Tomoki Matsuda

Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano

We report ultrasensitive Ca2+ indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca2+-sensing domain of a genetically encoded Ca2+ indicator, YC2.60 or YC3.60. Their high Ca2+ affinities (Kd = 15–140 nM) and large signal change (1,450%) enabled detection of subtle Ca2+ transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

Luminescent proteins for high-speed single-cell and whole-body imaging

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca2+, cyclic adenosine monophosphate and adenosine 5′-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

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.

Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein

All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 μm2/s up to ∼100 μm2/s, and found significant changes in free protein movement during cell-cycle progression.

Serum angiotensin I-converting enzyme is reduced in Crohn's disease and ulcerative colitis irrespective of genotype.

OBJECTIVES:Crohns disease (CD) is recognized to be a vascular endothelial-associated disease. Angiotensin I-converting enzyme (ACE) exists mainly in endothelial cells. There are some reports on serum ACE levels in patients with CD, but the ACE level is still controversial. Recently, genetic control of serum ACE levels by ACE gene polymorphisms (classified as II, ID, and DD) has been suggested. Although we must consider such polymorphisms to elucidate ACE levels in patients with CD, there is no report about this.METHODS:We studied 341 healthy controls (male/female = 178/162), 39 patients with CD (31/8), 43 patients with ulcerative colitis (UC) (22/21) and 19 patients with infectious enterocolitis (8/11). The polymorphism in intron 16 of the ACE gene was examined by PCR. Serum ACE levels were measured by the method of Kasahara.RESULTS:Serum ACE levels in patients with CD and UC were significantly lower than in healthy controls, irrespective of the genotype of ACE (genotype II: CD 7.0 ± 2.5 [mean ± SD], UC 7.1 ± 3.3, controls 11.8 ± 2.9, genotype ID: CD 9.7 ± 4.1, UC 11.4 ± 4.6, controls 15.2 ± 3.6, genotype DD: CD 13.9 ± 5.8, UC 10.7 ± 3.6, controls 19.3 ± 3.9 IU/L, controls vs CD, UC; p < 0.01, 0.05). However, there was no significant difference in serum ACE levels between CD and UC.CONCLUSIONS:Considering ACE gene polymorphism, serum ACE levels in patients with inflammatory bowel disease are lower than in controls. Serum ACE levels reflect a part of the pathogenesis of inflammatory bowel disease.

Increased Apoptosis Associated with Depressed Type of Early Intestinal Gastric Cancer

Early gastric cancer can be macroscopically classified into elevated and depressed types. To clarify the relationship between macroscopic appearance of early gastric cancer and apoptosis or cell proliferation, formalin‐fixed paraffin‐embedded tissue specimens of 44 intestinal‐type early gastric cancers were investigated by the TUNEL method and immunohistochemical techniques. Diffuse type was excluded in this study. When tissue sections of gastric cancer were vertically classified into the 3 compartments of laminar, intermediate and basal, the apoptosis index (%) was significantly higher in the basal compartment of depressed type (1.76±2.04, mean±SD) than in the basal compartment of elevated type (0.63±0.81, P=0.01). In depressed type, the apoptosis index (%) was significantly higher in the basal compartment than in the luminar compartment (0.76 ±0.85, P=0.03). Apoptosis‐inducing protein, Bax, was expressed more in each of the compartments of depressed type than in those of elevated type, while there were no significant differences in expression of anti‐apoptotic protein, Bcl‐2, between the two types. Moreover, the apoptosis index (%) of Bax‐positive gastric cancer was significantly higher in the basal compartment (P=0.03), compared to that of Bax‐negative gastric cancer, while there were no significant differences in apoptosis index (%) in any compartment between Bcl‐2‐positive and Bcl‐2‐negative gastric cancers. There were no significant differences in Ki‐67 expression, either between the two types, or among the compartments of depressed type. These results indicate that increased apoptosis with excessive expression of Bax in the basal compartment is involved in the morphogenesis of the depressed type in intestinal‐type early gastric cancer.

Development of ultra-sensitive Ca2+ indicators, yellow cameleon-nanos

There are so many Ca2+ indicators such as Oregon green 488 BAPTA-1 and cameleon, all of which have moderate Ca2+ affinities (Kd > 150 nM). These indicators successfully report changing in Ca2+ concentration in most of cells. However, it is known that some cells are estimated to have a very low resting [Ca2+] and display very small ▵[Ca2+]s, which is below the detection limit of the indicators. To develop high-affinity indicators, we modified a FRET-based Ca2+ indicator, yellow cameleon (YC) 2.60 (Kd = 100 nM) because of the brightness and the large dynamic range. By engineering the Ca2+ sensing domain, we obtained a series of ultra-sensitive Ca2+ indicators. Their high Ca2+ affinities (Kd = 15, 30, and 50 nM) enabled detection of subtle Ca2+ transients associated with spontaneous network activity in zebrafish. Our measurements revealed that both the resting Ca2+ level and the amplitude of Ca2+ transients significantly differed by cell type and stimulation, indicating that the selection of indicators with the appropriate Kd is essential for successful in vivo Ca2+ imaging. A lineup of such indicators with finely tuned Kd values optimized to detect [Ca2+] from 10 nM to 100 nM would enable precise and reliable Ca2+ imaging.

Imaging the dynamics of intracellular protein translocation by photoconversion of phamret-cybr/ROM.

Cybr/Reduced On‐random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein‐95/discs‐large/ZO‐1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C‐terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin‐1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells’ periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation‐mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.

Real time imaging study on injury-induced microglial activation in the hippocampal slice culture

Effects of regularity, familiarity, and congruity of stimuli were investigated on Auditory and Visually Evoked Magnetic Fields. Regularity was controlled by exponent n on a fluctuation of 1/fn, Familiarity was controlled by the score of word familiarity in a database of Lexical Properties of Japanese. Congruity was controlled by the semantic distance of the components of the constructed stimulus. Decrement of these three factors of stimuli enhanced the amplitude and prolonged the latency of the AEF activity of the N100m component of primary auditory aria. The increment of the three factors of visual stimuli enhanced the VEF of the 150 ms and 220 ms components. The three factors of stimuli play similar roles for activity enhancement of the auditory and visual cortex, and provoked expectation of auditory stimuli and figure ground segmentation for visual stimuli. The activities of the cortex reflected the basic function of the conventionalization of the pattern of a repetitive experience unit in the process of the emergence of linguistic structure.