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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005., Includes bibliographical references., The Drosophila brain is composed of many morphologically and functionally distinct processing centers and brain morphogenesis depends on the creation and maintenance of distinct boundaries between adjacent regions to prevent cells from mixing. In the Drosophila visual system, I have found that Slit and Roundabout (Robo) proteins function to prevent cells from adjacent compartments from mixing. I have defined a boundary between two distinct compartments, the lamina and lobula, and find that the secreted ligand Slit is present in the lamina, while the Robo receptors (Robo, Robo2 and Robo3) are expressed on lobula neurons. I examined the function of theses proteins by identifying a tissue-specific allele of slit and creating transgenic RNAi flies that inhibit the expression of the Robo proteins. Loss of Slit or all three Robo proteins in the visual system results in the invasion of lobula neurons into the lamina. Mixing of cells at the lamina/lobula boundary results in glial cell mispositioning and aberrant photoreceptor axon targeting. Thus, Slit and Robo proteins are required to restrict movement of cells across the lamina/lobula boundary. Additionally, I have characterized Ptpmeg, a highly conserved protein tyrosine phosphatase (PTP). In addition to the C-terminus PTP domain, Ptpmeg contains a central PDZ domain and an N-terminus FERM domain. The in vivo role of this family of proteins is unknown. To explore the function of Ptpmeg in flies, mutants were generated by targeted gene disruption. Examination of the adult nervous system of Ptpmeg mutants reveals a defect in the mushroom bodies (MB), brain structures required for olfactory learning and memory. In mutant animals, the MB lobes are disorganized and fail to elaborate their characteristic structure. I find, (cont.) that Ptpmeg is expressed on MB axons and targeted knockdown of Ptpmeg in the MB results in similar defects as seen in homozygous mutants. Thus, the MB neurons appear to require Ptpmeg for proper formation., by Timothy D. Tayler., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005., Includes bibliographical references., BEACH proteins comprise an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chediak-Higashi) domain of unknown function. They have been shown to play a role in a number of important cellular processes, ranging from cytokinesis to synaptic transmission, and implicated in human diseases, such as Chediak-Higashi Syndrome and cancer. Analysis of several BEACH proteins suggests that they may be involved in membrane trafficking; however, little insight has been gained into their molecular mechanism of function. We identified Drosophila Beach1 in a gain-of-function screen: beach1 overexpression in the photoreceptors drastically alters their growth cone morphology. In a subsequent genetic modifier screen, I identified rabll as a strong enhancer of the beach1 eye overexpression phenotype. Rabll is a small GTPase, which has been shown to regulate the delivery of vesicles and cargo to the plasma membrane via both the recycling and the biosynthetic pathways. Although beach1 loss-of-function mutants exhibit no obvious phenotypes, a sensitized background of a rabll mutant revealed a requirement for beach1 during development and in bristle extension., (cont.) I also found that Beach1 functionally antagonizes Rab11 at the neuromuscular junction by suppressing the rabll synaptic overgrowth phenotype. Subcellular fractionation and double-labeling experiments suggest that these proteins may function in the same subcellular compartment; however, further experiments are needed to determine whether Beach1 and Rab11 interact directly, function in the same protein complex, or closely cooperate in the same molecular pathway. The interaction I found between Beach1 and Rab11 suggests a mechanism by which other BEACH proteins may be involved in vesicle trafficking., by Rita Khodosh., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004., Includes bibliographical references., Cell migration and axon guidance are highly similar processes important for the development of the nervous system. Both processes involve the transduction of signals across the membrane, resulting in changes in the cytoskeleton. I have examined the roles of two receptors that are involved in axon guidance and cell migration in Drosophila. Ptp69D is a receptor tyrosine phosphatase required for axon guidance in the developing embryo and for layer-specific axon targeting in the developing visual system. Using a dominant-negative form of Ptp69D, I have identified several genes with which Ptp69D genetically interacts in photoreceptor axon targeting. Removing a single dose of the cytoplasmic tyrosine kinases Src64 or Abl or the repulsive axon guidance receptor robo enhanced the Ptp69D dominant-negative phenotype. In mammalian systems, Src plays a key role in the regulation of cell adhesion, and Abl is a known regulator of actin cytoskeletal dynamics. Removing a single dose of the EGF receptor or Ras85D suppressed the Ptp69D dominant negative phenotype. Interestingly, the cytoplasmic tyrosine kinase PR2 binds Ptp69D, and the C. elegans homolog of PR2 has been found to suppress the Egfr/Ras pathway. Other evidence suggests that the Egfr/Ras pathway may be required for axon outgrowth. In addition to PR2, I also identified the receptor tyrosine kinase Pvr in a biochemical screen for proteins that bind Ptp69D. I generated mutants in Pvr by gene disruption and EMS mutagenesis. These mutants have disruptions in the embryonic central nervous system, including mispositioning of axon tracts and the glia that wrap them. However, these defects are not inherent to the CNS., (cont.) Rather, they result from a failure of hemocytes to migrate out of the head and engulf dead cells in the CNS. To identify proteins that signal downstream of Pvr in cell migration, I used a yeast two-hybrid screen to identify 15 proteins that bind the intracellular domain of kinase-active Pvr. One of these proteins, drk, is required for Pvr-dependent Erk MAP kinase activation. None has defects in hemocyte migration when disrupted by RNA interference. Whether these proteins have redundant functions or function downstream of Pvr in other systems remains to be determined., by Heather C. Robertson Sears., Ph.D.

BEACH proteins comprise an evolutionarily conserved family characterized by the presence of a BEACH (Beige and Chediak-Higashi) domain of unknown function. They have been shown to play a role in a number of important cellular processes, ranging from cytokinesis to synaptic transmission, and implicated in human diseases, such as Chediak-Higashi Syndrome and cancer. Analysis of several BEACH proteins suggests that they may be involved in membrane trafficking; however, little insight has been gained into their molecular mechanism of function. We identified Drosophila Beach1 in a gain-of-function screen: beach1 overexpression in the photoreceptors drastically alters their growth cone morphology. In a subsequent genetic modifier screen, I identified rabll as a strong enhancer of the beach1 eye overexpression phenotype. Rabll is a small GTPase, which has been shown to regulate the delivery of vesicles and cargo to the plasma membrane via both the recycling and the biosynthetic pathways. Although beach1 loss-of-function mutants exhibit no obvious phenotypes, a sensitized background of a rabll mutant revealed a requirement for beach1 during development and in bristle extension., (cont.) I also found that Beach1 functionally antagonizes Rab11 at the neuromuscular junction by suppressing the rabll synaptic overgrowth phenotype. Subcellular fractionation and double-labeling experiments suggest that these proteins may function in the same subcellular compartment; however, further experiments are needed to determine whether Beach1 and Rab11 interact directly, function in the same protein complex, or closely cooperate in the same molecular pathway. The interaction I found between Beach1 and Rab11 suggests a mechanism by which other BEACH proteins may be involved in vesicle trafficking., by Rita Khodosh., Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005., Includes bibliographical references.