Fluorescence tools that illuminate biology and inspire translation

Project Summary: Our lab works at the interface of chemistry, biology, and medicine. I am drawn to new or complex biological systems whose chemistry and/or mechanism is incompletely understood. New and complex biology is replete with opportunities for fundamental discovery, and history has taught that fundamental discoveries are often accompanied by unexpected therapeutic insights. Efforts on two complex systems comprise my NIGMS portfolio. The first is the organelle interactome, the system of inter-membrane contacts that directly or indirectly control virtually all cell function]. Despite its unquestioned importance, the organelle interactome–especially its dynamics–remains poorly characterized because there are so few useful tools. Visualizing organelle dynamics in live cells for biologically relevant times, especially at resolutions < 100 nm, was impossible, let alone in multiple colors. We developed HIDE probes to fill this void. We discovered that tethering a fluorophore to an organelle membrane via a lipid (rather than a membrane protein) improves imaging time by as much as 50-fold. Even better, as HIDE probes are composed of cell-permeant small molecules, they interrogate cells that are difficult or impossible to modify genetically. We made great progress in the last period developing and applying HIDE probes for imaging. Here we broaden their utility for multi-organelle (>2) HIDE fluorescence lifetime imaging microscopy; to deliver photocatalyst proximity labeling tools; and to sequester and inactivate difficult to target disease-causing proteins. The second portion of my NIGMS portfolio focuses on understanding and improving macromolecule delivery, the single most important bottleneck hindering the development of next-gen biologic therapies. The delivery problem can be summed up in one phrase: inefficient endosomal escape. In previous NIGMS-funded work, we identified a mini-protein (ZF5.3) that is actively endocytosed and escapes the endocytic pathway with unprecedented efficiency. We learned much about ZF5.3 during the last period. Using fluorescence correlation spectroscopy (FCS), a state-of-the-art single molecule method, we learned that ZF5.3 escapes from late endosomes through a previously unrecognized portal (HOPS), and is most efficient when conjugated to cargo that are small or unfold easily. We also discovered that ZF5.3 itself unfolds in late endosomes and unfolding is required for HOPS-mediated endosomal escape. Here we build on these discoveries to develop scaffolds for genuinely cell-permeant protein interaction inhibitors; others that escape earlier along the endocytic pathway and deliver compact gene-editing tools; and probe the mechanism of endosomal escape in vitro and in cellulo.