Figure 3: Plin1 enhances Fsp27-mediated lipid exchange and transfer.
From: Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes
(a) Representative FRAP images showing that Plin1, but not Plin1ΔAM, accelerates Fsp27-mediated neutral lipid exchange. Scale bars, 2 μm. pEGFP-N1-Fsp27 was co-transfected with pCMV5-HA empty vector (upper panel), pCMV5-HA-Plin1 (middle panel) or pCMV5-HA-Plin1ΔAM (lower panel) into 3T3-L1 preadipocytes. Cells were incubated with 200 μM OA and Bodipy 558/568 C12 fatty acid. Fluorescent lipid signal (red) in one LD was bleached (white circle). Blue box highlighted faster fluorescence recovery in the presence of HA-Plin1. (b–d) Fluorescence recovery curves corresponding to FRAP experiments in a. MOI in bleached regions (white circle in a) and unbleached regions (blue circle in a) were plotted as the percentage of the initial intensity. Fluorescent recovery curves are calculated from four independent FRAP experiments (mean±s.d.). (e) Lipid exchange rate (mean±s.e.m.) in 3T3-L1 preadipocytes expressing indicated proteins. N represents the number of LD pairs used for FRAP data collection. (one-way ANOVA Tukey test; ***P<0.001; NS, no significant difference). (f) Western blot showing similar Fsp27-GFP expression and comparable protein levels of HA-Plin1 and HA-Plin1ΔAM in a–e. GAPDH was used as a loading control. (g) Western blot showing similar Fsp27-GFP expression and comparable protein levels of Plin1-Cherry and Plin1ΔAM-Cherry in h–k. (h) Representative time-lapse images showing lipid transfer from smaller to larger LD in a contacted LD pair with Fsp27 enriched at the LDCS. Fsp27-GFP (green) and Cherry (red) were co-expressed in 3T3-L1 preadipocytes. Scale bars, 4 μm. (i) Representative time-lapse images showing that Plin1-Cherry accelerates Fsp27-mediated lipid transfer and shortens the duration required for LD fusion when co-expressed with Fsp27-GFP in 3T3-L1 preadipocytes. (j) Representative time-lapse images showing that Plin1ΔAM-Cherry cannot accelerate Fsp27-mediated lipid transfer when co-expressed with Fsp27-GFP in 3T3-L1 preadipocytes. (k) Lipid transfer rate in 3T3-L1 preadipocytes co-expressing Plin1 and Fsp27 is significantly higher than that in cells expressing Fsp27 alone or Plin1 ΔAM and Fsp27. N represents the number of lipid transfer events used for calculation (mean±s.d., one-way ANOVA Tukey test, ***P<0.001).
