Molecular dynamics of endomembrane structure and function

Abstract

Emerging visualization technologies are playing an increasingly important role in the study of endomembrane structure and function, capturing membrane processes at the level of whole organisms down to single molecules. Recent developments in probes, techniques, microscopes and quantification are dramatically expanding the areas of that can be analyzed through imaging. Photoactivatable fluorescent proteins (PA‐FPs) have been particular fruitful in this regard. They become bright and visible upon being exposed to a pulse of UV light. This allows selected populations of proteins to be pulse‐labeled and tracked over time. Used for in cellulo pulse chase experiments, the PA‐FPs have helped clarify mechanisms for biogenesis, targeting, and maintenance of organelles as separate identities within cells. PA‐FPs have further permitted the development of single molecule‐based superresolution (SR) imaging, which dramatically improves the spatial resolution of light microscopy by over an order of magnitude (~10–20 nm resolution). Involving the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules, single molecule SR imaging offers exciting possibilities for obtaining molecule scale information on biological events occurring at variable time scales. Here, I discuss several examples where we have used these new fluorescent imaging techniques to dissect different membrane trafficking topics, including the formation of autophagosomes, the dynamics of mitochondria, and transport through the secretory pathway.