Molecular organization of intercellular junctions in the inner ear

为了发挥其众多组织和器官特定的作用，上皮片依赖于特殊的结构和特殊的机械性能，从内耳图案精美、力平衡的感觉上皮，到排列在肠道绒毛和隐窝内的高度动态和曲折的上皮。上皮片的形态和力学特性都是其组成细胞的紧急特性；上皮细胞顶端边界的细微收缩可以导致更大规模的堆积几何变化，甚至导致上皮片的戏剧性弯曲。然而，上皮细胞顶端收缩的分子和结构基础尚不完全清楚。非肌肉肌球蛋白II(NMII)被认为是驱动环绕上皮细胞内表面的周向肌动蛋白带的钱包状收缩的原因之一。目前的模型描绘了沿周向肌动蛋白带随机分布的NMII细丝，但尚不清楚单个NMII细丝的活动是如何整合和协调以产生和传递跨越上皮的力的。了解这些过程对于了解发育过程中的上皮动态，以及发育后维持上皮结构和张力内稳态至关重要。 我们一直在研究NMII异构体在上皮尖连接中的分布、组织和作用，并以哺乳动物上皮模式最显著的例子之一Corti的器官为模型系统。为了研究NMII抑制对顶端连接的影响，我们用NMII特异性抑制剂Blebbistatin处理Corti器官的外植体培养。这在细胞和组织水平上都引起了上皮尖表面的变化。形态计量学定量显示连接长度增加，顶端细胞表面积相应增加，顶端细胞形状在整个组织中变形，导致上皮片整体扩张。令人惊讶的是，这些变化在清除了起泡蛋白后完全逆转。我们的观察表明，在周向连接肌球蛋白带内，NMII在张力下动态地维持上皮细胞的顶端周长。 随着数据支持NMII在影响上皮根尖连接中的作用，我们接下来确定了每个NMII平行对数NMIIA、IIB和IIC沿细胞周长的分布模式。NMIIB和NMIIC的免疫荧光显示出明显的分布，呈规则间隔的点状，与交界周围的肌动蛋白有关。相对荧光强度测量显示，NMII荧光点的肌动蛋白密度较低，而它们之间的肌动蛋白密度较高，类似于肌原纤维中的肌节条纹。这一发现促使我们测试肌肉肌节的另一个标志性成分-肌动蛋白交联剂-肌动蛋白的存在。免疫荧光证实，-肌动蛋白1实际上沿连接线呈周期性分布，与NMII交替存在，与肌动蛋白重合，就像在肌节中一样。 肌小节组织的另一个基本特征是双极肌球蛋白细丝平行于肌动蛋白细丝的取向。为了确定NMII细丝沿上皮细胞顶端周长的极性和方向，我们在Corti培养的器官中外源表达了NMIIC，在N端和C端标记了不同的荧光团。我们还用抗NMIIC-GFP N端的抗体标记了NMIIC-GFP转基因小鼠的组织(C端的GFP-Tag)。这些实验证实，双极NMII细丝在小的规则肌节单位内以阵列的形式组织，端到端地组装在一起，形成沿细胞周长的连续带。像肌肉肌节一样，在交界周围肌动蛋白带的NMII肌节也是收缩的，在使用Blebbistatin治疗时，长度增加或松弛，当Blebbistatin被去除时，缩短或收缩。值得注意的是，肌节长度的变化同时影响顶端连接长度和表面积，并最终影响整个上皮的几何形状。 值得注意的是，一个细胞周围的NMII肌节与相邻细胞中的肌节配对。无论肌节的收缩状态如何，这一注册表都保持不变，这表明它们是跨越连接线机械连接的。我们认为，这种耦合依赖于一个尚未确定的跨膜连接组件，该组件将肌节整合到一个跨细胞网络中，力沿着该跨细胞网络传播到根尖上皮表面。有趣的是，与目前将NMII与粘连连接联系在一起的观点相反，我们发现NMII肌节带与紧密连接和粘连连接重叠。这一观察结果，除了交界线上肌节的排列，还推断了NMII与其他交界处成分的相互作用。最后，我们将我们的研究扩展到胃肠道的粘膜上皮细胞，并证实了沿顶端连接的肌节带的存在，以及跨越交界线的肌节的登记，表明这种跨细胞的肌节网络可能是一个普遍的上皮特征。

To fulfill their numerous tissue- and organ-specific roles, epithelial sheets rely on specialized architecture and specific mechanical properties, ranging from the exquisitely patterned, force-balanced sensory epithelia of the inner ear, to the highly dynamic and convoluted epithelia lining the villi and crypts of the intestinal tract. Both the morphology and mechanical characteristics of epithelial sheets are emergent properties of their constituent cells; subtle shrinking of the apical boundaries of epithelial cells can lead to larger scale changes in packing geometry, even causing dramatic bending of epithelial sheets. However, the molecular and structural underpinnings for the apical constriction of epithelial cells are not fully understood. Non-muscle myosin II (NMII) has been implicated in driving a purse string-like contraction of the circumferential actin belt that encircles the inner surface of epithelial cells. Current models depict NMII filaments randomly distributed along the circumferential actin belt, but it is unclear how the activities of individual NMII filaments are integrated and coordinated to generate and transmit force across the epithelium. Understanding these processes is crucial to understanding epithelial dynamics during development, as well as the post-developmental maintenance of epithelial architecture and tensional homeostasis. We have been studying the distribution, organization and role of NMII isoforms within epithelial apical junctions using the organ of Corti, one of the most striking examples of mammalian epithelial patterning, as a model system. Seeking to investigate the effects of NMII inhibition on apical junctions, we treated explant cultures of the organ of Corti with the NMII-specific inhibitor blebbistatin. This induced changes in the epithelial apical surface at both a cellular and tissue level. Morphometric quantification showed an increase in junctional-length, a