The Mechanism Of ARP2 And ARP3 Regulate Cellular Lipid Droplet Transport

Intramuscular fat and fatty acid composition are important economic traits for pigs.Lipid droplets are essential for the regulation of intramuscular fat and fatty acids.Lipid droplets are the storage organelles in the homeostasis of lipids and energy.Lipid droplets have a unique structure.Lipid droplets are composed of triglycerides,monolayers of phospholipids and many proteins on the surface of phospholipids that are involved in cell biological processes.Filamentous actin,tubular microtubules and intermediate filaments with relatively stable structure and function together form a colorful cytoskeleton network.The cytoskeleton is a kind of filamentous scaffold,which is distributed from the plasma membrane to the nucleus.The interaction of the three components of the cytoskeleton helps to maintain the stability of the internal structure of the cell.The movement of lipid droplets mainly depends on the cytoskeleton.The study of lipid droplet metabolism and movement plays an important role in analyzing the mechanism of intramuscular fat metabolism.However,the molecular mechanism of lipid droplet and cytoskeleton interaction and lipid droplet movement is still unclear.This study shows that the microfilament proteins ARP2 and ARP3 promote the contact of lipid droplets with the cytoskeleton,further through subcellular fluorescent labeling,laser confocal fluorescence microscopy observation,protein domain analysis,immunoprecipitation tandem western blot,lipid synthesis signal Pathway phosphorylation level detection,reverse transcription PCR quantitative detection,live cell workstation observation and other methods have revealed that filaggrin ARP2 and ARP3 regulate the binding of lipid droplets to the cytoskeleton.Thus,the recruitment of MYH9 on the surface of lipid droplets is promoted and the movement and the metabolism of lipid droplets is regulated.In this study,the molecular mechanism of lipid droplet movement was analyzed from the perspective of organelle interaction,and provided a new idea of intramuscular fat in pigs in the future.The main findings are as follows:(1)ARP3-MYH9 protein complex regulates the movement of lipid droplets.The ARP3-EGFP fluorescent vector was constructed,and the subcellular localization of ARP3,microfilament and lipid droplet was detected by immunofluorescence technology;the interaction of ARP3 and MYH9 was detected by immunofluorescence and CO-IP.Knock down MYH9,the lipid droplet movement is significantly slowed down.(2)ARP3 regulates the contact between lipid droplets and microfilaments.After interfering with ARP3,the intracellular lipid droplets were extracted,and the intracellular lipid droplets and microfilaments were incubated at 37°C in vitro.Compared with the control group,the binding ratio of Si ARP3 lipid droplets and microfilaments decreased.The key region where ARP3 regulates the contact between lipid droplets and microfilaments is amino acids 1-112.(3)The microfilament proteins ARP2 and ARP3 interact with the motor protein MYH9 on the surface of lipid droplets,and ARP3 promotes the recruitment of MYH9 on the surface of lipid droplets.WB detects cell separation lipid droplet components,and the expression of MYH9 protein in the lipid droplet component of the interference ARP3 group is significantly down(4)MYH9 regulates the contact of lipid droplets with mitochondria.The interference motor protein MYH9 promotes the increase of intracellular lipid droplet content;the increase of triglyceride content;the decrease of contact between lipid droplets and mitochondria(5)ARP2 and ARP3 regulate the expression of lipid droplet surface genes and lipid synthase.After interfering with ARP2 and ARP3,the phosphorylation level of ASK1,JNK,P38,and AKT protein increased significantly.ARP2 and ARP3 regulate the lipid level in the cell by regulating the lipid anabolic pathway and the expression of lipid synthase downstream of the pathway.The above results indicate that ARP3 is a key protein for lipid droplets to bind to microfilaments,and ARP3 can recruit the motor protein MYH9 to the surface of lipid droplets.MYH9 is the motor protein that lipid droplets move along the microfilaments.Knockdown of MYH9 reduces the movement of lipid droplets in cells,which reduces the contact between lipid droplets and mitochondria and increases the content of intracellular lipid droplets.ARP2 and ARP3 regulate cell proliferation by regulating the lipid content in the cell.This study preliminarily clarified the molecular mechanism of ARP2/ARP3 regulating intracellular lipid droplet metabolism and cell proliferation,providing a new idea for the study of lipid droplet dynamics.