IF1, the endogenous regulator of the F1Fo-ATPsynthase, defines mitochondrial volume fraction in HeLa cells by regulating autophagy

Abstract

The protein IF1 limits mitochondrial ATP consumption when mitochondrial respiration is impaired by inhibiting the 'reverse' activity of the F(1)F(o)-ATPsynthase. We have found that IF1 also increases F(1)F(o)-ATPsynthase activity in respiring mitochondria, promoting its dimerization and increasing the density of mitochondrial cristae. We also noted that IF1 overexpression was associated with an increase in mitochondrial volume fraction that was conversely reduced when IF1 was knocked down using small interfering RNA (siRNA). The volume change did not correlate with the level of transcription factors involved in mitochondrial biogenesis. However, autophagy was dramatically increased in the IF1siRNA treated cells (-IF1), assessed by quantifying LC3-GFP translocation to autophagosomes, whilst levels of autophagy were low in IF1 overexpressing cells (+IF1). The increase in LC3-GFP labelled autophagosomes in -IF1 cells was prevented by the superoxide dismutase mimetic, manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP). An increase in the basal rate of generation of reactive oxygen species (ROS) in -IF1 cells was demonstrated using the fluorescent probe dihydroethidium (DHE). Thus, IF1 appears to limit mitochondrial ROS generation, limiting autophagy which is increased by IF1 knockdown. Variations in IF1 expression level may therefore play a significant role in defining both resting rates of ROS generation and cellular mitochondrial content.

Assessing Actual Contribution of IF1, Inhibitor of Mitochondrial FoF1, to ATP Homeostasis, Cell Growth, Mitochondrial Morphology, and Cell Viability

“…IF1 Does Not Affect Mitochondria Morphology-Previous studies reported that knockdown of IF1 caused stimulation of autophagy, decrease in mitochondrial volume fraction in the cell, and decrease in cristae density in mitochondria (10,11,13,14). However, none of these were observed for IF1-KD cells with certainty in our study.…”

“…When cells are deprived of oxygen and glucose, IF1-suppressed cells lose cellular ATP and die more rapidly than control cells, suggesting a role of IF1 in maintaining ATP concentration ([ATP]) in energy crises (9). Several groups reported that IF1 facilitates dimer formation of F o F 1 in the inner mitochondrial membranes (10 -12) and contributes to cristae formation (9,(13)(14)(15)(16)(17)(18). This profound effect of IF1 on mitochondrial morphology remains controversial (19,20).…”

The pathways of mitophagy for quality control and clearance of mitochondria

“…Although mitochondria can be engulfed non-selectively along with other cytosolic contents during bulk autophagy, 31 32,33 Similarly, knockdown of an inhibitor of the mammalian F 0 F 1 ATPsynthase, IF1, causes a dramatic reduction in mitochondrial mass that correlates with increased autophagic activity. 34 Depolarization of mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) 35 or photo-irradiation 36 induces autophagic clearance of mammalian mitochondria while leaving other organelles such as peroxisomes intact. Recently, it has been reported that mitochondria carrying deleterious mutations in mtDNA can be selectively eliminated through mitophagy, 37 although additional activation of macroautophagy, such as rapamycin-mediated inhibition of TORC1, may be required for efficient clearance.…”

Regulation and Quantification of Cellular Mitochondrial Morphology and Content

“…For reasons explained above this strategy is less applicable to thicker specimens [222]. However, a "semi-3D" image can be created by collapsing multiple imagesections from a wide-field or confocal z-stack into a single 2D projection [224,261]. A more advanced strategy consists of calculating a 3D representation of the mitochondrial compartment from the z-stack and performing a 3D analysis of mitochondrial number, morphology (e.g.…”

“…respect to mitochondrial content, the fractional cellular volume occupied by mitochondria has been measured based on fluorescence detection of mitoGFP and calcein blue in the cytosol [224]. Alternatively, we and others have developed procedures allowing systematic analysis of mitochondrial shape, number and mass using fluorescence microscopy images ( Table 2).…”