• Engineering Optogenetic Control of Endocytic Recycling: Controlling Rab11 Function in Drosophila melanogaster using Engineered Light-Responsive Nanobodies

      Ward, Devin; Department of Biological Sciences
      The regulated transport of materials in cells is an essential function of all living organisms. In eukaryotes, one main family of transport regulators is the Rab GTPases. Rab GTPases utilize GTP to move materials throughout the cell by binding to the membrane of vesicles or endosomes, and trafficking distinct, membrane-associated components throughout the cell. One member of this large family of proteins is Rab11. Rab11 is responsible for endosome recycling: returning membrane proteins and receptors from intracellular recycling endosomes to the cell membrane, where these membrane proteins and receptors may be reused. Although the exact mechanism of Rab11 trafficking is not known, Rab11 appears to be critical for the development and survival of many organisms. Drosophila mutants for the Rab11 gene are not viable, where lethality manifests during embryonic development. This early lethality has imposed significant limitations on elucidating the immediate effects of Rab11 inhibition. Thus, the goal was to engineer a novel method of inhibiting Rab11 in vivo in Drosophila melanogaster. Specifically, the goal was to generate a genomically non-invasive construct (Opto-Nanobody) utilizing an optogenetic, light-sensitive Cryptochrome 2 (Cry2) fused to YFP-targeting nanobodies to bind functional, endogenous, YFP-tagged Rab11. This system promises to provide precise light-responsive spatio-temporal control of Rab11 function in response to blue-light exposure through homo-oligomeric clustering, which has been shown to inhibit Rab-dependent trafficking. Using the Drosophila embryo as a model system, these tools were applied to directly determine the effects of Rab11 inhibition on Notch signaling, and to determine the mechanisms that govern Rab11 trafficking. The Opto-Nanobody was tested in vitro in S2 cells, and was shown to form homo-oligomeric clusters in the presence of blue light and demonstrated the ability to bind to YFP-Rab11. This Opto-Nanobody construct has been inserted into a D. melanogaster injection vector, so that the Opto-Nanobody may be inserted into the D. melanogaster genome, and used to control YFP::Rab11 activity in vivo to elucidate the role of Rab11 in Notch signalling.
    • Investigating how Notch, and JAK/STAT Signaling Synergistically Regulate Intestinal Stem Cell Homeostasis Using Engineered Optogenetic Alleles

      Lidster, Taylor; Department of Biological Sciences
      Stem cells are a class of undifferentiated cells that have the unique ability to give rise to a variety of specialized cell types during tissue development and growth. These cells communicate amongst one another by sending and receiving signals from multiple pathways that regulate cell fate decisions by promoting either self-renewal or differentiation. The Notch, and JAK/STAT signaling pathways are central regulators of multicellular development and are vital for tissue maintenance. The interplay between the Notch, and JAK/STAT pathways required for tissue homeostasis has not been fully elucidated, particularly as it relates to the intestinal epithelium. I utilize the Drosophila melanogaster midgut as a model system to study stem cell dynamics and more specifically, how Notch and JAK/STAT signaling cooperatively regulate intestinal stem cell turnover. In order to accomplish this, I employed both traditional and optogenetic methods to elicit Notch blockade and ectopic activation of JAK/STAT signaling in the midgut. First, I demonstrated that ectopic JAK/STAT signaling paired with Notch knockdown causes substantial ISC overproliferation, leading to the formation of large ISC tumors spanning the entire midgut. Quantitative assessment of ISC pools confirmed that Notch and JAK/STAT signaling work in a synergistic manner, rather than an additive manner, to regulate ISC homeostasis. I also utilized two optogenetic alleles, Opto-Delta and Opto-Domeless, to recapitulate Notch blockade and JAK/STAT hyperactivation using light. Opto-Delta was tested in the intestinal epithelium and demonstrated the ability to block Notch signaling in the ISCs, resulting in the formation of stem cell clusters. Subsequently, an Opto-Domeless construct was created and expressed in the nervous system during embryogenesis to assess its efficacy to activate JAK/STAT signaling. The embryos that expressed Opto-Domeless exhibited a significant decrease in viability when subjected to the light condition, suggesting possible light-responsiveness. Expression of Opto-Domeless in the midgut was also achieved, however, ISCs expressing Opto-Domeless appeared to have JAK/STAT activity in the absence of light exposure, suggesting that Opto-Domeless is not behaving in a binary fashion and will require further validation. Altogether, these findings reveal great potential for light-gated proteins, as they provide a powerful approach to disentangle dynamic cellular signals by using light.