Reconstituted in vitro systems to reveal the roles and functions of septins.

Septins are essential cytoskeletal proteins involved in key cellular processes and have also been implicated in diseases from cancers to neurodegenerative pathologies. However, they have not been as thoroughly studied as other cytoskeletal proteins. In vivo, septins interact with other cytoskeletal proteins and with the inner plasma membrane. Hence, bottom-up in vitro cell-free assays are well suited to dissect the roles and behavior of septins in a controlled environment. Specifically, in vitro studies have been invaluable in describing the self-assembly of septins into a large diversity of ultrastructures. Given that septins interact specifically with membrane, the details of these septin-membrane interactions have been analyzed using reconstituted lipid systems. In particular, at a membrane, septins are often localized at curvatures of micrometer scale. In that context, in vitro assays have been performed with substrates of varying curvatures (spheres, cylinders or undulated substrates) to probe the sensitivity of septins to membrane curvature. This Review will first present the structural properties of septins in solution and describe the interplay of septins with cytoskeletal partners. We will then discuss how septins interact with biomimetic membranes and induce their reshaping. Finally, we will highlight the curvature sensitivity of septins and how they alter the mechanical properties of membranes.

Isothermal self-assembly of multicomponent and evolutive DNA nanostructures.

Thermal annealing is usually needed to direct the assembly of multiple complementary DNA strands into desired entities. We show that, with a magnesium-free buffer containing NaCl, complex cocktails of DNA strands and proteins can self-assemble isothermally, at room or physiological temperature, into user-defined nanostructures, such as DNA origamis, single-stranded tile assemblies and nanogrids. In situ, time-resolved observation reveals that this self-assembly is thermodynamically controlled, proceeds through multiple folding pathways and leads to highly reconfigurable nanostructures. It allows a given system to self-select its most stable shape in a large pool of competitive DNA strands. Strikingly, upon the appearance of a new energy minimum, DNA origamis isothermally shift from one initially stable shape to a radically different one, by massive exchange of their constitutive staple strands. This method expands the repertoire of shapes and functions attainable by isothermal self-assembly and creates a basis for adaptive nanomachines and nanostructure discovery by evolution.

A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization.

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Reversible Supra-Folding of User-Programmed Functional DNA Nanostructures on Fuzzy Cationic Substrates.

We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined µm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Risk assessment of fluoride and arsenic in groundwater and a scenario analysis for reducing exposure in Inner Mongolia.

In contrast to Mongolia, family-owned land in Inner Mongolia is separated by fences, preventing the free movement of nomads and leading people to rely heavily on the same source of groundwater for their domestic water needs. Therefore, it is important to clarify groundwater quality and understand the associated human health concerns. To evaluate the risks of drinking groundwater to human health in Inner Mongolia, we examined groundwater quality by field surveys, a human health risk analysis, and a scenario analysis. During the summer of 2015 in Inner Mongolia, we measured the concentrations of major ions, metals, metalloids, and rare earth metals in groundwater samples (n = 32) and river water samples (n = 10), for which there were no known anthropogenic contamination sources. In addition, as part of a scenario analysis, samples of tap water (n = 1), snowmelt (n = 1), and bottled water (n = 1) were also evaluated. We used our analytical results to calculate hazard quotient (HQ) ratios by means of a probabilistic risk assessment method. The results indicated that residents who drank groundwater every day might have risk concerns for F- (mean ± standard deviation, 2.51 ± 1.80 mg L-1; range, 0.07-7.70 mg L-1) and As (6.49 ± 9.64 µg L-1; 0.31-47.0 µg L-1). We observed no relationships between well depth or any geophysical variation and groundwater quality. On the basis of the scenario analysis results, we concluded that using snow as a source of drinking water in winter could reduce health risks associated with using groundwater for this population in Inner Mongolia.

Non-carcinogenic risk assessment of groundwater in South Gobi, Mongolia.

Thirty-nine groundwater samples were collected from wells near the Tavan Tolgoi and Oyu Tolgoi mines in Mongolia and at a relatively pristine site in northern Mongolia during August to September 2013, and analyzed for the concentrations of F-, NO3-, Hg, As, Al, V, Mn, Co, Ni, Cu, Zn, Se, Mo, Cd, Sb, and Pb. A probabilistic risk assessment found that >95% of the population in the areas was at risk from drinking well water. The hazard index (HI) was >1, indicating a non-carcinogenic risk to human health. At Oyu Tolgoi, the hazard quotient (HQ) of the As concentration (mean 6.63 µg/L) was >1. At the northern site, the 95th percentile HQ was <1 but the 95th percentile HI was >1. The ratios of nitrogen and oxygen stable isotopes indicated that NO3- contamination of groundwater at Oyu Tolgoi and Tavan Tolgoi was caused by livestock waste. Mercury accumulation in livestock was examined from concentrations in livestock forage and in hair and wool samples from livestock in the South Gobi region. Sheep wool had the same level of mercury as in Japan, but camel, horse, and goat hair had high levels.

