Biology subjects

Bily, T.

Publications and source records attributed to Bily, T..

5 recordsLinked to original sources

bioRxiv2026

Trypanosomal MICOS is assembled on non-respiring mitochondrial crista precursors and associates with two integral microproteins

The mitochondrial contact site and cristae organizing system (MICOS) is a multiprotein complex that shapes crista junctions and maintains inner and outer membrane contacts. MICOS coordinates the assembly of electron transport chain complexes, a prerequisite for cellular respiration. Indeed, MICOS is lost in eukaryotes that dispensed with cellular respiration, suggesting that its assembly depends on the presence of an active respiratory chain. Trypanosoma brucei provides a unique system to test this hypothesis as its mitochondrion undergoes developmentally regulated remodeling. In the insect stage, the mitochondrion contains cristae with an active electron transport chain, whereas the mammalian bloodstream form possesses precursor cristae with stub-like morphology that lack respiratory activity. MICOS has been characterized in the insect stage but remains unexamined in the bloodstream form. Here, we demonstrate that all MICOS subunits assemble onto precursor cristae, retaining conserved interactions with both outer and inner membrane protein machineries. This is somewhat unexpected given the co-occurrence of MICOS with active cellular respiration in nature. Furthermore, we identify novel MICOS-associated proteins that are dispensable for its stability, suggesting auxiliary rather than core roles in MICOS function. Together, our findings establish that MICOS assembly precedes cellular respiratory competence and expand its interaction landscape in trypanosomatids.

cell biology↗

bioRxiv2025

In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside living cells.

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. An InCellCryst pipeline was recently established for structure determination by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution structural studies on proteins with low numbers of crystals. To overcome this limitation, we combined the InCellCryst approach with the advantages of 3D electron diffraction and established the new IncellED method. Using microcrystals of the HEX-1 protein from Magnaporthe grisea, grown inside High Five insect cells, we demonstrate that electron diffraction data collected from a single crystal yields a high-resolution structure at 1.9 [A] resolution, comparable to 1.8 [A] achieved by serial X-ray crystallography. The IncellED methodology opens structure determination for proteins that crystallize with low efficiency and use widely available electron cryo-microscope infrastructure, paving the way for intracellular macromolecular electron crystallography at high resolution.

biophysics↗

bioRxiv2025

The core MICOS complex subunit Mic60 has been substituted by two cryptic mitofilin-containing proteins in Euglenozoa

Cristae enclose respiratory chain complexes, making them the bioenergetic subcompartments of mitochondria. The MICOS complex is among the inducers of membrane curvature needed for crista formation. Resembling the respiratory chain complexes, MICOS is organized around a core protein, the mitofilin-domain bearing Mic60, that was inherited from the alphaproteobacterial progenitor of mitochondria. Extant alphaproteobacteria express Mic60 to form their own bioenergetic subcompartments, demonstrating the permeance of Mic60s form and function during prokaryotic and eukaryotic evolution. Yet, unlike virtually all aerobic eukaryotes, Mic60 is not encoded within the genomes of the multifarious protists that comprise the phylum Euglenozoa, including trypanosomes. Here, we show that Mic60 has been replaced in euglenozoans by two cryptic mitofilin domain-containing MICOS subunits, Mic34 and Mic40. Contrasting alphaproteobacterial and mitochondrial Mic60, these are not integral membrane proteins. Mic34 and Mic40 are as diverged from each other as both are to canonical Mic60. Reverse genetics revealed they are intertwined with the oxidative protein folding pathway required for mitochondrial-and crista-biogenesis, veiling a potential membrane remodeling role. Nevertheless, Mic34 binds phospholipid bilayers in vitro. Mic34 and Mic40 heterologous expression remodels gammaproteobacterial cytoplasmic membranes, like Mic60. Unexpectedly, Mic34 overexpression elaborates the simplified tubular mitochondrion of a Trypanosoma brucei life cycle stage with repressed oxidative phosphorylation. Furthermore, this activity was ablated by mutations to Mic34s mitofilin domain that correspond to essential motifs found in yeast Mic60s mitofilin domain. Thus, the mitofilin protein family is more diverse than originally supposed, with two of its structurally most divergent members altering the core of euglenozoan MICOS.

molecular biology↗

bioRxiv2022

The development of intracytoplasmic membranes in alphaproteobacteria involves the conserved mitochondrial crista-developing Mic60 protein

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three -helices of mitochondrial Mic60s mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs, and therefore have improved our understanding of the nature of the mitochondrial ancestor.

evolutionary biology↗

bioRxiv2021

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

biochemistry↗