Search

Paraformaldehyde ( PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining. [ABSTRACT FROM AUTHOR]

The transmembrane recognition complex ( TRC40) pathway mediates the insertion of tail-anchored ( TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum ( ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells ( IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells. [ABSTRACT FROM AUTHOR]

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins‐A1‐3, endocytic adaptors with curvature‐sensing and curvature‐generating properties, in mouse IHCs. Single‐cell RT–PCR indicated the expression of endophilins‐A1‐3 in IHCs, and immunoblotting confirmed the presence of endophilin‐A1 and endophilin‐A2 in the cochlea. Patch‐clamp recordings from endophilin‐A‐deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin‐mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co‐immunoprecipitated with purified endophilin‐A1 protein, suggestive of a molecular interaction that might aid exocytosis–endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome‐like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation. Synopsis: Using a multidisciplinary approach, we show that endophilin‐A positively regulates presynaptic Ca2+ influx and interacts with otoferlin. Moreover, endophilin‐A supports vesicle endocytosis and clathrin‐dependent vesicle reformation at ribbon synapses of murine inner hair cells. Absence of endophilin‐A leads to reduced presynaptic Ca2+channel cluster size and attenuated Ca2+ influx at inner hair cell ribbon synapses.Otoferlin physically interacts with endophilin; loss of endophilin‐A1 and ‐A3 leads to a reduction in otoferlin levels of ˜25%.The number of cytosolic and ribbon‐associated SVs is decreased in mutants missing several endophilin‐A genes; consequently, IHC sustained exocytosis was found to be reduced.Absence of endophilin‐A leads to accumulations of endosome‐like vacuoles and clathrin‐coated organelles; the severity of the phenotype depends on the number of missing endophilin alleles. Endophilins regulate Ca2+ channel abundance, Ca2+ influx and synaptic vesicle recycling in inner hair cells by coupling exocytosis to endocytosis, contributing to membrane retrieval and synaptic vesicle reformation. [ABSTRACT FROM AUTHOR]

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca 2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca 2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca 2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation., (© 2019 The Authors.)