Safirstein, R. | Search bioRxiv

bioRxiv2025

Renalase activates mitochondrial leak metabolism in response to cellular stress and to repair damage after injury

A variety of mechanisms enhance cell stress response and repair; however, the role of mitochondria in this activity is unclear. Here we show that exogenous renalase (RNLS), an intracellular flavin-dependent NADH oxidase, activates intramitochondrial RNLS activity to promote cell survival. RNLS interacts with the ATP synthase alpha and beta subunits (ATP5 and ATP5{beta}) and opens the ATP synthase c-subunit leak channel to increase complex I and II activities and protein synthesis rate. RNLS causes a selective, sustained, time-dependent increase in cellular protein synthesis without affecting cell proliferation, whereas RNLS deletion or direct inhibition of the mitochondrial leak blocks RNLS-mediated protein synthesis. Functional analysis of newly and differentially synthesized proteins over 24 hours reveals rapid changes in one-carbon metabolism and ribosomal biogenesis pathways as early as one hour after RNLS exposure. Mitochondrial injury is more severe in the RNLS KO kidney after acute stress, related to decreased protein synthesis rate and increased mitophagy. RNLS KO mice exposed to the stress of chronic cardiac pressure overload fail to develop cardiac hypertrophy, the physiological response, and die of heart failure and cardiac rupture. These data highlight the critical role RNLS has in activating mitochondrial leak metabolism to induce selective protein synthesis and protect against acute and chronic stress. HIGHLIGHTSO_LIRenalase interacts with the ATP synthase alpha and beta subunits C_LIO_LIRenalase activates mitochondrial leak metabolism C_LIO_LIRenalase and leak metabolism increase complex I and II activities C_LIO_LILeak metabolism increases protein synthesis rate C_LIO_LIRenalase protects against cell stress and organ injury C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/671117v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@96ea7eorg.highwire.dtl.DTLVardef@1fcffbaorg.highwire.dtl.DTLVardef@f946cdorg.highwire.dtl.DTLVardef@197498a_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2025

Scalable automated segmentation quantifies mitochondrial proteins and morphology at the nanoscale

Ultrastructural changes of mitochondria are closely associated with metabolic dysfunction, leading to a variety of human disorders. These changes can be visualized by pan-Expansion Microscopy (pan-ExM), a 3D microscopy technique requiring only standard fluorescence microscopes, at a throughput exceeding that of the current gold standard, 3D electron microscopy, by orders of magnitude. However, a lack of tools that enable the characterization and quantification of the observed ultrastructural features in the acquired 3D datasets at a comparable throughput has hindered the widespread adoption of pan-ExM for quantitative imaging. Here, we present an automated deep-learning based segmentation approach that utilizes pan-ExMs power to acquire multi-channel images and uses specific labeling as the annotation for the training of the segmentation network. This molecular annotation reduces the required manual annotation effort for mitochondria substructures to just a few hours when setting up the experiment and thereby provides access to 3D suborganellar morphology of mitochondria at an unprecedented throughput. Our approach, which we term MAPS (Mitochondrial Automated Pan-ExM Segmentation), enables for the first time to quantify mitochondrial ultrastructural morphology at scale. We demonstrate this power by characterizing the 3D mitochondrial morphology at the organelle and sub-organelle level in tens of HeLa cells under different treatments and localizing mitochondrial proteins in the sub-organellar context. To demonstrate our technology in tissue, we compare the ultrastructural morphology of mitochondria in proximal tubules of kidneys of mice exhibiting acute kidney injury (AKI) with those of untreated mice, revealing striking differences in their cristae structure. MAPS can easily be adapted to different cell and tissue types, allowing the analysis of tens of samples per day, and therefore provides a versatile tool for a comprehensive understanding of mitochondrial ultrastructural changes in many disease contexts. Requiring only standard fluorescence microscopes and computer infrastructure, MAPS is readily adoptable by any lab. HighlightsO_LIMAPS utilizes specific labeling to train a segmentation model for 3D super-resolution pan-ExM images C_LIO_LISuborganellar mitochondrial features and their changes in disease models are quantified at high throughput C_LIO_LI3D protein distributions are correlated to ultrastructural features in mitochondria C_LIO_LIRequiring only standard lab infrastructure, MAPS is readily adoptable C_LI

cell biology↗