Periodic Reporting for period 4 - MitoCRISTAE (Mitochondrial Cristae Biogenesis)
Periodo di rendicontazione: 2024-04-01 al 2025-09-30
Every cell in our body needs energy to work properly. This energy is mainly produced in tiny structures inside the cell called mitochondria, often described as the cell’s “powerhouses.” Mitochondria have an outer and an inner membrane. The inner membrane is folded many times into thin layers, a bit like the folds of an accordion. These folds are called cristae, and they are where most of the energy-carrying molecule ATP is made. When mitochondria do not work well, cells run low on energy, and this is linked to many human diseases. Changes in the shape and folding of the inner membrane often go hand in hand with such mitochondrial problems, but we still do not fully understand why.
The MitoCRISTAE project aims to uncover how these inner membrane folds are formed, maintained, and changed in healthy and diseased cells. To do this, the team uses cutting-edge imaging technologies, especially super-resolution light microscopy, which allows scientists to see details inside cells that were previously invisible. For the first time, they will be able to watch how cristae appear, change shape, and reorganize over time in living cells. By linking these dynamic structural changes to how well mitochondria produce energy, MitoCRISTAE will help explain how mitochondrial architecture supports cell health—and how its disruption contributes to disease.
The MitoCRISTAE project aims to uncover how these inner membrane folds are formed, maintained, and changed in healthy and diseased cells. To do this, the team uses cutting-edge imaging technologies, especially super-resolution light microscopy, which allows scientists to see details inside cells that were previously invisible. For the first time, they will be able to watch how cristae appear, change shape, and reorganize over time in living cells. By linking these dynamic structural changes to how well mitochondria produce energy, MitoCRISTAE will help explain how mitochondrial architecture supports cell health—and how its disruption contributes to disease.
During the reporting period, the team developed new ways to see the fine structure of mitochondria, in particular the cristae, in both living and chemically fixed cells using super-resolution light microscopy. They also created many tailored cell models in which specific proteins were removed. Some of these proteins are known to be important for building and maintaining cristae, or are linked to mitochondrial diseases in patients. By comparing cells with and without these proteins, the team could study in detail how each protein contributes to the formation and stability of cristae.
These new methods have become so reliable that they are now used by many other research groups and have even been integrated into courses to teach students super-resolution microscopy. Using several cutting-edge imaging techniques—including the first applications of a very powerful method called MINFLUX nanoscopy to mitochondria—the team observed large-scale rearrangements of the inner membrane and striking changes in the position of many inner membrane proteins when normal mitochondrial architecture was restored. These findings show that many different factors must work together to build and maintain healthy cristae. The results have been published in a number of scientific papers and were also featured on German television, bringing the research to a wider audience.
These new methods have become so reliable that they are now used by many other research groups and have even been integrated into courses to teach students super-resolution microscopy. Using several cutting-edge imaging techniques—including the first applications of a very powerful method called MINFLUX nanoscopy to mitochondria—the team observed large-scale rearrangements of the inner membrane and striking changes in the position of many inner membrane proteins when normal mitochondrial architecture was restored. These findings show that many different factors must work together to build and maintain healthy cristae. The results have been published in a number of scientific papers and were also featured on German television, bringing the research to a wider audience.
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.
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.