Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex.

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

Super-resolved 3D tracking of cargo transport through nuclear pore complexes.

Nuclear pore complexes (NPCs) embedded within the nuclear envelope mediate rapid, selective and bidirectional traffic between the cytoplasm and the nucleoplasm. Deciphering the mechanism and dynamics of this process is challenged by the need for high spatial and temporal resolution. We report here a multicolour imaging approach that enables direct three-dimensional visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring structure of the NPC scaffold. The success of this approach is enabled by the high positional stability of NPCs within permeabilized cells, as verified by a combined experimental and simulation analysis. Hourglass-shaped translocation conduits for two cargo complexes representing different nuclear transport receptor pathways indicate rapid migration through the permeability barrier on or near the NPC scaffold. Binding sites for cargo complexes extend more than 100 nm from the pore openings, which is consistent with a wide distribution of the phenylalanine-glycine polypeptides that bind nuclear transport receptors.

Tuning axial and lateral localization precision in 3D super-resolution microscopy with variable astigmatism.

Astigmatism imaging is a three-dimensional (3D) single molecule fluorescence microscopy approach that yields super-resolved spatial information on a rapid time scale from a single image. It is ideally suited for resolving structures on a sub-micrometer scale and temporal behavior in the millisecond regime. While traditional astigmatism imaging utilizes a cylindrical lens, adaptive optics enables the astigmatism to be tuned for the experiment. We demonstrate here how the precisions in x, y, and z are inter-linked and vary with the astigmatism, z-position, and photon level. This experimentally driven and verified approach provides a guide for astigmatism selection in biological imaging strategies.

Quantifying Surface-Height Change Over a Periglacial Environment With ICESat-2 Laser Altimetry.

We use Ice, Cloud, and land Elevation Satellite 2 (ICESat-2) laser altimetry crossovers and repeat tracks collected over the North Slope of Alaska to estimate ground surface-height change due to the seasonal freezing and thawing of the active layer. We compare these measurements to a time series of surface deformation from Sentinel-1 interferometric synthetic aperture radar (InSAR) and demonstrate agreement between these independent observations of surface deformation at broad spatial scales. We observe a relationship between ICESat-2-derived surface subsidence/uplift and changes in normalized accumulated degree days, which is consistent with the thermodynamically driven seasonal freezing and thawing of the active layer. Integrating ICESat-2 crossover estimates of surface-height change yields an annual time series of surface-height change that is sensitive to changes in snow cover during spring and thawing of the active layer throughout spring and summer. Furthermore, this time series exhibits temporal correlation with independent reanalysis datasets of temperature and snow cover, as well as an InSAR-derived time series. ICESat-2-derived surface-height change estimates can be significantly affected by short length-scale topographic gradients and changes in snow cover and snow depth. We discuss optimal strategies of post-processing ICESat-2 data for permafrost applications, as well as the future potential of joint ICESat-2 and InSAR investigations of permafrost surface-dynamics.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation.

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 µM), and this monomer binds reversibly to spTorA (KD ≈ 1 µM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Migratory earthquake precursors are dominant on an ice stream fault.

Simple fault models predict earthquake nucleation near the eventual hypocenter (self-nucleation). However, some earthquakes have migratory foreshocks and possibly slow slip that travel large distances toward the eventual mainshock hypocenter (migratory nucleation). Scarce observations of migratory nucleation may result from real differences between faults or merely observational limitations. We use Global Positioning System and passive seismic records of the easily observed daily ice stream earthquake cycle of the Whillans Ice Plain, West Antarctica, to quantify the prevalence of migratory versus self-nucleation in a large-scale, natural stick-slip system. We find abundant and predominantly migratory precursory slip, whereas self-nucleation is nearly absent. This demonstration that migratory nucleation exists on a natural fault implies that more-observable migratory precursors may also occur before some earthquakes.

Rigorous Lower Bounds for the Ground State Energy of Molecules by Employing Necessary N-Representability Conditions.

