probes for cell biology
trafficking probes: LUCID
The complexity of anterograde and retrograde traffic between the Golgi apparatus, the plasma membrane, autophagic vesicles, and the endolysosomal system is daunting. We are developing new probes and methods to quantitatively assess cargo flux through these pathways.
The first of these probes is the Luciferase reporter of intraluminal deposition, or LUCID. This probe facilitates the quantitative assay of integram membrane protein targeting to the lumen of the vacuole, via the late endosomal multivesicular body (MVB). Failure to package the luciferase-marked cargo into intraluminal vesicles of the MVB, or failure of the MVB to fuse with the hydrolytic vacuole, results in accumulation of luciferase in the cytoplasmic compartment.
Our first description of the LUCID probe and assay was published in Nickerson, 2012, and additional cargo probes are under active development.
In collaboration with the Baker Lab, we are using protein engineering to design novel protein probes for the detection of specific lipids. Key technologies used in this project include protein active site redesign using Rosetta, assembly of bilayer nanodiscs, synthesis of new fluorescent lipids, and S. cerevisiae surface display.