Fig. 8
Extracellular lipidosomes contain mitochondria. A-D Native (A-C) or COX-8-mCherry-transfected (D) FEMX-I cells growing on fibronectin-coated supports were processed for CLSM. Cells preincubated with MitoTracker™ Red CMXRos (A) or MitoView™ Fix 640 (C) were PFA-fixed and co-stained with fluorescent dyes LipidSpot™ 610 (A) or BODIPY™ 493/503 (C) or saponin-permeabilized, immunolabeled with an anti-60-kDa mitochondrial antigen antibody and stained with BODIPY™ 493/503 (B). Alternatively, COX-8-mCherry-transfected cells were permeabilized and immunolabeled with an anti-adipophilin antibody (D). Samples were also co-stained with fluorescence-conjugated WGA to highlight glycoconjugates at the cell membrane. E–G Native FEMX-I cells were incubated without (DMSO, control) or with 1 or 4 µM of CCCP for 8 h and for the 2 last hours were additionally co-incubated with MitoView™ Fix 640. After fixation and staining with BODIPY™ 493/503, cells were co-stained with fluorescence-conjugated WGA and DAPI to highlight and glycoconjugates at the cell membrane and nuclei, respectively. In all cases, composite images of all x–y optical sections are shown and a given marker was pseudo-colored as indicated. Cell extremities (upper panels) and the extracellular lipidosomes (lower panels) are shown (E). Note that in drug-treated cells, mitochondria are rounded (asterisk). Mitochondria-containing and mitochondria-free cell extremities (F) and extracellular particles (G) were quantified under various conditions as indicated. More than 200 cell extremities and 100 extracellular particles per condition were analyzed from 2 and 3 independent experiments, respectively. Each individual data in panel F corresponds to the percentage of cell extremities found in an area of 0.056 mm2. Extracellular particles were classified as containing or not lipid droplets (LD) and mitochondria (M), as indicated (G). Mean ± S.D. are presented. Although not significant, a slight reduction in extracellular particles without mitochondria (green and white, red bracket) is observed after incubation with CCCP. N.s. not significant, Scale bars are indicated