UC Berkeley

Small-molecule synthetic tools are immensely useful in biological experimentation. However, these small molecules are not inherently targeted to genetically defined cell populations, thus hindering their use in complex living tissues. In my dissertation work, I adapted and developed two novel genetic constructs that traffic small molecule tethering proteins HaloTag1 and SNAPf 2 to the extracellular surface in cell lines and Drosophila brain tissue. I then applied these novel targeting systems toward two distinct applications: functional voltage imaging in live Drosophila brains using synthetic voltage-sensitive dyes and registration of live-fly brains to standard anatomical templates for comparison with existing databases.Voltage imaging in intact brains offers the tantalizing promise of watching, in real-time, the electrical changes that underlie physiology. To enable voltage imaging in Drosophila melanogaster, we combined a chemically synthesized rhodamine voltage reporter (RhoVR) with a genetically encoded, self-labeling enzyme, HaloTag. We generated a Drosophila reporter line that expressed the HaloTag enzyme on the extracellular surface. We validated the voltage sensitivity of this approach in cell culture before driving expression of HaloTag in specific neurons in flies. We showed that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in larval neuromuscular junction (NMJ) and whole adult brains. We validated the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, showing the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and performed voltage imaging of carbachol-evoked depolarizations and osmolarity-evoked hyperpolarizations in projection neurons and in interoceptive suboesophageal zone neurons in fly brain explants following in vivo labeling. The turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila. The Drosophila neurobiology community has developed a multitude of open-source neuron anatomy databases for comparative analysis of anatomy across samples. Unfortunately, fixation and permeabilization of the sample are often required which precludes their use in live tissue. Our novel extracellularly targeted tethering platforms comprised of SNAPf and HaloTag fusion proteins allowed for multispectral, in vivo anatomical analysis at the single-cell and whole-brain levels. We labeled the neuronal expression patterns of various Gal4 driver lines in live Drosophila brains recapitulating histological staining. Expressing SNAPf pan-neuronally, we registered brains to an existing anatomical template. From directly registered live brain tissue, we performed bridging registrations and a neuronal morphology similarity search (NBLAST)4. We predict that these extracellular platforms will become a valuable complement to existing anatomical methods and prove useful for future genetic targeting of other small molecule probes, drugs, and actuators.