Characterizing the Activity of PPE Proteins in the frizzled/PCP Signaling Pathway

The frizzled/starry night signaling pathway is a non-canonical Wnt signaling pathway which controls planar cell polarity in both vertebrates and invertebrates. Genetic studies using Drosophila melanogaster identified a set of genes which control planar cell polarity and those genes were placed into three phenotypic and epistasis group in the frizzled/starry night signaling pathway. This dissertation reports on the molecular and genetic studies of downstream components – the planar cell polarity effector genes, inturned (in), fuzzy (fy) and fritz (frtz) and their relationship with upstream planar cell polarity core genes, such as disheveled (dsh).

In chapter 2, I describe the context dependent epistatic relationship between downstream planar cell polarity effector genes and upstream core genes. I have found that when over expressed the planar cell polarity effector proteins can alter both the subcellular location and level of accumulation of the upstream proteins. I also showed that In, Fy and Frtz form a protein complex within the wing cells and modulate the accumulation of each other. I further found that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

In Chapter 3, I describe a set of experiments to map the residues required for the interaction between In and Frtz WD40 fragment. Also, I found a direct interaction between Frtz or Fy with planar cell polarity core protein Dsh, suggesting the asymmetric accumulations of planar cell polarity effector proteins are mediated by physical interactions with Dsh. The interaction between Frtz and Dsh requires multiple fragments in the Frtz protein. Furthermore, I describe a newly generated in null mutant, in precise deletion (inPD) using the CRISPR/Cas9 genome editing technique and found that Frtz has both In independent and dependent functions in Drosophila wing cells as the mild overexpression of Frtz partially rescued the In loss of function mutant.

In Chapter 4, I describe a newly generated frtzmNeonGreen stock (made using the CRISPR/Cas9 genome editing technique) for studying the Frtz protein activity in Drosophila. I modified the frtz gene in the Drosophila genome by adding a green fluorescent protein mNeonGreen on the carboxyl terminus of frtz. This modified gene provides full rescue of frtz and is good for live imaging of the Frtz protein in various tissues, such as wing, thorax, abdomen and arista. Time-lapse imaging data shows Frtz protein is preferentially distributed to discrete membrane subdomains (“puncta”), and the puncta are very dynamic at the subcellular level¬. Fluorescence recovery after photo-bleaching shows Frtz protein in various tissues has a similar recovery rate.