MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation

Abstract

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F 1 F o ‐ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super‐resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re‐expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre‐existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60‐subcomplex is sufficient for CJ formation, whereas the Mic10‐subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F 1 F o ‐ATP synthase, fine‐tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.

Deuterated rhodamines for protein labelling in nanoscopy

“…2B), which has been used to study mitochondrial ultrastructure in live cells. [15,16] As for SNAP-GLP1R, we observed clean labelling with all dyes, and for both colors with an observable increase in brightness for the d12 derivatives. With this enhanced performance in microscopy, we wanted to quantify brightness by fluorescence activated cell sorting (FACS) to obtain robust values over large sample sizes.…”

“…SNAPf was expressed and purified as described previously [16] and complete amino acid sequences for constructs used can be found in the were maintained in DMEM supplemented with 10% FCS, 1% penicillin/streptomycin, 500 μg/mL G418, 25 mM HEPES, 1% nonessential amino acids and 2% L-glutamine. HeLa cells stably expressing SNAP-Cox8A [15] (Cox8A-SNAP:HeLa) were maintained in DMEM supplemented emission ratio.…”

Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins

“…Upon expression of Mic10-FLAG-T2A-EGFP, the self-cleaving T2A peptide caused the release of EGFP into the cytoplasm, whereas Mic10-FLAG was imported into the mitochondria. Induction of the expression of the Mic10-FLAG fusion protein fully rescued the wild-type cristae morphology from the aberrant cristae architecture of Mic10-depleted cells, demonstrating that Mic10-FLAG compensates for the lack of endogenous Mic10 (15). We used an anti-FLAG antibody (directly labeled with CF680) to decorate Mic10-FLAG in induced Mic10-TO cells and an anti-Mic60 antibody (directly labeled with Alexa Fluor 647) to label the endogenous Mic60.…”

Cristae Membrane Dynamics – A Paradigm Change

“…This motif is present in both the transmembrane segments of Mic10 and is essential to induce membrane curvature in baker's yeast. The de novo formation of CJs was shown recently by drug-controlled expression of MIC60 or MIC10 in respective knockout (KO) HeLa cells [36]. Owing to their key functions in IM organization, it is not surprising that the MICOS complex subunits Mic10 and Mic60 are evolutionarily well conserved [37,38].…”

“…On one hand, the latter could be rate-limiting for metabolite exchange but, on the other, may be beneficial for trapping protons inside CVs to promote ATP production by OXPHOS provided that sufficient ADP is available. Consistently, loss of MICOS subunits is known to lead to impaired cellular respiration [13,36,39,40,[63][64][65]. In addition, proton trapping may also result from increased concentration of protons at the cristae tip resulting from strong membrane curvature induced by rows of ATP synthase dimers [72], and from the capacity of CL to buffer protons at the IM [90].…”