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.
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