Molecular organization of intercellular junctions in the inner ear

基于复制品的冷冻断裂和冷冻蚀刻电子显微镜(EM)方法提供了表面形貌信息，特别适合于研究天然环境中的膜蛋白复合体。然而，金属复制品的保真度和分辨率受到金属原子结晶固有性质的限制。为了克服金属复制品的局限性，我们将非晶碳复制品与相差电子显微镜相结合。利用这种新的方法，我们首次提供了TJ膜内纤维具有双链形态的直接证据。直接观察紧密连接原纤维的这一基本结构特征有助于阐明原纤维是如何形成和重塑的，是什么导致了其内在的灵活性，以及原纤维间接触、分支和退火机制的性质。 我们之前发现，肌动蛋白和非肌肉肌球蛋白II(NMII)沿着上皮细胞的顶端连接形成一条肌小带，调节细胞的形状和组织几何形状。作为这项研究的后续，我们致力于阐明在上皮细胞顶端表面不同但相互连接的NMII网络的组织和动力学。为此，我们与Roberto Weigert(NCI)合作开发了一种改进的分辨率活体显微镜方法，结合光漂白后荧光恢复(FRAP)，以研究活体小鼠上皮组织中NMII的组织、动力学和周转。 去年，我们报道了一项冷冻蚀刻和电子断层扫描的研究，以表征小鼠肠道糖萼的纳米结构。我们发现，糖萼由微绒毛尖端出现的微米长的粘蛋白细丝组成，通过锯齿状的横向相互作用形成三维柱状网络，网状结构为30 nm。丝状末端会聚成相距30 nm的球状结构，这些结构是液晶填充在单一平面内的。通过活体成像，我们还使用活体显微镜评估了糖唇的变形性和孔隙率。我们的数据表明，柱状的网络结构和丝状末端的液晶填充允许糖萼作为管腔内容物的可变形的尺寸排除过滤器。 与Roberto Weigert(NCI)合作，我们使用改进的分辨率活体显微镜显示，在胞吐过程中，NMII和肌动蛋白组装成先前未描述的产生力量的多面体晶格围绕分泌颗粒。肌动球蛋白网络是细胞的主要作用力机制，通过组装成不同的具有不同的力产生特性的结构，在无数的动态过程中重塑细胞膜。虽然线性和分支肌球蛋白结构在细胞培养和无细胞系统中具有很好的特征，但目前还不知道肌动蛋白和肌球蛋白网络是如何形成和作用于复杂的三维哺乳动物组织中的膜的。在这里，我们使用带有图像去卷积的四维旋转圆盘共聚焦显微镜来获取活体小鼠外分泌腺中动态肌球蛋白网络的大分子尺度细节。我们讨论了肌动蛋白和肌球蛋白如何围绕着大的膜结合的分泌小泡组织，并产生完成胞吐所需的力量。我们发现，肌动蛋白和非肌肉肌球蛋白II(NMII)在囊泡膜周围组装成先前未描述的多面体样晶格。NMII晶格由双极细丝和非正则三条腿构型组成。利用体内的光漂白和药理学扰动，我们证明了肌动球蛋白的收缩能力和肌动蛋白聚合共同推动下面的囊泡膜，以克服能量障碍和完成胞吐。因此，我们的成像方法揭示了一个能产生力量的肌动球蛋白晶格，它调节着活动物外分泌器官的分泌。

Replica-based freeze-fracture and freeze-etch electron microscopy (EM) methods provide surface topography information, particularly suited to studying membrane protein complexes in their native context. However, the fidelity and resolution of metal replicas is limited by the inherent property of metal atoms to crystallize. To overcome the limitations of metal replicas, we combined amorphous carbon replicas with phase-contrast electron microscopy. Using this novel approach, we provided the first direct evidence that TJ intramembrane fibrils have a double stranded morphology. Direct visualization of this fundamental structural feature of the tight junction fibril can help elucidate how the fibrils are formed and remodel, what leads to their intrinsic flexibility, and the nature of the inter-fibril contacts, branching, and annealing mechanisms. We previously revealed that actin and non-muscle myosin II (NMII) along apical junctions of epithelial cells form a sarcomeric belt that regulates cell shape and tissue geometry. As a follow up to this study, we are focusing on elucidating the organization and dynamics of distinct but interconnected NMII networks at the apical surface of epithelial cells. To this end, we developed in collaboration with Roberto Weigert (NCI), an improved resolution intravital microscopy approach, combined with fluorescence recovery after photobleaching (FRAP), to study the organization, dynamics, and turnover of NMII in epithelial tissues in live mice. We reported last year a freeze-etching and electron tomography study to characterize the nanoarchitecture of the murine enteric glycocalyx. We found that the glycocalyx consist of micrometer-long mucin filaments that emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures 30 nm apart that are liquid-crystalline packed within a single plane. Using intravital imaging we also assessed glycocalyx deformability and porosity using intravital microscopy. Our data suggest that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents. In collaboration with Roberto Weigert (NCI), we used improved resolution intravital microscopy to show that NMII and actin assemble into previously undescribed force-generating, polyhedral lattices around secretory granules during exocytosis. Actomyosin networks, the cells major force production machineries, remodel cellular membranes during in a myriad of dynamic processes by assembling into various architectures with distinct force generation properties. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning disc confocal microscopy with image deconvolution to acquire macromolecular- scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis. We found that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice consisted of bipolar minifilaments as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.