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

摘要 细胞迁移是许多生理和病理过程的核心，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期间。移徙是一种周期性的现象， 细胞在前缘建立整合素介导的粘附的多步骤过程，使用这些粘附 把自己向前拉，并在细胞后部分解它们，允许向 运动许多研究表明，细胞后部的粘附解体需要钙离子内流，但我们的研究表明， 对如何控制这一过程的理解在很大程度上仍然是不完整的。 在大多数迁移细胞中，STIM 1/奥赖复合物是Ca 2+内流的重要介质。这两个 传感器/通道在其感测时在内质网-质膜（ER-PM）接触处组装 减少ER中的Ca 2+并触发细胞外Ca 2+向胞质溶胶的局灶性内流。STIM 1和奥赖是 在多种转移性癌症中协同上调，药理学抑制阻断迁移 和转移。在构成这一提议基础的初步研究中，我们发现， 在ER-PM接触处的STIM 1/奥赖激活与粘着斑解体之间的信号级联， 介导的小GTdR Arf 5，其鸟嘌呤核苷酸交换因子（GEF）IQSec 1和ORP 3，一个成员 氧固醇结合蛋白介导内质网和内质网之间直接脂质交换的相关蛋白质家族 和其他细胞器。我们发现，响应于STIM 1/奥赖，ORP 3被募集到ER/PM接触部位 激活，以Ca 2+依赖性方式与IQSec 1牢固结合，并且对于Ca 2+诱导的激活至关重要 Arf5引人注目的是，ORP 3、IQSec 1或Arf 5的单独敲低产生相同的表型：扁平细胞 伴随着扩大的粘着斑、减缓的粘着解体和显著降低的细胞运动性。基于 根据这些观察，我们假设后缘的钙信号触发了局部ORP 3依赖性的 IQSec 1激活Arf 5，Arf 5活性对粘着斑解体和细胞运动性至关重要。 该模型将在三个特定目的中进行测试：1）确定Ca 2+内流如何触发ORP 3的募集， ER/PM接触位点，2）以确定Ca 2+内流如何控制ORP 3与IQSec 1的相互作用并调节 其鸟嘌呤核苷酸交换活性，和3）确定Arf 5如何促进粘着斑转换， 细胞运动性在这种情况下，已知Arf是I型PI 5-激酶PIPKIβ的变构激活剂，其具有 通过招募基于网格蛋白的内吞机制参与粘着斑解体。 据报道，Arf GEF IQSec 1促进乳腺癌和黑色素瘤的转移， 但其作用机制知之甚少。这些研究的完成将提供一个机制 了解IQSec 1如何促进肿瘤转移（和其他情况下的迁移），并将提供 对ER/PM接触位点在细胞运动控制中的新作用的新见解。

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 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.