Molecular organization of intercellular junctions in the inner ear

我们使用Corti器官，哺乳动物上皮模式最显著的例子之一，来检查与顶端连接复合体（AJC）相关的非肌肉肌球蛋白II （NMII）的组织模式。极化上皮细胞的AJC富含肌动蛋白，并含有NMII，其收缩特性介导上皮结构的发育、功能和稳态变化。然而，沿着连接线产生力所需的收缩肌动球蛋白装置的精确结构组织尚未阐明。利用先进的显微镜和遗传学，我们发现NMII纤维的周期性组装与周结肌动蛋白相互交织，形成一个肌肉状的收缩带，动态地影响顶端细胞的周长和上皮的几何形状。通过免疫荧光、外源表达标记蛋白和NMIIC- gfp小鼠，我们发现NMIIB和NMIIC沿感觉和非感觉内耳上皮细胞的AJC有规律地定位。它们形成平行的双极细丝，与-肌动蛋白交联的f -肌动蛋白交替，形成一条类似肌肉肌节的重复单元带。用50 &#956；M blebbistatin抑制NMII可使肌节长度可逆地增加，同时也增加了连接长度，这为这种连接肌节装置的收缩性提供了证据。相邻细胞的肌节常处于登记状态，表明它们的组装或定位是由细胞间连接成分介导的。NMII异构体在连接线上的分布在同质连接中是对称的，在异质连接中是不对称的。同种异构体的缺失揭示了至少部分的补偿。最后，我们发现NMII的周期性定位发生在其他上皮组织中，这表明肌合成肌动球蛋白带是AJC的普遍组成部分。

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.