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A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction
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DOI：10.1083/jcb.202311126.png)
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Abstract
En 中文
Li and Gamuyao et al. engineer a synthetic lipid droplet (LD)-targeting motif and develop a tool kit that detects contact sites between LDs and other organelles. This method utilizes a reversible, split fluorescence protein (splitFAST) that enables the investigation of dynamic organelle interactions under various metabolic conditions in living cells. Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact 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 regulation.
Keywords:
ENDOPLASMIC-RETICULUM
COA SYNTHETASE
CONTACT SITES
PROTEIN
ER
SEIPIN
INHIBITION
DYNAMICS
REPORTER
CELLS
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