“Opening a new window into the secretory cell with in situ cryo imaging”

Regulation of secretion is critical in signaling, cell metabolism, growth and differentiation. However, most studies of cellular secretion have been constrained by limited resolution in vivo or non-native conditions when conducted in vitro. As a result, we have developed new imaging methods to visualize the distribution and trafficking of secretory vesicles under near native conditions within the cell. To do so, we have combined and integrated a number of innovative new cryo-imaging methods that we adapted for use in mammalian cells including cryo-electron microscopy, cryo-electron tomography, cryo-focused beam milling and cryo-correlative light and electron microscopy (cryo-CLEM). Significantly, we recently discovered an organelle sharing morphology with secretory vesicles but whose membranes are associated with 80S ribosomes we have termed Ribosome Associated Vesicles (RAVs). These RAVs appear to be novel ER-derived organelles that associate with mitochondria and establish direct connections with them. We also show that RAVs exist across different secretory cell types and species. Using computational approaches to establish averages of the biological structures picked within the imaged tomograms, we have also been able to establish the structures of the individual constituents of the macromolecular complexes associated with the RAV membrane in situ. In parallel, we have also begun applying these imaging methods to primary human fibroblasts to dissect the molecular mechanisms associated with mitochondrial disease. Taken together, the combination of novel imaging and computational approaches opens the door to the study of disease and cell development at a fundamental molecular level within the native cellular context for the first time.