The role of Cornichon Homologue proteins in cytosolic calcium homeostasis through sorting and activity of plant ionotropic- Glutamate Receptor-Like channels

Cornichon 同源蛋白通过植物离子型谷氨酸受体样通道的分选和活性在胞质钙稳态中的作用

• 批准号: 10480922
• 负责人: Jose A Feijo
• 金额: $ 33.99万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480922
• 关键词: Address; Affect; Animals; Apoptosis; Arabidopsis; Area; Calcium; Calcium Signaling; Cell model; Cell physiology; Cells; Cognitive; Complement; Cryoelectron Microscopy; Custom; Data; Electrophysiology (science); Eukaryotic Cell; Family; Feedback; Financial compensation; Genetic; Glutamate Receptor; Growth; Homeostasis; Homologous Gene; Homologous Protein; Image; Integral Membrane Protein; Ion Channel; Ions; Knowledge; Lead; Ligands; Link; Location; Maintenance; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Modeling; Molecular; Morphogenesis; Mosses; Mutation; Nature; Nerve Degeneration; Pathologic; Pathology; Permeability; Phenotype; Plants; Pollen; Pollen Tube; Proteins; Protoplasts; Public Health; Regimen; Regulation; Research; Rest; Role; Ryanodine Receptors; Schizophrenia; Signal Transduction; Sorting - Cell Movement; Specificity; Tertiary Protein Structure; Testing; Vesicle; Work; Yeasts; base; design; dosage; male; mathematical model; member; mutant; neurotransmission; novel; overexpression; phosphoric diester hydrolase; recruit; screening; trafficking

Project Summary Calcium signaling is fundamental in all eukaryotic cells. Its existence relies on the homeostatic maintenance of sub-micromolar levels of cytosolic calcium ([Ca2+]cyt). Abnormal perturbation of this basal level triggers a number of pathologies, from cancer to neurodegeneration and apoptosis. This proposal will focus on the provocative hypothesis that vesicular traffic and protein targeting by plant CORNICHON-homologue (CNIH) proteins have a direct regulatory role in [Ca2+]cyt homeostasis and signalling. My group was pioneer in showing that GLUTAMATE RECEPTOR-like (GLR) proteins are Ca2+ permeable ion channels in plants. CORNICHON proteins are ER cargo adaptors mediating the recruitment of integral membrane proteins into COPII vesicles. Here we present evidence that pairs of CNIHs are a necessary condition for the selective targeting of GLRs to specific endomembrane compartments, resulting in their differential localization to different Ca2+ stores. These results made us hypothesize that CNIHs themselves have a feed-back role in Ca2+ homeostasis by controlling the quantity and types of channels that are targeted to these stores. This hypothesis was further substantiated by our finding that the interaction between GLRs and CNIHs gate substantial ion currents in the absence of a ligand. We will test this hypothesis by a combination of genetics, quantitative Ca2+ imaging, mathematical modelling, electrophysiology and protein structural analysis, focusing on three specific aims. (1) We will manipulate CNIH action by over-expression, by changing molecular determinants of cargo sorting and domain swaps within the 5 CNIHs expressed in Arabidopsis. This will allow us to re-address or retain specific GLRs to different subcellular locations. We predict this will produce growth phenotypes by crossing [Ca2+]cyt homeostasis boundaries, which will inform us of the functional hierarchy of the trafficking mechanisms affected. (2) We will develop mathematical models to simulate the relevance of each sub-cellular location to [Ca2+]cyt. We will calibrate these models by screening a vast array of multiple, combined mutations in the GLR/CNIH families, and quantify their Ca2+ choreography changes and GLR localization. This approach will result in phenotypes that will reveal hierarchical contributions of each GLR/location set. Finally (3) we will study the physical interaction of CNIHs and GLRs by electrophysiology after heterologous expression in mammalian cells and by Cryo-EM. Results should enable us to establish a novel model of [Ca2+]cyt regulation based on vesicular trafficking mediated by CNIHs. We argue that this mechanism may be more visible and relevant in plants because they lack all the molecular machinery that animal cells evolved for coordinating small ligand operated Ca2+ stores (such as IP3 and ryanodine receptors, cyclases and phosphodiesterases). However, similar functional interactions between these two classes of proteins exist in yeast and animal cells, and thus we posit they may have an important role in animal cell physiology and pathology, thus potentially challenging the current paradigm of [Ca2+]cyt regulation in eukaryotic cells.

