Light microscopy of mitochondria at the nanoscale.

Mitochondria are essential for eukaryotic life. These double-membrane organelles often form highly dynamic tubular networks interacting with many cellular structures. Their highly convoluted contiguous inner membrane compartmentalizes the organelle, which is crucial for mitochondrial function. Since the diameter of the mitochondrial tubules is generally close to the diffraction limit of light microcopy, it is often challenging, if not impossible, to visualize submitochondrial structures or protein distributions using conventional light microscopy. This renders super-resolution microscopy particularly valuable, and attractive, for studying mitochondria. Super-resolution microscopy encompasses a diverse set of approaches that extend resolution, as well as nanoscopy techniques that can even overcome the diffraction limit. In this review, we provide an overview of recent studies using super-resolution microscopy to investigate mitochondria, discuss the strengths and opportunities of the various methods in addressing specific questions in mitochondrial biology, and highlight potential future developments. Expected final online publication date for the Annual Review of Biophysics, Volume 49 is May 6, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates

Functional tat transport of unstructured, small, hydrophilic proteins

The twin-arginine translocation ( Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level

Effect of management on rangeland phytomass, cover and condition in two biomes in South Africa

In rangelands, grazing management is a main driver of rangeland condition. Due to masking effects of seasonal climate fluctuations, little is known about (dis)similarity of management effects on rangeland condition and forage provision across major dryland biomes. Taking a macro-ecological perspective, we analysed if management effects differed between South Africa's central grassland and Kalahari savanna biomes. We recorded proxies of forage provision (phytomass, vegetation cover and their ratio) over five seasons, annual rainfall to account for seasonal climate fluctuations, and rangeland condition (through relative abundances of increaser and decreaser species). Regarding forage provision, we found effects of management for the savanna, where, irrespective of rainfall, rotational grazing management resulted in higher phytomass and phytomass-cover ratios than management with continuous grazing. In the grassland, however, this difference was only discernible for phytomass-cover ratio in two years with above-average antecedent rainfall. This suggests that management effects are biome-dependent and that modulating effects of annual rainfall are stronger in the grassland. In either biome, management effects on the dominance of increaser and decreaser species were negligible, i.e. rangeland condition did not differ across management types in either biome. We conclude that investigations on management effects should account for interactions with biome and rainfall

Discrimination and characterization of management systems in semi-arid rangelands of South Africa using RapidEye time series

In South African grasslands, rangeland management is strongly related to land tenure. Communal farms are reported to exhibit less desirable vegetation conditions for livestock than commercial farms. Time series of high spatial and temporal resolution imagery may be useful for improved evaluation of these rangelands as they provide information at a spatial scale similar to the typical scale of field assessments and may thus overcome the limited spatio-temporal representativeness of field measurements. A time series of 13 RapidEye images over one growing season (2010-2011) was used to explore spectral differences between and within two management systems (commercial vs. communal). Isomap ordination was applied to map continuous spectral dissimilarities of sample plots. Using regression with simultaneous autoregressive models (SAR), dissimilarities were subsequently related to ecological variables of plant and soil, including indicators for grazing effects. The largest differences were found between sample plots of communal and commercial farms. Vegetation attributes were significantly related to dissimilarities in reflectance, both from the growing season and the dormant period. However, these relationships did not suggest vegetation degradation on communal farms. They further suggest that a management-related pattern of grazing disturbance in the summer months led to spectral differences between farms but could have impaired the detailed characterization of spectral dissimilarities related to differences in vegetation composition

Are There Consistent Grazing Indicators in Drylands? Testing Plant Functional Types of Various Complexity in South Africa's Grassland and Savanna Biomes

