Establishment and Expression of Polarity in Pelvetia Zygotes

本工作将探讨极性在岩藻受精卵中建立和表达的机制。大部分工作将集中在细胞骨架的作用上。关于细胞骨架的三个主要目标将被追求。(1)深入表征微丝和微管网络的结构，特别是这些网络之间的相互作用。这些实验将依靠共聚焦显微镜。肌动蛋白结合蛋白和微管相关蛋白的初步研究将使用生化方法和特异性抗体探针进行。(2)通过向活细胞内微注射荧光探针，实时研究皮层微丝和内质微管的动态重排。抑制剂将用于研究胚胎发生中细胞骨架重排的功能。(3)细胞内运输的机制也将通过显微注射进行检查。两种类型的细胞内运输将被研究，核原迁移和分泌囊泡运输。研究的第二部分将发展原生质体作为实验工具在两方面的研究。首先，根状细胞的原生质体提供了一个独特的机会来研究极性的丧失，并将作为“去极化”的模型进行研究。其次，研究极性的分子方法将从原生质体cDNA文库的生产和初步表征开始发展。

This work will investigate the mechanism(s) by which polarity is established and expressed in fertilized eggs of fucoid algae. Much of the work will focus on the role of the cytoskeleton. Three major objectives concerning the cytoskeleton will be pursued. (1) An in-depth characterization of the structure of microfilament and microtubule networks, and especially interactions between these networks. These experiments will rely on confocal microscopy. Preliminary investigations of actin-binding proteins and microtubule-associated proteins will be conducted using biochemical approaches and specific antibody probes. (2) Dynamic rearrangements of cortical microfilaments and endoplasmic microtubules will be investigated in real time by microinjecting fluorescent probes into living cells. Inhibitors will be use to investigate the function of cytoskeletal rearrangements in embryogenesis. (3) Mechanisms of intracellular transport will also be examined using microinjection. Two types of intracellular transport will be studied, pronuclear migration and secretory vesicle transport. The second part of the research will develop the use of protoplasts as an experimental tools in two lines of research. First, protoplasts of the rhizoid cell provide a unique opportunity to investigate the loss of polarity, and will be investigated as models of 'depolarization'. Second, molecular approaches to studying polarity will be developed beginning with production and initial characterization of a cDNA library from protoplasts.