The paracellular passage of ions and small molecules across epithelia is controlled by tight junctions, complex meshworks of claudin polymers that form tight seals between neighboring cells. How the nanoscale architecture of tight junction meshworks enables paracellular passage of specific ions or small molecules without compromising barrier function is unknown. Here we combine super-resolution stimulated emission depletion microscopy in live and fixed cells and tissues, multivariate classification of super-resolution images and fluorescence resonance energy transfer to reveal the nanoscale organization of tight junctions formed by mammalian claudins. We show that only a subset of claudins can assemble into characteristic homotypic meshworks, whereas tight junctions formed by multiple claudins display nanoscale organization principles of intermixing, integration, induction, segregation, and exclusion of strand assemblies. Interestingly, channel-forming claudins are spatially segregated from barrier-forming claudins via determinants mainly encoded in their extracellular domains also known to harbor mutations leading to human diseases. Electrophysiological analysis of claudins in epithelial cells suggests that nanoscale segregation of distinct channel-forming claudins enables barrier function combined with specific paracellular ion flux across tight junctions.

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, is internalized by macrophages and processed in phagolysosomes. Phagosomal compaction, a crucial step in phagolysosome maturation, is driven by contact of Rab5a-positive vesicles with the phagosomal coat. We show that the sorting nexin SNX3 is transported with Rab5a vesicles and that its PX domain enables vesicle–phagosome contact by binding to PI(3)P in the phagosomal coat. Moreover, the C-terminal region of SNX3 recruits galectin-9, a lectin implicated in protein and membrane recycling, which we identify as a further regulator of phagosome compaction. SNX3 thus forms a hub for two distinct vesicle populations, constituting a convergence point for the endosomal recycling machinery, to contribute to phagosome maturation and intracellular processing of borreliae. These data also suggest that the helical shape of B. burgdorferi itself, providing sites of high curvature and thus local PI(3)P enrichment at phagosomes, may be one of the driving elements underlying the efficient elimination of spirochetes by immune cells.

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

Rhodamine molecules are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, which improves photophysical parameters...

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.