Biology subjects

Jullie, D.

Publications and source records attributed to Jullie, D..

4 recordsLinked to original sources

bioRxiv2022

Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling

Opioid tolerance is well described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here we delineate a cellular mechanism underlying this longer-term form of opioid tolerance. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the -opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptors cytoplasmic tail, and that partial or biased agonist drugs which have reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the {delta}-opioid receptor (DOR) conforms to the same general paradigm except that endocytosis of DOR from the presynapse, in marked contrast to MOR, does not require phosphorylation of the receptors cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.

neuroscience↗

bioRxiv2019

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric bias into the signaling system. However, the core underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of downstream readouts through which receptor-proximal interactions are typically inferred. Here we describe a scalable cell-based assay to overcome this challenge, based on the use of engineered GPCR-interacting proteins as orthogonal biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between protein probe recruitment produced by chemically distinct opioid ligands in living cells. We then show how the selective recruitment applies to GRK2, a biologically relevant opioid receptor regulator protein, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

cell biology↗

bioRxiv2018

Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming

Somatic cells can be reprogrammed into pluripotent stem cells by the forced expression of the OCT4, SOX2, KLF4 and c-MYC transcription factors. This process requires the reshaping of not only epigenetic landscapes, but the global remodeling of cell identity, structure, and function including such basic processes of metabolism and organelle form and function. Cellular reprogramming is a stochastic process with only a marginally measureable fraction of cells successfully crossing these, and many other, cellular epitomes to acquire the fully pluripotent state. We hypothesize that this variation is due, in part, by variable regulation of the proteostasis network and its influence upon the protein folding environment within cells and their organelles upon the remodeling process. We find that the endoplasmic reticulum unfolded protein response (UPRER), the heat-shock response (HSR) and the mitochondrial unfolded protein response (UPRmt), which monitor and ensure the quality of the proteome of, respectively, the ER, the cytosol and the mitochondria during stress, are activated during cellular reprogramming. Particularly, we find that the UPRER is essential for reprograming, and ectopic, transient activation of the UPRER, either genetically or pharmacologically, enhances the success of cells to reach a pluripotent state. Finally, and most revealing, we find that stochastic activation of the UPRER can predict the reprogramming efficiency of naive cells. The results of these experiments indicate that the low efficiency and stochasticity of cellular reprogramming is partly the result of the inability to initiate a proper ER stress response for remodeling of the ER and its proteome during the reprogramming process. The results reported here display only one aspect of the proteostasis network and suggest that proper regulation of many more components of this network might be essential to acquire the pluripotent state.

cell biology↗

bioRxiv2018

A genetically encoded biosensor reveals location bias of opioid drug action

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. One way that drugs can differ is by imposing selective effects on the conformational equilibrium of individual ORs. We wondered if drugs can also impose selective effects on the location of OR activation in individual OR-expressing neurons. Here we develop a genetically encoded biosensor that directly localizes ligand-induced activation and deactivation of ORs in living cells, and use it to generate the first real-time map of the spatiotemporal organization of - and {delta}-OR activation in neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by uniquely driving OR activation in the somatic Golgi apparatus and extending throughout the dendritic arbor in Golgi outposts. These results demonstrate a new approach to probe the cellular basis of neuromodulation and reveal that drugs profoundly distort the spatiotemporal landscape of neuronal OR activation.

neuroscience↗