Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years

2de: The Phoenix Of Proteomics

Given the rapid developments in mass spectrometry (MS) in terms of sensitivity, mass accuracy, and throughput, some have suggested that two-dimensional gel electrophoresis (2DE) may no longer be a method of choice for proteomic analyses. However, as recognition of issues with these newer shotgun-MS approaches grows, there is a fresh and growing regard for the maturity of 2DE-MS as a genuine top-down analytical approach, particularly as it resolves thousands of intact protein species in a single run, enabling the simultaneous analysis of total protein complement, including isoforms and post-translational modifications. Given the strengths of both, it is most appropriate to view these as complementary or at least parallel approaches: as proteins encompass a myriad of physico-chemical properties, and the real aim is to explore proteomes as deeply as possible, all available resolving strategies must be considered in terms of the complexity encountered. It is time to critically and constructively focus on the optimization and integration of existing techniques rather than simplistically suggesting that one should replace the other. Our intention here is thus to present an overview of protein resolving techniques, focusing on milestones associated with 2DE, including pros, cons, advances and variations, in particular relative to shotgun proteomic approaches. Biological significance: Proteomic researchers recognize the importance of 2DE in the history of proteomics. But the latest developments in mass spectrometry-based techniques have led some researchers to retire 2DE in their labs. However, we argue here that 2DE-MS is a genuine top-down analytical approach. 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Long-Term Potentiation of Exocytosis and Cell Membrane Repair in Fibroblasts

We previously found that a microdisruption of the plasma membrane evokes Ca(2+)-regulated exocytosis near the wound site, which is essential for membrane resealing. We demonstrate herein that repeated membrane disruption reveals long-term potentiation of Ca(2+)-regulated exocytosis in 3T3 fibroblasts, which is closely correlated with faster membrane resealing rates. This potentiation of exocytosis is cAMP-dependent protein kinase A dependent in the early stages (minutes), in the intermediate term (hours) requires protein synthesis, and for long term (24 h) depends on the activation of cAMP response element-binding protein (CREB). We were able to demonstrate that wounding cells activated CREB within 3.5 h. In all three phases, the increase in the amount of exocytosis was correlated with an increase in the rate of membrane resealing. However, a brief treatment with forskolin, which is effective for short-term potentiation and which could also activate CREB, was not sufficient to induce long-term potentiation of resealing. These results imply that long-term potentiation by CREB required activation by another, cAMP-independent pathway

HIV-1 Envelope Proteins Complete Their Folding into Six-helix Bundles Immediately after Fusion Pore Formation

Fusion proteins of many viruses, including HIV-1 envelope protein (Env), fold into six-helix bundle structures. Fusion between individual Env-expressing cells and target cells was studied by fluorescence microscopy, and a temperature jump technique, to determine whether folding of Env into a bundle is complete by the time fusion pores have formed. Lowering temperature to 4°C immediately after a pore opened halted pore growth, which quickly resumed when temperature was raised again. HIV gp41-derived peptides that inhibit bundle formation (C34 or N36) caused the cold-arrested pore to quickly and irreversibly close, demonstrating that bundle formation is not complete by the time a pore has formed. In contrast, lowering the temperature to an intermediate value also halted pore growth, but the pore was not closed by the bundle-inhibiting peptides, and it enlarged when temperature was again elevated. This latter result shows that bundle formation is definitely required for the fusion process, but surprisingly, some (if not all) bundle formation occurs after a pore has formed. It is concluded that an essential function of the bundle is to stabilize the pore against collapse and ensure its growth