Ippei Kotera

An ultramarine fluorescent protein with increased photostability and pH insensitivity

We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concentration and caspase-3 activation in the same apoptotic cells.

Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response

Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities. Our thermometric method enabled a fast tracking of the temperature change with a time resolution of 50 ms. We used this method to observe the spatiotemporal temperature change between the cytoplasm and nucleus in cells, and quantified thermogenesis from the mitochondria matrix in a single living cell after stimulation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which was an uncoupler of oxidative phosphorylation. Moreover, exploiting the wide temperature range of sensitivity from 5°C to 50°C of gTEMP, we monitored the temperature in a living medaka embryo for 15 hours and showed the feasibility of in vivo thermometry in various living species.