Validation and application of a novel set of “self-blinking” dyes for use in super-resolution (PALM) microscopy. This project is initiated by Fadi Jradi in collaboration with T3, the Advanced Imaging Center and others.
Nitric Oxide chemical sensor
In vitro validation of several iterations of fluorescent dye sensors for the gaseous cellular messenger nitric oxide (NO). Yoshi Aso is attempting to use these in Drosophila to elucidate the roles of NO in learning and memory. This project was initiated by Fadi Jradi.
Localized Reactive Oxygen generating tags
Fluorescent dyes were designed to produce damaging reactive oxygen species (ROS) upon light-activation (Chad Binns, Luke Lavis). When coupled to protein-specific HaloTag, this reagent is used to target the ROS damage to a particular cell or protein and can be used to ablate a cell or induce turnover of a specific protein. T3 has assisted in validating these molecules in vitro. Janelia investigators are continuing their use to ablate specific neurons in zebrafish.
Enzymatic In vivo cell surface protein labeling
T3 has spearheaded the expression and purification of an enzyme (phosphopantetheinyl transferase) and conjugation and purification of JF549-Coenzyme A. The goal is to use these reagents to resurrect a method to enzymatically label specific membrane proteins in the brain, and to assess protein localization and dynamics in vivo. Ultimately, the tools may be used to assess the functional contribution of each protein to neural computation.
Single-chain Variable Fragment (scFv) cell surface protein labeling
T3 is optimizing scFv reagents to specifically label neuronal membrane proteins as an alternative and complementary strategy to the enzymatic method outlined above. This is also a gateway project to develop additional applications of scFv technologies within T3.
Creating CaMPARI2 expressing Drosophila strains
CaMPARI2 is a second-generation calcium sensitive neuronal activity integrator (Eric Schreiter). T3 has generated a panel of 15 Drosophila strains which have pan-neuronal drivers of CaMPARI2 variants in various chromosomal landing sites, along with CsChrimson which can be used to light-activate specific subsets of neurons. The availability of these strains will lower the threshold to CaMPARI2 employment.
Light-activated protein dimerization reagents
Manipulation of protein interaction with high temporal and spatial temporal control can be formidable tools for investigating cell functions. T3 is working with collaborators to improve technologies based on the use of photoreceptor domains which use light to trigger targeted protein interaction.