Characterization of the gravitaxis-related protein Yuri Gagarin through genetic and biochemical approaches
Texada, Michael James
Beckingham, Kathleen M.
Doctor of Philosophy
The Drosophila melanogaster gene yuri gagarin was first identified in a screen for aberrant behavioral responses to the mechanosensory stimulus provided by the force of gravity. This novel gene encodes Yuri isoforms of four sizes; the three larger isoforms are predicted to be composed largely of coil-forming domains, which are common protein interaction domains. The four isoforms are expressed in varying ratios throughout the animal, at all stages of development. Notably, Yuri is present in muscle tissue, localized to Actin-containing regions of the sarcomere. A male-sterile deletion allele of yuri, yuriF64, was identified and sequenced. This deletion removes roughly 500 base pairs of sequence upstream of the Yuri coding region; in these animals, the mid-sized Yuri isoforms are not expressed in any tissue, and the smallest isoform is no longer expressed in the testis. In wild-type animals, Yuri protein is present throughout the testis, and it assumes a dynamic microtubule-dependent nuclear localization pattern during the process of nuclear condensation, appearing to be a component of the "dense complex" described in ultrastructural studies of spermatogenesis. A previously undescribed filamentous Actin network co-localizes with Yuri on the nuclear surface; Yuri may act as a linker between the Actin and microtubule cytoskeletons. In yuriF64 homozygotes, no Yuri-staining or filamentous Actin structures are detected on the condensing sperm nuclei, and the basal body and centriolar adjunct are mis-localized or absent. Axonemal defects are apparent in heterozygotes and homozygotes, and the Actin-dependent sperm individualization does not take place, leaving immature sperm bound in bundles of 64. Yuri physically interacts with components of the Actin cytoskeleton (Actin, Tropomyosin 1, and Troponin T) in an in vivo pull-down assay. Tropomyosin 1 (Tm1) is the interacting protein purified in greatest abundance in the assay, suggesting that it and Yuri interact directly; this coil-forming molecule wraps actin filaments, stabilizes them against enzymatic or physical degradation, and blocks the interaction of the filament with other proteins. Tm1 is present on the condensing sperm nucleus, and Yuri co-localizes with it in muscle tissue. Thus, the defects seen in yuriF64 animals may reflect aberrant Actin dynamics arising from a lack of Yuri-Tm1 interaction.