Drosophila melanogaster - UMKC Research Groups Resources

Sleep is a phenomenon that is ubiquitous in the animal kingdom and can be found in nearly every evolutionary niche that has come about. Humans cannot live without it, and it is required for optimal physical and mental health. Despite its importance, we have yet to discover the reason or reasons for which animals require sleep, making the study of sleep a rich vein of inquiry. Sleep is regulated by the circadian clock and the sleep homeostat, which primarily reside in the brain and communicate via complex neurocircuitry. Though difficult to study in mammalian systems, Drosophila melanogaster, the fruit fly, has become a tractable model for this kind of study. With its high degree of homology to mammals and unparalleled genetic toolbox, it is an invaluable asset in studying the neurobiological and molecular aspects of many behaviors, including sleep. In the Drosophila sleep field, the 23E10-GAL4 driver is the most widely used tool to modulate sleep. 23E10-GAL4 expresses in the dorsal Fan-Shaped Body (dFB), a brain region whose associated neurons are believed to be a major sleep regulating center. However, because the dFB contains a group of 31 neurons and the 23E10-GAL4 driver expresses in many other neurons of the fly's nervous system, it is difficult to link individual neurons to observed sleep phenotypes when manipulating 23E10-GAL4 neurons. To identify which neurons in the 23E10-GAL4 driver modulate sleep, we undertook a Split-GAL4 approach to genetically dissect out the individual neurons expressed in the 23E10-GAL4 driver. We screened 22 Split-GAL4 lines based on 23E10-GAL4 and identified a pair of neurons in the ventral nerve cord, the equivalent of the fly's spinal cord, that modulate sleep when manipulated. Through immunohistochemical experiments and RNAi knockdown, we found that these cells use acetylcholine as their primary neurotransmitter. Furthermore, we found that the number of dFB neurons contained in a Split-GAL4 line did not indicate whether it promoted sleep. Therefore, this dissertation has both identified a novel pair of sleep regulating neurons in the fly and has also raised new questions concerning past and future studies done using the 23E10-GAL4 driver in Drosophila sleep research.

In this dissertation, the research focus is on elucidating the mechanism behind BRIDE OF DOUBLETIME (BDBT) puncta/foci formation. During this process it was discovered that both the circadian and visual photoreceptors have roles for normal cycles of BDBT foci formation. This BDBT foci cycling is needed for normal changes in subcellular localization of the circadian regulators PERIOD (PER) and DOUBLETIME (DBT) in the Drosophila eye. Finally, BDBT is demonstrated to have a role with the eye clock in altering circadian color preference in flies.

The kinase DBT is a regulator of the Drosophila circadian clock and phosphorylates PER during the day, targeting PER for degradation. BDBT interacts with DBT, enhancing DBT kinase activity towards PER, and accumulates during the dark of a 12 hr light/12 hr dark cycle in PER- and DBT-dependent cytosolic foci. This accumulation of BDBT is shown here to be constitutively high when flies are raised under constant dark (circadian time: CT), but decreases during constant light conditions (LL), demonstrating a light/dark responsive mechanism.

Analysis of circadian loss of function CRYPTOCHROME (cry) mutants and mutants of the RHODOPSIN 1(ninaE) visual transduction pathway indicated that disappearance of cytosolic BDBT foci in response to light is dependent upon both the CRY and the RHODOPSIN-1 visual transduction pathways. The ARR1 (Arr1) and ARR2 (Arr2) mutants eliminated the wild-type accumulation of BDBT foci under dark conditions in LD and constant darkness (DD) conditions, indicating a novel mechanism in which ARRESTINS are able to regulate accumulation of BDBT cytosolic foci. Arr1 and Arr2 mutants also lead to increased nuclear accumulation of PER protein during stimulated day and night, likely affecting circadian regulation in the Drosophila eye.

As arrestins have been shown to affect endocytosis of RHODOPSIN, I examined the effect of overexpression of a temperature sensitive dynamin GTPase in the eye SHIBIRE (SHI) and observed increased membrane vesicle formation at restrictive temperatures. Knock-down of BDBT specifically in the eye was achieved producing a loss of circadian molecular oscillations for both PER and DBT with uncoupling of their sub cellular localizations: PER was constitutively nuclear and DBT was constitutively cytosolic. The loss of eye-specific molecular rhythm was reflected in an altered rhythm of visual preference that has been observed with other loss of circadian function mutations. The research put forth in this dissertation has illustrated the importance of BDBT foci formation to maintaining a functional eye clock in Drosophila and novel role where the eye clock can influence behavior.

In this dissertation, I examined the function of the Drosophila melanogaster protein Tribbles (Trbl) within the insulin/insulin-like signaling (IIS) pathway. Trbl was initially discovered as a regulator of mitosis during embryonic development and cell migration in developing egg chambers. This discovery was soon followed by the identification of mammalian Tribbles homologs, thus establishing the Tribbles (Trib) family of pseudokinases.

