Dynamics Of CIDE-mediated Lipid Droplet Fusion Complex And Its Screen Application

Lipid droplet(LD)is a crucial organelle in cells,its abundance and size reflecting the energy fluctuation of cells.Aberrant LD is closely linked to the development of metabolic diseases,such as obesity and fatty liver.By forming LD fusion complex(LDFC),CIDE family proteins mediate the fusion of a donor LD into an acceptor LD to form a larger LD.This process plays an important role in lipid accumulation in livers and adipocytes.However,the micro-visualization and characterization of LDFC dynamics remain lack.We investigate the dynamic changes of LDFC during LD fusion to reveal the mechanism of LDs growth.We investigate the external width and inner effective fusion pore size of LDFC to understand the micro-visualization and dynamics of LDFC.Using high-resolution and lipid transfer assays,we find the widths of LDFC and lipid transfer rate are dependent on donor LD sizes but are acceptor LD size-independent.The effective pore size of LDFC which is estimated by Hagen-Poiseuille equation is dynamics in the fusion process.The dynamics of effective pore size go through two stages: 1)expanding and reaching the maximum,2)narrowing until fusion finish.In the second stage,the pore size is limited to donor LD size.The super-resolution imaging shows one LDFC may be consist of irregular small pores.It is reported that Plin1 promotes Cidec-mediated LD fusion but the details of the mechanism are still unknown.We find Plin1 increases the width of LDFC,reduces the amount requirement of Cidec and enlarges the size of fusion pore to accelerate the LD fusion.Collectively,We find the LDFC is a dynamics channel of lipid transfer.The effective pore size of LDFC is dependent on donor LD size.These results indicate the inherent insight that donor LD size-dependent dynamics of LDFC regulate the LD fusion.Then we try to use the yeast knockout library and drugs to screen the regulatory genes and compounds of LDs growth.In most cells,the LD size is small which limit investigation of the smaller LDs phenotype,due to the limitation of microscopy.We express CIDE protein in cells to induce large LDs.We search for the perturbation that leads to the small LD phenotypes.In this strategy,we find twelve genes involved in LD growth using the yeast knockout library.In HeLa cells,we find eight compounds that affect the LD morphology.These high-content screens prove the validation of our strategy that utilizes CIDE fusion ability to enhance the morphology variation.The strategy has the application value for disease investigation and drug development.