Biology subjects

Stephan, T.

Publications and source records attributed to Stephan, T..

8 recordsLinked to original sources

bioRxiv2026

AMPK reinforces mitochondrial metabolism and suppresses pathological remodeling in Complex V-deficient cardiomyocytes

TMEM70 variants represent the most common nuclear cause of mitochondrial ATP synthase (Complex V) deficiency and are associated with particularly severe cardiac manifestations. Yet, how TMEM70 deficiency disrupts cardiomyocyte metabolic maturation and function remains poorly understood, in part because suitable human disease models are lacking. Here, we model TMEM70-related Complex V deficiency using CRISPR-engineered human induced pluripotent stem cells differentiated into cardiomyocytes. While TMEM70-deficient pluripotent cells retain mitochondrial function, differentiated cardiomyocytes develop reduced mitochondrial membrane potential, impaired respiratory capacity, and pathological remodeling, revealing a differentiation-dependent failure of metabolic maturation. Chronic activation of AMP-activated protein kinase (AMPK) restores mitochondrial respiratory capacity despite persistent Complex V deficiency. Proteomic and metabolomic analyses reveal that AMPK activation in TMEM70-deficient cardiomyocytes reinforces mitochondrial and fatty-acid metabolism while suppressing pathological structural remodeling, accompanied by improved cardiac function. Together, these findings identify AMPK-dependent metabolic remodeling as a state-dependent mechanism of functional rescue in mitochondrial cardiomyopathy.

cell biology↗

bioRxiv2025

Detergent-free reconstitution of transmembrane proteins in giant liposomes of complex curvature by the Synthetic Membrane Transfer

Transmembrane proteins perform many crucial functions in the cell, including transfer of matter and information across the membrane. Giant Unilamellar Vesicles (GUVs) are the ideal tool for studying these processes; yet functional transmembrane protein reconstitution in GUVs still represents a bottleneck in bottom-up synthetic biology. Here, we developed a novel approach that we call Synthetic Membrane Transfer (SMT), where transmembrane proteins reconstituted into styrene-maleic acid particles in a detergent-free environment can be transferred to the GUV membrane. The SMT approach is a one-step, facile and general method that works for structurally diverse proteins, and it does not require any specific lipid or buffer composition. Moreover, the SMT is fully compatible with the Synthetic Membrane Shaper (SMS) technology, which allows to deform GUVs in a controlled fashion, obtaining dumbbell-shaped GUVs exhibiting a catenoid-like geometry. The combination of SMT and SMS results in functional reconstitution of transmembrane proteins in catenoid membrane necks. Using this approach, we demonstrate that Mic10, a component of the MICOS complex, directly senses membrane curvature and localizes at the neck of dumbbells GUVs, a geometry that recapitulates the shape of mitochondria cristae junctions. This paves the way for bottom-up reconstitution of the MICOS complex on a physiological membrane geometry.

synthetic biology↗

bioRxiv2024

Mitochondrial inner membrane remodeling as a driving force of organelle shaping

Mitochondria are dynamic organelles exhibiting diverse shapes. While the variation of shapes, ranging from spheres to elongated tubules, and the transition between them, are clearly seen in many cell types, the molecular mechanisms governing this morphological variability remain poorly understood. Here, we propose a novel shaping mechanism based on the interplay between the inner and outer mitochondrial membranes. Our biophysical model suggests that the difference in surface area, arising from the pulling of the inner membrane into cristae, correlates with mitochondrial elongation. Analysis of live cell super-resolution microscopy data supports this correlation, linking elongated shapes to the extent of cristae in the inner membrane. Knocking down cristae shaping proteins further confirms the impact on mitochondrial shape, demonstrating that defects in cristae formation correlate with mitochondrial sphericity. Our results suggest that the dynamics of the inner mitochondrial membrane are important not only for simply creating surface area required for respiratory capacity, but go beyond that to affect the whole organelle morphology. This work explores the biophysical foundations of individual mitochondrial shape, suggesting potential links between mitochondrial structure and function. This should be of profound significance, particularly in the context of disrupted cristae shaping proteins and their implications in mitochondrial diseases.

cell biology↗

bioRxiv2023

The Drosophila MIC10 orthologue has a propensity to polymerize into cristae-shaping filaments

Mitochondria are essential eukaryotic double-membrane organelles. The convoluted mitochondrial inner membrane forms highly organized invaginations, termed cristae, which are crucial for energy metabolism. Cristae formation requires MICOS, a conserved hetero-oligomeric inner membrane complex. The MICOS core subunit MIC10 is a small transmembrane protein that oligomerizes through highly conserved glycine-rich motifs to control cristae formation. Sequence alignments show that D. melanogaster exhibits three MIC10-like proteins with different tissue-specific expression patterns. Here, we show that the ubiquitously expressed Dmel_CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Loss of DmMIC10b disturbs cristae architecture of mitochondria and reduces the life-span and fertility of flies. Moreover, using fluorescence nanoscopy and electron tomography, we demonstrate that despite its high similarity to the MIC10 proteins from yeast and humans, DmMIC10b exhibits the unique ability to polymerize into elongated filaments upon overexpression. DmMIC10b filaments form bundles which accumulate in the intermembrane space and alter the shape of mitochondrial cristae membranes. We show that the formation of the filaments relies on conserved glycine and cysteine residues and is suppressed by co-expression of other MICOS proteins. Thereby, our findings provide new insights into the regulation of MICOS in flies.

cell biology↗

bioRxiv2023

Deep learning enables fast, gentle STED microscopy

STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that denoising STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

bioengineering↗

bioRxiv2022

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Capturing mitochondrias intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to 2D single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene to a benzo-fused cyanine dye, we report a mitochondrial inner-membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability and markedly reduced phototoxicity. PKMO enables super-resolution recordings of inner-membrane dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell 3D STED nanoscopy of mitochondria for three-dimensional analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED, we demonstrate that imaging with PKMO can capture the sub-mitochondrial localization of proteins, or interactions of mitochondria with different cellular components, such as the ER or the cytoskeleton at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial inner-membrane architecture and dynamics in a multiplexed manner.

cell biology↗

bioRxiv2021

Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

biophysics↗

bioRxiv2019

Live-cell STED microscopy of mitochondrial cristae

Mitochondria are highly dynamic organelles that exhibit a complex inner architecture. They exhibit a smooth outer membrane and a highly convoluted inner membrane that forms invaginations called cristae. Imaging cristae in living cells poses a formidable challenge for light microscopy. Relying on a cell line stably expressing the mitochondrial protein Cox8A fused to the SNAP-tag and using STED (stimulated emission depletion) super-resolution microscopy, we demonstrate the visualization of cristae dynamics in cultivated human cells.

cell biology↗