RESUMO
Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.
Assuntos
Encéfalo/metabolismo , Imagem Molecular/métodos , Receptores de Orexina/genética , Orexinas/análise , Proteínas Recombinantes/metabolismo , Animais , Comportamento Animal , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Receptores de Orexina/metabolismo , Orexinas/genética , Orexinas/farmacologia , Fótons , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Sono/fisiologiaRESUMO
The kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to ß-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor-biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.
Assuntos
Técnicas Biossensoriais , Receptores Opioides kappa , Proteínas de Ligação ao GTP/metabolismo , Ligantes , beta-ArrestinasRESUMO
Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.
Assuntos
Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/química , Caveolina 1/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Cavéolas/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/genética , Deleção de Genes , Expressão Gênica , Inativação Gênica , Células HeLa , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Class-B1 G-protein-coupled receptors (GPCRs) are an important family of clinically relevant drug targets that remain difficult to investigate via high-throughput screening and in animal models. Here, we engineered PAClight1P78A, a novel genetically encoded sensor based on a class-B1 GPCR (the human PAC1 receptor, hmPAC1R) endowed with high dynamic range (ΔF/F0 = 1100%), excellent ligand selectivity, and rapid activation kinetics (τON = 1.15 s). To showcase the utility of this tool for in vitro applications, we thoroughly characterized and compared its expression, brightness and performance between PAClight1P78A-transfected and stably expressing cells. Demonstrating its use in animal models, we show robust expression and fluorescence responses upon exogenous ligand application ex vivo and in vivo in mice, as well as in living zebrafish larvae. Thus, the new GPCR-based sensor can be used for a wide range of applications across the life sciences empowering both basic research and drug development efforts.
Assuntos
Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Camundongos , Humanos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Células HEK293 , Técnicas Biossensoriais/métodos , Engenharia de Proteínas/métodos , LigantesRESUMO
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.
Assuntos
Neurônios , Nociceptina , Peptídeos Opioides , Receptores Opioides , Animais , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides/genética , Neurônios/metabolismo , Humanos , Camundongos , Masculino , Área Tegmentar Ventral/metabolismo , Receptor de Nociceptina , Células HEK293 , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Ligantes , Técnicas Biossensoriais/métodosRESUMO
Intracellular G protein-coupled receptors (GPCRs) can be activated by permeant ligands, which contributes to agonist selectivity. Opioid receptors (ORs) provide a notable example, where opioid drugs rapidly activate ORs in the Golgi apparatus. Our knowledge on intracellular GPCR function remains incomplete, and it is unknown whether OR signaling in plasma membrane (PM) and Golgi apparatus differs. Here, we assess the recruitment of signal transducers to mu- and delta-ORs in both compartments. We find that Golgi ORs couple to Gαi/o probes and are phosphorylated but, unlike PM receptors, do not recruit ß-arrestin or a specific Gα probe. Molecular dynamics simulations with OR-transducer complexes in bilayers mimicking PM or Golgi composition reveal that the lipid environment promotes the location-selective coupling. We then show that delta-ORs in PM and Golgi have distinct effects on transcription and protein phosphorylation. The study reveals that the subcellular location defines the signaling effects of opioid drugs.
Assuntos
Analgésicos Opioides , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismoRESUMO
The µ-opioid receptor (µOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of µOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the µOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded µOR ligand and uncover the molecular basis for µOR antagonism by solving the cryo-EM structure of the NbE-µOR complex. NbE displays a unique ligand binding mode and achieves µOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a ß-hairpin loop formed by NbE that deeply inserts into the µOR and centers most binding contacts, we design short peptide analogues that retain µOR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel µOR ligands that can serve as a basis for therapeutic developments.
RESUMO
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.
RESUMO
We analyzed the assembly of caveolae in CV1 cells by following the fate of newly synthesized caveolin-1 (CAV1), caveolin-2 and polymerase I and transcript release factor (PTRF)/cavin-1 biochemically and using live-cell imaging. Immediately after synthesis in the endoplasmic reticulum (ER), CAV1 assembled into 8S complexes that concentrated in ER exit sites, due to a DXE sequence in the N-terminal domain. The coat protein II (COPII) machinery allowed rapid transport to the Golgi complex. Accumulating in the medial Golgi, the caveolins lost their diffusional mobility, underwent conformational changes, associated with cholesterol, and eventually assembled into 70S complexes. Together with green fluorescent protein-glycosyl-phosphatidylinositol (GFP-GPI), the newly assembled caveolin scaffolds underwent transport to the plasma membrane in vesicular carriers distinct from those containing vesicular stomatitis virus (VSV) G-protein. After arrival, PTRF/cavin-1 was recruited to the caveolar domains over a period of 25 min or longer. PTRF/cavin-1 itself was present in 60S complexes that also formed in the absence of CAV1. Our study showed the existence of two novel large complexes containing caveolar coat components, and identified a hierarchy of events required for caveolae assembly occurring stepwise in three distinct locations--the ER, the Golgi complex and the plasma membrane.
Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Proteínas de Ligação a RNA/metabolismoRESUMO
Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin beta (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin beta. The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Vírus da Hepatite B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Replicação Viral/fisiologia , Animais , Núcleo Celular/virologia , Células HeLa , Humanos , Imunoprecipitação , RNA Interferente Pequeno , Xenopus laevisRESUMO
Modulation of neuronal circuit activity is key to information processing in the brain. G protein-coupled receptors (GPCRs), the targets of most neuromodulatory ligands, show extremely diverse expression patterns in neurons and receptors can be localized in various sub-neuronal membrane compartments. Upon activation, GPCRs promote signaling cascades that alter the level of second messengers, drive phosphorylation changes, modulate ion channel function, and influence gene expression, all of which critically impact neuron physiology. Because of its high degree of complexity, this form of interneuronal communication has remained challenging to integrate into our conceptual understanding of brain function. Recent technological advances in fluorescence microscopy and the development of optical biosensors now allow investigating neuromodulation with unprecedented resolution on the level of individual cells. In this review, we will highlight recent imaging techniques that enable determining the precise localization of GPCRs in neurons, with specific focus on the subcellular and nanoscale level. Downstream of receptors, we describe novel conformation-specific biosensors that allow for real-time monitoring of GPCR activation and of distinct signal transduction events in neurons. Applying these new tools has the potential to provide critical insights into the function and organization of GPCRs in neuronal cells and may help decipher the molecular and cellular mechanisms that underlie neuromodulation.
Assuntos
Técnicas Biossensoriais , Imagem Molecular , Neurônios , Receptores Acoplados a Proteínas G , Microscopia de Fluorescência , Neurônios/química , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.
RESUMO
Protein-protein and protein-lipid interactions play important roles in the assembly of protein coats that regulate membrane organization, signaling, and trafficking in eukaryotic cells. Caveolae are plasma membrane invaginations that are formed by a protein coat consisting of caveolin and cavin protein complexes. The biochemical and structural principles of membrane binding by coat components can be studied through in vitro reconstitution of purified proteins and lipid vesicles. In this chapter, we describe a method to isolate peripheral cavin coat complexes and to subsequently bind purified cavin to chemically defined liposomes. The cavin proteoliposomes can be further analyzed to gain insights into lipid binding specificity, membrane-remodeling properties, and structural characteristics of the cavin family members.
Assuntos
Cavéolas/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Animais , Células HEK293 , Humanos , Lipossomos/síntese química , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Ligação a RNA/isolamento & purificaçãoRESUMO
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 underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.
About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell's outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor's shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell's activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR's preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/metabolismo , Animais , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiologia , Receptores Opioides/fisiologia , Proteínas RecombinantesRESUMO
A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.
Assuntos
Endocitose/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Sinápticas/metabolismo , Analgésicos Opioides/farmacologia , Animais , Células Cultivadas , Endocitose/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neurotransmissores/agonistas , Receptores de Neurotransmissores/metabolismo , Vesículas Sinápticas/efeitos dos fármacosRESUMO
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. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living 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 additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.
Assuntos
Analgésicos Opioides/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Neurônios/metabolismo , Peptídeos/metabolismo , Receptores Opioides/metabolismo , Animais , Técnicas Biossensoriais , Ala(2)-MePhe(4)-Gly(5)-Encefalina/metabolismo , D-Penicilina (2,5)-Encefalina/metabolismo , Leucina Encefalina-2-Alanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Espaço Intracelular , Microscopia de Fluorescência , Morfina/metabolismo , Naloxona , Antagonistas de Entorpecentes , Ratos , Análise Espaço-TemporalRESUMO
Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.
Assuntos
Caveolina 1/metabolismo , Lisossomos/metabolismo , Proteínas Ubiquitinadas/metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , UbiquitinaçãoRESUMO
Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.