Mechanisms of Cytoplasmic Localization

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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.