation of the Ih conductance-voltage relationship is consistent with a reduction in HCN1 with no change in HCN2 channel expression. Behavioral analysis using the Morris water maze indicates that GABAAγ2(R43Q) mice are unable to learn as effectively as their wildtype littermates suggesting a deficit in hippocampal-based learning. SWD-free mice harboring the R43Q mutation had no learning deficit. We conclude that SWDs reduce hippocampal HCN1 expression and function, and that the reduction associates with a spatial learning deficit.

Genetic and pharmacological modulation of giant depolarizing potentials in the neonatal hippocampus associates with increased seizure susceptibility.

The expression of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is responsible for high intracellular Cl(-) resulting in the excitatory action of GABA(A) receptor activation in the developing brain. Giant depolarizing potentials (GDPs) are spontaneous network oscillations that involve GABA(A) receptors and are thought to be important in establishing neuronal circuit wiring. Earlier work established that seizure susceptibility in the GABA(A) γ2(R43Q) epilepsy mouse is impacted by developmental consequences of impaired GABA(A) receptor function. We investigated the potential mechanism of the developmental influence by recording GDPs in the CA3 pyramidal neurons from brain slices of the neonatal GABA(A) γ2(R43Q) mouse. Interestingly, the number of GPDs was significantly lower in slices from mutant mouse compared with wild-type control, suggesting an involvement in setting seizure susceptibility. To test this idea we blocked NKCC1 with bumetanide in neonatal mice and reduced the number of GDPs to a level similar to that seen in the mutant mice. We found that neonatal treatment with bumetanide resulted in a similar level of susceptibility to thermally induced seizures as described for the GABA(A) γ2(R43Q) mouse. These results provide evidence that a human GABA(A) receptor epilepsy mutation exerts a developmental influence by modulating the number of GDPs. It also draws attention to the potential risk of early treatment with bumetanide.

Perturbations in cortical development and neuronal network excitability arising from prenatal exposure to benzodiazepines in mice.

During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.

DOCU-CLIM: A global documentary climate dataset for climate reconstructions.

Documentary climate data describe evidence of past climate arising from predominantly written historical documents such as diaries, chronicles, newspapers, or logbooks. Over the past decades, historians and climatologists have generated numerous document-based time series of local and regional climates. However, a global dataset of documentary climate time series has never been compiled, and documentary data are rarely used in large-scale climate reconstructions. Here, we present the first global multi-variable collection of documentary climate records. The dataset DOCU-CLIM comprises 621 time series (both published and hitherto unpublished) providing information on historical variations in temperature, precipitation, and wind regime. The series are evaluated by formulating proxy forward models (i.e., predicting the documentary observations from climate fields) in an overlapping period. Results show strong correlations, particularly for the temperature-sensitive series. Correlations are somewhat lower for precipitation-sensitive series. Overall, we ascribe considerable potential to documentary records as climate data, especially in regions and seasons not well represented by early instrumental data and palaeoclimate proxies.

Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain.

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins.

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.

Cytotoxic effect induced by retinoic acid loaded into galactosyl-sphingosine containing liposomes on human hepatoma cell lines.