corresponding increase in apical cell-surface area, and a deformation of apical cell shape across the tissue, resulting in an overall expansion of the epithelial sheet. Strikingly, the changes were completely reversed upon washout of the blebbistatin. Our observations suggested that apical perimeters of epithelial cells are dynamically maintained under tension by NMII within the circumferential junctional actomyosin belt. With data supporting a role for NMII in influencing epithelial apical junctions, we next determined the pattern of distribution of each NMII paralog, NMIIA, IIB and IIC, along the cell perimeter. Immunofluorescence of NMIIB and NMIIC showed a striking distribution as regularly spaced puncta associated with perijunctional actin. Measurements of relative fluorescence intensity revealed low actin density at NMII fluorescence puncta and higher actin density between them, resembling the sarcomeric striations in myofibrils. This finding prompted us to test for the presence of another hallmark component of muscle sarcomeres, the actin cross-linker α-actinin. Immunofluorescence confirmed that α-actinin1 is in fact present along the junctional line in a periodic distribution, alternating with NMII and coinciding with actin, just like in sarcomeres. Another essential feature of sarcomeric organization is the orientation of bipolar myosin filaments parallel to actin filaments. To determine the polarity and orientation of NMII filaments along the apical perimeter of epithelial cells we exogenously expressed, in organ of Corti cultures, NMIIC tagged with different fluorophores at the N-terminus and C-terminus. We also labeled tissue from an NMIIC-GFP transgenic mouse (GFP-tag at the C-terminus), with antibody against the NMIIC N-terminus. These experiments confirmed that bipolar NMII filaments organize as arrays within small regular sarcomeric units assembled end-to-end to form a continuous belt along the cellular perimeter. Like muscle sarcomeres, the NMII sarcomeres at the perijunctional actin belt are also contractile, increasing in length, or relaxing, on treatment with blebbistatin, and shortening, or contracting when blebbistatin was removed. Remarkably, the changes in sarcomere length concomitantly influence apical junctional length and surface area, and ultimately the geometry of the entire epithelium. Notably, NMII sarcomeres along the perimeter of one cell were paired with sarcomeres in the adjacent cell. This registry was maintained regardless of the contractile state of the sarcomeres, suggesting that they are mechanically coupled across the junctional line. We propose that this coupling depends on a yet to be identified transmembrane junctional component that integrates the sarcomeres into a transcellular network along which forces are propagated across the apical epithelial surface. Interestingly, contrary to current views that associate NMII with the adherens junction, we showed that the NMII sarcomeric belt overlaps with both the tight and adherens junctions. This observation, in addition to the alignment of the sarcomeres across the junctional line, also infers the interaction of NMII with other junctional components. Finally, we extended our investigation to the mucosal epithelia of the gastrointestinal tract, and confirmed the presence of the sarcomeric belt along apical junctions, and the registry of the sarcomeres across the junctional line, suggesting that this transcellular sarcomeric network may be a universal epithelial feature.