项目摘要 钙信号在所有真核细胞中都是基本的。它的存在依赖于稳态维护 胞质钙的亚微摩尔水平水平（[Ca2+] Cyt）。这种基础水平的异常扰动会触发A 从癌症到神经变性和凋亡的病理数量。该建议将重点放在 挑衅性的假设是植物Cornichon-Hologogue（CNIH）的水泡交通和蛋白质靶向 蛋白质在[Ca2+] Cyt稳态和信号传导中具有直接调节作用。我的小组是先驱 表明谷氨酸受体样（GLR）蛋白是植物中的Ca2+渗透性离子通道。 Cornichon蛋白是ER货物适配器，将整体膜蛋白募集到 Copii囊泡。在这里，我们提供证据表明，CNIH对是选择性的必要条件 将GLR靶向特定的内膜隔室，从而导致其定位差异 不同的Ca2+存储。这些结果使我们假设CNIHS本身在反馈中起作用 通过控制针对这些商店的通道的数量和类型，CA2+稳态。这 我们发现GLR和CNIHS门之间的相互作用进一步证实了假设 在没有配体的情况下，大量离子电流。我们将通过结合 遗传学，定量Ca2+成像，数学建模，电生理学和蛋白质结构 分析，重点是三个特定目标。 （1）我们将通过过度表达来操纵CNIH动作 改变货物排序和域交换的分子决定因素 拟南芥。这将使我们能够重新添加或保留特定的GLR到不同的亚细胞位置。我们 预测这将通过跨越[Ca2+] Cyt稳态边界来产生生长表型，这将告知 我们的运输机制功能层次结构受到影响。 （2）我们将发展数学 模拟每个亚细胞位置与[Ca2+] Cyt的相关性。我们将通过 筛选GLR/CNIH家族中的大量多个合并突变，并量化其Ca2+ 编舞变化和GLR定位。这种方法将导致表型揭示 每个GLR/位置集的分层贡献。最后（3）我们将研究 在哺乳动物细胞和低温EM中，电生理学的CNIH和GLR通过电生理学。 结果应使我们能够基于囊泡运输建立[Ca2+] Cyt调节的新型模型 由CNIHS介导。我们认为这种机制在植物中可能更明显和相关，因为 他们缺乏动物细胞进化用于协调小配体操作Ca2+的所有分子机制 商店（例如IP3和Ryanodine受体，环化酶和磷酸二酯酶）。但是，相似 这两类蛋白质之间的功能相互作用存在于酵母和动物细胞中，因此我们 认为它们可能在动物细胞生理和病理学中起重要作用，因此可能具有挑战性 真核细胞中[Ca2+] Cyt调节的当前范例。

项目成果

Ethylene-independent signaling by the ethylene precursor ACC in Arabidopsis ovular pollen tube attraction

• DOI: 10.1038/s41467-020-17819-9
• 发表时间: 2020-08-14
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Mou, Wangshu;Kao, Yun-Ting;Chang, Caren
• 通讯作者: Chang, Caren

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells.

AMEBaS：偏振单细胞比率荧光延时的自动中线提取和背景扣除。

• DOI: 10.3791/64857
• 发表时间: 2023
• 期刊: Journal of visualized experiments : JoVE
• 作者: Badain,Rafael;Damineli,DanielSC;Portes,MariaTeresa;Feijó,José;Buratti,Stefano;Tortora,Giorgia;NevesdeOliveira,Hugo;CesarJr,RobertoM
• 通讯作者: CesarJr,RobertoM

Analyzing Intracellular Gradients in Pollen Tubes.

分析花粉管中的细胞内梯度。

• DOI: 10.1007/978-1-0716-0672-8_14
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Damineli,DanielSC;Portes,MariaTeresa;Feijó,JoséA
• 通讯作者: Feijó,JoséA

Plasma membrane H+-ATPases sustain pollen tube growth and fertilization

• DOI: 10.1038/s41467-020-16253-1
• 发表时间: 2020-05-14
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Hoffmann, Robert D.;Portes, Maria Teresa;Palmgren, Michael
• 通讯作者: Palmgren, Michael

The Surprising Dynamics of Electrochemical Coupling at Membrane Sandwiches in Plants.

• DOI: 10.3390/plants12010204
• 发表时间: 2023-01-03
• 期刊: Plants (Basel, Switzerland)
• 作者: Dreyer I;Vergara-Valladares F;Mérida-Quesada F;Rubio-Meléndez ME;Hernández-Rojas N;Riedelsberger J;Astola-Mariscal SZ;Heitmüller C;Yanez-Chávez M;Arrey-Salas O;San Martín-Davison A;Navarro-Retamal C;Michard E
• 通讯作者: Michard E