The mechanism of cell size regulation by polycystins

多囊蛋白调节细胞大小的机制

• 批准号: 10345768
• 负责人: Qian Chen
• 金额: $ 32.45万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10345768
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Animal Model; Autosomal Dominant Polycystic Kidney; Biomass; Calcineurin; Calcium; Calcium Signaling; Calcium Spikes; Calmodulin; Cell Cycle; Cell Proliferation; Cell Separation; Cell Size; Cell division; Cell membrane; Cell physiology; Cells; Collaborations; Cytokinesis; Cytoskeleton; Endocytosis; Environment; Evolution; Family; Fission Yeast; Genes; Genetic Diseases; Genetic Screening; Growth; Human; Human Genetics; Image; In Vitro; Intracellular Transport; Ion Channel; Ions; Kidney Diseases; Lead; Lipids; Mediating; Michigan; Microfilaments; Microfluidics; Modeling; Molecular; Morphogenesis; Mutation; Myosin Type I; Myosin Type V; Osmotic Pressure; Pathway interactions; Pattern; Permeability; Phosphotransferases; Play; Polycystic Kidney Diseases; Process; Quantitative Microscopy; Regulation; Regulation of Cell Size; Reproducibility; Role; Signal Pathway; Signaling Molecule; Stimulus; Structure; Techniques; Tennessee; Testing; Universities; Work; Yeasts; base; cell growth; constriction; daughter cell; driving force; imaging modality; innovation; loss of function mutation; mechanical force; mechanical signal; mechanotransduction; multidisciplinary; novel; patch clamp; public health relevance; reconstitution; response

The mechanism of mechanosensing by polycystins during cell growth Cytokinesis is the last stage of cell division when two daughter cells separate, but it is equally important for the transition to cell growth including cell size expansion. The mechanism regulating such a transition is poorly understood. We identified the role of calcium and the polycystin channel Pkd2p in this process while studying cytokinesis of the model organism fission yeast. Pkd2p is essential to regulate the cell size and it mediates calcium influx. Polycystins are evolutionally conserved ion channels. Loss of function mutations of human polycystins lead to the genetic disorder, Autosomal Polycystic Kidney Disorder (ADPKD). The cellular function of this highly conserved family of channels is not well understood. This study will determine how Pkd2p and calcium regulate the transition to cell growth. Aim 1. Determine how the Hippo signaling pathways regulate Pkd2p. Our genetic screen has identified the fission yeast Hippo pathways SIN and MOR as crucial for the regulation of Pkd2p. Both are highly conserved kinase cascades that are essential for cell proliferation. SIN antagonizes Pkd2p activity, while MOR synergizes with Pkd2p. However, the mechanism is unclear. We will 1) Determine how MOR and SIN regulate cellular calcium during cytokinesis through calcium-imaging. 2) Determine how MOR and SIN regulate Pkd2p in cell size expansion. 3) Determine how MOR promotes the cell cycle-dependent localization of Pkd2p. 4) Determine whether Pkd2p is a direct substrate of the MOR kinase Orb6p. Aim 2. Determine how the Pkd2p channel is activated. Pkd2 channel opens in response to mechanical force in vitro, but the mechanism is unclear. We will test the proposal that Pkd2p is a channel sensitive to the force driving the yeast cell growth. We will 1) Determine how Pkd2p regulates calcium when the cells are stimulated by external force. 2) Probe how Pkd2p senses osmotic stimuli and the lipid environment in vitro through a collaboration with Allen Liu’s group (University of Michigan). 3) Determine whether Pkd2p channel allows ions other than calcium to pass through, using patch clamp, through a collaboration with Du Jianyang’s group (University of Tennessee). Aim 3 Determine how Pkd2p regulates the actin re-organization during cytokinesis. Calcium can activate two highly conserved molecules Cam1p and Ppb1p. We will determine how they contribute to the role of Pkd2p in cytokinesis. We will determine 1) how Pkd2p regulates the Cam1p-dependent endocytosis during cytokinesis. 2) how Pkd2p regulates the Cam1p-dependent intracellular transport. 3) how Pkd2p regulates the enzymatic activity of Ppb1p. Through this study, we expect to uncover a novel cell size regulation pathway mediated by Pkd2p channel. We will demonstrate how both internal signaling pathways and external environment play a vital role in activating this channel in cytokinesis. We will employ novel imaging methods combined with innovative in vitro techniques in our study. Our works shall help us better understand the cellular functions of the human polycystins.

多囊蛋白在细胞生长过程中的机械敏感机制 胞质分裂是细胞分裂的最后阶段，此时两个子细胞分离，但它对细胞分裂同样重要。 向细胞生长的过渡，包括细胞尺寸扩大。调节这种转变的机制很差 明白我们在研究中发现了钙离子和多囊蛋白通道Pkd2p在这一过程中的作用。 模式生物裂殖酵母的胞质分裂。PKD2p对调节细胞大小是必需的，它介导 钙内流多囊蛋白是进化上保守的离子通道。人类功能缺失突变 多囊蛋白导致遗传疾病，常染色体多囊肾疾病（ADPKD）。细胞功能 对这个高度保守的通道家族的了解还不是很清楚。这项研究将确定Pkd2p和 钙调节向细胞生长的过渡。 目标1.确定Hippo信号通路如何调节Pkd2p。我们的基因筛选已经确定了 裂殖酵母Hippo途径SIN和莫尔对于Pkd2p的调节至关重要。两者都高度保守 对细胞增殖至关重要的激酶级联。SIN拮抗Pkd 2 p活性，而莫尔有协同作用 pkd2p然而，其机制尚不清楚。我们将1）确定莫尔和SIN如何调节细胞内 通过钙成像检测胞质分裂过程中的钙。2)确定莫尔和SIN如何调节细胞大小中的Pkd2p 扩张. 3)确定莫尔如何促进Pkd2p的细胞周期依赖性定位。4)确定 Pkd2 p是否是莫尔激酶Orb6 p的直接底物。目标2.确定Pkd2p通道如何 激活pkd 2通道在体外响应机械力而开放，但机制尚不清楚。我们将 测试Pkd2p是对驱动酵母细胞生长的力敏感的通道的提议。1）确定 当细胞受到外力刺激时，Pkd2p如何调节钙。2)探索Pkd2p如何感知 通过与艾伦刘的研究小组（纽约大学）的合作， 密歇根州）。3)使用贴片确定Pkd2p通道是否允许钙离子以外的离子通过 钳，通过与杜建阳的小组（田纳西大学）合作。目标3确定如何 Pkd2p调控胞质分裂过程中肌动蛋白的重组。钙可以激活两个高度保守的 分子Cam1p和Pb1p。我们将确定它们如何有助于Pkd2p在胞质分裂中的作用。我们将 确定1）在胞质分裂期间Pkd2p如何调节Cam1p依赖的内吞作用。2)如何Pkd2p 调节Cam 1p依赖性细胞内转运。3)Pkd2p如何调节Ppb1p的酶活性。 通过本研究，我们期望发现一条新的由Pkd2p通道介导的细胞大小调节途径。我们 将展示内部信号通路和外部环境如何在激活 细胞质分裂中的这个通道。我们将采用新的成像方法结合创新的体外技术 在我们的研究中。我们的工作将有助于我们更好地了解人类多囊蛋白的细胞功能。