For macromolecular structure determination at synchrotron sources, radiation damage remains a major limiting factor. Estimation of the absorbed dose (J kg−1) during data collection at these sources by programs such as RADDOSE‐3D has allowed direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has enabled prediction of roughly when radiation damage will manifest so it can potentially be avoided. X‐ray free‐electron lasers (XFELs), which produce intense X‐ray pulses only a few femtoseconds in duration, can be used to generate diffraction patterns before most of the radiation damage processes have occurred and hence hypothetically they enable the determination of damage‐free atomic resolution structures. In spite of this, several experimental and theoretical studies have suggested that structures from XFELs are not always free of radiation damage. There are currently no freely available programs designed to calculate the dose absorbed during XFEL data collection. This article presents an extension to RADDOSE‐3D called RADDOSE‐XFEL, which calculates the time‐resolved dose during XFEL experiments. It is anticipated that RADDOSE‐XFEL could be used to facilitate the study of radiation damage at XFELs and ultimately be used prior to data collection so that experimenters can plan their experiments to avoid radiation damage manifesting in their structures.

Acid sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid-sensitivity on all 30 subunits of the C. elegans DEG/ENaC family using Two-Electrode Voltage Clamp (TEVC) in Xenopus oocytes. We found two groups of acidsensing DEG/ENaCs characterised by being inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified four new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acidsensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to .

No abstract

Acid sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid-sensitivity on all 30 subunits of the C. elegans DEG/ENaC family using Two-Electrode Voltage Clamp (TEVC) in Xenopus oocytes. We found two groups of acid-sensing DEG/ENaCs characterised by being inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified four new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass-spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding. Using these tools in vivo, we show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell-guidance, with the potential for wide- ranging biomedical implications in development and cancer biology.