A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction

Abstract

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based onFluorogen-ActivatedBimolecular complementation atCONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

A tunable and versatile chemogenetic near infrared fluorescent reporter

“…The bisection of members of the FAST family previously led to chemogenetic tools enabling the functional study of protein-protein interactions 6, 15, 18–20 . Bisection led to either systems displaying low self-complementation in presence of the fluorogen, well suited for reporting protein-protein interactions 18 and contact sites between organelles 23, 24 , or to systems displaying high self-complementation in presence of the fluorogen, which were used to develop CATCHFIRE (chemically assisted tethering of chimera through fluorogenic induced recognition) 19 , a chemically induced dimerization tool with intrinsic fluorescence readout for controlling and visualizing protein proximity. Bisection of nirFAST allowed us to generate a CATCHFIRE system with NIR fluorescence readout, in which HPAR-3OM and HPAR-3,5DOM act as fluorogenic molecular glues promoting the dimerization of the two fragments.…”