Figure 6: Disruption of CIDE-N homodimerization abolishes Fsp27-mediated LD growth.
From: Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes
(a,b) Mutations at the interaction interface (E87Q/D88N, QQN, R46E, R55E and R46E/R55E) disrupt the CIDE-N homodimerization of Fsp27. Flag and HA-tagged CIDE-N domain of Fsp27 (a.a. 1–135) containing indicated mutations were co-expressed in 293T cells. Anti-Flag M2 beads were used for immunoprecipitation. QQN represents E86Q/E87Q/D88N triple mutations; IP, immunoprecipitation; IB, immunoblot; L, light chain. (c) Representative images showing that CIDE-N homodimerization-defective mutants of full-length Fsp27-GFP (green) fail to induce large LD formation when expressed in 3T3-L1 preadipocytes. LDs were stained with Bodipy 558/568 C12 fatty acid (red). Scale bars, 5 μm. (d) Quantitative analysis showing that CIDE-N dimerization is critical for Fsp27’s activity. Large LDs are defined as LDs with a diameter ≥2.5 μm in c. ***P<0.001 represents statistical analysis between wild-type Fsp27 (WT-Fsp27) and mutant forms of Fsp27 (one-way ANOVA Dunnett test, mean±s.d., n≥1,000).
