Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location.

G protein-coupled receptors (GPCRs) are membrane bound signaling molecules that regulate many aspects of human physiology. Recent advances have demonstrated that GPCR signaling can occur both at the cell surface and internal cellular membranes. Our findings suggest that cannabinoid receptor 1 (CB1) signaling is highly dependent on its subcellular location. We find that intracellular CB1 receptors predominantly couple to Gαi while plasma membrane receptors couple to Gαs. Here we show subcellular location of CB1, and its signaling, is contingent on the choice of promoters and receptor tags. Heterologous expression with a strong promoter or N-terminal tag resulted in CB1 predominantly localizing to the plasma membrane and signaling through Gαs. Conversely, CB1 driven by low expressing promoters and lacking N-terminal genetic tags largely localized to internal membranes and signals via Gαi. Lastly, we demonstrate that genetically encodable non-canonical amino acids (ncAA) offer a solution to the problem of non-native N-terminal tags disrupting CB1 signaling. We identified sites in CB1R and CB2R which can be tagged with fluorophores without disrupting CB signaling or trafficking using (trans-cyclooctene attached to lysine (TCO*A)) and copper-free click chemistry to attach fluorophores in live cells. Together, our data demonstrate the origin of location bias in cannabinoid signaling which can be experimentally controlled and tracked in living cells through promoters and novel CBR tagging strategies.

A genetically encoded sensor for real-time monitoring of poly-ADP-ribosylation dynamics in-vitro and in cells.

ADP-ribosylation, the transfer of ADP-ribose (ADPr) from nico-tinamide adenine dinucleotide (NAD+) groups to proteins, is a conserved post-translational modification (PTM) that occurs most prominently in response to DNA damage. ADP-ribosylation is a dynamic PTM regulated by writers (PARPs), erasers (ADPr hy-drolases), and readers (ADPR binders). PARP1 is the primary DNA damage-response writer responsible for adding a polymer of ADPR to proteins (PARylation). Real-time monitoring of PARP1-mediated PARylation, especially in live cells, is critical for under-standing the spatial and temporal regulation of this unique PTM. Here, we describe a genetically encoded FRET probe (pARS) for semi-quantitative monitoring of PARylation dynamics. pARS feature a PAR-binding WWE domain flanked with turquoise and Venus. With a ratiometric readout and excellent signal-to-noise characteristics, we show that pARS can monitor PARP1-dependent PARylation temporally and spatially in real-time. pARS provided unique insights into PARP1-mediated PARylation kinetics in vitro and high-sensitivity detection of PARylation in live cells, even under mild DNA damage. We also show that pARS can be used to determine the potency of PARP inhibitors in vitro and, for the first time, in live cells in response to DNA damage. The robustness and ease of use of pARS make it an important tool for the PARP field.

In vivo and ex vivo confocal microscopy for the evaluation of surgical margins of melanoma.

We report the first series of melanomas (MMs) where the surgical margins were evaluated both by ex vivo confocal microscopy (EVCM) and in vivo reflectance confocal microscopy (RCM). We evaluated the surgical margins of 42 cutaneous MMs of lentigo maligna/lentigo maligna melanoma type and 2 mucosal MMs with EVCM. Cutaneous MMs also underwent RCM mapping. Imaging results were compared with histopathology. The rate of correct identification of the tumor margins (invaded or not invaded) was 97.6% for RCM (evaluations of cutaneous MMs) and 95.5% for EVCM (evaluations of both cutaneous and mucosal MMs). Our study showed that the MM extension is visible under EVCM and that the combination of in vivo RCM and EVCM can be a new strategy for the evaluation of surgical margins of MMs.