Molecular organization of intercellular junctions in the inner ear

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We use the organ of Corti, one of the most striking examples of mammalian epithelial patterning, to examine the mode of organization of nonmuscle myosin II (NMII) associated with the apical junctional complex (AJC). The AJC of polarized epithelial cells is rich in actin and contains NMII, whose contractile properties mediate developmental, functional, and homeostatic changes of epithelial architecture. However, the precise structural organization of the contractile actomyosin apparatus required to generate force along the junctional-line has not been elucidated. Using cutting-edge microscopy and genetics we show that periodic assemblies of NMII filaments interlace with perijunctional actin to form a sarcomeric, muscle-like contractile belt that dynamically influences apical cell perimeter and epithelial geometry. Using immunofluorescence, exogenous expression of tagged-proteins, and an NMIIC-GFP mouse, we found that NMIIB and NMIIC localize at regular intervals along the AJC of both sensory and non-sensory inner-ear epithelial cells. They form parallel bipolar filaments, which alternate with α-actinin cross-linked F-actin, to form a belt of repeating units that resemble muscle sarcomeres. Inhibition of NMII with 50 μM blebbistatin produces a reversible increase in sarcomere-length matched by an increase in junctional-length, providing evidence for contractility of this junctional sarcomeric apparatus. The sarcomeres of adjacent cells are often in register, suggesting that their assembly or positioning is mediated by intercellular junctional components. NMII isoform distribution across the junctional-line is symmetric in homomeric junctions and asymmetric in heteromeric junctions. Isoform deletion reveals at least partial compensation. Finally, we show that periodic localization of NMII occurs in other epithelial tissues suggesting that the sarcomeric actomyosin belt is a universal component of the AJC. Uncovering the presence of an NMII sarcomeric-belt at the interface of the tight and adherens junction, and the in-register alignment of the sarcomeres of adjacent cells across the junctional-line, provides a new level of detail and a novel specific target to investigate the role of NMII in AJC homeostasis and epithelial dynamics. Knowledge pertaining to the differential expression of NMII paralogs can be used to explore differences in biomechanical properties in the AJC of various tissues. Considering that defects in NMII paralogs are linked to the onset and progression of a number of human diseases, including hearing loss, and that cancer and metastasis in epithelial tissues depend on NMII mediated contractility and cell adhesion dynamics, the relevance of our findings across various biomedical disciplines is axiomatic. To better visualize fluorescently labeled samples we developed a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.