Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

Abstract

During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs. During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs. Lipid droplets (LDs) are lipid-rich structures which are found in many prokaryotic and eukaryotic cells. Depending on the cell type, their sizes can vary greatly, with diameters ranging from some tens of nanometers in most cells to several tens of microns in adipocytes. Despite these size differences, the general structure of LDs is similar: A core of lipids, which can also contain a minor amount of proteins (1Welte M.A. Proteins under new management: lipid droplets deliver.Trends Cell Biol. 2007; 17: 363-369Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), is shielded against the cell plasma by a membrane consisting of a monolayer of phospholipids. The phospholipid membrane itself contains a variety of proteins, which can be bound by different mechanisms (2Walther T.C. Farese Jr, R.V. Lipid droplets and cellular lipid metabolism.Annu. Rev. Biochem. 2012; 81: 687-714Crossref PubMed Scopus (987) Google Scholar). Proteins associated with the LD membrane mediate many different processes ranging from triacylglyceride (TAG) synthesis (3Kuerschner L. Moessinger C. Thiele C. Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets.Traffic. 2008; 9: 338-352Crossref PubMed Scopus (304) Google Scholar) to lipolysis (4Brasaemle D.L. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). The most important function of LDs, however, is storage of lipids required for membrane synthesis and energy generation. Work of the last decade thus has shown that LDs can be regarded as true cellular organelles (5Martin S. Parton R.G. Lipid droplets: a unified view of a dynamic organelle.Nat. Rev. Mol. Cell Biol. 2006; 7: 373-378Crossref PubMed Scopus (914) Google Scholar, 6Goodman J.M. The gregarious lipid droplet.J. Biol. 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