Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells

Background

Lipid droplets (LDs) play a key role in maintaining lipid homeostasis (Farese and Walther, 2009; Yang et al., 2012). Defective LD maturation and growth are closely associated with the development of metabolic diseases such as obesity, fatty liver disease, cardiovascular disease, and type II diabetes (Krahmer et al., 2013; Rosen and Spiegelman, 2014; Gluchowski et al., 2017). LDs are dynamic organelles which budded from the endoplasmic reticulum (ER) (Gross et al., 2011; Choudhary et al., 2015) and continue to grow via triglyceride synthesis and lipid transfer from the ER (Fujimoto et al., 2007; Wilfling et al., 2013; Xu et al., 2018) or LD fusion (Gong et al., 2011; Somwar et al., 2011). LD-associated cell death–inducing DNA fragmentation factor alpha-like effector (CIDE) family proteins including CIDEA, CIDEB, and CIDEC/Fsp27 (Gao et al., 2017) are crucial regulators in the lipid homeostasis by governing atypical LD fusion and growth for lipid storage. Previously, we reported that CIDEC mediates LD fusion through directional lipid transfer from small (donor) to large (acceptor) LDs (Gong et al., 2011; Somwar et al.). The enrichment of CIDEC at the LD-LD contact site (LDCS) and the formation of the fusion pore are two essential steps for lipid exchange and transfer to occur. To evaluate the fusion ability of each LD pair in cells mediated by the CIDE family proteins and their regulators such as Perilipin1 (Sun et al., 2013a) or Rab8a (Wu et al., 2014), lipid-exchange rate assay was first proposed and performed as previously described (Gong et al., 2011; Sun et al., 2013b). However, the analytic process reported in the previous methodology neither eliminated the photobleaching effect upon laser exposure nor provided a detailed procedure to ensure experimental reproducibility and accuracy.

Here, we detailed the protocol of our renewed lipid-exchange rate assay used in Wang et al. (2018). The three key steps in the assay including cell preparation, fluorescence recovery after photobleaching (FRAP) image acquisition, and data analysis using mean of intensities (MOI) and BODIPY-C12-stained size measurement of LDs were reported. Upon performing a three-cycle photobleaching process and the pre-estimation of exchange rate based on the linear criteria in the data analysis, we can ensure the accuracy and repetitiveness of the fitted exchange rates by using our proposed equation underlying molecular thermodynamics in the Theory section. This lipid-exchange rate assay is also applicable for the evaluation of a nanometer size channel connecting two vesicles when the micrometer volumes of the vesicles are measured in advance.