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组分，以提供强度并在植物细胞生长和发育中起关键作用。作为“地球上最丰富的生物聚合物”，纤维素还为生物燃料和可再生生物材料提供了巨大的潜在资源，以实现负碳经济。尽管其重要性，在植物中纤维素生物合成的详细分子机制仍然很难理解。该项目通过应用先进的显微成像方法来捕捉活植物中纤维素合酶的动态活性，通过植物生物学家和光学物理学家之间的协同合作来解决这一知识差距。该研究小组将利用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，特别是在亚细胞和分子水平上负责纤维素合成的酶。这些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。该项目还旨在促进密歇根州立大学和南达科他州矿业与技术学院当前教育和推广计划中的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。（PM）通过含有多种纤维素酶（CESA）的纤维素合酶复合物（CSC）。在拟南芥基因组中，鉴定了10个推定的CESA基因，并且生物化学和遗传研究已经揭示，植物中纤维素合成需要至少3种不同的CESA同工型，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。本项目旨在开发和发展显微镜方法，以相关图像的组装和动态的CSC在植物和纤维素微纤维结构在原位。推定的CESA结构域，可能发挥关键作用的CSC组装和运输过程中纤维素生物合成已被改造为表达荧光蛋白标签在相应的cesa敲除背景中的植物成像，使用超分辨率显微镜。具体而言，晶格光片（LLSM）和斜选择性平面照明显微镜系统与增强的光子效率用于3D/4D单分子跟踪，以提高定位精度和深层组织成像，和荧光共振能量转移（FRET）和时间相关的单光子计数方法用于测量CESA-CESA相互作用。此外，3D/4D轨迹数据进行了分析，与其他初步的成像结果，如纤维素微纤丝的超微结构的AFM成像和细胞壁的物理化学性质的受激拉曼散射显微镜成像。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，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。