Molecular organization of intercellular junctions in the inner ear

我们使用的Corti器官，哺乳动物上皮图案的最引人注目的例子之一，检查与顶端连接复合体（AJC）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，产生沿着连接线的力所需的收缩性肌动球蛋白装置的精确结构组织尚未阐明。使用尖端的显微镜和遗传学，我们表明，周期性组装的NMII丝与连接周围肌动蛋白交织，形成一个肌节，肌肉样收缩带，动态影响顶端细胞周长和上皮几何形状。使用免疫荧光，外源性表达的标记蛋白，和NMIIC-GFP小鼠，我们发现，NMIIB和NMIIC定位在规则的间隔沿着AJC的感觉和非感觉内耳上皮细胞。它们形成平行的双极丝，与辅肌动蛋白交联的F-肌动蛋白交替，形成类似肌肉肌节的重复单位带。用50 M blebbistatin抑制NMII产生了与连接长度增加相匹配的肌节长度的可逆增加，为这种连接肌节装置的收缩性提供了证据。相邻细胞的肌节通常是对齐的，这表明它们的组装或定位是由细胞间连接成分介导的。跨连接线的匪II同种型分布在同聚连接中是对称的，在异聚连接中是不对称的。同种型缺失揭示至少部分补偿。最后，我们发现，周期性本地化的NMII发生在其他上皮组织，这表明肌节肌动球蛋白带是一个普遍的组成部分的AJC。 揭示了在紧密连接和粘附连接的界面处存在NMII肌节带，以及跨连接线的相邻细胞的肌节的对齐，提供了一个新的细节水平和一个新的特异性靶点来研究NMII在AJC稳态和上皮动力学中的作用。有关NMII旁系同源物的差异表达的知识可用于探索各种组织的AJC中生物力学特性的差异。考虑到NMII旁系同源物的缺陷与许多人类疾病的发作和进展有关，包括听力损失，并且上皮组织中的癌症和转移取决于NMII介导的收缩性和细胞粘附动力学，我们的发现在各种生物医学学科中的相关性是不言自明的。 为了更好地可视化荧光标记的样品，我们开发了一种简单实用的方法来产生基于点定位的超分辨率图像，该方法不需要光活化或光开关探针。称为漂白/闪烁辅助定位显微镜（BaLM），该技术依赖于所有常用荧光探针的固有漂白和闪烁行为特征。我们还表明，BaLM工程与荧光分子在同一样品中的光谱。我们还表明，BaLM工程与荧光分子在同一样品中的光谱。因此，BaLM将基于单分子的超分辨定位扩展到用多种常规荧光探针标记的样品。

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.