Journal: Nature Communications
Article Title: Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein
Figure Lengend Snippet: A53T hNs transmit mitochondrial pathology to corrected hNs. a , b Micrographs of co-cultured GFP +ve -corrected and GFP −ve -A53T-mutant hNs antigenically labeled for ubiquitin (Ubq) and PS129; scale bar: 10 μm ( a ). Enlarged regions of GFP +ve neurites and GFP −ve neurites show that both display equal Ubq/PS129 colocalization. Boolean operation was employed to label regions of colocalization in blue, scale bar: 50 μm. Quantifications of PS129 +ve neurons within the GFP +ve and GFP −ve populations ( b ). Data represent mean ± s.e.m. P = 0.8919 by t -test, 10 coverslips over 3 independent differentiations, DIV: 60. c , d Micrographs of GFP +ve -corrected and GFP −ve -A53T-mutant hNs expressing mitoDSRed and antigenically labeled for endogenous LC3. GFP +ve neurites ( i ) and GFP −ve neurites ( ii ) are enlarged and show LC3 punctate colocalizes with mitochondria. Arrows show areas where LC3 colocalizes with MitoDSRed. Boolean operation was employed to label regions of colocalization in blue, scale bar: 10 μm. d Quantification of percentage of total GFP +ve and GFP −ve hNs that have fragmented mitochondria. Data represent mean ± s.e.m. P = 0.1479 by t -test, n = 8 coverslips over 3 independent differentiations, DIV: 60. e Co-cultures were labeled with 200 nM TMRE, and mitochondrial potential (Δ Ψ m ) was measured by flow cytometry using the dequench method in GFP +ve (red trace) and GFP −ve (black trace) populations. Representative plot illustrating that GFP +ve corrected cells within co-cultures have adopted a pattern indicative of mitochondrial depolarization. Representative trace from 3 independent experiments (10,000 events per experiment), DIV: 60. f Corrected cells expressing CL-GFP and RPRE-RFP were differentiated in co-cultured with either RFP -ve /GFP −ve Corrected or RFP -ve /GFP −ve A53T hNs. Translocation of cardiolipin from the IMM to the OMM was assessed by visualizing the redistribution of RPRE-RFP from the plasma membrane to the cardiolipin-GFP +ve mitochondria. Magnified insets show CL-GFP colocalization with RPRE-RFP in Corrected hNs when differentiated in co-culture, scale bar: 10 μm. g , h Fluorescence resonance energy transfer (FRET) from CL-GFP to RPRE-RFP in hiPSC-derived A53T and corrected hNs or hESC-derived WT, A53T and E46K hNs was assessed ( g ) and mean FRET intensity was quantified ( h ). Data represent mean ± s.e.m. ** P
Article Snippet: Phosphoserine 129 (81A) (1:1000) LC3 (1:500), beta-III-Tubulin and Ubiquitin (1:500) were purchased from BioLegend (Cat. no. 825701, 827101, 801202, 840501), while total α-synuclein (1:500) was from BD Biosciences (Cat. no. 610787).
Techniques: Cell Culture, Mutagenesis, Labeling, Expressing, Flow Cytometry, Cytometry, Translocation Assay, Co-Culture Assay, Fluorescence, Förster Resonance Energy Transfer, Derivative Assay