Molecular and Cellular Basis of Oriented Cellulose Microfibril Deposition in Boergesenia

巨藻细胞（Boergesenia forbesii）将用于研究定向纤维素微纤维沉积的分子和细胞基础。该模式系统的优点是：(a)大规模培养无性系；(b)细胞对损伤作出反应，同步形成大量原生质体；(c)蛋白质合成开始，允许分离发育阶段进行生化分析；(d)高度结晶的纤维素微纤维的组装是由有序的质膜颗粒的线性聚集引起的，称为末端络合物（TCs）；(e) TC尺寸足够大，可以进行显微检测。将采用先进的生化技术（包括自由流动电泳）纯化年轻原生质体和薄壁无平面孢子的质膜。蛋白质模式的差异将与纤维素生物合成的开始有关。将产生针对纯化质膜蛋白的单克隆抗体。由于TC大，结合抗体可以用荧光显微镜筛选。蛋白质将通过western blotting鉴定，并通过免疫亲和柱纯化。抗体将用于研究TC在体内和体外的功能。通过抗体方法鉴定纤维素蛋白，可以对其他植物、原核生物和可能合成纤维素的动物的纤维素蛋白进行比较分析。纤维素壁是如何形成的问题是植物生物学中最基本的，也是最不为人所知的问题之一。植物细胞壁生长问题是植物细胞生长分裂乃至整个植物发育问题的关键。在这个奖项下进行的工作也可能导致我们对纤维素合成的理解的进步，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.