Toru Matsu-ura

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.

TIP49b, a New RuvB-like DNA Helicase, Is Included in a Complex Together with Another RuvB-like DNA Helicase, TIP49a

We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB. In this study, we isolated a newTIP49a-related gene, termed TIP49b, from human and yeast cells. TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family. Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase. Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5′ to 3′) was the opposite to that of TIP49a. TIP49b and TIP49a bound to each other and were included in the same complex of ∼700 kDa in a cell. We found thatTIP49b was an essential gene for the growth ofSaccharomyces cerevisiae, as is the TIP49agene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+-induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

Efficient gene editing in Neurospora crassa with CRISPR technology

BackgroundEfficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, Neurospora crassa.FindingsIn this report, we demonstrate efficient gene replacement in a model filamentous fungus, Neurospora crassa, with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of clr-2 with the β-tubulin promoter, and (2) introduce a codon optimized fire fly luciferase under the control of the gsy-1 promoter at the csr-1 locus. CLR-2 is one of the core transcription factors that regulate the expression of cellulases, and GSY-1 regulates the conversion of glucose into glycogen. We show that the β-tubulin promoter driven clr-2 strain shows increased expression of cellulases, and gsy-1-luciferase reporter strain can be easily screened with a bioluminescence assay.ConclusionCRISPR/Cas9 system works efficiently in Neurospora crassa, which may be adapted to Neurospora natural isolates and other filamentous fungi. It will be beneficial for the filamentous fungal research community to take advantage of CRISPR/Cas9 tool kits that enable genetic perturbations including gene replacement and insertions.

Receptor-Selective Diffusion Barrier Enhances Sensitivity of Astrocytic Processes to Metabotropic Glutamate Receptor Stimulation

An mGluR5-selective diffusion barrier enriches mGluR5 in astrocytic processes, enabling compartmentalized calcium signaling. Keeping Calcium Signals in the Processes Although astrocytes, the most numerous form of glial cell in the brain, are electrically inexcitable, their ability to release chemical messengers and respond to such messengers with propagated calcium signals allows them to participate actively in the regulation of local blood flow and of synaptic efficacy. Here, Arizono et al. expressed a genetically encoded calcium indicator in neuron-astrocyte cocultures and hippocampal slices and found that, compared to the soma, astrocyte processes showed enhanced calcium responses to stimulation of the metabotropic glutamate receptor (mGluR). The enhanced calcium response observed in processes resulted from an increased density of mGluRs, rather than from differences in the distribution or sensitivity of the calcium release machinery. Analysis of the movement of single mGluR5s revealed a membrane barrier that selectively blocked the movement of mGluR5 between astrocyte somata and their processes. Noting that various neurological disorders are associated with abnormal calcium signaling in astrocytes, the authors speculate that the existence of this barrier—and thereby of compartmentalized calcium signals—could allow individual processes to regulate associated partners (synapses or blood vessels) independently, in the absence of a somatic calcium signal. Metabotropic glutamate receptor (mGluR)–dependent calcium ion (Ca2+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca2+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca2+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca2+ responses. Immunocytochemistry indicated that these polarized Ca2+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca2+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca2+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent proteins chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects.

Control of Neuronal Growth Cone Navigation by Asymmetric Inositol 1,4,5-Trisphosphate Signals

Measurements of its spatial profile reveal the crucial role of asymmetric IP3 signals in growth cone navigation. IP3 Points the Way During nervous system development, neurites navigate to their targets by responding to cues in the environment. Attractive or repulsive turning depends on the generation of asymmetric changes in Ca2+ within the growth cone of the developing neurite. The source of these intracellular Ca2+ signals is critical to determining whether the response is attractive (with the growth cone turning toward the side with increased Ca2+ concentration) or repulsive (with the growth cone turning away from the side with increased Ca2+ concentration). Here, Akiyama et al. show that attractive turning to nerve growth factor (NGF) depends on phospholipase C (PLC)–dependent generation of asymmetric changes in inositol 1,4,5-trisphosphate (IP3) and the ensuing IP3-induced Ca2+ release from the endoplasmic reticulum. Dissection of the pathway showed that the turning response required basal cAMP signaling upstream of asymmetric IP3-induced Ca2+ release and required basal phosphatidylinositol 3-kinase (PI3K)–dependent signaling downstream. Inositol 1,4,5-trisphosphate (IP3) is generally viewed as a global messenger that increases cytosolic calcium ion (Ca2+) concentration. However, the spatiotemporal dynamics of IP3 and the functional significance of localized IP3 production in cell polarity remain largely unknown. Here, we demonstrate the critical role of spatially restricted IP3 signals in axon guidance. We found that IP3 and ensuing Ca2+ signals were produced asymmetrically across growth cones exposed to an extracellular gradient of nerve growth factor (NGF) and mediated growth cone turning responses to NGF. Moreover, photolysis-induced production of IP3 on one side of a growth cone was sufficient to initiate growth cone turning toward the side with the higher concentration of IP3. Thus, locally produced IP3 encodes spatial information that polarizes the growth cone for guided migration.

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular IP3-gated calcium (Ca2+) release channel and plays important roles in regulation of numerous Ca2+-dependent cellular responses. Many intracellular modulators and IP3R-binding proteins regulate the IP3R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP3R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP3R. The interaction was favored by the cytosolic Ca2+ concentration and it functionally inhibited IP3R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP3R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP3R-mediated Ca2+ signal through a direct interaction with IP3R in a cytosolic Ca2+-dependent manner.

Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

Sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA) is an intracellular Ca2+ pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca2+ stores is pivotal for maintaining intracellular Ca2+ homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca2+ pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca2+ imaging confirmed that the FRET signal changes directly reflect the Ca2+ pump activity. Dual imaging of cytosolic Ca2+ and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca2+ concentration changes evoked by ATP stimulation. The Ca2+ pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca2+ of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca2+ dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca2+ stores in living cells for shaping the intracellular Ca2+ dynamics. F-L577 will be useful for future studies on Ca2+ signaling involving SERCA2a activity.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular Ca2+ release channel, and its opening is controlled by IP3 and Ca2+. A single IP3 binding site and multiple Ca2+ binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca2+ is unknown. In this study, we visualized conformational changes in IP3R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP3 and Ca2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca2+ alone from the FRET changes evoked by both ligands together. A mutant IP3R containing a single amino acid substitution at K508, which is critical for IP3 binding, did not exhibit this bell-shaped Ca2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca2+ sensor, resulted in ∼10-fold reduction in the Ca2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP3R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca2+ binding affinity, as the mechanism underlying the IP3-dependent regulation of the bell-shaped relationship between the IP3R activity and cytosolic Ca2+.