Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

B0AT1 Amino Acid Transporter Complexed With SARS-CoV-2 Receptor ACE2 Forms a Heterodimer Functional Unit: In Situ Conformation Using Radiation Inactivation Analysis.

The SARS-CoV-2 receptor, angiotensin-converting enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared with all other human tissues. Enterocyte ACE2 is coexpressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2:B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2:B0AT1] heterodimer pair constitutes a physiological "functional unit." This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on radiation target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size molecular weight 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2:B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2:B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance understanding the physiology of B0AT1 interaction with ACE2 in the gut, and thereby contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul postacute sequelae of SARS-CoV-2.

Desiccation resistance in embryos of the killifish, Fundulus heteroclitus: single embryo measurements.

Northern killifish, Fundulus heteroclitus macrolepidotus, spawn in estuaries at high tides. Embryos may be stranded in air at stream margins as the water recedes. These aerially incubated embryos are exposed to desiccation stress and may survive and develop normally to hatching at ∼14 days post-fertilization (dpf). We developed a technique to quantitatively measure the kinetics of water loss at various developmental stages from single embryos in controlled relative humidities (RHs). Embryos were able to tolerate short periods (2 hr) of severe desiccation and survive to hatching. Mid-stage (7 dpf) embryos showed the highest degree of desiccation tolerance compared to early-stage (2 dpf) and late-stage (14 dpf) embryos. We classified the patterns of water loss into four phases, the perivitelline space (PVS) phase, the resistance phase, the desiccation phase, and the equilibration phase. In the PVS phase, water loss was rapid at all developmental stages and all RHs (∼25% of total embryo weight). The water loss rate was slower during the resistance phase. It decreased as RH increased and length of this phase was longer in mid-stage than in early- and late-stage embryos. The water loss rate and length of the desiccation phase also depended on RH. These data support the hypothesis that low permeability embryonic compartment surface membranes retard water loss significantly and promote prolonged survival of these embryos during desiccation. We also show this mechanism cannot completely account for the survival of severely desiccated embryos (especially in 23% RH) and that there must also be complementary cellular responses.

Energetics of osmoregulation: I. Oxygen consumption by Fundulus heteroclitus.

We have developed a flow-through method for measuring oxygen consumption in fish which allows continuous monitoring over periods of days with good accuracy. Our goal was to determine the changes in basal metabolic rate in estuarine fish as a function of salinity. We show that in Fundulus heteroclitus, the oxygen consumption drops by 50% during the first 12 hr in the respirometer, as the fish cease exploratory movements. We have determined the influence of temperature and body size on resting respiratory rate, but failed to find any circadian or tidal rhythm in aerobic respiration. With these variables controlled, we determined that changing from 10 to 30 ppt water had no demonstrable effect on oxygen uptake. Since there must be a large change in osmotic flux due to this change in salinity, it appears that the fish might be diverting energy from other uses rather than increasing aerobic energy production to meet the increased osmoregulatory work load.

Energetics of osmoregulation: II. Water flux and osmoregulatory work in the euryhaline fish, Fundulus heteroclitus.

Teleost fish experience passive osmotic water influx in fresh water (FW) and water outflux in salt water, which is normally compensated by water flow driven by active ion transport mechanisms. Euryhaline fish may also minimize osmotic energy demand by "behavioral osmoregulation", seeking a medium isotonic with their body fluids. Our goal was to evaluate the energy requirement for osmoregulation by the euryhaline fish Fundulus heteroclitus, to determine whether it is of sufficient magnitude to favor behavioral osmoregulation. We have developed a method of weighing small fish repetitively for long periods without apparent damage, which was used to assess changes in water content following changes in external salinity. We found that cold (4 degrees C) inhibits osmoregulatory active transport mechanisms in fish acclimated to warmer temperatures, leading to a net passive water flux which is reversed by rewarming the fish. A sudden change of salinity at room temperature triggers a transient change in water content and the initial slope can be used to measure the minimum passive flux at that temperature. With some reasonable assumptions as to the stoichiometry of the ion transport and ATP-generating processes, we can calculate the amount of respiration required for ion transport and compare it to the oxygen uptake measured previously under the same conditions. We conclude that osmoregulation in sea water requires from 6% to 10% of the total energy budget in sea water, with smaller percentages in FW, and that this fraction is probably sufficient to be a significant selective driving force favoring behavioral osmoregulation under some circumstances.