Collaborative Research: Reducing complexity in vivo enables investigation of Cellulose Synthase-like D complex formation, trafficking and function

纤维素作为木材、纸张和纺织纤维的组分具有重要的经济价值。它产生于植物细胞壁，在那里它控制生长和发育。制造大多数植物纤维素（CESA）的酶产生18组葡萄糖链，并将它们捆绑在一起形成坚固耐用的微纤维。相关酶（CSLDs）也产生葡萄糖链，但这些链是否捆绑在一起以及这如何影响最终产品的特性尚不清楚。CLDs是某些植物细胞正常分裂和生长所必需的，这些细胞形成长纤维，这表明CLDs产生的纤维素的特殊性质对这些过程很重要。CESA产生的大量纤维素阻碍了研究CLDD产生的纤维素的尝试。虽然大多数植物不能在没有CESA的情况下存活，但可以产生缺乏CESA的苔藓植物，从而仅依赖于CSSD获得纤维素。这些植物将被用于研究CSLDs及其产生的纤维素的结构和性质。植物细胞通过控制CESA和CSOD在时间和空间上的分布来调节其生长发育。细胞如何在正确的时间将这些酶传递到正确的位置，将通过向CESA和CSLD添加荧光标签来研究。这项研究将有助于我们了解植物生长以及如何改善商业植物纤维的性能。本项目的目的是检验纤维素合成酶样D（CSLD）蛋白质形成纤维素合成酶复合物并合成不同形式的微纤维纤维素，并表征CSLDs的功能分化和细胞靶向，以阐明它们在支持顶端生长的特化细胞壁结构域沉积中的作用和胞质分裂。这项研究是通过敲除所有纤维素合成酶（CESA）基因的藓类立碗藓（以前的立碗藓）的品系实现的。这种独特的资源将用于研究体内CSLD活性，而不受背景CESA活性的干扰。第一个目标是使用所有cesa敲除线，通过冷冻断裂电子显微镜对含有CLDD的膜复合物进行结构表征，并分离和分析CLDD产生的纤维素的结构。第二个目标是通过亚细胞蛋白定位和突变分析来表征CSLDs在原丝体顶端生长和胞质分裂中的作用。最终的目标是使用可能在P.patens中的强大的遗传和活细胞成像工具来确定将CSLD和CESA靶向质膜的不同区域的细胞运输途径。通过定义CESA和CSLDs之间的分工，这项研究将增强对有助于调节尖端生长和胞质分裂的特化细胞壁结构域的合成、结构和机械特性的理解，以及这些如何对整个生物体形态发生做出贡献。该奖项反映了NSF的法定使命，并通过利用基金会的智力价值和能力进行评估，被认为值得支持更广泛的影响审查标准。

Cellulose has important economic value as a component of wood, paper, and textile fibers. It is produced in the walls of plant cells, where it controls growth and development. The enzymes that make most plant cellulose (CESAs) produce chains of glucose in groups of 18 and bundle them together to form strong, durable microfibrils. Related enzymes (CSLDs) also produce chains of glucose, but whether these chains are bundled together and how this affects the properties of the final product is unknown. CSLDs are required for normal cell division and growth of certain plant cells that form long filaments, suggesting that special properties of the cellulose produced by CSLDs are important for these processes. Attempts to study the cellulose produced by CSLDs have been hampered by the abundance of cellulose produced by CESAs. Although most plants cannot survive without CESAs, it is possible to produce moss plants lacking CESAs thereby solely relying on CSLDs for cellulose. These plants will be used to study CSLDs and the structure and properties of the cellulose they produce. Plant cells regulate their growth and development by controlling the distribution of CESAs and CSLDs in time and space. How cells deliver these enzymes to the right place at the right time will be studied by adding fluorescent tags to CESAs and CSLDs. This research will contribute to our understanding of plant growth and how we might improve the properties of commercial plant fibers. It will also build the scientific workforce by training undergraduate and graduate students.The aims of this project are to test the hypothesis that Cellulose Synthase-like D (CSLD) proteins form cellulose synthase complexes and synthesize a distinct form of microfibrillar cellulose and to characterize the functional differentiation and cellular targeting of CSLDs to elucidate their roles in deposition of the specialized cell wall domains that support tip growth and cytokinesis. This research is enabled by lines of the moss Physcomitrium (formerly Physcomitrella) patens with all Cellulose Synthase (CESA) genes knocked out. This unique resource will be used to investigate CSLD activity in vivo without interference from background CESA activity. The first objective is to use the all cesa knockout lines to structurally characterize CSLD-containing membrane complexes by freeze fracture electron microscopy and to isolate and analyze the structure of the cellulose produced by CSLDs. The second objective is to characterize the roles of CSLDs in protonemal tip growth and cytokinesis by sub-cellular protein localization and mutation analysis. The final objective is to determine cellular trafficking pathways that target CSLDs and CESAs to distinct regions of the plasma membrane using the powerful genetic and live cell imaging tools possible in P. patens. By defining the division of labor between CESAs and CSLDs, this research will enhance understanding of the synthesis, structure and mechanical properties of the specialized cell wall domains that contribute to the regulation of tip growth and cytokinesis, and how these contribute to whole organism morphogenesis.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.