Two retinoids, ATRA and 13cisRA, were incorporated into liposomes of different composition and charge and added to two hepatoma cell lines with different degree of transformation to measure cytotoxicity by MTT assay. Retinoid-free cationic liposomes were more toxic than the other kinds (anionic and made only of PC) but were also the best delivery system for retinoic acid to induce specific cytotoxic effects on these tumor hepatoma cell lines. Galactosyl-sphingosine containing cationic liposomes increased the cytotoxic effect induced by ATRA on Hep3B cells when compared to glucosyl-sphingosine cationic liposomes, but did not improve the effect induced by free retinoid or ATRA loaded into liposomes without glycolipids. This suggests that in this cell line, ATRA is being incorporated by a mechanism mediated by the asialoglycoprotein receptor, but at the same time, non-specific sugar-independent capture is also taking place as well as free diffusion of ATRA directly through the membrane. Galactose-specific effect was not observed in HepG2 cells treated with ATRA or both cell lines treated with 13cisRA. In fact, treatment of HepG2 cells with retinoids entrapped into liposomes likely induces proliferation instead of cytotoxicity, a result that interferes with the measurement of cell death by MTT. Compared to the specific effect of ATRA entrapped into cationic liposomes, vesicles made only by PC, did not mediate a specific mechanism, since differences between ATRA in galactosyl- and glucosyl-shpingosine PC-liposomes were not statistically significant. The specific mechanism was not present in the myoblastic cell line C2C12, where ATRA incorporated into galactosyl- and glucosyl-sphingosine containing cationic and PC-liposomes, was able to induce cytotoxicity at the same extent. Micelles containing ATRA and galactosyl-sphingosine had a significantly more toxic effect than the retinoid administered together with glucosyl-sphingosine, in Hep3B cells. Also, micelles containing ATRA were more toxic than glycolipid-containing liposomes with ATRA, for both kinds of sphingosines. The same effect was not observed in C2C12 cells, where glycolipid-containing liposomes worked better than micelles, and a sugar-specific mechanism was not seen. This suggests that, even though galactose-containing cationic liposomes could be a promising approach, a galactose-specific emulsion system could be the best strategy to specifically deliver retinoic acid to liver tumor cells, since it shows tissue specificity (perhaps induced by ASGPR-mediated internalization) and a stronger cytotoxic effect than the retinoid incorporated into liposomes.

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2.

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 microM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase-8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteases.

Reduction of p75 neurotrophin receptor ameliorates the cognitive deficits in a model of Alzheimer's disease.

Alzheimer's disease (AD) is an extremely prevalent cause of dementia. It is characterized by progressive memory loss, confusion, and other behavioral and physiological problems. The amyloid-ß (Aß) protein is thought to be involved in the pathogenesis of AD, and there is evidence that Aß may act through the p75 neurotrophin receptor (p75) to mediate its pathogenic effects. This raises the possibility that reducing levels of p75 could be a treatment for AD by preventing the effects of Aß. In this study, we have crossed the transgenic AD model mice, Tg2576, with p75(-/-) mice to generate Tg2576/p75(+/-) mice with reduced levels of p75. These mice are rescued from the deficits in learning and memory and hippocampal function which were found in the Tg2576 mice. These findings suggest that reduction of p75 can ameliorate some of the primary symptoms of AD.

Nano-positioning system for structural analysis of functional homomeric proteins in multiple conformations.

Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target-site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a positioning method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states, with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one per subunit), relative to a single fluorescent acceptor placed in a static site. As first application, the method is used to determine the position of a functional voltage-gated potassium channel's voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.

An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations.

Developing an understanding of the mechanism of voltage-gated ion channels in molecular terms requires knowledge of the structure of the active and resting conformations. Although the active-state conformation is known from x-ray structures, an atomic resolution structure of a voltage-dependent ion channel in the resting state is not currently available. This has motivated various efforts at using computational modeling methods and molecular dynamics (MD) simulations to provide the missing information. A comparison of recent computational results reveals an emerging consensus on voltage-dependent gating from computational modeling and MD simulations. This progress is highlighted in the broad context of preexisting work about voltage-gated channels.

In search of a consensus model of the resting state of a voltage-sensing domain.

Voltage-sensing domains (VSDs) undergo conformational changes in response to the membrane potential and are the critical structural modules responsible for the activation of voltage-gated channels. Structural information about the key conformational states underlying voltage activation is currently incomplete. Through the use of experimentally determined residue-residue interactions as structural constraints, we determine and refine a model of the Kv channel VSD in the resting conformation. The resulting structural model is in broad agreement with results that originate from various labs using different techniques, indicating the emergence of a consensus for the structural basis of voltage sensing.