Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+ release and CaMKII signaling.

Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the consequences of Arc ubiquitination on other mGluR-mediated signaling events is poorly characterized. Here we find that pharmacological activation of Group I mGluRs with S-3,5-dihydroxyphenylglycine (DHPG) increases Ca2+ release from the endoplasmic reticulum (ER). Disrupting Arc ubiquitination on key amino acid residues enhances DHPG-induced ER-mediated Ca2+ release. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination altered Arc self-assembly and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII in HEK293 cells. Colocalization of Arc and CaMKII was altered in cultured hippocampal neurons, with the notable exception of secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca2+ signaling that may support mGluR-LTD, which in turn, may regulate CaMKII and its interactions with Arc.

Tuning Protein Dynamics to Sense Rapid Endoplasmic-Reticulum Calcium Dynamics.

Multi-scale calcium (Ca2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca2+ indicator, R-CatchER, which showed superior kinetics in vitro (koff ≥2×103  s-1 , kon ≥7×106  M-1 s-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca2+ indicators with a single Ca2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca2+ dynamics, and drug discovery.

Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER.

The precise spatiotemporal characteristics of subcellular calcium (Ca2+) transients are critical for the physiological processes. Here we report a green Ca2+ sensor called "G-CatchER+" using a protein design to report rapid local ER Ca2+ dynamics with significantly improved folding properties. G-CatchER+ exhibits a superior Ca2+ on rate to G-CEPIA1er and has a Ca2+-induced fluorescence lifetimes increase. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER+-JP45), we report a rapid local Ca2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER+ in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca2+ dynamics. G-CatchER+ will be an invaluable tool to examine local ER/SR Ca2+ dynamics and facilitate drug development associated with ER dysfunction.

A High-content Assay for Monitoring AMPA Receptor Trafficking.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Synergistic potential of papaya and strawberry nectar blends focused on specific nutrients and antioxidants using alternative thermal and non-thermal processing techniques.

Traditional processing has detrimental effects on nutrient value of fruit nectars; however, combining fruit nectars prior to processing can result in synergistic outcomes, e.g., a combination of nutrients providing a greater effect than they would individually, thus offsetting these losses. To examine this food synergism, papaya and strawberry nectars and their respective blends (25P:75S, 50P:50S, 75P:25S) were processed using ultra high temperature (UHT) and irradiation and examined for ascorbic acid concentration, carotenoid concentration, and antioxidant capacity. Ascorbic acid concentration was best retained after UHT processing, with synergistic relationships in all blends. Synergistic relationships were observed for ß-cryptoxanthin concentration after irradiation. ß-Carotene experienced both antagonistic and additive relationships whereas lycopene concentration encountered synergistic relationships in the 25P:75S blend for both techniques. All blends exhibited synergistic relationships for antioxidant capacity after UHT processing. These findings demonstrate the benefits of blending fruit nectars; producing a superior product than either fruit processed individually.

Relationship between pulp structure breakdown and nutritional value of papaya (Carica papaya) and strawberry (Fragaria x ananassa) nectars using alternative thermal and non-thermal processing techniques.

BACKGROUND: Papaya and strawberry contain a wide array of nutrients that contribute to human health; however, availability of these fruits is limited due to their short shelf lives and seasonal nature. In this study, the effect of alternative techniques including ultra high temperature (UHT, 20-135 °C, 1-3 s) and irradiation (0-10 kGy) on carotenoid concentration, antioxidant capacity and changes to pulp structure using transmission electron microscopy were determined for papaya and strawberry nectars. RESULTS: UHT had moderate effects on antioxidant capacity, but the greatest overall release of carotenoids from the pulp matrix (34.2%, 6.26%, 8.31%; ß-cryptoxanthin, ß-carotene, and lycopene, respectively). Irradiation resulted in the greatest increase in antioxidant capacity [19.22 to 24.32 µmol Trolox equivalents kg(-1) (papaya), 190.51 to 287.68 µmol Trolox equivalents kg(-1) (strawberry)], with moderate effects on carotenoid concentration. CONCLUSION: This research demonstrates that decreases in nutrient value and antioxidant capacity can result from processing, but that regeneration of these losses can be seen corresponding to the apparent breakdown of pulp structure. © 2015 Society of Chemical Industry.