Biology subjects

Graef, M.

Publications and source records attributed to Graef, M..

6 recordsLinked to original sources

bioRxiv2026

Mechanical stress-induced release of mitochondrial double-stranded RNA activates RIG-I to drive osteogenic differentiation in aortic disease

Background: Gain-of-function mutations in RIG-I and MDA5 (RIG-I-like receptors, RLR) cause Singleton-Merten syndrome, an interferonopathy with early aortic calcification. Here we set out to study a possible involvement of RIG-I-like receptor signaling in the formation of aortic valve stenosis in general. Methods and results: We investigated whether mechanical stress-induced RLR activation promotes osteogenic differentiation and calcification of primary human aortic cells, whether such RLR activation drives the development of aortic valve stenosis in vivo, and whether inhibition of downstream JAK/STAT activation ameliorates aortic valve stenosis. We found that activation of primary human aortic interstitial cells by exogenous RIG-I- and MDA5 agonists induced a type I interferon response and an osteogenic program including increased expression of BMP-2 and BSP2, and promoted calcification. These effects were RIG-I/MDA5 or MAVS-dependent, as assessed by CRISPR/Cas9 knockouts. The stress-granule protein G3BP1 and the Wnt receptor FZD5 were induced downstream of RLR activation and were required for osteogenic gene expression and calcification. To assess a potential endogenous source of RIG-I activation, mechanical stretch mimicking valvular tissue stress was applied in cell cuture, which induced a significant RIG-I-dependent interferon response. Sequencing of highly purified preparations of double-stranded RNA from stretched cells demonstrated a predominant mitochondrial origin of double-stranded RNA, and depleting mitochondrial RNA abolished the stretch-dependent response. The JAK/STAT inhibitor ruxolitinib suppressed the RIG-I-induced response including the expression of osteogenic factors in vitro. In a murine wire-injury model of aortic stenosis, MAVS deficiency attenuated valvular morphological changes and improved hemodynamic parameters. Ruxolitinib attenuated hemodynamic progression, prevented aortic stenosis development, and improved survival. Conclusions: Altogether, our results demonstrate that the mechanical-stress-induced release of mitochondrial double-stranded RNA activates RIG-I signaling in aortic interstitial cells, thereby driving osteogenic differentiation and calcification via a MAVS- and G3BP1-dependent pathway. JAK/STAT inhibition with ruxolitinib mitigates this process in a mouse model of aortic valve stenosis, identifying a candidate therapeutic strategy.

cell biology↗

bioRxiv2026

In situ visualization of autophagy suggests vesicle fusion can contribute to phagophore expansion

The autophagy core machinery mediates the enclosure of cytosolic cargo destined for degradation in the lysosome. The Atg9-Atg2-Atg18 complex coordinates phagophore expansion via directed lipid transfer until closure of the phagophore rim. Using an Atg2 variant (Atg2-PM4) as a model of decelerated autophagosome biogenesis, we visualized the morphological states prior to autophagosome closure by cryogenic correlative light and electron microscopy in S. cerevisiae. Using in situ cryo-electron tomography, we find an enlarged rim morphology of an expanding phagophore in Atg2-PM4 cells in comparison with Atg2 wildtype condition. Analysis of segmented rim membrane features as well as surrounding and attached vesicles suggest that the enlarged rims are a result of cytosolic vesicles fusing with the growing phagophore. High-resolution imaging in this study shows that, apart from the initial nucleation phase, vesicle fusion can also contribute to phagophore expansion during later stages of autophagosome biogenesis.

cell biology↗

bioRxiv2023

Homeostatic mitophagy scales mitochondrial networks

Cells scale organelle sizes to ensure physiological function. Here, we uncover a dual role for autophagy in controlling mitochondrial network size with a key function for homeostatic mitophagy. During starvation, non-selective autophagy sustains mitochondrial biogenesis in non-dividing yeast cells resulting in mitochondrial network expansion. Strikingly, Atg32/Bcl2L13-mediated mitophagy scales mitochondria back to pre-starvation size. Without mitophagy, mitochondria size increases two-fold while retaining wildtype-like structural, compositional, and metabolic features. In turn, synthetically elevated mitophagy only mildly reduces mitochondria size, suggesting cells maintain a minimal network size by compensatory biogenesis. Single-cell analysis predicts two metabolically tunable setpoints, mitochondria-to-cell and mitochondria-to-cytosol volume ratios, for mitochondria scaling by autophagy-driven biogenesis and degradation, respectively. Our work reveals how cells use autophagy to scale mitochondria to metabolic and cellular size parameters. One Sentence SummaryA homeostatic form of mitophagy controls the network size of mitochondria during starvation.

cell biology↗

bioRxiv2022

A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here, we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, we find core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 during pexophagy when TORC1 remains active under phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits not only bind selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.

cell biology↗

bioRxiv2022

Parallel phospholipid transfer by Vps13 and Atg2 determines autophagosome biogenesis dynamics

During autophagy, rapid membrane assembly expands small phagophores into large double-membrane autophagosomes. Theoretical modelling predicts the majority of autophagosomal phospholipids is derived from highly efficient non-vesicular phospholipid transfer (PLT) across phagophore-ER contacts (PERCS). Currently, the phagophore-ER tether Atg2 is the only PLT protein known to drive phagophore expansion in vivo. Here, our quantitative live-cell-imaging analysis reveals poor correlation between duration and size of forming autophagosomes and number of Atg2 molecules at PERCS of starving yeast cells. Strikingly, we find Atg2-mediated PLT is non-rate-limiting for autophagosome biogenesis, because membrane tether and PLT protein Vps13 localizes to the rim and promotes expansion of phagophores in parallel with Atg2. In the absence of Vps13, the number of Atg2 molecules at PERCS determines duration and size of forming autophagosomes with an apparent in vivo transfer rate of ~200 phospholipids per Atg2 molecule and second. We propose conserved PLT proteins cooperate in channeling phospholipids across organelle contact sites for non-rate-limiting membrane assembly during autophagosome biogenesis.

cell biology↗

bioRxiv2020

The RNA-binding protein Puf5 buffers mRNA levels against chromatin-mediated changes in nascent transcription

Gene expression is a dynamic process regulated at all stages, starting with opening of chromatin, transcription, and continuing with mRNA export, translation and, finally, degradation. While there are feedback mechanisms within the system, it is not clear whether these extend to crosstalk between chromatin architecture and mRNA decay. Here, we show that changes in nascent transcription, mediated by mutating H3K56 to alanine, are post-transcriptionally buffered by the Pumilio protein Puf5, which stabilizes transcripts in a context-dependent manner. Depleting Puf5 in an H3K56A background leads to synthetic lethality. This genetic interaction can be explained by a decrease in translation due to downregulation of its direct mRNA targets, largely consisting of ribosomal protein genes. Importantly, we show that this post-transcriptional buffering is not only linked to H3K56A, but may be a more widespread phenomenon that also buffers against an increase in nascent RNA transcription in order to maintain physiological mRNA levels and cellular homeostasis.

molecular biology↗