Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy

モノクローナル抗体の用途を広げる革新技術 --多重超解像可視化プローブへの迅速変換法--. 京都大学プレスリリース. 2022-09-21.Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging

Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium

SummaryGABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses

Melting of excitonic insulator phase by an intense terahertz pulse in Ta2_2NiSe5_5

In this study, the optical response to a terahertz pulse was investigated in the transition metal chalcogenide Ta$_2$NiSe$_5$, a candidate excitonic insulator. First, by irradiating a terahertz pulse with a relatively weak electric field (0.3 MV/cm), the spectral changes in reflectivity near the absorption edge due to third-order optical nonlinearity were measured and the absorption peak characteristic of the excitonic phase just below the interband transition was identified. Next, by irradiating a strong terahertz pulse with a strong electric field of 1.65 MV/cm, the absorption of the excitonic phase was found to be reduced, and a Drude-like response appeared in the mid-infrared region. These responses can be interpreted as carrier generation by exciton dissociation induced by the electric field, resulting in the partial melting of the excitonic phase and metallization. The presence of a distinct threshold electric field for carrier generation indicates exciton dissociation via quantum-tunnelling processes. The spectral change due to metallization by the electric field is significantly different from that due to the strong optical excitation across the gap, which can be explained by the different melting mechanisms of the excitonic phase in the two types of excitations.Comment: 66 pages, 11 figures, 2 table

ISR-DEPENDENT METABOLIC REGULATION

The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation‐dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand‐activated skeletal muscle–specific derivative of the eIF2α protein kinase R‐like ER kinase revealed the expected up‐regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small‐molecule ISR activator that promoted Fgf21 expression in cell‐based screens and by implication of the ISR‐inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell‐autonomous proteostasis and amino acid metabolism, but also affects non‐cell‐autonomous metabolic regulation by induced expression of a potent myokine.—Miyake, M., Nomura, A., Ogura, A., Takehana, K., Kitahara, Y., Takahara, K., Tsugawa, K., Miyamoto, C., Miura, N., Sato, R., Kurahashi, K., Harding, H. P., Oyadomari, M., Ron, D., Oyadomari, S. Skeletal muscle‐specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21‐mediated non‐cell‐autonomous energy metabolism

Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs

In most species, fertilization induces Ca(2+) transients in the egg. In mammals, the Ca(2+) rises are triggered by phospholipase Czeta (PLCzeta) released from the sperm; IP3 generated by PLCzeta induces Ca(2+) release from the intracellular Ca(2+) store through IP3 receptor, termed IP3-induced Ca(2+) release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 muM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca(2+) and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca(2+) sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca(2+) wave. [IP3] stayed at the elevated level with small peaks followed after Ca(2+) spikes through Ca(2+) oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca(2+) spike, suggesting that Ca(2+)-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca(2+) oscillations in fertilized eggs

Skeletal muscle–specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21–mediated non–cell-autonomous energy metabolism

The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.Ministry of Education, Culture, Sports, Science and Culture (MEXT) of Japan Inoue Foundation for Science Mitsubishi Foundation Uehara Memorial Foundation Naito Foundation Cell Science Research Foundation Takeda Science Foundation Sankyo Foundation Ono Medical Research Foundation Mochida Memorial Foundation Ube Foundation Kowa Life Science Foundation Suzuken Memorial Foundation Kanae Foundation Japan Diabetes Foundation Japan Society for Promotion of Science (JSPS) EU FP7. Grant Number: 277713 Wellcome Trust. Grant Number: 084812/Z/08/

Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy

Summary: Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Evaluation of three-dimensional glenoid structure using MRI

The tilting angle and the shape of the glenoid cavity are considered to relate closely to shoulder stability. They are also important when planning arthroplasty and developing new designs. This study examines the glenoid cavity using 3-dimensional MRI. Forty volunteers (20 men, 20 women; average age 21.4; range 18–35 y) were enrolled in the study. The tilting angles of the glenoid bone were measured in 5 consecutive axial planes perpendicular to the glenoidal long axis. Cross sections were divided into 3 types (concave, flat, convex) according to the shape on each plane. The average tilting angles for the 5 planes from the bottom to the top were 3.3±4.1, 1.4±3.8, −0.6±1.9, −1.4±3.3, and −6.2±3.3 degrees anteriorly, indicating that the 3-dimensional bony structure of the glenoid was twisted anteriorly to posteriorly. Images on the bottom plane consisted of 82.5% concave type, 15% flat type and 2.5% convex type, while only 3 cases (7.5%) showed concave at the top plane. The shape of the glenoid cavity is thought to be conducive to glenohumeral motion and stability