Mechanisms of Cytoplasmic Localization

Fucus的受精卵和幼胚提供了一个模型系统来研究合子/细胞不对称的产生和局部细胞质成分向子细胞的分配的基本机制。这些极性过程在许多不同的生物中都很常见；从酵母菌、两栖动物和线虫，到脊椎动物、哺乳动物和植物。然而，要了解细胞如何建立极轴并定向地将细胞质物质运送到由轴定义的预定位置，在大多数这些生物体中很难进行研究和实验操作。相反，fucale的卵是极性的，卵中没有明显的空间图案，“极性决定因素”不局限于卵中，也不受单侧光等矢量的旋转。因此，在单细胞受精卵的同步培养中，极轴在表观遗传上响应这些梯度而产生，并且易于实验分析。本研究的目标是：1)在极性生长位点表征轴稳定复合物中的蛋白质，并制备针对这些蛋白质的多克隆抗体，用于：a)测试这些基因产物在极轴固定中的作用；b)筛选cDNA表达文库，以分离和表征这些不对称定位的关键结构成分的编码基因；2)使用转录和表达载体构建根状体和菌体特异性cDNA文库，并鉴定其克隆；A)转录本在根状细胞或菌体细胞中局部表达，b)作为轴稳定复合体的一部分，蛋白质产物可能在轴固定或根状细胞中发挥作用；3)通过确定上述确定的局部基因产物的作用；a)反义RNA技术，首先需要发展瞬时转化技术（如微注射、电穿孔和微射体）来鉴定在受精卵中表达的嵌合基因结构，b)抗体微注射来确定特定蛋白质在极性过程中的作用。这个项目试图确定生物体从最初的径向对称卵子发展到不对称的机制。

Zygotes and young embryos of Fucus provide a model system to study the basic mechanisms involved in the generation of zygotic/cell asymmetry and partitioning of localized cytoplasmic components to the resulting daughter cells. These polar processes are common to many different organisms; from yeasts, amphibians, and nematodes, to vertebrates, mammals and plants. However, to understand how a cell establishes a polar axis and directionally transports cytoplasmic material to a predetermined site defined by the axis, is difficult to study and experimentally manipulate in most of these organisms. In contrast, eggs of the Fucales are apolar, there is no apparent spatial patterning in the egg, and "polar determinants" are not localized in the egg and rotated by such vectors such as unilateral light. Hence the polar axis arises epigenetically in response to these gradients, in synchronous cultures of single-celled zygotes, and is amenable to experimental analysis. The proposed goals are to: 1) characterize the proteins in the axis stabilizing complex at the site of polar growth, and prepare polyclonal antibodies to these proteins for use in: a) testing the role of these gene products in polar axis fixation, and b) screening a cDNA expression library to isolate and characterize the genes coding for these critical structural components that are asymmetrically localized, 2) construct rhizoid and thallus specific cDNA libraries, using both transcriptional and expression vectors, and identify clones whose; a) transcripts are locally expressed in either the rhizoid or thallus cell, and b) protein products may play a role in axis fixation or rhizoid function as a part of the axis stabilizing complex; 3) determine the role of localized gene products identified above by; a) antisense RNA techniques, which first requires the development of transient transformation techniques (e.g. microinjection, electroporation and microprojectiles) to identify chimeric gene constructs that are expressed in zygotes, and b) microinjection of antibodies to determine the role of specific proteins in polar processes. %%% This project attempts to determine the mechanisms involved in the development of asymmetry in an organism from a initially radially symmetric egg.