Zygotes (fertilized eggs) of fucoid brown algae are ideal for investigating the mechanisms that establish polarity in developing organisms. Dr. Kropf's lab has for some time been investigating the cellular and physiological mechanisms that establish a growth axis and determine the division plane in these zygotes. From this work they have formulated hypotheses to be tested during this award period. The working hypotheses are as follows: Growth axis specification. Dr. Kropf proposes that polarity is first set in place at fertilization with the site of sperm entry specifying the rhizoid pole of the growth axis. This site is marked by local assembly of a cytoskeletal element, F-actin. The fertilized egg then settles onto the rocks in the intertidal zone, attaches and begins to monitor its environment for spatial information to help determine the direction of the rocky substratum. If relevant vectors are perceived, the sperm-induced axis is abandoned and a new axis is generated such the new rhizoid pole is toward the rock surface. Concomitantly, the actin patch at the sperm entry site is disassembled and a new patch is assembled at the nascent rhizoid site. Realignment of the axis can occur repeatedly over several hours. Finally, about 12 hours after fertilization, growth ensues at the current rhizoid pole; the rhizoid formed attaches the zygote firmly to rocks and eventually forms the holdfast of the alga. Division plane alignment. Dr. Kropf speculates that division plane is determined at telophase of mitosis such that the new cell wall is deposited transverse to the axis defined by the two telophase nuclei. The orientation of the telophase nuclei is determined by centrosomal position. Centrosomes are inherited paternally and separate to opposite sides of the zygotic nucleus. Initially, the axis defined by the two centrosomes is randomly aligned with the growth axis, but prior to cytokinesis the centrosomal axis becomes aligned with the growth axis by a combination of nuclear rotation and spindle elongation. Research in this proposal will test and extend the most important aspects of the models. The primary objectives are to 1) investigate the requirement for dynamic actin in specifying the growth axis, 2) identify actin nucleating molecules, 3) determine the role of asymmetric cell shape in division plane alignment, 4) assess whether telophase spindle position determines division plane, and 5) investigate the role of microtubules in defining the division plane.
