dissection-local-ca2-signals-cultured-cells-membrane-targeted-ca2

Dissection of Local Ca<sup>2+</sup> Signals in Cultured Cells by Membrane-targeted Ca<sup>2+</sup> Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

RIKEN Center for Brain Science

The calcium ion (Ca) is a ubiquitous intracellular signaling molecule that regulates diverse physiological and pathological processes, including cancer. Increasing evidence indicates that oncogenes and tumor suppressors regulate the Ca transport systems. Inositol 1,4,5-trisphosphate (IP) receptors (IPRs) are IP-activated Ca release channels located on the endoplasmic reticulum (ER). They play pivotal roles in the regulation of cell death and survival by controlling Ca transfer from the ER to mitochondria through mitochondria-associated ER membranes (MAMs). Optimal levels of Ca mobilization to mitochondria are necessary for mitochondrial bioenergetics, whereas excessive Ca flux into mitochondria causes loss of mitochondrial membrane integrity and apoptotic cell death. In addition to well-known functions on outer mitochondrial membranes, B-cell lymphoma 2 (Bcl-2) family proteins are localized on the ER and regulate IPRs to control Ca transfer into mitochondria. Another regulatory protein of IPR, IPR-binding protein released with IP (IRBIT), cooperates with or counteracts the Bcl-2 family member depending on cellular states. Furthermore, several oncogenes and tumor suppressors, including Akt, K-Ras, phosphatase and tensin homolog (PTEN), promyelocytic leukemia protein (PML), BRCA1, and BRCA1 associated protein 1 (BAP1), are localized on the ER or at MAMs and negatively or positively regulate apoptotic cell death through interactions with IPRs and regulation of Ca dynamics. The remodeling of Ca signaling by oncogenes and tumor suppressors that interact with IPRs has fundamental roles in the pathology of cancers.

Remodeling of Ca signaling in cancer: Regulation of inositol 1,4,5-trisphosphate receptors through oncogenes and tumor suppressors.

Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca release from the type 1 inositol 1,4,5-trisphosphate receptor (IPR1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca homeostasis in PCs, through IPR1 signaling.

Ca signaling and spinocerebellar ataxia.

Spinocerebellar ataxia type 29 (SCA29) is autosomal dominant congenital ataxia characterized by early-onset motor delay, hypotonia, and gait ataxia. Recently, heterozygous missense mutations in an intracellular Ca channel, inositol 1,4,5-trisphosphate (IP) receptor type 1 (IPR1), were identified as a cause of SCA29. However, the functional impacts of these mutations remain largely unknown. Here, we determined the molecular mechanisms by which pathological mutations affect IPR1 activity and Ca dynamics. Ca imaging using IPR-null HeLa cells generated by genome editing revealed that all SCA29 mutations identified within or near the IP-binding domain of IPR1 completely abolished channel activity. Among these mutations, R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, and E497K impaired IP binding to IPR1, whereas the T579I and N587D mutations disrupted channel activity without affecting IP binding, suggesting that T579I and N587D compromise channel gating mechanisms. Carbonic anhydrase-related protein VIII (CA8) is an IPR1-regulating protein abundantly expressed in cerebellar Purkinje cells and is a causative gene of congenital ataxia. The SCA29 mutation V1538M within the CA8-binding site of IPR1 completely eliminated its interaction with CA8 and CA8-mediated IPR1 inhibition. Furthermore, pathological mutations in CA8 decreased CA8-mediated suppression of IPR1 by reducing protein stability and the interaction with IPR1. These results demonstrated the mechanisms by which pathological mutations cause IPR1 dysfunction, i.e., the disruption of IP binding, IP-mediated gating, and regulation via the IPR-modulatory protein. The resulting aberrant Ca homeostasis may contribute to the pathogenesis of cerebellar ataxia.

Aberrant IP receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29.

Extracellular stimuli evoke a robust increase in the concentration of intracellular Ca ([Ca]) throughout the cell to trigger various cellular responses, such as gene expression and apoptosis. This robust expansion of [Ca] is called Ca propagation. To date, it is thought that intracellular second messengers, such as inositol 1,4,5-trisphosphate (IP) and intracellular Ca, and clusters of IP receptors (IPRs) regulate Ca propagation. However, little is known about how the elevation in the [Ca] spreads throughout the cell, especially in non-polar cell, including HeLa cell. In this study, we developed a novel multi lines analysis tool. This tool revealed that the velocity of Ca propagation was inconstant throughout cell and local concentration of intracellular Ca did not contribute to the velocity of Ca propagation. Our results suggest that intracellular Ca propagation is not merely the result of diffusion of intracellular Ca, and that, on the contrary, intracellular Ca propagation seems to be regulated by more complicated processes.

A novel multi lines analysis tool of Ca dynamics reveals the nonuniformity of Ca propagation.

In most species, fertilization induces Ca transients in the egg. In mammals, the Ca rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP generated by PLCζ induces Ca release from the intracellular Ca store through IP receptor, termed IP-induced Ca release. Here, we developed new fluorescent IP sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) 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 and IP, using IRIS-2s as the IP sensor, we developed a new single fluorophore Ca sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP concentration ([IP]) starts to increase before the onset of the first Ca wave. [IP] stayed at the elevated level with small peaks followed after Ca spikes through Ca oscillations. We detected delays in the peak of [IP] compared to the peak of each Ca spike, suggesting that Ca-induced regenerative IP production through PLC produces small [IP] rises to maintain [IP] over the basal level, which results in long lasting Ca oscillations in fertilized eggs.

Article	Year
Remodeling of Ca signaling in cancer: Regulation of inositol 1,4,5-trisphosphate receptors through oncogenes and tumor suppressors. Advances in biological regulation\| PubMed ID: 29287955	2018
Ca signaling and spinocerebellar ataxia. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 29777722	2018
Aberrant IP receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30429331	2018
A novel multi lines analysis tool of Ca dynamics reveals the nonuniformity of Ca propagation. Cell calcium\| PubMed ID: 30669073	2019
Dual-FRET imaging of IP and Ca revealed Ca-induced IP production maintains long lasting Ca oscillations in fertilized mouse eggs. Scientific reports\| PubMed ID: 30886280	2019

Katsuhiko Mikoshiba

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