FIG. 15.

FLIP analysis of single mitochondrion in HEK293 cells. HEK293 cell lines were created that stably expressed mitochondria-targeted GFP (COX8-AcGFP1) or a GFP-tagged version of the NDUFS3 subunit of CI (NDUFS3-AcGFP1). Analysis of single mitochondria by FRAP revealed that AcGFP1 was fully mobile, whereas NDUFS3-AcGFP1 was partially immobilized in the mitochondrial matrix (85). Here, we used FLIP analysis to determine the submitochondrial localization of immobile NDUFS3-AcGFP1. (A) In COX8-AcGFP1 cells, repetitive bleaching of mitochondrial AcGFP1 using a 1.4 μm square bleach region induced complete loss of fluorescence in the bleach and FLIP region within 12 s. Visual inspection revealed that AcGFP1 fluorescence was completely lost from the mitochondrial filament (inset). (B) In contrast, for NDUFS3-AcGFP1 cells, repetitive AcGFP1 bleaching induced complete fluorescence loss in the bleach region but not in the FLIP region (arrowhead). In this case, visual inspection confirmed that NDUFS3-AcGFP1 fluorescence was absent in the bleach region but not outside this region. (C) Explanation of the obtained results with NDUFS3-AcGFP1 cells. Repetitive photobleaching induces loss of fluorescence for the mobile fraction of NDUFS3-AcGFP1 but not for the immobile fraction of NDUFS3-AcGFP1, which appears to be localized (arrowheads). (D) Magnification of a typical FLIP experiment illustrating the localization of the immobile NDUFS3-AcGFP1 fluorescence fraction (arrowhead). This might represent local compartments of the mitochondrial matrix that restrict NDUFS3-AcGFP1 diffusion and/or NDUFS3-AcGFP1 that is membrane bound in CI (sub)complexes or ETC supercomplexes. (C) and (D) were taken from (83). ETC, electron transport chain; FLIP, fluorescence loss in photobleaching. Color images are available online.