UCLA

Cappuccino (Capu) is an actin assembly factor that is necessary to establish Drosophila oocyte polarity. Disrupting normal polarity leads to female sterility. It is thought that Capu helps establish oocyte polarity by creating a mesh-like actin structure that spans the oocyte during early stages of development. Disappearance of this actin mesh in later stages of oogenesis coincides with rapid coordinated flows of the cytoplasm, referred to as cytoplasmic streaming. When cytoplasmic streaming starts prematurely, as occurs in capu null mutants, many polarity determinants fail to localize properly. In this dissertation, I describe the discovery and analysis of autoinhibitory regulation of Capu using both in vitro and in vivo approaches. The N-terminal half of Capu (Capu-NT, aa1-466) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (aa467-1059). We identified residues 1-222 as the Capu Inhibitory Domain (CID) using various biochemical techniques such as pyrene polymerization, limited proteolysis and polarization anisotropy assays. This domain is sufficient to bind the short sequence C-terminal to the FH2 domain called Capu-Tail and inhibits the FH2 domain. Based on our biochemical data, we over-expressed a constitutively active form of Capu, CapuδN (Capu271-1059), in Drosophila oocytes. Only 18% of eggs laid by flies expressing CapuδN hatched, compared to 47% fertility for flies over-expressing full-length Capu. From this we concluded that loss of autoinhibition is deleterious to the developing egg. Closer examination of CapuδN-expressing oocytes revealed that the actin mesh persisted beyond stage 10B and the onset of cytoplasmic streaming was delayed. We quantified cytoplasmic streaming using Particle Image Velocimetry and found that streaming is significantly slowed or absent in flies expressing CapuδN. Supporting that CapuδN lost its autoinhibitory regulation, trans-expression of CapuδN and Capu-NT rescued fertility, actin mesh and cytoplasmic streaming phenotypes associated with expressing CapuδN alone. We also found that the decrease in fertility is due to disruption in the late delivery of polarity factors due to the persistent mesh. Several classic polarity factors, such as oskar, nanos, and bicoid are improperly localized in the oocyte. Thus autoinhibition of Capu is critical to oogenesis and our ongoing work is elucidating the mechanisms underlying this observation.