Establishment and Expression of Polarity in Pelvetia Zygotes

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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.