Figure S3.
Mid51 interacts with Fis1 in a Mid51/Fis1 complex and regulates mitochondrial and lysosomal tethering. (A) IP of myc-tagged WT Mid51 and co-IP of Flag-tagged WT Fis1, with corresponding input, with and without Mid51, confirming that IP of Mid51 and co-IP of Fis1 is dependent on the presence of Mid51 (lane 2). See Fig. 4, A and B, for quantification. *, Nonspecific bands. (B) IP of myc-tagged WT Mid51 and co-IP of Flag-tagged WT Fis1, with corresponding input, with and without Fis1, confirming that co-IP of Fis1 is dependent on the presence of Fis1 (lane 2). See Fig. 4, C and D, for quantification. *, Nonspecific bands. (C and D) Confocal time-lapse microscopy showing lysosomal cluster of inter-lysosomal tethers which are also tethered to mitochondria (white arrows, inset in D) in live HeLa cells expressing Mid51 (mitochondria mCherry-Mid51; lysosome LAMP1-mGFP). Scale bars, 5 μm (C); 0.5 μm (D). (E and F) Confocal time-lapse microscopy showing few inter-lysosomal tethers or mitochondria–lysosome tethers (inset in F) in live HeLa cells expressing Mff (mitochondria mCherry-Mff; lysosome LAMP1-mGFP). Scale bars, 5 μm (E); 0.5 μm (F). (G–I), Confocal time-lapse microscopy showing prolonged inter-lysosomal (L-L) tethering (white arrows; inset in H; I) in live HeLa cells expressing Mid51 (lysosome LAMP1-mGFP). Scale bars, 5 μm (G); 0.5 μm (H and I). Video 8 corresponds to H. Source data are available for this figure: SourceData FS3.

Mid51 interacts with Fis1 in a Mid51/Fis1 complex and regulates mitochondrial and lysosomal tethering. (A) IP of myc-tagged WT Mid51 and co-IP of Flag-tagged WT Fis1, with corresponding input, with and without Mid51, confirming that IP of Mid51 and co-IP of Fis1 is dependent on the presence of Mid51 (lane 2). See Fig. 4, A and B, for quantification. *, Nonspecific bands. (B) IP of myc-tagged WT Mid51 and co-IP of Flag-tagged WT Fis1, with corresponding input, with and without Fis1, confirming that co-IP of Fis1 is dependent on the presence of Fis1 (lane 2). See Fig. 4, C and D, for quantification. *, Nonspecific bands. (C and D) Confocal time-lapse microscopy showing lysosomal cluster of inter-lysosomal tethers which are also tethered to mitochondria (white arrows, inset in D) in live HeLa cells expressing Mid51 (mitochondria mCherry-Mid51; lysosome LAMP1-mGFP). Scale bars, 5 μm (C); 0.5 μm (D). (E and F) Confocal time-lapse microscopy showing few inter-lysosomal tethers or mitochondria–lysosome tethers (inset in F) in live HeLa cells expressing Mff (mitochondria mCherry-Mff; lysosome LAMP1-mGFP). Scale bars, 5 μm (E); 0.5 μm (F). (G–I), Confocal time-lapse microscopy showing prolonged inter-lysosomal (L-L) tethering (white arrows; inset in H; I) in live HeLa cells expressing Mid51 (lysosome LAMP1-mGFP). Scale bars, 5 μm (G); 0.5 μm (H and I). Video 8 corresponds to H. Source data are available for this figure: SourceData FS3.

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