[en] Highlights: • The role of calpain-1 in neutrophil shape change was investigated. • Neutrophils were obtained from knock-out mice and assessed ex vivo. • These cells failed to migrate through endothelial monolayers. • Spontaneous cell spreading was grossly abberant. • Calpain-1 null cells failed to spread in response to IP₃ -induced Ca²⁺ signals. It has been proposed that Ca²⁺ activation of calpain-1 is an important trigger for rapid cell spreading by neutrophils. In this paper, we have investigated this by assessing the ex vivo functioning of neutrophils from calpain-1 null mice, Calpain-1 null neutrophils failed to migrate through TNF-activated endothelial monolayers. The failure to transmigrate through endothelial monolayers was therefore unlikely to be due to a failure of chemotaxis as chemotaxis by adherent calpain-1 null neutrophils towards fMLP was unpaired. In contrast, the capacity of calpian-1 neutrophils to spontaneously spread was limited to smaller diameters than for wild type cells. Photolytic uncaging of IP₃ with Individual wild type neutrophils resulted in a large Ca²⁺ signal and rapid cell spreading. In contrast, calpain-1 neutrophils failed to spread in response to the IP₃-induced Ca²⁺ signal. This work has therefore demonstrated that the presence of calpain-1 was required for effective rapid cell spreading by neutrophils.

[en] Neuroblastoma is the second most common paediatric cancer. It develops from undifferentiated simpatico-adrenal lineage cells and is mostly sporadic; however, the aetiology behind the development of neuroblastoma is still not fully understood. Intracellular calcium ([Ca"2"+]_i) is a secondary messenger which regulates numerous cellular processes and, therefore, its concentration is tightly regulated. This review focuses on the role of [Ca"2"+]_i in differentiation, apoptosis and proliferation in neuroblastoma. It describes the mechanisms by which [Ca"2"+]_i is regulated and how it modulates intracellular pathways. Furthermore, the importance of [Ca"2"+]_i for the function of anti-cancer drugs is illuminated in this review as [Ca"2"+]_i could be a target to improve the outcome of anti-cancer treatment in neuroblastoma. Overall, modulations of [Ca"2"+]_i could be a key target to induce apoptosis in cancer cells leading to a more efficient and effective treatment of neuroblastoma

[en] Highlights: • Drosophila TRP 783–862 is a novel calmodulin binding site. • The shortest Ca²⁺-CaM interaction region and core CaM binding sequences in TRP 783–862 were dissected. • Ca²⁺-CaM binding induces helical structure formation of Drosophila TRP 783–862. Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in photo-transduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca²⁺ signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca²⁺ dependent CaM binding site (TRP 783–862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783–862 and Ca²⁺-CaM is 0.10 ± 0.04 μM and their binding stoichiometry is 1:1. In addition, the shortest Ca²⁺-CaM interaction region and core CaM binding sequences in TRP 783–862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca²⁺-CaM binding, the TRP 783–862 fragment showed Ca²⁺-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, our findings provided a biochemical and structural basis for further in vivo functional studies of Ca²⁺-mediated TRP channel regulation through CaM/TRP interaction.

[en] Highlights: • Phosphorylation by Plk2 regulates Ras and Rap specificity of synGAP's GAP activity. • Phosphorylation by both Plk2 and CDK5 additively increases RasGAP activity by 230%. • Together, CaMKII, Plk2, and CDK5 act like a rheostat to finely tune GAP specificity. SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40–50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca²⁺/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.

[en] Highlights: • We performed dual-color in vivo two-photon Ca²⁺ imaging of neurons and astrocytes. • α1-adrenoblocker prazosin substantially reduced global astrocytic activities. • Astrocytes showed weak but reliable visual responses in the awake mouse visual cortex. • Astrocytic visual responses were delayed by 5 s relative to the neuronal ones. Astrocytes are known to contact with a great number of synapses and may integrate sensory inputs. In the ferret primary visual cortex, astrocytes respond to a visual stimulus with a delay of several seconds with respect to the surrounding neurons. However, in the mouse visual cortex, it remains unclear whether astrocytes respond to visual stimulations. In this study, using dual-color simultaneous in vivo two-photon calcium imaging of neurons and astrocytes in the awake mouse visual cortex, we examined the visual response of astrocytes and their precise response timing relative to the surrounding neurons. Neurons reliably responded to visual stimulations, whereas astrocytes often showed neuromodulator-mediated global activities, which largely masked small visual responses. Administration of the selective α1-adrenergic receptor antagonist prazosin substantially reduced such global astrocytic activities without affecting the neuronal visual responses. In the presence of prazosin, astrocytes showed weak but consistent visual responses mostly at their somata. Cross-correlation analysis estimated that the astrocytic visual responses were delayed by approximately 5 seconds relative to the surrounding neuronal responses. In conclusion, our research demonstrated that astrocytes in the primary visual cortex of awake mice responded to visual stimuli with a delay of several seconds relative to the surrounding neurons, which may indicate the existence of a common mechanism of neuron–astrocyte communication across species.

[en] Highlights: • Wild-type ABP34 protein induces formation of actin bundles at low calcium concentrations, and this activity is diminished at higher calcium concentrations. • The actin bundling activity of ABP34 is activated when calcium ion is not bound to the protein through its EF2 calcium-binding motif. • The presence of calcium ion in the EF2 motif affects the stability and conformation of the EF-hand pair. Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell–cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.

