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Experiments were conducted involving the mammalian gene encoding N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAP) and SNAP receptors. Further cloning and sequencing of the mammalian gene encoding NSF have resulted in the identification of the protein as 48% identical to SEC18 gene product from yeast. Both proteins were found to be members of a heterogeneous protein group known as adenosine triphosphatase.

Aims Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. Methods We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age-matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. Results Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. Conclusions This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia. [ABSTRACT FROM AUTHOR]

Ca2+ regulates a multitude of cellular processes and does so by partitioning its actions in space and time. In this review, we discuss how Ca2+ responses are constructed from small quantal (elementary) events that have the potential to propagate to produce large pan-cellular responses. We review how Ca2+ is compartmentalized both physically and functionally, and describe how each organelle has its own distinct Ca2+-handling properties. We explain how coordination of the movement of Ca2+ between organelles is used to shape and hone Ca2+ signals. Finally, we provide a number of specific examples of where compartmentation and localization of Ca2+ are crucial to cell function. [ABSTRACT FROM AUTHOR]

Abstract: Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP3R, and establish that the indicator is capable of reliably reporting the Ca2+ release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (≤100μl). [Copyright &y& Elsevier]

We describe the actions of 2,3-butanedione monoxime (BDM) on calcium responses in secretory cells. Our studies were prompted by the widespread use of BDM as a myosin-ATPase inhibitor. Application of 10 mM BDM almost completely inhibited agonist-evoked amylase secretion from mouse pancreatic acinar cells. This action might be interpreted as indicating a role for myosin in secretion. However, BDM alone elicited a calcium response in single cells and this calcium signal was sufficient to activate calcium-dependent chloride currents. Furthermore, in some cases, BDM potentiated agonist-evoked calcium signals but almost always blocked agonist-evoked calcium oscillations. These effects of BDM were not due to an action on calcium influx pathways but rather to direct effects on IP3-sensitive stores. We conclude that BDM cannot be used for unequivocal identification of the involvement of myosin motors in a cellular response. Further, our evidence suggests that BDM can act directly to modify the opening of IP3 receptors. [ABSTRACT FROM AUTHOR]

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured bioimage data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable bioimage data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing bioimage data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). Here, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse bioimage data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made generating community standard practices for imaging Quality Control (QC) and metadata (Faklaris et al., 2022; Hammer et al., 2021; Huisman et al., 2021; Microscopy Australia , 2016; Montero Llopis et al., 2021; Rigano et al., 2021; Sarkans et al., 2021). We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location., Competing Interests: Conflict of Interest Statements Below are the statements shared by contributors indicating potential conflict of interest. NameStatementUlrike BoehmUB’s contribution to this manuscript is a result of her voluntary involvement with QUAREP-LiMi and BINA, and does not reflect the position of Carl Zeiss AG on this matter.Josh Mooreholds equity in Glencoe Software.Denis SchapiroDS reports funding from GSK and received honorariums from Immunai, Noetik, Alpenglow and Lunaphore.Damir SudarDSu is employed by Quantitative Imaging Systems, a commercial entity developing imaging software.Siyuan (Steven) WangFounder, shareholder, consultant of Translura, Inc

Cyanine dyes, as used in super-resolution fluorescence microscopy, undergo light-induced "blinking", enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio-conjugate cyanine dye (AF-647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light-induced electron transfer and intermediates formed via the metastable, triplet excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF-647 undergoes reversible photo-induced isomerization to at least two long-lived dark species. These photo-isomers are characterized spectroscopically and their interconversion probed by computational means. The first-formed isomer is light sensitive and transforms to a longer-lived species in modest yield that could be involved in dSTORM related blinking. Permanent photobleaching of AF-647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The epithelial Na + -coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (P i ). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent K m for P i ([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to P i coordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of P i and Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates P i and its slightly larger analog, arsenate, as well as the cosubstrate, Na + Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating P i directly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data., (Copyright © 2016 the American Physiological Society.)

The synthesis of a series of adenophostin A analogues modified at C-6 and C-2 of adenine is described. The target compounds were synthesized by a convergent route involving a modified Vorbrüggen condensation of either 6-chloropurine or 2,6-dichloropurine with a protected disaccharide, yielding two versatile intermediates capable of undergoing substitution with a range of nucleophiles. The new analogues showed a range of abilities to mobilize Ca(2+) from the intracellular stores of permeabilized hepatocytes and are among the first totally synthetic compounds to approach the activity of adenophostin A. In agreement with the biological results, docking studies of adenophostin A using the recently reported X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor binding core suggested that, in likely binding modes of adenophostin A, the area around N(6) may be relatively open, identifying this region of the adenophostin A molecule as a promising target for further elaboration. The docking results also point to specific interactions involving residues within the binding domain of the Ins(1,4,5)P(3) receptor that may be involved in the molecular recognition of the adenophostins.