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SUMMARY Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10 μm. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases.

Quantitative analysis of Ca 2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca 2+ -dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca 2+ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca 2+ release kinetics with an off-rate estimated at around 700 s −1 and appropriate Ca 2+ binding affinity, likely attributable to local Ca 2+ -induced conformational changes around the designed Ca 2+ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca 2+ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca 2+ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca 2+ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca 2+ homeostasis.

Activated carbon fibers (ACFs) were surface modified with oxygen plasma at low pressure. The novel effects of the plasma treatment on the microstructural properties of the ACFs were characterized using the Brunauer, Emmett, and Teller method and scanning electron microscopy. Micropores developed on the ACFs. Moreover, the specific surface area and micropore volume increased by 10% at a certain plasma treatment time and power. The changes in the structural properties of the ACFs are discussed in detail with the respect of plasma etching. X-ray photoelectron spectroscopy revealed new oxygen-containing groups, such as CsO, CdO, and OsCdO, had formed on the surface of the ACFs after plasma treatment. Plasma surface oxidative reactions such as the generation of radicals, the combination of the radicals and active oxygen species in the plasma chamber, and the generation of the various oxygen-containing groups are believed to have occurred. The effect of the plasma treatment parameters such as plasma treatment time and power was examined from the perspective of both surface structure and chemistry. It was observed that the micropores and surface functionalities of the ACFs were increased under moderate treatment conditions (50 s and 100 W).

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