7/🔬 Using TEM, cryo-ET and fluorescence microscopy, we describe a phenotype that is specific to a mutant of these four ASR-identified residues, which features box-like shaped cristae and longer average cristae lengths.

6/🔬 Several TEM studies have shown that Mic60 and Mitofilin deletions result in detached cristae. We observe the same phenotype for our knockout variants. ✅

🤯 Surprisingly, Mitofilin ancestors which rescue respiratory growth like wildtype have knockout-like detached cristae.

5/🎯 Comparing reconstructed ancestors, we identified four residues in the Mitofilin domain that are sufficient for rescuing respiration.

4/🧫 We found that common Mitofilin ancestors as far back as the putative last opisthokont common ancestor #LOCA rescues respiration in yeast. ✅

3/❓How did Mitofilin-supported respiration diverge between yeast 🍺 and humans 👤?

💻🦖🧪 We used ancestral sequence reconstruction #ASR to reconstruct ancestral Mitofilin sequences and resurrected them for in vivo characterization.

2/ 🧫 Using respiratory growth assays, we found that swapping the yeast Mitofilin domain with that of another yeast rescued respiration. ✅

Replacing it with human or bacterial homologues at the endogenous locus did not rescue respiration. 💀

❗This is in contrast to previous plasmid-based work.

1/ ❓Mic60, a core component of the #MICOS complex in the #mitochondrial inner membrane, is crucial for the organization of #cristae and their functions. It's unclear, how Mic60's #Mitofilin domain encodes cristae-supported functions like cellular respiration.