MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking (2021)
Figure 5

Figure 5. MICAL2PV interacts with Miro1/2

A Lysates of H1299 cells expressing Flag-MICAL2PV were immunoprecipitated using an anti-Flag antibody, and immunoprecipitated proteins were analyzed by Western blotting using different antibodies as indicated.

B H1299 cells were co-transfected with different combinations of plasmids as indicated. The cell lysates were immunoprecipitated using an anti-Myc antibody. Immunoprecipitated proteins were analyzed by Western blotting using different antibodies as indicated.

C The monooxygenase (MO) domain of MICAL2PV interacts with Miro2. A schematic representation of protein domain organization in MICAL2PV and its truncation mutants was shown in (panel a). H1299 cells were co-transfected with Myc-Miro2 and different HA-tagged MICAL2PV mutants. Following transfection, cells were lysed and immunoprecipitated using an anti-HA antibody and subjected to Western blotting with different antibodies (panel b).

D MICAL2PV interacts with N-GTPase, EF hand and C-terminal domains of Miro2, but not with the C-GTPase domain. A schematic representation of protein domain organization in Miro2 and its truncation mutants was shown in (panel a). H1299 cells were co-transfected with Flag-MICAL2PV and different Myc-tagged Miro2 mutants. Transfected cells were lysed, and immunoprecipitation was performed using an anti-Myc antibody and subjected to Western blotting with different antibodies as indicated (panel b). It should be noted that the upper band (marked by "*") migrating closely above the Flag-M2 band in the top section of panel b is a non-specific species because it is present in anti-Myc immunoprecipitates of cells only expressing the vector control, not expressing any Myc-Miro2 (lane 1).

E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.

F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.