Studying the effects of Hsp27 phosphorylation on viability and fertility. Emily Furbee¹, Joseph Ayoob², Jonathan Minden¹. 1) Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 2) Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

   The small heat shock protein Hsp27 plays many roles within the cell. Some of Hsp27s chaperone roles are to regulate caspase activity and actin dynamics. Although the exact molecular details underlying each of these functions are poorly understood, phosphorylation appears to play a central role in Hsp27 regulation. To further study the role of Hsp27 and its phosopho-regulation in vivo, we used C31 site-directed transgenesis to express either unphosphorylatable (Hsp27^SA) or phosphomimetic (Hsp27^SD) versions of Hsp27 under the control of Hsp27s endogenous promoter. We were surprised to find that ectopic expression of phospho-mutant proteins in an Hsp27 wild-type background produced distinct phenotypes. Embryos that are homozygous for wild-type Hsp27 and Hsp27^SA die in late embryogenesis or as early larvae. These mutants exhibit fragmented or absent trachea, suggesting defects in tracheal morphogenesis, which is an actin-dependant process. In contrast, embryos that are homozygous for wild-type Hsp27 and Hsp27^SD survive to adulthood, but the males are completely sterile. Interestingly, these males display defects in the caspase-dependant individualization stage of spermatogenesis, featuring disrupted actin-based individualization complexes. Here we present the initial molecular dissection of both of these phenotypes that includes live microscopy and histochemical analysis with a focus on how each isoform affects caspase activation and actin dynamics.