Hidekazu Tsutsui

Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62‐Tyr 63‐Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra‐) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green‐to‐red photoconvertibility. Substitution of the His 62‐Tyr 63‐Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi‐rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several‐fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two‐photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

Improving membrane voltage measurements using FRET with new fluorescent proteins

We used two new coral fluorescent proteins as fluorescence resonance energy transfer (FRET) donor and acceptor to develop a voltage sensor, named Mermaid, that displays ∼40% changes in emission ratio per 100 mV, allowing for direct visualization of electrical activities in cultured excitable cells. Notably, Mermaid has fast on-off kinetics at warm (∼33 °C) temperatures and can report voltage spikes comparable to action potentials.

mKikGR, a Monomeric Photoswitchable Fluorescent Protein

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes

Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103+DCs, including Langerin+CD103+dermal DCs (DDCs), remained in the dLN for 4–4.5 days after migration from the skin, while CD103−DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103−DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103−DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103−DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103−DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Visualizing voltage dynamics in zebrafish heart

The zebrafish heart provides a useful vertebrate cardiovascular model with outstanding advantages, including genetic manipulatability, optical accessibility and rapid development. In addition, an emerging topic in cardiotoxicity assay and drug discovery is its use in phenotype‐based chemical screening. Here, we report a technique that allows non‐invasive voltage mapping in beating heart using a genetically encoded probe for transmembrane potential. Application of the anti‐allergy compound astemizole resulted in aberrant propagation of excitation, which accounted for a lack of ventricular contraction. This optical method will provide new opportunities in broad areas of physiological, developmental and pharmacological cardiovascular research.

Improved detection of electrical activity with a voltage probe based on a voltage-sensing phosphatase

•  The use of genetically encoded voltage probes has been expected to enable sensitive detection of spatiotemporal electrical activities in excitable cells. •  However, existing probes suffer from low signal amplitude and/or kinetics too slow to detect fast electrical activity. •  We have developed an improved voltage probe named Mermaid2. •  Mermaid2 provides ratiometric readouts of electrical activity with fast kinetics and great sensitivity, and was able to detect single‐event electrical activity both in vitro and in vivo. •  Mermaid2 will expand our chances to analyse electrical events that have been less accessible by using other techniques.

The E1 Mechanism in Photo-Induced β-Elimination Reactions for Green-to-Red Conversion of Fluorescent Proteins

KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with ultraviolet or violet light. Similar to Kaede and EosFP, two naturally occurring photoconvertible proteins, KikGR contains a His(62)-Tyr(63)-Gly(64) tripeptide sequence, which forms a green chromophore that can be photoconverted to a red one via formal beta-elimination and subsequent extension of a pi-conjugated system. Using a crystallizable variant of KikGR, we determined the structures of both the green and red state at 1.55 A resolution. The double bond between His(62)-C(alpha) and His(62)-C(beta) in the red chromophore is in a cis configuration, indicating that rotation along the His(62) C(alpha)-C(beta) bond occurs following cleavage of the His(62) N(alpha)-C(alpha) bond. This structural rearrangement provides evidence that the beta-elimination reaction governing the green-to-red photoconversion of KikGR follows an E1 (elimination, unimolecular) mechanism.

Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Background/Aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

Strategies for Sperm Chemotaxis in the Siphonophores and Ascidians: A Numerical Simulation Study

Chemotactic swimming behaviors of spermatozoa toward an egg have been reported in various species. The strategies underlying these behaviors, however, are poorly understood. We focused on two types of chemotaxis, one in the siphonophores and the second in the ascidians, and then proposed two models based on experimental data. Both models assumed that the radius of the path curvature of a swimming spermatozoon depends on [Ca2+]i, the intracellular calcium concentration. The chemotaxis in the siphonophores could be simulated in a model that assumes that [Ca2+]i depends on the local concentration of the attractant in the vicinity of the spermatozoon and that a substantial time period is required for the clearance of transient high [Ca2+]i. In the case of ascidians, trajectories similar to those in experiments could be adequately simulated by a variant of this model that assumes that [Ca2+]i depends on the time derivative of the attractant concentration. The properties of these strategies and future problems are discussed in relation to these models.

A diffraction-quality protein crystal processed as an autophagic cargo

Crystallization of proteins may occur in the cytosol of a living cell, but how a cell responds to intracellular protein crystallization remains unknown. We developed a variant of coral fluorescent protein that forms diffraction-quality crystals within mammalian cells. This expression system allowed the direct determination of its crystal structure at 2.9 Å, as well as observation of the crystallization process and cellular responses. The micron-sized crystal, which emerged rapidly, was a pure assembly of properly folded β-barrels and was recognized as an autophagic cargo that was transferred to lysosomes via a process involving p62 and LC3. Several lines of evidence indicated that autophagy was not required for crystal nucleation or growth. These findings demonstrate that in vivo protein crystals can provide an experimental model to study chemical catalysis. This knowledge may be beneficial for structural biology studies on normal and disease-related protein aggregation.