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）方法提供表面形貌信息，特别适合于在其原生环境中研究膜蛋白复合物。然而，金属复制品的保真度和分辨率受到金属原子结晶的固有性质的限制。为了克服金属复制品的局限性，我们结合了无定形碳复制品与相衬电子显微镜。使用这种新的方法，我们提供了第一个直接的证据表明，TJ膜内纤维具有双链形态。紧密连接原纤维的这种基本结构特征的直接可视化可以帮助阐明原纤维是如何形成和重塑的，是什么导致其内在的柔性，以及原纤维间接触、分支和退火机制的性质。 我们以前发现，肌动蛋白和非肌肉肌球蛋白II（NMII）沿沿着上皮细胞顶端连接形成一个肌节带，调节细胞形状和组织几何形状。作为本研究的后续，我们专注于阐明上皮细胞顶端表面不同但相互关联的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，结合光漂白后的荧光恢复（FRAP），以研究活体小鼠上皮组织中NMII的组织，动力学和营业额。 我们去年报道了一项冷冻蚀刻和电子断层扫描研究，以表征小鼠肠道糖萼的纳米结构。我们发现，糖萼由微米长的粘蛋白丝从微绒毛尖端出现，并通过标记的横向相互作用形成一个三维的柱状网络与30 nm的网格。细丝末端会聚成间隔30 nm的球状结构，其是在单个平面内填充的液晶。使用活体成像，我们还评估了糖萼变形性和孔隙率使用活体显微镜。我们的数据表明，柱状网络结构和液晶包装的细丝终端允许糖萼作为一个可变形的尺寸排阻过滤器的内腔内容物。 与Roberto Weigert（NCI）合作，我们使用分辨率更高的活体显微镜来显示NMII和肌动蛋白在胞吐过程中围绕分泌颗粒组装成以前未描述的产生力的多面体晶格。 肌动球蛋白网络是细胞主要的力产生机制，在无数的动态过程中通过组装成具有不同力产生特性的各种结构来重塑细胞膜。虽然线性和分支的肌动球蛋白架构在细胞培养和无细胞系统中得到了很好的表征，但肌动蛋白和肌球蛋白网络如何形成和发挥作用以重塑复杂三维哺乳动物组织中的膜尚不清楚。在这里，我们使用四维旋转圆盘共聚焦显微镜与图像去卷积获得大分子尺度的细节动态肌动球蛋白网络在外分泌腺的活小鼠。我们解决肌动蛋白和肌球蛋白如何组织周围的大型膜结合的分泌囊泡，并产生所需的力量完成胞吐。我们发现，肌动蛋白和非肌肉肌球蛋白II（NMII）组装成以前未描述的多面体状晶格周围的囊泡膜。NMII晶格由双极微丝以及非正则的三腿结构组成。使用光漂白和药理学扰动在体内，我们表明，肌动球蛋白的收缩性和肌动蛋白聚合在一起推动底层囊泡膜，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种产生力的肌动球蛋白晶格，它调节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.