MCA: Understanding cellulose synthase complex in planta using single molecule methods

MCA：使用单分子方法了解植物中的纤维素合酶复合物

• 批准号: 2321398
• 负责人: Shi-You Ding
• 金额: $ 37.35万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2321398&HistoricalAwards=false
• 关键词: MCA; Understanding; cellulose; synthase; complex

Cellulose is predominantly produced by plants as the main load-bearing component of the plant cell wall to provide strength and play critical roles in plant cell growth and development. As “the most abundant biopolymer on earth,” cellulose also provides a great potential resource for biofuels and renewable biomaterials towards a carbon-negative economy. Despite its importance, the detailed molecular mechanism underlying cellulose biosynthesis in plants remains largely elusive. This project addresses this knowledge gap by applying advanced microscopic imaging methods to capture the dynamic activity of cellulose synthase in living plants, achieved through a synergistic collaboration between a plant biologist and an optical physicist. The research team will exploit and develop new microscopy techniques to allow real-time visualization of cell wall biosynthesis in growing plants, specifically the enzymes that are responsible for cellulose synthesis at the subcellular and the molecular levels. The results will provide new insights into cellulose biosynthesis and critical information required for further rational design and production of bio-based materials. The project also aims to foster collaboration and multidisciplinary team training of young researchers and students in the current education and outreach programs at Michigan State University and South Dakota School of Mines and Technology to explore their interests in plant science and discover their passion in science and engineering.The biosynthesis of cellulose has been described as a spatially and temporally controlled process carried out in the plasma membrane (PM) by a cellulose synthase complex (CSC) containing multiple cellulose synthases (CESAs). In the Arabidopsis genome, ten putative cesa genes are identified and biochemical and genetic studies have revealed that at least three different CESA isoforms at a 1:1:1 ratio are required for cellulose synthesis in planta. However, the architecture of CSC and its dynamic function in synthesizing cellulose has been largely elusive. This project aims to exploit and develop microscopy approaches to correlatively image the assembly and dynamics of CSCs in planta and cellulose microfibril structure in situ. The putative CESA domains that may play critical roles in CSC assembly and trafficking during cellulose biosynthesis have been engineered to express fluorescence protein tags in corresponding cesa knockout backgrounds for in planta imaging using super resolution microscopy. Specifically, the lattice light-sheet (LLSM) and oblique selective plane illumination microscopy systems with enhanced photon efficiency are used for 3D/4D single molecule tracking to improve localization accuracy and deep tissue imaging, and fluorescence resonance energy transfer (FRET) and time correlated single photon counting methods are used to measure CESA-CESA interactions. Furthermore, the 3D/4D trajectory data are analyzed to correlate with other preliminary imaging results, such as ultrastructure of cellulose microfibrils imaged by AFM and physicochemical properties of cell walls imaged by stimulated Raman scattering microscopy. The findings from this project will allow us to test our working hypotheses and formulate future research directions.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.

纤维素主要由植物产生，作为植物细胞壁的主要承重成分，为植物细胞的生长和发育提供强度并发挥关键作用。作为“地球上最丰富的生物聚合物”，纤维素也为植物细胞提供了巨大的潜在资源。尽管生物燃料和可再生生物材料对碳负经济具有重要意义，但植物中纤维素生物合成的详细分子机制仍然在很大程度上难以捉摸，通过应用先进的显微成像方法来捕获纤维素的动态活动。通过植物生物学家和光学物理学家之间的协同合作，研究小组将开发和开发新的显微镜技术，以实时可视化生长植物中的细胞壁生物合成，特别是负责的酶。研究结果将为纤维素生物合成提供新的见解，并为进一步合理设计和生产生物基材料提供关键信息。在密歇根州立大学和南达科他州矿业与技术学院当前的教育和外展项目中促进年轻研究人员和学生的合作和多学科团队培训，以探索他们对植物科学的兴趣并发现他们对科学和工程的热情。纤维素的生物合成已被描述为由含有多种纤维素合酶（CESA）的纤维素合酶复合物（CSC）在质膜（PM）中进行的空间和时间控制过程。在拟南芥基因组中，有十种假定的纤维素合酶。基因的鉴定以及生化和遗传学研究表明，植物中的纤维素合成至少需要三种不同的 CESA 异构体（比例为 1:1:1）。然而，CSC 的结构及其在合成纤维素中的动态功能在很大程度上是难以捉摸的。该项目旨在开发和开发显微镜方法，对植物中 CSC 的组装和动态以及可能发挥关键作用的纤维素微纤维结构进行相关成像。纤维素生物合成过程中 CSC 组装和运输的作用已被设计为在相应的 cesa 敲除背景中表达荧光蛋白标签，用于使用超分辨率显微镜进行植物成像，特别是具有增强功能的晶格光片 (LLSM) 和倾斜选择性平面照明显微镜系统。光子效率用于 3D/4D 单分子跟踪，以提高定位精度和深层组织成像，以及荧光共振能量转移 (FRET) 和时间相关单光子此外，还使用计数方法测量 CESA-CESA 相互作用，分析 3D/4D 轨迹数据以与其他初步成像结果相关联，例如通过 AFM 成像的纤维素微纤维的超微结构和通过受激拉曼散射显微镜成像的细胞壁的物理化学特性。该项目的研究结果将使我们能够测试我们的工作假设并制定未来的研究方向。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力评估进行评估，认为值得支持优点和更广泛的影响审查标准。