Evolution of a Fundamental Morphogenetic Process in the Volvocine Algae

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0131565KirkOur goal is to determine how an essential morphogenetic process requiring extensive intercellular coordination arose as multicellular Volvox evolved from a unicellular ancestor resembling its closest unicellular relative, Chlamydomonas. In order to be able to swim, at the end of embryogenesis Volvox must turn right-side-out in a gastrulation-like process called "inversion," which involves coordinated cytoskeletal rearrangements and changes in cell shape and motility. Transposon tagged mutants with a variety of inversion (inv) defects have been generated. The first of these to be analyzed was Inv61, in which inversion is arrested at the halfway point as a result of a transposon insertion in invA, a gene encoding a novel kinesin that is localized in the embryo near the cytoplasmic bridges that play a key role in inversion. We have cloned the C. reinhardtii orthologue of invA (iarA) and found that it encodes a kinesin (IarA) that is 90 % identical in aa sequence to InvA in both its motor and tail domains. Because invA encodes a microtubule motor, whereas invA mutants resemble wild-type embryos exposed to actomyosin inhibitors, we will explore the possibility that InvA acts as a link between the actin- and tubulin-based cytoskeletons in Volvox. Meanwhile, we will study expression and localization of its orthologue, IarA, throughout the Chlamydomonas cell cycle, and will study the effects of blocking IarA function, using a dominant negative construct, antibody inhibition, or RNA interference. We will use nuclear transformation to determine whether iarA can rescue the invA mutant. If it does not, we will test chimeric iarA-invA constructs to define regions of InvA that are required to confer an inversion function on IarA. The functional significance of such structural differences will then be tested with appropriate kinesin binding and/or motility assays. If iarA does rescue invA, we will compare the proteins with which IarA interacts in Chlamydomonas versus those with which it interacts in Volvox.