A free intravesicular C-terminal of otoferlin is essential for synaptic vesicle docking and fusion at auditory inner hair cell ribbon synapses.

Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof+/GFP mice exhibited a normal hearing function, homozygote OtofGFP/GFP mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof GFP/GFP IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.

Mitofusin-mediated contacts between mitochondria and peroxisomes regulate mitochondrial fusion.

Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.

Staying on track - Keeping things running in a high-end scientific imaging core facility.

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.

CAVIN1-Mediated hERG Dynamics: A Novel Mechanism Underlying the Interindividual Variability in Drug-Induced Long QT.

BACKGROUND: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (IKr) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. METHODS: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. RESULTS: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and IKr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1, essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin. CONCLUSIONS: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.