Photoinduced Bilayer-to-Nonbilayer Phase Transition of POPE by Photoisomerization of Added Stilbene Molecules.

The photocontrol of a bilayer-to-nonbilayer phase transition (the liquid-crystalline Lα phase to the inverted hexagonal HII phase) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by the photoisomerization of incorporated stilbene molecules was examined by utilizing differential scanning calorimetry, small-angle X-ray diffraction, ultraviolet (UV)/visible absorption, and attenuated total reflectance Fourier transform infrared spectroscopies. cis-Stilbene lowered the transition temperature, Th, to a greater extent than did trans-stilbene, and the difference was at most ca. 10 °C. At temperatures higher than the Th of POPE/cis-stilbene but lower than that of POPE/trans-stilbene, the photoisomerization from the trans to the cis form of the stilbene molecules by irradiation with UV light caused a Lα-HII phase transition. The UV irradiation partially induced the HII phase at a constant temperature because of the incomplete photoisomerization of stilbene (ca. 60%). The reduction in Th by the incorporation of stilbenes was caused mainly by the reduction in the spontaneous radius of curvature of the lipid monolayer, R0. The greater bulkiness of cis-stilbene as compared to the trans form resulted in a more effective reduction in R0 and stabilization of the HII phase.

Human health risk assessment of mercury vapor around artisanal small-scale gold mining area, Palu city, Central Sulawesi, Indonesia.

Emissions of elemental mercury, Hg(0), from artisanal small-scale gold mining activities accounted for 37% of total global Hg(0) emissions in 2010. People who live near gold-mining areas may be exposed to high concentrations of Hg(0). Here, we assessed the human health risk due to Hg(0) exposure among residents of Palu city (Central Sulawesi Province, Indonesia). The area around the city has more than 60t of gold reserves, and the nearby Poboya area is the most active gold-mining site in Indonesia. Owing to its geography, the city experiences alternating land and sea breezes. Sampling was done over a period of 3 years (from 2010 Aug. to 2012 Dec.) intermittently with a passive sampler for Hg(0), a portable handheld mercury analyzer, and a mercury analyzer in four areas of the city and in the Poboya gold-processing area, as well as wind speeds and directions in one area of the city. The 24-h average concentration, wind speed, and wind direction data show that the ambient air in both the gold-processing area and the city was always covered by high concentration of mercury vapor. The Hg(0) concentration in the city was higher at night than in the daytime, owing to the effect of land breezes. These results indicate that the inhabitants of the city were always exposed to high concentrations of Hg(0). The average daytime point-sample Hg(0) concentrations in the city, as measured with a handheld mercury analyzer over 3 days in July 2011, ranged from 2096 to 3299ngm(-3). In comparison, the average daytime Hg(0) concentration in the Poboya gold-processing area was 12,782ngm(-3). All of these concentrations are substantially higher than the World Health Organization air-quality guideline for annual average Hg exposure (1000ngm(-3)). We used the point-sample concentrations to calculate hazard quotient ratios by means of a probabilistic risk assessment method. The results indicated that 93% of the sample population overall was at risk (hazard quotient ratio ≥1 and cut off at the 95th percentile value of the sample population) of mercury toxicity, that is, damage to the central nervous system due to chronic exposure. The corresponding percentages for the northern, central, southern, and western areas of the city were 83%, 84%, 95%, and 95%, respectively. Our results indicate that the residents of Palu city are at serious risk from exposure to high concentrations of atmospheric Hg(0).

Effect of n-alkanes on lipid bilayers depending on headgroups.

Phase behavior and structural properties were examined for phospholipid bilayers having different headgroups (DMPC, DMPS and DMPE) with added n-alkanes to study effect of flexible additives. Change in the temperatures of main transition of the lipid/alkane mixtures against the length of added alkanes depends largely on the headgroup. Theoretical analysis of the change of the temperature of transition indicates that the headgroup dependence is dominantly originated in the strong dependence of total enthalpy on the headgroups. The results of X-ray diffraction show that the enthalpic stabilization due to enhanced packing of acyl chains of the lipid by alkanes in the gel phase causes the headgroup-dependent change in the phase transition behavior. The enhanced packing in the gel phase also leads to easy emergence of the subgel phase with very short relaxation time at room temperature in the DMPE-based bilayers.