MitoCRISTAE set out to develop tools to analyse mitochondrial cristae dynamics and to establish a quantitative framework for crista biogenesis in health and disease. These aims have been fully achieved. The project has generated an extensive toolbox – including >40 genome-edited cell lines, new fluorescent dyes, advanced imaging protocols and quantitative analysis pipelines – and has led to major conceptual advances in crista biology. To date, this work has produced 27 peer-reviewed original articles, a review, a book chapter and nine preprints, with additional manuscripts in preparation.
Methodologically, the project has transformed crista imaging from a niche capability into a robust, broadly accessible technology. We established cell lines expressing SNAP-tag fusion proteins targeted to inner mitochondrial membranes and combined them with STED and related super-resolution modalities, enabling routine visualization of crista dynamics in living cells. In parallel, we co-developed highly specific crista-targeted dyes that perform reliably across diverse cell types, including neurons and primary human cells, eliminating the need for genome editing in many contexts. These approaches are now sufficiently mature to be used in teaching super-resolution microscopy and have been widely disseminated through several comprehensive reviews.
We further pushed the state of the art in nanoscale imaging by pioneering MINFLUX nanoscopy for mitochondrial proteins, particularly components of the MICOS complex and ATP synthase. This allowed counting and tracking of individual molecules at crista junctions and contributed directly to the technological evolution of MINFLUX in close collaboration with the group of Stefan Hell. Complementary use of RESOLFT, 4Pi-STORM, MINSTED, FIB-SEM and electron tomography provided multi-scale views of crista architecture across developmental stages and in defined perturbations. Mass-spectrometry–based interactomics and complexome profiling revealed a surprisingly stable composition of key crista-forming complexes, even during pronounced morphological remodeling, highlighting a robust molecular scaffold underlying dynamic inner-membrane restructuring.
Conceptually, MitoCRISTAE has delivered a refined model of lamellar crista biogenesis in higher eukaryotes. Our data support a sequence in which MICOS-independent inner-membrane infoldings are remodelled by MICOS into mature lamellar cristae with secondary crista junctions. Junction position and morphology are further tuned by the Mic10 subcomplex, OPA1 and F₁F₀-ATP synthase. Stacked cristae appear to arise from single precursor membranes, a mechanism that may generalise to mitochondria with alternative architectures. We demonstrated how mutations in these pathways, including disease-associated OPA1 variants, derail crista formation and impair mitochondrial function.
The project has also catalysed translation and capacity building. Foundational results enabled a major public–private HRDS consortium grant to exploit high-resolution imaging for drug discovery, and supported an ERC Synergy application on single-molecule nanoscopy. Team members have moved into leading positions in academia and core facilities, ensuring continued impact, and the work has attracted substantial public interest, including a prime-time television documentary.
