Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a , b Quantification of fission (Fis.) and fusion (Fus.) events associated with ER. Cells were transfected with mCherry-ER and mito-GFP. Each point represents an individual cell, with 15 cells quantified in 4 independent experiments. a ER-Positive events (%), ( b ) Total events per cell. Bars show the average ± SD. c Representative fusion event showing the enrichment of AC-ER at the fusion site (arrowhead). Cells were transfected with mCherry-mito (mitochondria, magenta) and AC-ER (actin, green). Scale bar 2 µm. d , e Quantification of fission (Fis.) and fusion (Fus.) events positive for AC-mito. Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. d ER-Positive events (%), ( e ) Total events per cell. f AC-ER signal enrichment at fusion (Left) and fission (Right) sites in cells transfected as in ( c ). Signal intensity at the event site relative to an adjacent site on the ER network was quantified for AC-ER (AC) and mCherry-ER (mCh). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. two-sided t-test. g Quantification of AC-ER signal associated with each of the fusing mitochondria (the stationary mitochondrion (Rec) and the mobile mitochondrion (Fus)) as well as with the fusion site one frame post-fusion (Post). Each point represents an individual cell, with 13 cells quantified in 3 independent experiments. Bars show the average ± SD. One-way ANOVA. h , i Kinetics of recruitment of AC-mito and AC-ER at the fusion site showing AC-ER recruitment relative to AC-mito ( g ) and recruitment of either marker relative to the time of fusion ( h ). Each point represents an individual event (20 events total in 4 cells within 3 independent experiments). two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a Representative images showing the loss of AC-mito signal in primary fibroblasts transfected with AC-mito (green) and mCherry-Fis1 (magenta), and treated with the Arp2 inhibitor CK-666. Scale bar 10 µm ( b ) Quantification of AC-mito signal accumulation in cells transfected as in ( a ). Each point represents an individual cell, with 15 cells quantified per condition in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. ( c ) Quantification of the number of fusion (Left, blue) and fission (Right, orange) events, as well as the fusion/fission ratio ( d ) in cells transfected as in ( a ) and treated as indicated. Each point represents an individual cell, with 12 (control) and 9 (CK-666) cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test. e Quantification of fusion events as tip-to-side (Side) or tip-to-tip (End). The total number of events for 9 cells in 3 experiments is shown for each condition. f Quantification of mitochondrial length (Left) and connectivity (Right) in cells from ( c ). Each point represents an individual cell. Bars show the average ± SD. One-way ANOVA. g – i Mitochondrial fusion assay. Human primary fibroblasts were transfected with photoactivatable-GFP (PA-GFP), treated as indicated and imaged before (pre) and after activation with the 405 nm laser. Fluorescence traces ( g , Left) and the quantification of the loss of fluorescence at 3 min relative to the initial time post-activation ( g , Right) are shown. Each point ( g , Right) represents an individual cell, with 14 ctrl and 16 CK-666 cells quantified in 4 independent experiments. Bars show the average ± SD. Two-sided t-test. Fusion was also directly quantified by measuring the events in which PA-GFP was transferred from one mitochondrion to another upon fusion (H, separation by fusion type; I, total fusion events). Each point represents an individual cell, with 12 ctrl and 11 CK-666 cells quantified in 3 independent experiments. Bars show the average ± SD. Two-sided t-test.

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Control, Single Vesicle Fusion Assay, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Mitochondria- and ER-associated actin are required for mitochondrial fusion

doi: 10.1038/s41467-024-55758-x

Figure Lengend Snippet: a – d Temporal relationship between mitochondrial actin and the fusion dynamin MFN2. Cells were transfected with mCherry-MFN2 and AC-mito while mitochondria were visualised with Mitotracker Deep red. ( a ) Presence of AC-mito and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. b – d Kinetics of recruitment of AC-mito and mCherry-MFN2 at fission ( b ) and fusion ( c ) sites showing recruitment of either marker relative to the time of fusion, as well as mCherry-MFN2 recruitment relative to AC-mito ( d ). Each point represents an individual event in 4 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. e – h Temporal relationship between the fusion dynamin MFN2 and the fission Dynamin DRP1. Cells were transfected with mCherry-MFN2 and GFP-DRP1 while mitochondria were visualised with Mitotracker Deep red. e Presence of GFP-DRP1 and mCherry-MFN2 at sites of mitochondrial fission, tip-to-side fusion and tip-to-tip fusion. ( f – h ) Kinetics of recruitment of mCherry-MFN2 andGFP-DRP1 at fission ( f ) and fusion ( g ) sites showing recruitment of either marker relative to the time of fusion, as well as GFP-DRP1 recruitment relative to mCherry-MFN2 ( d ). Each point represents an individual event in 3 cells within 3 independent experiments). Bars show the average ± SD. two-sided t-test. ( i ) Schematic representation of the timing of MFN2 and DRP1 recruitment to tip-to-side fusion (Top) and fission (Bottom) relative to actin recruitment (AC-mito-positive).

Article Snippet: These cells were stably transfected with mitochondria-targeted GFP (CCOeGFP, addgene, pLVX-EF1a-CCO-IRES, #134861) using Metafectene (Biontex).

Techniques: Transfection, Marker