Genetically encodable calcium ion (Ca 2+ ) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca 2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

Functional selectivity at the opioid receptor (R), a prototypical G-protein-coupled receptor that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. Functional selectivity is a cumulative effect of the magnitudes of individual signaling pathways, e.g., the G-mediated and the arrestin-mediated pathways for R. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface control the magnitude of arrestin signaling, and therefore functional selectivity, atR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for R, lengthens surface lifetimes of receptor-arrestin clusters significantly compared with morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail ofR. Mutation of these leucines to alanines decreased the magnitude of arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetimes by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on R to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.

6Genetically encodable calcium ion (Ca 2+ ) indicators (GECIs) based on green fluorescent 1 7 proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model 1 8organisms. Following almost two decades of steady improvements in the Aequorea victoria GFP 1 9(avGFP)-based GCaMP series of GECIs, the performance of the most recent generation (i.e., 2 0GCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort 2 1to sustain the steady progression towards ever-improved GECIs, we undertook the 2 2 development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The 2 3resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides 2 4 comparable performance as demonstrated by imaging Ca 2+ dynamics in cultured cells, primary 2 5 neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising 2 6 next-generation GECI that could inherit the mantle of GCaMP and allow the steady 2 7improvement of GECIs to continue for generations to come. 2 8

G protein‐coupled receptors (GPCRs) are flexible scaffolds that allow cells to detect thousands of environmental signals. After binding ligand, receptor endocytosis enables cellular desensitization, after which the internalized receptor is sorted in the endolysosomal system for either degradation or recycling back to the plasma membrane. Throughout the postendocytic pathway, GPCRs continue to signal with different signals being attributed to differential endosomal localization of receptors during this process. Attempts to measure a given GPCR's recycling pathway have required perturbation of postendocytic dynamics through overexpression of specific marker proteins. We sought to understand receptor trafficking through the postendocytic pathway by looking for quantitative differences in individual recycling events that contained specific receptor cargos. Using three exemplar cargo –the GPCRs mu opioid (MOR) and B2‐adrenergic receptor (B2AR), and the classic endocytic cargo transferrin receptor (TfR) – we used total internal fluorescence microscopy to visualize individual exocytic events. We then analyzed these events by developing an automated analysis program which used supervised machine learning to discern and quantify discrete receptor exocytic events. We compared overall fluorescence intensity, event duration, and other quantitative characterizations of individual events. We discovered that TfR and B2AR recycle in a similar manner, while MOR recycled in a distinct manner with markedly less heterogeneity in its recycling population. These findings show that GPCR recycling offers another dimension through which receptors distinguish themselves, and suggest the value of measuring recycling as a potential means of dissecting a given receptor's signaling profile. Further, the tools developed in this study can be used to easily characterize the recycling phenotype of novel receptors, and can be extended to a variety of exocytic cargos.Support or Funding InformationSupported by University of MichiganThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.