Abstract
Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.
Highlights
Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells
Design of the post-correlation on-lamella cryo-Correlative light and EM (CLEM) workflow. The aim of this correlation workflow is (i) to be aplicable for adherent cells, directly grown on cryo-electron microscopy (EM) grids, (ii) to be compatible with commercially available widefield cryo-light microscopy (LM) setups, and (iii) to enable target validation by correlating the fluorescent signal derived from the lamella position with the cryo-transmission electron microscopy (TEM) map of the lamella
All scripts and software plugins necessary are provided. The goal of this on-lamella cryo-CLEM workflow is to precisely localize the fluorescence signal derived from the lamella with the cryo-TEM map both in the X–Y plane and in the Z-dimension. While the former is straightforward, the latter is challenging because (i) the Z-dimension of the lamella is reduced to under 5% of the original Z-dimension of the cell and (ii) the cryo-LM has limited Z-resolution
Summary
Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. AEC2 fatty acid metabolism and LB ultrastructure are severely disrupted by the pandemic influenza strain H1N19 and the highly pathogenic H7N9 strain[10] In spite of their importance in health and disease, many questions remain open about LB biogenesis, structure, and secretion. In the current model of LB biogenesis, lipids are flipped by ABCA3 from the cytosolic to the luminal leaflet and are imported into the LB core[16], where SP-B and SP-C are responsible for further lipid rearrangement into tightly packed membrane sheets[17] This model has been difficult to validate. A study employing cryo-electron microscopy (EM) of vitrified sections (CEMOVIS) on rat lungs enabled imaging of frozen-hydrated LBs and showed smooth concentric membranes[18]. Available in situ cryo-CLEM workflows involving cryo-FIB milling are aimed at site-specific cryo-FIB milling[29,30] but do not offer target validation
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