Technology development
The development and application of novel scientific tools and approaches is a key part of our research. Here are examples of assays that we have developed:
Optogenetic assay for site-specific cAMP generation- Optogenetic approaches have become a valuable tool for manipulating and deciphering the activities of complex networks. We have developed an optogenetic assay, which gives precise temporal and spatial control over the generation of the second messenger cAMP in cells (Figure 1). We are applying the assay to probe the functional consequences of site-specific signaling.
For reference, see:
Tsvetanova NG and von Zastrow M. (2014). Spatial encoding of cAMP signaling specificity by GPCR endocytosis. Nature Chemical Biology 10: 1061-1065.
Tsvetanova NG, Trester-Zedlitz M, Newton BW, Peng GP, Johnson JR, Jimenez-Morales D, Krogan NJ, von Zastrow M. (2021) Endosomal cAMP production broadly impacts the cellular phosphoproteome. The Journal of Biological Chemistry 297;100907.doi: 10.1016/j.jbc.2021.100907.
Figure 1- Site-specific generation of cAMP in cells. A light-activated adenylyl cyclase is targeted to the plasma membrane (PM), endosomes, or cytosol for local generation of cAMP using light.
cAMP reporter for genomic screening- We routinely use genomics and functional genomics approaches in our research. We have optimized a novel fluorescent transcriptional reporter for cAMP that enables high-throughput CRISPR-based genomic screening (Figure 2). We are applying it to identify novel modulators of GPCR signaling for further characterization.
For reference, see:
Semesta KM, Tian R, Kampmann M, von Zastrow M, Tsvetanova NG. (2020). A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling. PLoS Genetics 16(10): e1009103.
Figure 2- Screen for regulators of GPCR/cAMP signaling. Cells expressing a fluorescent transcriptional CREB reporter are transduced with a genomic CRISPRi library, treated with drug, and separated by FACS based on ligand effects on reporter expression. Genomic DNA is isolated from each population, and analyzed by deep sequencing to identify regulators.