To understand the mechanism of black-pearl and Ddok functioning in Drosophila, this thesis project is comprised of two separate studies: a) Role of black-pearl in Drosophila

In Drosophila, black pearl (Blp) is required for growth and development. EMS- induced blp-null mutants are lethal and fail to develop beyond the first instar larval stage Genetic screening for zygotic lethal mutations that are associated with maternal-effect lethal phenotypes showed that blp GLC mutants are embryonic lethal with segmentation defects. Blp flies homozygous for P-element insertion are semi-lethal, and the escapers exhibit melanotic inclusions in the hemocoel, similar to those observed in innate immune defects. The mammalian Blp homologue, Magmas, is a highly conserved and an essential inner mitochondrial membrane protein. Magmas expression is highly regulated during murine embryogenesis and is responsive to growth factor stimulation. Studies in yeast have shown that the Blp homolog, Magmas/Pam 16, is required for mitochondrial protein transport, growth and survival.

In the present study, we showed that blp-null clones in Drosophila eye imaginal discs exhibit a cell proliferation defect, then investigated the mechanisms underlying the blp phenotype using Blp-RNAi knockdown in Schneider cells. Lowering of Blp led to mitochondrial membrane depolarization, decreased ATP levels, increased amounts of reactive oxygen species (ROS), cell cycle arrest and activation of autophagy. These Blp effects were associated with a selective loss of oxidative phosphorylation complex IV (cytochrome c oxidase) activity, indicating that the cell cycle arrest and activation of autophagy are secondary to the decreased ATP levels and the elevated production of ROS. Consistent with these observations, homozygous blp hypomorph larvae have decreased numbers of plasmatocytes with reduced active mitochondria, indicating that the blp melanotic lesions result from a defective immune system. Further, we have shown that treatment of S2 cells with a recently developed small molecule inhibitor that blocks Magmas dimerization more rapidly recapitulates the effects of Blp-RNAi knock-down. Thus these studies demonstrate a primary role of Blp in oxidative phosphorylation.

Dorsal closure (DC) occurs during mid-embryogenesis in Drosophila and involves the bilateral dorsal movement of the epidermis until it sutures dorsally. DC is widely used as a model system to study concerted epithelial sheet movement during development, morphogenesis and wound healing. A non-receptor, Syk family tyrosine kinase, SH2 domain a&barbelow;nkyrin r&barbelow;epeat k&barbelow;inase (Shark), is required for DC, upstream of the Jun kinase (INK) pathway. A yeast-two-hybrid screen identified the essential adaptor protein, Ddok, as a Shark-interacting protein. Ddok activates Shark by localizing it to the cell periphery and to the actomyosin cable (AC) in the leading edge (LE) cells of the epidermis. Ddok is also essential for the maintenance of F-actin cytoskeleton organization both in the LE cells and the lateral epidermis during DC.

We used the yeast-two-hybrid system to screen for Ddok-interacting proteins from a Drosophila embryonic cDNA library and have identified: 1) Spaghetti squash (Sqh) - the regulatory light chain of non-muscle myosin II, and 2) Cdep, a protein that contains a spectrin-actin binding band 4.1 and an RhoGEF domain. The non-muscle myosin II heavy chain (MHC) is required for DC and is an essential component of the AC. Our studies showed that Ddok constitutively associates with Sqh in a tyrosine phosphorylation-independent manner in S2 cells. Conversely, Ddok association with the myosin heavy chain (MHC) and actin is positively regulated by tyrosine phosphorylation. In contrast to the Ddok-Sqh interaction, Ddok-Cdep binding is inhibited by tyrosine phosphorylation. Preliminary evidence indicates that Cdep tyrosine phosphorylation is also mediated, directly or indirectly, via Src family kinases. Interestingly, Cdep also associates with MHC in a tyrosine phosphorylation dependent manner. Further studies are underway to identify the minimal Ddok regions of interaction with Sqh and Cdep and to validate the requirement of these interactions for the completion of DC.