INV-induced mitochondrial aggregation as an assay for TPD54 binding to INVs. (A) Schematic diagram of vesicle capture at mitochondria and their subsequent aggregation. MitoTrap is an FRB domain targeted to mitochondria, XFP-FKBP-TPD54 (XFP is GFP or mCherry) is coexpressed, and, when rapamycin is added, the INVs associated with TPD54 become trapped at the mitochondria, eventually causing aggregation of mitochondria. (B) CLEM experiment to demonstrate INVs between aggregated mitochondria. Cells expressing GFP-FKBP-TPD54 and mCherry-MitoTrap were imaged before light microscopy (LM), before (Pre) and after (Post) rapamycin 200 nM addition for 3 min. An ultrathin section of the same cell is shown by transmission EM. Inset: 4× zoom. Scale bars, 500 nm or 50 nm (insets). (C) Representative confocal micrographs of HeLa cells expressing dark MitoTrap and either mCherry-FKBP-TPD54 WT (1–206) or R159E mutant, treated with 200 nM rapamycin. Inset: 5× zoom. Scale bars, 10 µm or 1 µm (inset). (D) Quantification of mitochondrial aggregation in cells expressing mCherry-FKBP–tagged TPD54 constructs and GFP-MitoTrap, treated with 200 nM rapamycin for 30 min. Dots show average mitochondrial shape (high values are more aggregated) per cell (see Materials and methods). Box plots show interquartile range (IQR), bar represents the median, and whiskers show 9th and 91st percentiles. Dunnett’s post hoc test was done using mCherry-FKBP as control; blue indicates P < 0.05.