[en] Highlights: • Spontaneous CBF oscillation was found in ciliated mouse nasal epithelial cells. • The CBF oscillation was caused by the [Ca²⁺]i oscillation. • The [Ca²⁺]i oscillations were generated by the periodic Ca²⁺ release from internal stores via interactions between IP₃ and IP₃ receptor. • The CBF oscillation may play a crucial role for maintaining an efficient mucociliary clearance in nasal epithelia. Ciliary beating frequency (CBF) was investigated in ciliated nasal epithelial cells (cMNECs) isolated from mice using video microscopy equipped with a high-speed camera. In cMNECs, a spontaneous CBF oscillation was observed. The CBF oscillation was abolished by BAPTA-AM but not by Ca²⁺-free solution. The addition of thapsigargin, which depletes Ca²⁺ from internal stores, also abolished CBF oscillation. Moreover, the intracellular Ca²⁺ concentration [Ca²⁺]_i, spontaneously oscillated even with the Ca²⁺-free solution. Moreover, 2APB (an inhibitor of the IP₃ receptor) abolished CBF oscillation in cMNECs. Overall, these findings suggest that the CBF oscillation in cMNECs is triggered by the release of Ca²⁺ from the IP₃-sensitive internal stores. Moreover, IBMX, an inhibitor of phosphodiesterase, did not affect CBF oscillation in cMNECs, although it slightly increased CBF. These results suggest that CBF oscillations were induced by [Ca²⁺]_i oscillation controlled via the release of Ca²⁺ from IP₃-sensitive stores, rather than via cAMP accumulation. CBF oscillation possibly plays a crucial role in maintaining an efficient mucociliary clearance in the nasal epithelia.

[en] Excessive increase of cytosolic Ca²⁺ through the activation of L-type Ca²⁺ channels (LTCCs) via β adrenergic receptor induces apoptosis of cardiomyocytes. Canstatin, a cleaved fragment of collagen type IV α2 chain, is abundantly expressed in normal heart tissue. We previously reported that canstatin inhibits β adrenergic receptor-stimulated apoptosis in cardiomyoblasts. Here, we tested the hypothesis that canstatin regulates LTCCs activity in ventricular cardiomyocytes. Collagen type IV α2 chain (COL4A2) small interfering (si) RNA (for canstatin suppression) or control siRNA was injected via jugular vein in Wistar rats. Two days after the injection, electrocardiogram (ECG) was recorded and the left ventricular tissue was isolated using Langendorff apparatus. Immunofluorescence staining was performed to clarify the distribution of canstatin in cardiomyocytes. The knockdown efficiency was confirmed by Western blotting. The L-type Ca²⁺ channel current (I_CaL) of ventricular cardiomyocyte was measured by a whole-cell patch clamp technique. In immunofluorescence staining, colocalization of canstatin and α_v integrin was observed in the isolated ventricular cardiomyocytes. The I_CaL of ventricular cardiomyocyte isolated from COL4A2 siRNA-injected rats was significantly enhanced compared with control siRNA-injected rats. Recombinant canstatin (250 ng/ml) significantly reversed it. ECG analysis showed that QT interval tended to be shortened and amplitude of T wave was significantly increased in the COL4A2 siRNA-injected rats. In summary, we for the first time clarified that suppressing canstatin expression increases the basal I_CaL in ventricular cardiomyocytes. It is proposed that canstatin might play a role in the stabilization of cardiac function through the modulation of LTCC activity in cardiomyocytes.

[en] High precision computer controlled tracings of bright Ca⁺-mottles were performed during 1974 and 1975 at the Locarno Observatory of Gottingen to study solar differential rotation and to search for giant cell circulation pattern. The method consists of measuring the position of 5-15 bright Ca⁺- mottles with respect to the center of the solar disc every 10 to 15 min during 4h every day. From a linear least square fit of the observed positions the solar-latitude and longitude were computed for the beginning and the end of the daily 4h observation period. From this the components in latitude and longitude of the proper motions were derived which result from the differential rotation, possible giant cell circulation and the small scale random walk of these features. (Auth.)

[en] Highlights: • Inhibition of NMDA promoted axonal outgrowth at the early stages. • GluN2B but not GluN2A inhibited axonal outgrowth in immature hippocampal neurons. • NMDA inhibited axonal outgrowth by inactivating Akt and activating GSK-3β signaling. Neurons are highly polarized cells with an axon and dendritic arbors. It is still not well studied that how formation and elaboration of axon and dendrites is controlled by diffusible signaling factors such as glutamate via specific receptors. We found that N-methyl-D-aspartate (NMDA) receptors were enriched (stage 2–3) but decreased expression (stage 4–5) at tip of axon of cultured hippocampal neurons during distinct development stages. Inhibition of NMDA receptor activity by competitive antagonist DL-2-amino-5-phosphonovalerate (APV) or channel blocker MK801 promoted axonal outgrowth at the early stages, whereas inhibited dendritic development in later stages. Meanwhile, knockdown of NMDA receptors also promoted axonal outgrowth and branch in immature neurons. Furthermore, GluN2B but not GluN2A subunit inhibited axonal outgrowth in immature hippocampal neurons. Finally, we found that NMDA receptors inhibited axonal outgrowth by inactivating Akt and activating GSK-3β signaling in a calcineurin-dependent manner. Taken together, our results demonstrate that stabilization GSK-3β activation in the axon growth cone by Ca²⁺ influx through NMDA receptors may be involved in regulation of axon formation in immature neurons at early stages.