Molecular and Cellular Basis of Oriented Cellulose Microfibril Deposition in Boergesenia

巨大的藻细胞，Boergesenia forbesii，将被用来研究定向纤维素微纤维沉积的分子和细胞基础。这个模型系统的优点是：(A)大规模无菌培养；(B)细胞对伤害做出反应，同时形成大量原生质体；(C)启动蛋白质合成，允许分离发育阶段进行生化分析；(D)高结晶纤维素微纤维组装由称为末端复合体(TC)的有序质膜颗粒的线性聚集启动；以及(E)TC尺寸足够大，可用于显微镜检测。利用先进的生化技术(包括自由流动电泳法)对幼嫩原生质体和薄壁无节孢子的质膜进行纯化。蛋白质模式的差异将与纤维素生物合成的开始相关。将产生针对纯化的质膜蛋白的单抗。由于TC较大，结合抗体可以用荧光显微镜进行筛选。蛋白质将通过免疫印迹和免疫亲和层析进行鉴定和纯化。抗体将用于体外和体内研究TC的功能。通过抗体方法鉴定TC中的蛋白质，可以比较分析其他植物、原核生物，可能还有一些合成纤维素的动物的TC蛋白质。如何铺设纤维素壁的问题是植物生物学中最基本、也最不为人所知的问题之一。壁的生长问题是植物细胞生长和分裂以及整个植物发育问题的关键。根据这一奖项开展的工作也可能导致我们对纤维素合成的理解有所进步，这可能具有商业意义。

The giant algal Cell, Boergesenia forbesii, will be used to investigate the molecular and cellular basis of oriented cellulose microfibril deposition. Advantages of this model system are: (a) large scale cultivation of axenic cultures; (b) the cell responds to wounding to synchronously form numerous protoplasts; (c) protein synthesis is initiated, allowing segregation of developmental stages for biochemical analysis; (d) highly crystalline cellulose microfibril assembly is initiated by linear aggregation of ordered plasma membrane particles known as terminal complexes (TCs); and (e) TC size is sufficiently large for microscopic detection. Plasma membranes of young protoplasts and thin walled aplanospores will be purified by advanced biochemical techniques (including free flow electrophoresis). Differences in protein patterns will be correlated with onset of cellulose biosynthesis. Monoclonal antibodies will be produced against purified plasma membrane proteins. Due to large TC size, binding antibodies can be screened using fluorescence microscopy. Protein will be identified by western blotting and purified by immunoaffinity columns. Antibodies will be used to study TC function in vitro and in vivo. The antibody approach leading to identification of proteins in the TC will allow comparative analysis of TC proteins from other plants, prokaryotes, and possibly some animals which synthesize cellulose. The question of how cellulosic wall is laid down is one of the most fundamental, and least well understood, problems in plant biology. The question of wall growth is key to problems of plant cell growth and division, as well as whole-plant development. The work to be carried out under this award may also result in advances in our understanding of cellulose synthesis which may be of commercial significance.