Electronic structure calculations, in particular the computation of the ground state energy, lead to challenging problems in optimization. These problems are of enormous importance in quantum chemistry for calculations of properties of solids and molecules. Minimization methods for computing the ground state energy can be developed by employing a variational approach, where the second-order reduced density matrix defines the variable. This concept leads to large-scale semidefinite programming problems that provide a lower bound for the ground state energy. Upper bounds of the ground state energy can be calculated for example with the Hartree-Fock method or numerically more exact for a given basis set by full CI. However, Nakata et al. ( J. Chem. Phys.200111482828292) observed that due to numerical errors the semidefinite solver produced erroneous results with a lower bound significantly larger than the full CI energy. For the LiH, CH-, NH-, OH, OH-, and HF molecules violations within one mhartree were observed. We applied the software VSDP which takes all numerical errors due to floating-point arithmetic operations into consideration. For two test libraries VSDP provides tight rigorous error bounds lower than full CI energies reported with an accuracy of 0.1 to 0.01 mhartree. Only little computation work must be spent in order to compute close rigorous error bounds for the ground state energy.

The Tat protein transport system: intriguing questions and conundrums.

The Tat machinery catalyzes the transport of folded proteins across the cytoplasmic membrane in bacteria and the thylakoid membrane in plants. Transport occurs only in the presence of an electric field (Δψ) and/or a pH (ΔpH) gradient, and thus, Tat transport is considered to be dependent on the proton motive force (pmf). This presents a fundamental and major challenge, namely, that the Tat system catalyzes the movement of large folded protein cargos across a membrane without collapse of ion gradients. Current models argue that the active translocon assembles de novo for each cargo transported, thus providing an effective gating mechanism to minimize ion leakage. A limited structural understanding of the intermediates occurring during transport and the role of the pmf in stabilizing and/or driving this process have hindered the development of more detailed models. A fundamental question that remains unanswered is whether the pmf is actually 'consumed', providing an energetic driving force for transport, or alternatively, whether its presence is instead necessary to provide the appropriate environment for the translocon components to become active. Including addressing this issue in greater detail, we explore a series of additional questions that challenge current models, and, hopefully, motivate future work.

Response of Pacific-sector Antarctic ice shelves to the El Niño/Southern Oscillation.

Satellite observations over the past two decades have revealed increasing loss of grounded ice in West Antarctica, associated with floating ice shelves that have been thinning. Thinning reduces an ice-shelf's ability to restrain grounded-ice discharge, yet our understanding of the climate processes that drive mass changes is limited. Here, we use ice-shelf height data from four satellite altimeter missions (1994-2017) to show a direct link between ice-shelf-height variability in the Antarctic Pacific sector and changes in regional atmospheric circulation driven by the El Niño-Southern Oscillation. This link is strongest from Dotson to Ross ice shelves and weaker elsewhere. During intense El Niño years, height increase by accumulation exceeds the height decrease by basal melting, but net ice-shelf mass declines as basal ice loss exceeds lower-density snow gain. Our results demonstrate a substantial response of Amundsen Sea ice shelves to global and regional climate variability, with rates of change in height and mass on interannual timescales that can be comparable to the longer-term trend, and with mass changes from surface accumulation offsetting a significant fraction of the changes in basal melting. This implies that ice-shelf height and mass variability will increase as interannual atmospheric variability increases in a warming climate.

A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation.

The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.

Investigating molecular crowding within nuclear pores using polarization-PALM.

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC's internal organization consists of multiple dynamic environments with different local properties.

Variability of upper firn processes in West Antarctica observed with GPS reflectometry, 2007-2017.

Land ice loss from Antarctica is a significant and accelerating contribution to global sea-level rise; however, Antarctic mass-balance estimates are complicated by insufficient knowledge of surface mass-balance processes such as snow accumulation. These variables are challenging to observe on a continental scale and in situ data are sparse, so we largely rely on estimates from atmospheric models. Here, we employ a novel method, GPS interferometric reflectometry (GPS-IR), to measure upper (<2 m) firn-column thickness changes across a 23-station GPS array in West Antarctica. We compare the results with antenna heights measured in situ to establish the method's daily uncertainty (0.06 m) and with output from two atmospheric reanalysis products to categorize spatial and temporal variability of near-surface processes. GPS-IR is an effective method for monitoring surface mass-balance processes that can be applied to both historic GPS datasets and future experiments to provide critical in situ observations of processes driving surface-height evolution.

Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches.

