SummaryThe neuromuscular junction (NMJ) plays a fundamental role in transferring information from lower motor neuron to skeletal muscle to generate movement. It is also an experimentally accessible model synapse routinely studied in animal models to explore fundamental aspects of synaptic form and function. Here, we combined morphological techniques, super-resolution imaging, and proteomic profiling to reveal the detailed cellular and molecular architecture of the human NMJ. Human NMJs were significantly smaller, less complex, and more fragmented than mouse NMJs. In contrast to mice, human NMJs were also remarkably stable across the entire adult lifespan, showing no signs of age-related degeneration or remodeling. Super-resolution imaging and proteomic profiling revealed distinctive distribution of active zone proteins and differential expression of core synaptic proteins and molecular pathways at the human NMJ. Taken together, these findings reveal human-specific cellular and molecular features of the NMJ that distinguish them from comparable synapses in other mammalian species.

SummarySignaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains.

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Planar polarity refers to cellular polarity in an orthogonal plane to apicobasal polarity, and is seen across scales from molecular distributions of proteins to tissue patterning. In many contexts it is regulated by the evolutionarily conserved ‘core' planar polarity pathway that is essential for normal organismal development. Core planar polarity pathway components form asymmetric intercellular complexes that communicate polarity between neighbouring cells and direct polarised cell behaviours and the formation of polarised structures. The core planar polarity pathway consists of six structurally different proteins. In the fruitfly Drosophila melanogaster, where the pathway is best characterised, an intercellular homodimer of the seven-pass transmembrane protein Flamingo interacts on one side of the cell junction with the seven-pass transmembrane protein Frizzled, and on the other side with the four-pass transmembrane protein Strabismus. The cytoplasmic proteins Diego and Dishevelled are co-localised with Frizzled, and Prickle co-localises with Strabismus. Between these six components there are myriad possible molecular interactions, which could stabilise or destabilise the intercellular complexes and lead to their sorting into polarised distributions within cells. Post-translational modifications are key regulators of molecular interactions between proteins. Several post-translational modifications of core proteins have been reported to be of functional significance, in particular phosphorylation and ubiquitination. In this review, we discuss the molecular control of planar polarity and the molecular ecology of the core planar polarity intercellular complexes. Furthermore, we highlight the importance of understanding the spatial control of post-translational modifications in the establishment of planar polarity.

Drug development may include extensive screening for crystalline forms of active pharmaceutical ingredients. Crystal engineering aims to apply supramolecular knowledge to simplify such a task. The failure of such a strategy may result in overlooking potentially interesting compounds. Here, the advantages of such a knowledge-based approach is compared to a systematic crystallization screening for pharmaceutical cocrystals. This work indicates that a screening simply based on known synthons and their relative frequency as reported in the database is as effective as a random screening exercise, potentially missing 25% of the successful cocrystallization. Readily available computational methods perform better, enabling the identification of all of the observed cocrystals with a reduction of 24% of the experimental attempts.