Despite our growing knowledge on plants' functional responses to grazing, there is no consensus if an optimum level of functional aggregation exists for detecting grazing effects in drylands. With a comparative approach we searched for plant functional types (PFTs) with a consistent response to grazing across two areas differing in climatic aridity, situated in South Africa's grassland and savanna biomes. We aggregated herbaceous species into PFTs, using hierarchical combinations of traits (from single-to three-trait PFTs). Traits relate to life history, growth form and leaf width. We first confirmed that soil and grazing gradients were largely independent from each other, and then searched in each biome for PFTs with a sensitive response to grazing, avoiding confounding with soil conditions. We found no response consistency, but biome-specific optimum aggregation levels. Three-trait PFTs (e.g. broad-leaved perennial grasses) and two-trait PFTs (e. g. perennial grasses) performed best as indicators of grazing effects in the semi-arid grassland and in the arid savanna biome, respectively. Some PFTs increased with grazing pressure in the grassland, but decreased in the savanna. We applied biome-specific grazing indicators to evaluate if differences in grazing management related to land tenure (communal versus freehold) had effects on vegetation. Tenure effects were small, which we mainly attributed to large variability in grazing pressure across farms. We conclude that the striking lack of generalizable PFT responses to grazing is due to a convergence of aridity and grazing effects, and unlikely to be overcome by more refined classification approaches. Hence, PFTs with an opposite response to grazing in the two biomes rather have a unimodal response along a gradient of additive forces of aridity and grazing. The study advocates for hierarchical trait combinations to identify localized indicator sets for grazing effects. Its methodological approach may also be useful for identifying ecological indicators in other ecosystems

PspF binding domain PspA 1 144 and the PspA center dot F complex New insights into the coiled coil dependent regulation of AAA plus proteins

Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ54 enhancer binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ54-dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiological tasks via coiled-coil domains

Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of ≈25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains ≈15 mobile TatA complexes and a pool of ≈100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport

Response of community-aggregated plant functional traits along grazing gradients: insights from African semi-arid grasslands

Questions: Plant communities fulfil key functions in the ecosystem, which can be characterized by their plant functional traits. In functional ecology, plant communities are considered to hold a set of trait attributes reflecting a specific plant strategy adapted to persist in the environment to which they are exposed. In semi-arid grasslands of the Republic of South Africa, we addressed the following questions: how are community-aggregated plant functional traits (CPFT) shaped by grazing gradients; which plant strategies are associated with the response of CPFTs; and are environmental factors, such as soil properties and grazing management, interrelated with the functional response of vegetation to grazing gradients? Location: Semi-arid grasslands close to Thaba Nchu, Free State (Republic of South Africa). Methods: Piosphere transects from a water point into the field were established to portray grazing gradients on two communal grazing areas with continuous grazing and two commercial farms with rotational grazing. Along each transect, six plots (5 x 5 m) were evenly distributed. The trait-transect sampling was applied to record 12 CPFT related to light capture and forage quality. A redundancy analysis was performed to derive relationship between CPFTs, grazing gradients and environmental conditions. Results: Grazing intensity decreased along piosphere transects, from the water point into the field. Most CPFTs responded to this decreasing gradient of grazing intensity and so allowed derivation of trait syndromes that clearly reflect plant strategies of ruderal and competitive vegetation. Close to water points, plants had higher nitrogen concentrations, fewer cell wall components and higher specific leaf area, hence light capture might be faster and more efficient per leaf area and leaf mass. Plant communities exposed to intensive grazing were well adapted to defoliation, trampling and nutrient accumulation through fast growth rates and a quick return strategy. Conclusions: In the sacrifice zone around water points, there is an ecological niche for vegetation communities exhibiting a strategy of fast growth, which is well adapted to intense and frequent grazing and is also associated with forage of high nutritional quality

A SmartBed for Non-obtrusive Physiological Monitoring During Sleep: The LAID Project

The individual experience of inadequate or insufficient sleep is one of the most common health issues in the industrialized world. The 65% of Italian population reports disturbed sleep experiences, while chronic sleep disorders affect about 10% of the population. The people with inadequate and unsatisfactory sleep often suffers drowsiness during the day associated with both somatic and mental disorders. For these reasons, the systematic and continuative monitoring of sleep is one of the main objectives in preventive, personalized and participatory sleep medicine. The purpose of this paper is to describe the architecture of a “smart mattress’’ (SmartBed) that is the main outcome of the Italian R&D project called LAID. SmartBed will be able to non-obtrusively collect physiological and environmental parameters and signals, to processing them and to provide information about the quality of sleep, the levels of stress, and more generally the well-being of an individual. Specifically, SmartBed will be able to estimate data relating to cardiorespiratory activity, movements, body position, snoring and environmental parameters. SmartBed aims to obtain a continuative and ecological assessment of sleep and well-being of a person, in order to improve his quality of life. SmartBed will be a fundamental tool for carrying out both longitudinal and epidemiological studies on the quality of sleep and life on general population