The fly protein and mammalian Trib3 both inhibit the phosphorylation of Akt, a central regulator in the IIS pathway, and in humans a naturally-occurring single nucleotide polymorphism in Trib3 (Q84R) is associated with type 2 diabetes. In my first research project, I proposed that the functional relevance of this residue is conserved. In support of this, I observed that misexpression of a TrblR141Q mutant in the larval fat body increased phospho-dAkt, decreased circulating glucose, and increased storage metabolites. These data indicate that this conserved residue is required for the function of Trib protein in regulating IIS-mediated growth and homeostasis.

Next, I studied Trbl subcellular trafficking and turnover in the larval fat body, an important site of insulin signaling in the developing fly. Akt subcellular trafficking is dynamic at the plasma membrane (PM) in response to nutritional context, and I proposed that Trbl localization in insulin-responsive cells under different dietary states might also be nutrition-dependent. To test this, I compared Trbl subcellular localization in fed and fasted larvae and observed increased Trbl protein levels and membrane accumulation of Trbl in fasted cells compared to fed, suggesting a molecular mechanism for the targeting of dAkt by Trbl.

Next, I found that found that misexpression of a Trbl mutant protein with a site-directed mutation in the conserved SLE motif resulted in strong localization to the membrane, conferred stability, and enhanced binding to wild-type Trbl and dAkt. To explore the association of Trbl with the membrane, I tested the ability of Trbl to interact with bio-active lipids and identified phosphatidylserine, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate as membrane phospholipids that Trbl selectively bound. These data suggest a complex relationship between Trbl, dAkt, and phospholipids at the PM physically interacting in response to developmental and homeostatic cues.

Members of the Tribbles (Trb) family of pseudokinases have roles in development, cell cycle control, insulin signaling, and the immune response and have been linked to many human diseases. The structure of Drosophila Tribbles includes a conserved kinase-like core that binds targets and an N-terminal region that contains a PEST domain, and a Carboxy-terminal tail that binds the E3 ligase, constitutive photomorphogenic I (COP1, Murphy et al., 2015; Jamieson et al., 2018). The bound COP1 ubiquitin-ligase ubiquitinates the target protein, leading to degradation of the target by the proteolytic machinery (Murphy. et al., 2015; Jamieson et al., 2018). The C-terminal tail of Tribbles is responsible for recruiting ubiquitination complexes that target proteins such as String (Stg) and Slow border cells (Slbo) for proteolytic degradation. While Drosophila Tribbles (Trbl) retains this conserved function, it lacks a C-terminal COP1 binding site found in most Trb family members that have been shown necessary for target protein destruction. To uncover the cryptic binding site, we misexpressed Tribbles and mutant variants in Drosophila tissue, including the wing and egg chamber to determine function.

Multiple sequence alignments of closely and distantly related Tribbles proteins in arthropods determined that the divergence from a COP1 binding site in fly Tribble is a recent evolutionary event. Motif scans led to the discovery of a PEST domain in the C-terminal tail and site-directed mutagenesis of the PEST domain revealed its importance for Trbl function. Genetic interaction with the Cullin 3 proteome adaptor protein has shed light on a possible mechanism of Trbl tail function.

The Lar-family of receptor protein tyrosine phosphatases (RPTPs); including Lar, RPTPσ and RPTPδ, are utilized in signal transduction pathways during neural development. Fundamentally, the processes of axon guidance and synaptogenesis are carried out by rearrangements of the actin cytoskeleton for extension and maturation of structures. To determine whether this regulation is conserved in other tissues, we utilized interdisciplinary approaches for a structure/function analysis of Lar-RPTPs in the Drosophila musculature. We find that the single fly ortholog, Dlar, is localized to the muscle costamere in wandering L3 larvae. The costamere has important functions in muscle integrity and force transmission during contractions. Further, depletion of Dlar in the musculature causes aberrant sarcomeric actin patterning and mislocalization of the major transmembrane receptor of the costamere, integrin dimers. Ablation of two additional proteins from the musculature; including the guanine nucleotide exchange factor, Trio, and the basement membrane protein, Glutactin (Glt), results in similar disruptions to the muscle architecture. Thus, Trio and Glt provide links to actin through the Rho family of small GTPases and the BM membrane which is intimately involved in integrin signaling. We show that the actin cytoskeletal aberrations cause deficits in larval locomotor function. Additionally, we find that the cytosolic domains of Dlar are particularly important for muscle function and have implications in integrin signaling versus physical receptor/ligand interactions which agree with x-ray crystallographic analysis of the FN4-6 repeats. A proteomic approach was utilized to find novel binding partners and resulted in identification of basement membrane proteins as conserved Lar-RPTP ligands. Finally, we offer a model for Dlar signaling that results in the low affinity integrin conformation induced by inside-out signals arising from actin remodeling.