Dynamin-related GTPase proteins (DRPs) are main players in membrane remodelling. Conserved DRPs called mitofusins (Mfn1/Mfn2/Fzo1) mediate the fusion of mitochondrial outer membranes (OM). OM fusion depends on self-assembly and GTPase activity of mitofusins as well as on two other proteins, Ugo1 and Mdm30. Here, we define distinct steps of the OM fusion cycle using in vitro and in vivo approaches. We demonstrate that yeast Fzo1 assembles into homo-dimers, depending on Ugo1 and on GTP binding to Fzo1. Fzo1 homo-dimers further associate upon formation of mitochondrial contacts, allowing membrane tethering. Subsequent GTP hydrolysis is required for Fzo1 ubiquitylation by the F-box protein Mdm30. Finally, Mdm30-dependent degradation of Fzo1 completes Fzo1 function in OM fusion. Our results thus unravel functions of Ugo1 and Mdm30 at distinct steps during OM fusion and suggest that protein clearance confers a non-cycling mechanism to mitofusins, which is distinct from other cellular membrane fusion events.

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, frequent early relapse and a high mortality rate ^1–3 . Recent evidence has suggested that innervation has an important role in the development and progression of several types of cancer ^4,5 . Cancer-to-neuron synapses have been reported in gliomas ^6,7 , but whether peripheral tumours can form such structures is unknown. Here we show that SCLC cells can form functional synapses and receive synaptic transmission. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified neuronal, synaptic and glutamatergic signalling gene sets in mouse and human SCLC. Further experiments revealed the ability of SCLC cells to form synaptic structures with neurons in vitro and in vivo. Electrophysiology and optogenetic experiments confirmed that cancer cells can receive NMDA receptor- and GABA _A receptor-mediated synaptic inputs. Fitting with a potential oncogenic role of neuron–SCLC interactions, we showed that SCLC cells derive a proliferation advantage when co-cultured with vagal sensory or cortical neurons. Moreover, inhibition of glutamate signalling had therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells seem to hijack synaptic signalling to promote tumour growth, thereby exposing a new route for therapeutic intervention.

Background: Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. Results: In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. Conclusions: The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions. See Commentary: http://www.biomedcentral.com/1741-7007/8/99

Obesity during pregnancy is a known health risk for mother and child. Since obesity is associated with increased inflammatory markers, our objectives were to determine interleukin-6 (IL-6) levels in obese mice and to examine the effect of IL-6 on placental endothelial cells. Placentas, blood, and adipose tissue of C57BL/6N mice, kept on high fat diet before and during pregnancy, were harvested at E15.5. Serum IL-6 levels were determined and endothelial cell markers and IL-6 expression were measured by qRT-PCR and western blot. Immunostaining was used to determine surface and length densities of fetal capillary profiles and placental endothelial cell homeostasis. Human placental vein endothelial cells were cultured and subjected to proliferation, apoptosis, senescence, and tube formation assays after stimulation with hyperIL-6. Placental endothelial cell markers were downregulated and the percentage of senescent endothelial cells was higher in the placental exchange zone of obese dams and placental vascularization was strongly reduced. Additionally, maternal IL-6 serum levels and IL-6 protein levels in adipose tissue were increased. Stimulation with hyperIL-6 provoked a dose dependent increase of senescence in cultured endothelial cells without any effects on proliferation or apoptosis. Diet-induced maternal obesity led to an IUGR phenotype accompanied by increased maternal IL-6 serum levels. In the placenta of obese dams, this may result in a disturbed endothelial cell homeostasis and impaired fetal vasculature. Cell culture experiments confirmed that IL-6 is capable of inducing endothelial cell senescence.

Mitochondrial DNA (mtDNA) is exposed to multiple insults produced by normal cellular function. Upon mtDNA replication stress, the mitochondrial genome transfers to endosomes for degradation. Using proximity biotinylation, we found that mtDNA stress leads to the rewiring of the mitochondrial proximity proteome, increasing mitochondria’s association with lysosomal and vesicle-related proteins. Among these, the retromer complex, particularly VPS35, plays a pivotal role by extracting mitochondrial components. The retromer promotes the formation of mitochondrial-derived vesicles shuttled to lysosomes. The mtDNA, however, directly shuttles to a recycling organelle in a BAX-dependent manner. Moreover, using a Drosophila model carrying a long deletion on the mtDNA (ΔmtDNA), we found that ΔmtDNA activates a specific transcriptome profile to counteract mitochondrial damage. Here, Vps35 expression restores mtDNA homoplasmy and alleviates associated defects. Hence, we demonstrate the existence of a previously unknown quality control mechanism for the mitochondrial matrix and the essential role of lysosomes in mtDNA turnover to relieve mtDNA damage.

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30⁺hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30⁺tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30^–tumor cells to provide binding sites for BV. Notably, CD30⁺EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30^–cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30⁺EVs. Since BV is only toxic toward CD30^–DLBCL cells when CD30⁺EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30⁺tumor cell but also CD30^–DLBCL cells in the presence of CD30⁺EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30^–DLBCL and warrants confirming studies in animal models.