Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.

The affinity of cytokine-receptor complexes on the cell surface is often poorly predictive of functional potency. To address this conundrum, we explored the inter-relationships of receptor binding to a wide range of downstream functional metrics for a prototypical cytokine, Interleukin-13 (IL-13), through structure-based engineering of agonists covering a spectrum of binding strengths for IL-13Rα1. Surprisingly, engineered IL-13 agonists spanning a broad affinity range exhibited similar potencies of STAT6 phosphorylation, while delays in STAT6 activation and nuclear translocation were only apparent for ligands with dramatically lower affinities. From this data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis plays a key role in buffering STAT6 phosphorylation potencies, while the lifetime of signaling complexes at the plasma membrane determines the long-term functional potency. The surprisingly complex inter-relationships between extracellular ligand binding and function highlight the importance of feedback mechanisms in modulating receptor responsiveness, and suggest new mechanism-based strategies to enhance the therapeutic efficacy of cytokine therapy.

The target of rapamycin (TOR) protein kinase forms multi-subunit TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which exhibit distinct substrate specificities. Sin1 is one of the TORC2-specific subunit essential for phosphorylation and activation of certain AGC-family kinases. Here, we show that Sin1 is dispensable for the catalytic activity of TORC2, but its conserved region in the middle (Sin1CRIM) forms a discrete domain that specifically binds the TORC2 substrate kinases. Sin1CRIM fused to a different TORC2 subunit can recruit the TORC2 substrate Gad8 for phosphorylation even in the sin1 null mutant of fission yeast. The solution structure of Sin1CRIM shows a ubiquitin-like fold with a characteristic acidic loop, which is essential for interaction with the TORC2 substrates. The specific substrate-recognition function is conserved in human Sin1CRIM, which may represent a potential target for novel anticancer drugs that prevent activation of the mTORC2 substrates such as AKT.DOI: http://dx.doi.org/10.7554/eLife.19594.001

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand–receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

Background COVID-19 is a lung disease, and there is medical evidence that air pollution is one of the external causes of lung diseases. Fine particulate matter is one of the air pollutants that damages pulmonary tissue. The combination of the coronavirus and fine particulate matter air pollution may exacerbate the coronavirus’ effect on human health. Research question This paper considers whether the long-term concentration of fine particulate matter of different sizes changes the number of detected coronavirus infections and the number of COVID-19 fatalities in Germany. Study design Data from 400 German counties for fine particulate air pollution from 2002 to 2020 are used to measure the long-term impact of air pollution. Kriging interpolation is applied to complement data gaps. With an ecological study, the correlation between average particulate matter air pollution and COVID-19 cases, as well as fatalities, are estimated with OLS regressions. Thereby, socioeconomic and demographic covariates are included. Main findings An increase in the average long-term air pollution of 1 μg/m 3 particulate matter PM 2.5 is correlated with 199.46 (SD = 29.66) more COVID-19 cases per 100,000 inhabitants in Germany. For PM 10 the respective increase is 52.38 (SD = 12.99) more cases per 100,000 inhabitants. The number of COVID-19 deaths were also positively correlated with PM 2.5 and PM 10 (6.18, SD = 1.44, respectively 2.11, SD = 0.71, additional COVID-19 deaths per 100,000 inhabitants). Conclusion Long-term fine particulate air pollution is suspected as causing higher numbers of COVID-19 cases. Higher long-term air pollution may even increase COVID-19 death rates. We find that the results of the correlation analysis without controls are retained in a regression analysis with controls for relevant confounding factors. Nevertheless, additional epidemiological investigations are required to test the causality of particulate matter air pollution for COVID-19 cases and the severity.