NSF/MCB-BSF: Development of novel CRAC channel photoswitches

本项目将提供新的分子工具和了解钙释放激活钙（CRAC）通道。 这些是影响细胞中钙进入和信号传导的关键蛋白质。 钙是活细胞中最重要的信号分子之一。 了解钙离子进入和信号传导对理解许多细胞过程，包括迁移，增殖和基因表达具有重要意义。 这个NSF-BSF项目由美国缅因州大学的研究人员领导，与以色列海法理工学院的研究人员合作。 作为这项国际合作的一部分，研究生和本科生将在有机化学，分子生物学和生物物理学的接口进行跨学科研究的培训。 将通过定期视频会议和前往以色列的合作者实验室促进互动和培训。 作为一个补充目标，该提案的更广泛影响将增加缅因州大学STEM领域传统上代表性不足的群体的研究参与，特别是女性和第一代大学生。该项目的总体目标是更好地了解和控制CRAC通道的功能，这是钙库操纵的钙进入（SOCE）的重要组成部分，在后生动物中调节细胞内钙水平的进化保守机制。 该提案的第一个目标将结合联合收割机合成化学和分子生物学，以开发小分子光操纵CRAC通道抑制剂（LOCI），其将提供对天然和基因工程CRAC通道的功能的快速和可逆控制。将使用电生理学和活细胞成像技术合成和评估一组具有不同生物物理特性的LOCI。 由于存在多种CRAC通道同种型，因此该项目的第二个目的将结合联合收割机合成化学和基因工程以开发具有双组分光遗传学构建体的新型LOCI，其将使LOCI能够共价连接到细胞膜或直接连接到不同的CRAC通道组分。这些构建体将使得能够以细胞类型和同种型特异性方式并且以高空间和时间精度控制CRAC通道活性。 最后，LOCI将被用作钙依赖性生物过程的光激活调节剂，特别是细胞迁移和基因表达。这一美国/以色列合作项目得到了美国国家科学基金会和以色列两国科学基金会的支持。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will provide new molecular tools and understanding about Calcium Release Activated Calcium (CRAC) channels. These are key proteins that affect calcium entry and signaling in cells. Calcium is one of the most important signaling molecules in living cells. Understanding calcium entry and signaling has important implications in understanding numerous cellular processes including migration, proliferation, and gene expression. This NSF-BSF project is led by researchers at the University of Maine in the US, in collaboration with researchers at the Technion in Haifa, Israel. As part of this international collaboration, graduate and undergraduate students will be trained in interdisciplinary research at the interface of organic chemistry, molecular biology, and biophysics. Interactions and training will be facilitated through regular video meetings and travel to the collaborator’s laboratory in Israel. As a complimentary goal, the Broader Impacts of this proposal will increase research participation of traditionally underrepresented groups in STEM at the University of Maine, particularly women and first-generation college students.The overarching goal of this project is to better understand and control the function of CRAC channels, which are an essential component of store-operated calcium entry (SOCE), an evolutionary conserved mechanism that regulates intracellular calcium levels in metazoans. The first aim of this proposal will combine synthetic chemistry and molecular biology in order to develop small molecule Light Operated CRAC Channel Inhibitors (LOCIs), which will provide rapid and reversible control of the function of both native and genetically engineered CRAC channels. A set of LOCIs with different photophysical properties will be synthesized and evaluated using electrophysiological and live cell imaging techniques. As there are multiple CRAC channel isoforms, the second Aim of this project will combine synthetic chemistry and genetic engineering to develop new types of LOCIs with two-component optogenetic constructs that will enable covalent tethering of LOCIs to the cell membrane or directly to different CRAC channel components. These constructs will enable the control of CRAC channel activity in a cell-type and isoform specific manner and with high spatial and temporal precision. Finally, LOCIs will be used as light activated modulators of calcium dependent biological processes, particularly cell migration and gene expression.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.