Pluronic F-68, an 80% hydrophilic member of the Pluronic family of polyethylene-polypropylene-polyethylene tri-block copolymers, protects non-neuronal cells from traumatic injuries and rescues hippocampal neurons from excitotoxic and oxidative insults. F-68 interacts directly with lipid membranes and restores membrane function after direct membrane damage. Here, we demonstrate the efficacy of Pluronic F-68 in rescuing rat hippocampal neurons from apoptosis after oxygen-glucose deprivation (OGD). OGD progressively decreased neuronal survival over 48 h in a severity-dependent manner, the majority of cell death occurring after 12 h after OGD. Administration of F-68 for 48 h after OGD rescued neurons from death in a dose-dependent manner. At its optimal concentration (30 M), F-68 rescued all neurons that would have died after the first hour after OGD. This level of rescue persisted when F-68 administration was delayed 12 h after OGD. F-68 did not alter electrophysiological parameters controlling excitability, NMDA receptor-activated currents, or NMDA-induced increases in cytosolic calcium concentrations. However, F-68 treatment prevented phosphatidylserine externalization, caspase activation, loss of mitochondrial membrane potential, and BAX translocation to mitochondria, indicating that F-68 alters apoptotic mechanisms early in the intrinsic pathway of apoptosis. The profound neuronal rescue provided by F-68 after OGD and the high level of efficacy with delayed administration indicate that Pluronic copolymers may provide a novel, membrane-targeted approach to rescuing neurons after brain ischemia. The ability of membrane-active agents to block apoptosis suggests that membranes or their lipid components play prominent roles in injury-induced apoptosis.

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

Xenopus laevis oocytes are used to study membrane proteins because of their ability to translate exogenous mRNA, but their high intrinsic fluorescence limits fluorescence recordings. Lee and Bezanilla present two methods to increase the amount of melanin and reduce background fluorescence in oocytes.

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization.Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.