Role of ARF5 and ER/plasma membrane contacts in the control of cell migration

ARF5 和 ER/质膜接触在细胞迁移控制中的作用

• 批准号: 10387031
• 负责人: James E. Casanova
• 金额: $ 12.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-11 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387031
• 关键词: Adhesions; Binding; Biological Models; Breast Melanoma; Calcium; Calcium Signaling; Cell membrane; Cells; Clathrin; Complex; Cytosol; Data; Disseminated Malignant Neoplasm; Embryonic Development; Endoplasmic Reticulum; Focal Adhesions; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Individual; Inflammation; Integrins; Lipids; Mediating; Mediator of activation protein; Membrane; Modeling; Monomeric GTP-Binding Proteins; Movement; Neoplasm Metastasis; Organelles; Pathologic Processes; Periodicity; Phenotype; Phosphotransferases; Physiological; Physiological Processes; Process; Protein Family; Publishing; Reporting; Role; STIM1 gene; Signal Transduction; Site; Testing; base; cancer pharmacology; cell motility; extracellular; insight; knock-down; malignant breast neoplasm; member; migration; novel; oxysterol binding protein; recruit; response; sensor; tissue regeneration; tumor; wound healing

ABSTRACT Cell migration is central to many physiological and pathological processes, including morphogenetic movements during embryogenesis, wound healing, tissue regeneration and tumor metastasis. Migration is a cyclical, multistep process in which cells establish integrin-mediated adhesions at the leading edge, use those adhesions to pull themselves forward, and disassemble them at the cell rear, allowing translocation in the direction of movement. Many studies have shown that adhesion disassembly at the cell rear requires calcium influx, but our understanding of how this process is controlled is still largely incomplete. In most migrating cells, the STIM1/Orai complex is an essential mediator of Ca2+ influx. This bipartite sensor/channel assembles at endoplasmic reticulum-plasma membrane (ER-PM) contacts when it senses decreased Ca2+ in the ER and triggers focal influx of extracellular Ca2+ to the cytosol. STIM1 and Orai are coordinately upregulated in a wide variety of metastatic cancers, and pharmacological inhibition blocks migration and metastasis in model systems. In preliminary studies that form the basis of this proposal, we have uncovered a signaling cascade between STIM1/Orai activation at ER-PM contacts and focal adhesion disassembly that is mediated by the small GTPase Arf5, its guanine nucleotide exchange factor (GEF) IQSec1 and ORP3, a member of the Oxysterol binding protein Related (ORP) family of proteins that mediate direct lipid exchange between the ER and other organelles. We find that ORP3 is recruited to ER/PM contact sites in response to STIM1/Orai activation, binds robustly to IQSec1 in a Ca2+-dependent manner and is essential for the Ca2+-induced activation of Arf5. Strikingly, individual knockdown of ORP3, IQSec1, or Arf5 yields the same phenotype; flattened cells with enlarged focal adhesions, slowed adhesion disassembly and dramatically reduced cell motility. Based on these observations, we hypothesize that calcium signaling at the trailing edge triggers the local ORP3-dependent activation of Arf5 by IQSec1, and that Arf5 activity is essential for focal adhesion disassembly and cell motility. This model will be tested in three Specific Aims: 1) to determine how Ca2+ influx triggers recruitment of ORP3 to ER/PM contact sites, 2) to determine how Ca2+ influx controls the interaction of ORP3 with IQSec1 and modulates its guanine nucleotide exchange activity, and 3) to determine how Arf5 promotes focal adhesion turnover and cell motility. In this context, Arfs are known to be allosteric activators of the type I PI5-kinase PIPKIβ, which has been implicated in focal adhesion disassembly through its recruitment of the clathrin-based endocytic machinery. The Arf GEF IQSec1 has been reported to promote metastasis in both breast cancers and melanomas, but its mechanism of action is poorly understood. Completion of these studies will provide a mechanistic understanding of how IQSec1 promotes tumor metastasis (and migration in other contexts) and will also provide new insight into a novel role for ER/PM contact sites in the control of cell motility.

摘要 细胞迁移是许多生理和病理过程的中心，包括形态发生运动。 在胚胎发生、伤口愈合、组织再生和肿瘤转移过程中。移民是一种周期性的、 细胞在前沿建立整合素介导的粘连的多步骤过程，使用这些粘连 将自身向前拉，并在细胞后部将其分解，从而允许向 有动静。许多研究表明，细胞后部的黏附分解需要钙离子内流，但我们的 对于这一过程是如何控制的，人们在很大程度上仍不了解。 在大多数迁移细胞中，STIM1/Orai复合体是钙离子内流的重要中介。这个二重奏 感觉器/通道在内质网-质膜(ER-PM)接触时组装 减少内质网中的钙离子，并触发细胞外钙离子向胞浆内的局部内流。STIM1和ORAI是 在多种转移性癌症中协同上调，药物抑制可阻断迁移 以及模型系统中的转移。在构成这项提案基础的初步研究中，我们发现 ER-PM接触处STIM1/ORAI激活和局部粘连分解之间的信号级联，即 在小GTP酶Arf5的介导下，其鸟核苷酸交换因子IQSec1和成员ORP3 氧甾醇结合蛋白相关(ORP)家族的一种蛋白质，它介导 ER和其他细胞器。我们发现ORP3被招募到ER/PM联系地点以响应STIM1/ORAI 激活，以钙依赖的方式牢固地与IQSec1结合，是钙诱导的激活所必需的 是Arf5的。引人注目的是，单独敲除ORP3、IQSec1或Arf5会产生相同的表型；扁平的细胞 随着局灶性粘连的扩大，粘连解体速度减慢，细胞运动性显著降低。基于 这些观察，我们假设，钙信号在后缘触发局部ORP3依赖 IQSec1激活Arf5，Arf5活性是局部黏附解体和细胞运动所必需的。 该模型将在三个具体目标中进行测试：1)确定钙离子内流如何触发ORP3的招募 ER/PM接触部位，2)确定钙离子内流如何控制ORP3与IQSec1的相互作用并调节 其鸟核苷酸交换活性，以及3)确定Arf5如何促进局灶性黏附转换和 细胞运动性。在这种情况下，ARF是已知的I型PI5-激酶PIPKIβ的变构激活剂，它具有 通过招募基于笼蛋白的内吞机制参与局部黏附的分解。 据报道，Arf环境基金IQSec1可促进乳腺癌和黑色素瘤的转移， 但人们对其作用机制知之甚少。这些研究的完成将提供一个机械的 了解IQSec1如何促进肿瘤转移(以及在其他情况下的迁移)，并将提供 对ER/PM接触部位在控制细胞运动中的新作用的新见解。

项目成果

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and cilia to regulate ciliogenesis and ciliary protein traffic.

• DOI: 10.1091/mbc.e21-09-0443
• 发表时间: 2022-02-01
• 期刊: MOLECULAR BIOLOGY OF THE CELL
• 影响因子: 3.3
• 作者: Turn, Rachel E.;Hu, Yihan;Dewees, Skylar, I;Devi, Narra;East, Michael P.;Hardin, Katherine R.;Khatib, Tala;Linnert, Joshua;Wolfrum, Uwe;Lim, Michael J.;Casanova, James E.;Caspary, Tamara;Kahn, Richard A.
• 通讯作者: Kahn, Richard A.