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Screening for Arabidopsis mutants with altered Ca²⁺ signal response using aequorin-based Ca²⁺ reporter system.

ABSTRACT: Environmental stimuli evoke transient increases of the cytosolic Ca²⁺ level. To identify upstream components of Ca²⁺ signaling, we have optimized two forward genetic screening systems based on Ca²⁺ reporter aequorin. AEQsig6 and AEQub plants were used for generating ethyl methanesulfonate (EMS)-mutagenized libraries. The AEQsig6 EMS-mutagenized library was preferably used to screen the mutants with reduced Ca²⁺ signal response due to its high effectiveness, while the AEQub EMS-mutagenized library was used for screening of the mutants with altered Ca²⁺ signal response. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020) and Zhu et al. (2013).

SUBMITTER: Sun S

PROVIDER: S-EPMC8144734 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system.

Sun Shujing S Zhang Xiaoyan X Chen Kong K Zhu Xiaohong X Zhao Yang Y

STAR protocols 20210523 2

Environmental stimuli evoke transient increases of the cytosolic Ca2+ level. To identify upstream components of Ca2+ signaling, we have optimized two forward genetic screening systems based on Ca2+ reporter aequorin. AEQsig6 and AEQub plants were used for generating ethyl methanesulfonate (EMS)-mutagenized libraries. The AEQsig6 EMS-mutagenized library was preferably used to screen the mutants with reduced Ca2+ signal response due ...[more]

PMID: 34041505

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Project description:Mutations in either of the polycystic kidney disease genes, PKD1 or PKD2, engender the growth of cysts, altering renal function. Cystic growth is supported by major changes in cellular metabolism, some of which involve the mitochondrion, a major storage site for Ca2+ and a key organelle in cellular Ca2+ signaling. The goal here was to understand the role of components of the mitochondrial Ca2+ uptake complex in PC1-mutant cells in autosomal dominant polycystic kidney disease (ADPKD). We found that the mitochondrial Ca2+ uniporter (MCU) and voltage-dependent anion channels 1& 3 (VDAC) were down-regulated in different mouse and cell models of ADPKD along with the Ca2+-dependent enzyme, pyruvate dehydrogenase phosphatase (PDHX). The release of Ca2+ from the endoplasmic reticulum, and Ca2+ uptake by the mitochondria were upregulated in PC1(polycystin)-null cells. We also observed an enhanced staining with MitoTracker Red CMXRos in PC1-null cultured cells than in PC1-containing cells and a substantially higher increase in response to ER Ca2+ release. Increased colocalization of the Ca2+ sensitive dye, rhodamine2, with MitoTracker Green suggested an increase Ca2+ entry into the mitochondria in PC1 null cells subsequent to Ca2+ release from the ER or from Ca2+ entry from the extracellular solution. These data clearly demonstrate abnormal release of Ca2+ by the ER and corresponding alterations in Ca2+ uptake by the mitochondria in PC1-null cells. Importantly, inhibiting mitochondrial Ca2+ uptake with the specific inhibitor Ru360 inhibited cyst growth and altered both apoptosis and cell proliferation. We further show that the decrease in mitochondrial proteins and abnormally high Ca2+ signaling can be reversed by application of the cystic fibrosis (CFTR) corrector, VX-809. We conclude that enhanced Ca2+ signaling and alterations in proteins association with the mitochondrial Ca2+ uptake complex are associated with malfunction of PC1. Finally, our results identify novel therapeutic targets for treating ADPKD.

Dataset Information

Screening for Arabidopsis mutants with altered Ca²⁺ signal response using aequorin-based Ca²⁺ reporter system.

Publications

Screening for <i>Arabidopsis</i> mutants with altered Ca<sup>2+</sup> signal response using aequorin-based Ca<sup>2+</sup> reporter system.

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