Lipid droplet monolayer protein binding and complexation

The mechanisms by which the lipid droplet membrane contributes to the regulation of lipolysis remains a key unknown in our efforts to understand and treat metabolic disorders. We developed artificial lipid droplets (aLDs), confocal imaging analysis, and four-color fluorescence cross-correlation spectroscopy (FCCS) to reveal the lipid and protein behaviors correlated with triglyceride storage and mobilization. Confocal microscopy was used to assess the competitive binding and stability of lipolysis-associated proteins on coverslip-adhered aLDs caps. The focal plane was set to a few microns above the widest part of a cap and the bound protein densities were observed versus time. We observed the association of perilipin 5 (PLIN5) and alpha beta hydrolase domain containing protein 5 (ABHD5) to the aLD caps with binding rates and saturation values dependent on the solution protein concentrations. In our cost effective, four-color FCCS, the excitation wavelengths were chosen from a single, wide-band laser source and spectral fitting of the emitted light revealed the interactions for up to four fluorophores simultaneously. The fluorescence emission was dispersed onto a sCMOS or EMCCD camera at 10 kHz for spectral fitting of each fluorophore's intensity vs time. The auto- and cross-correlations revealed the diffusion rates and binding partners in complex environments. In particular, we observed the ligand-stimulated association between ABHD5 and adipose triglyceride lipase (ATGL/PNPLA2) coincident with the dissociation of ABHD5 from PLIN5 on the aLD membrane. These studies will provide further understanding of how lipolysis is regulated via protein interactions with the lipid droplet membrane.