Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down‐regulation concomitant to MICOS clustering

Abstract

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/ topogenesis of mitochondrial outer membrane b-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-b indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs. monomers and state-3/ state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMMdetached cristae of OPA1-and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-

Regulation of glucose‐stimulated insulin secretion by ATPase Inhibitory Factor 1 (IF1)

“…C). We propose that this cluster organization of IF1 is due to localization of IF1 within certain microdomains, presumably matrix compartments of mitochondrial cristae, as also mitochondrial ATP synthase and other proteins of intracristal membrane have been shown to form similar clusters .…”

Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation

“…OPA1 cooperates with the MICOS subunits Mic60/Mitofilin and Mic19/CHCHD3 to maintain cristae morphology (26), which is crucial to adapt cells to physiological changes. It has recently been demonstrated that MICOS-OPA1 synergy regulates the response to hypoxia by modulating cristae outlets and decreasing the number of ATP synthases dimers (50). Moreover, hDISC1 has been reported previously to inhibit glycogen synthase kinase 3b (GSK3b) (51), which in turn phosphorylates Drp1, increasing its GTPase activity and mitochondrial recruitment, with the resulting trigger of mitochondrial fragmentation (52).…”

Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites

“…Moreover, Blue Native (BN)-PAGE followed by western blotting with Miro1 and Miro2 antibodies revealed the appearance of complexes at 1000 /1200 and 500 -700 kDa that are similar to complexes detected with Mic19/CHCHD3, Sam50 or Mic60/Mitofilin antibodies ( Fig. 3F) 50,53,55,56 . Thus, Miro proteins can be detected in a complex with key components of the MICOS complex on mitochondria.…”