Due to its central role in macromolecular trafficking and nucleocytoplasmic information transfer, the nuclear pore complex (NPC) has been studied in great detail using a wide spectrum of methods. Consequently, many aspects of its architecture, general function, and role in the life cycle of a cell are well understood. Over the last decade, fluorescence microscopy methods have enabled the real-time visualization of single molecules interacting with and transiting through the NPC, allowing novel questions to be examined with nanometer precision. While initial single-molecule studies focused primarily on import pathways using permeabilized cells, it has recently proven feasible to investigate the export of mRNAs in living cells. Single-molecule assays can address questions that are difficult or impossible to answer by other means, yet the complexity of nucleocytoplasmic transport requires that interpretation be based on a firm genetic, biochemical, and structural foundation. Moreover, conceptually simple single-molecule experiments remain technically challenging, particularly with regard to signal intensity, signal-to-noise ratio, and the analysis of noise, stochasticity, and precision. We discuss nuclear transport issues recently addressed by single-molecule microscopy, evaluate the limits of existing assays and data, and identify open questions for future studies. We expect that single-molecule fluorescence approaches will continue to be applied to outstanding nucleocytoplasmic transport questions, and that the approaches developed for NPC studies are extendable to additional complex systems and pathways within cells.

High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors.

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.

Subglacial Lake Whillans microbial biogeochemistry: a synthesis of current knowledge.

Liquid water occurs below glaciers and ice sheets globally, enabling the existence of an array of aquatic microbial ecosystems. In Antarctica, large subglacial lakes are present beneath hundreds to thousands of metres of ice, and scientific interest in exploring these environments has escalated over the past decade. After years of planning, the first team of scientists and engineers cleanly accessed and retrieved pristine samples from a West Antarctic subglacial lake ecosystem in January 2013. This paper reviews the findings to date on Subglacial Lake Whillans and presents new supporting data on the carbon and energy metabolism of resident microbes. The analysis of water and sediments from the lake revealed a diverse microbial community composed of bacteria and archaea that are close relatives of species known to use reduced N, S or Fe and CH4 as energy sources. The water chemistry of Subglacial Lake Whillans was dominated by weathering products from silicate minerals with a minor influence from seawater. Contributions to water chemistry from microbial sulfide oxidation and carbonation reactions were supported by genomic data. Collectively, these results provide unequivocal evidence that subglacial environments in this region of West Antarctica host active microbial ecosystems that participate in subglacial biogeochemical cycling.

Impact of placement and restoration timing on single-implant esthetic outcome - a randomized clinical trial.

AIM: The objective of this randomized clinical trial was to investigate the influence of the time of implant placement (immediate vs. early) and the time of restoration (immediate vs. early) on esthetic outcome in maxillary anterior single implants. MATERIAL AND METHODS: Forty-eight patients with a single failing incisor in the maxilla and a natural contralateral site were randomly distributed into four groups. Treatment variations affected the time of implant placement (immediate or early) as well as the time of restoration (immediate or early) - in detail, group 1a with immediate implant placement and immediate temporary restoration, group 1b with immediate implant placement and early restoration, group 2a with early implant placement and immediate temporary restoration, and group 2b with early implant placement and early restoration. All patients received the final prosthetic restoration 10-12 weeks after implant placement. Standardized photographs were taken eight months after tooth extraction. Five competent observers analyzed the esthetic outcome according to the PES after Fürhauser. For statistical analysis, the Kruskal-Wallis test and Dunn's post hoc test were applied. Interobserver reliability was evaluated by Krippendorff's alpha. RESULTS: The overall scores of the four treatment groups revealed PES values of 8.47 (SD 2.08, group 1a), 7.93 (SD 3.21, group 1b), 6.62 (SD 3.24, group 2a), and 8.10 (SD 3.25, group 2b). The differences between groups 2a and 1a and between groups 2a and 2b were statistically significant (P = 0.015 and P = 0.047). The single parameter analysis displayed a certain range of fluctuation and heterogeneity. CONCLUSIONS: Immediate implant placement and restoration appear to be a viable alternative to early implant placement if an experienced surgeon is entrusted with the implantation procedure.

Large cargo transport by nuclear pores: implications for the spatial organization of FG-nucleoporins.

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine-glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non-uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG-NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.

Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores.

Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles.

Effect of cargo size and shape on the transport efficiency of the bacterial Tat translocase.

The Tat machinery translocates fully-folded and oligomeric substrates. The passage of large, bulky cargos across an ion-tight membrane suggests the need to match pore and cargo size, and therefore that Tat transport efficiency may depend on both cargo size and shape. A series of cargos of different sizes and shapes were generated using the natural Tat substrate pre-SufI as a base. Four (of 17) cargos transported with significant (>20% of wild-type) efficiencies. These results indicate that cargo size and shape significantly influence Tat transportability.