Collaborative Research: SPK1-ROP Signaling at the ER surface: Implications for ERES Assembly and Morphogenesis

拟议活动的智力价值植物的结构和生长特性对现代食品和可再生能源生产系统至关重要。然而，由于缺乏对蛋白质机制和细胞生长机制的了解，因此几乎不可能设计出适合作物生长和组成的作物。在植物中，叶表皮是一张机械敏感的薄片，它决定了器官的生长特性和结构。这项研究的长期目标是了解蛋白质复合体和细胞聚合物如何协调表皮生长。对信息流和生长的细胞机制的直接了解将推动未来的作物改良战略。这个项目的目标是确定一种进化上保守的信号蛋白SPIKE1在生长过程中如何协调原材料的生产、传递和组装。Szymanski和Stahelin实验室将检验他们的中心假设，即SPIKE1膜结合和内质网(ER)特定区域的小GTP酶激活协调货物的出口和生长。他们将追求三个研究目标。目标1是确定SPK1依赖的小GTP酶的激活及其在内质网亚区的定位如何与囊泡运输和细胞形态发生有关。他们的工作假设是，SPK1环境基金的活性促进了内质网退出位点的组装和内质网和高尔基体之间有效的蛋白质循环。第二个目标是发现SPK1被限制在内质网表面点状分布的细胞机制。他们的工作假设是SPK1与磷脂酰丝氨酸结合对膜流动性敏感，并介导活细胞内ER的定位。目的#3是了解保守的高尔基体定位的蛋白质复合体是否是SPIKE1细胞形状控制途径的中介。我们的工作假设是，SPIKE1促进调节货物运输的高尔基体定位蛋白复合体的小GTP酶激活。他们的初步数据表明，SPIKE1信号促进内质网的形成，并起源于内质网的专门区域。这些内质网出口部位通常被认为是分泌途径中顺行蛋白运输的入口点。这项研究有望为ER结构域定义一个新的重要性，该结构域包括小GTP酶信号和细胞内生长控制系统的整合。鉴于棘突蛋白及其信号靶标的保守性，这项研究可能会对细胞形态发生领域产生广泛影响。广泛影响我们的研究探索具有挑战性的植物细胞生物学的新领域，旨在提供能够对改良作物进行工程设计的知识库。该项目是跨学科的，将研究和学习活动紧密结合在一起。例如，普渡大学和巴黎圣母院的博士后研究员和研究生将开发和指导本科生研究项目。本科生研究人员将学习科学写作，并将在地方和国家科学会议上展示他们的工作。团队学习和解决问题是研究计划的核心。研究项目包括斯塔赫林和希曼斯基实验室之间的跨学科研究。研究发现将在每两个月举行一次的面对面会议上进行讨论，这些会议与芝加哥细胞骨架会议同时举行。研究生和博士后将接受广泛的培训，包括适当的数据管理、有效的口头报告和科学写作。该项目的研究活动将发表在备受瞩目的期刊上，纳入植物细胞生物学研究生课程，在网上发布新闻稿，并将在一个关于尖峰信号蛋白的新网络资源中进行特色报道。像过去一样，新的突变株将捐赠给ABRC库存中心。该项目还将产生与细胞器特定磷脂修饰相关的载体和菌株，这些载体和菌株将被植物细胞生物界广泛使用。

Intellectual Merit of the Proposed ActivitiesThe architecture and growth properties of plants are fundamentally important to modern food and renewable energy production systems. However, a lack of knowledge about the protein machineries and cellular mechanisms of growth makes it nearly impossible to engineer crop plants for optimal growth and composition. In plants, the leaf epidermis is a mechanosensitive sheet that dictates the growth properties and architecture of the organ. The long-range goal of this research is to understand how protein complexes and cellular polymers coordinate epidermal growth. Direct knowledge about the cellular mechanisms of information flow and growth will drive future crop improvement strategies. The goal for this project is to determine how an evolutionarily conserved signaling protein termed SPIKE1 coordinates the production, delivery, and assembly of raw materials during growth. The Szymanski and Stahelin laboratories will test their central hypothesis that SPIKE1 membrane-binding and small GTPase activation at specific domains of the endoplasmic reticulum (ER) coordinates cargo export and growth. They will pursue three research objectives.Objective #1 is to determine how SPK1-dependent small GTPase activation and its localization to subdomains of the ER relate to vesicle trafficking and cell morphogenesis. Their working hypothesis is that SPK1 GEF activity promotes ER exit site assembly and efficient protein recycling between the ER and the Golgi. Objective #2 is to discover the cellular mechanisms by which SPK1 is restricted to a punctate distribution on the surface of the ER. Their working hypothesis is that SPK1 binding to phosphatidylserine is sensitive to membrane fluidity and mediates ER localization in living cells. Objective #3 is to learn if conserved Golgi-localized protein complexes are intermediaries in a SPIKE1 cell shape control pathway. Our working hypothesis is that SPIKE1 promotes small GTPase activation of Golgi-localized protein complexes that regulate cargo trafficking. Their preliminary data indicate that SPIKE1 signals promote the formation of, and arise from specialized domains of the ER. These ER exit sites are commonly known as the entry point for anterograde protein trafficking in the secretory pathway. This research is expected to define a new importance for an ER domain that includes small GTPase signaling and the integration of intracellular growth control systems. Given the conserved nature of SPIKE-like proteins and their signaling targets, this research is likely to have a broad impact on the cell morphogenesis field.Broader ImpactsOur research explores challenging new areas of plant cell biology and aims to provide the knowledge base that will enable the engineering of improved crops. The project is interdisciplinary and closely integrates research and learning activities. For example, post-doctoral fellows and graduate students at Purdue and Notre Dame will develop and supervise undergraduate research projects. Undergraduate researchers will learn scientific writing and will present their work at both local and national scientific meetings. Team learning and problem solving are central to the research plan. The research projects include cross-disciplinary research between the Stahelin and Szymanski labs. Research discoveries will be discussed in bi-monthly face-to-face meetings that are held in concert with the Chicago cytoskeleton meetings. The graduate students and post-docs will receive broad training that includes proper data management, effective oral presentations, and scientific writing. The research activities from this project will be published in high profile journals, incorporated into the a graduate plant cell biology course, featured in web-based news releases, and will be featured in a new web resource on SPIKE-like signaling proteins. As done in the past, new mutant strains will be donated to the ABRC stock center. The project will also generate vectors and strains associated with organelle-specific phospholipid modification that will be broadly used by the plant cell biology community.