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Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells*

  • Luc Veya
    Footnotes
    Affiliations
    Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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  • Joachim Piguet
    Correspondence
    To whom correspondence may be addressed.
    Footnotes
    Affiliations
    Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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  • Horst Vogel
    Correspondence
    To whom correspondence may be addressed: EPFL-ISIC, Station 6, CH-1015 Lausanne, Switzerland. Tel.: 41-21-693-31-55
    Affiliations
    Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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  • Author Footnotes
    * This work was supported by grants of the Ecole Polytechnique Fédérale de Lausanne, the Swiss National Science Foundation, the National Centre of Competence in Research Chemical Biology, and the European Community (Project SynSignal, Grant FP7-KBBE-2013-613879). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Movies S1–S3.
    1 Both authors contributed equally to this work.
Open AccessPublished:September 11, 2015DOI:https://doi.org/10.1074/jbc.M115.666677
      Lateral diffusion enables efficient interactions between membrane proteins, leading to signal transmission across the plasma membrane. An open question is how the spatiotemporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue by studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor, during its different phases of cellular signaling. Attaching a single quantum dot to individual neurokinin-1 receptors enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm2/s comprising receptors freely diffusing and others confined in 100–600-nm-sized membrane domains as well as immobile receptors. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction and the other showing low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we found that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the canonical pathway activity of the receptors. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity.

      Introduction

      Membrane receptors are of utmost importance for cellular signaling, transferring the information of extracellular stimuli into intracellular responses. In this context, their lateral distribution and mobility in the plasma membrane play a critical role as random or directed movements in the membrane plane bring signaling partners efficiently into transient or stable contact (
      • Adam G.
      • Delbrück M.
      ,
      • Saffman P.G.
      • Delbrück M.
      Brownian motion in biological membranes.
      ,
      • Singer S.J.
      • Nicolson G.L.
      The fluid mosaic model of the structure of cell membranes.
      ,
      • Nicolson G.L.
      The fluid-mosaic model of membrane structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years.
      ). A fundamental issue of modern quantitative cell biology is to understand how the complex, highly dynamic spatial distribution of components of the plasma membrane influences central cellular signaling processes (
      • Kusumi A.
      • Tsunoyama T.A.
      • Hirosawa K.M.
      • Kasai R.S.
      • Fujiwara T.K.
      Tracking single molecules at work in living cells.
      ,
      • Grecco H.E.
      • Schmick M.
      • Bastiaens P.I.
      Signaling from the living plasma membrane.
      ,
      • Lingwood D.
      • Simons K.
      Lipid rafts as a membrane-organizing principle.
      ). Single molecule optical imaging, more specifically single particle tracking (SPT),
      The abbreviations used are: SPT
      single particle tracking
      GPCR
      G protein-coupled receptor
      CME
      clathrin-mediated endocytosis
      NK1R
      neurokinin-1 receptor
      SP
      substance P
      CCP
      clathrin-coated pit
      Qdot
      quantum dot
      ACP
      acyl carrier protein
      NCS
      newborn calf serum
      mβCD
      methyl-β-cyclodextrin
      ROCK
      Rho-associated coiled coil kinase.
      is ideally suited to establish a tomogram of the distribution of individual plasma membrane components over time and space, revealing the full complexity of individual signaling reactions that would be hidden in ensemble measurements (
      • Eggeling C.
      • Ringemann C.
      • Medda R.
      • Schwarzmann G.
      • Sandhoff K.
      • Polyakova S.
      • Belov V.N.
      • Hein B.
      • von Middendorff C.
      • Schönle A.
      • Hell S.W.
      Direct observation of the nanoscale dynamics of membrane lipids in a living cell.
      ).
      Here we concentrate on seven-transmembrane domain receptors, also known as G protein-coupled receptors (GPCRs). GPCRs establish the largest family of cell surface receptors converting extracellular signals into intracellular responses. As they are involved in many central physiological processes, they are also among the most important targets for drug development (
      • Heng B.C.
      • Aubel D.
      • Fussenegger M.
      An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases.
      ,
      • Salon J.A.
      • Lodowski D.T.
      • Palczewski K.
      The significance of G protein-coupled receptor crystallography for drug discovery.
      ,
      • Shoichet B.K.
      • Kobilka B.K.
      Structure-based drug screening for G-protein-coupled receptors.
      ,
      • Venkatakrishnan A.J.
      • Deupi X.
      • Lebon G.
      • Tate C.G.
      • Schertler G.F.
      • Babu M.M.
      Molecular signatures of G-protein-coupled receptors.
      ) After activation by extracellular stimuli, GPCRs are typically desensitized, internalized, and recycled. These processes occur from seconds (phosphorylation) over minutes (endocytosis) to hours (down-regulation) (
      • Ferguson S.S.
      Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling.
      ). In this context, the clathrin-mediated endocytosis (CME) machinery is essential for maintaining proper function of GPCRs on the cell surface (
      • Hanyaloglu A.C.
      • von Zastrow M.
      Regulation of GPCRs by endocytic membrane trafficking and its potential implications.
      ). All this yields an amazingly diverse network of intracellular signaling reactions and in turn a complex receptor pharmacology.
      Here we used the human neurokinin-1 receptor (NK1R) as a prototypical GPCR to investigate its lateral distribution in living cells at different states before and after activation. The NK1R is activated by tachykinin neuropeptides and belongs structurally to the rhodopsin-like GPCR family (
      • Takahashi K.
      • Tanaka A.
      • Hara M.
      • Nakanishi S.
      The primary structure and gene organization of human substance P and neuromedin K receptors.
      ). It mediates a wide variety of neuronal and metabolic processes and is an important drug target for treating diseases such as depression and cancer (
      • Steinhoff M.S.
      • von Mentzer B.
      • Geppetti P.
      • Pothoulakis C.
      • Bunnett N.W.
      Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.
      ).
      The NK1R mediates classical membrane signaling reactions as summarized in Fig. 1. After binding its natural agonist, the undecapeptide substance P (SP), the NK1R activates its G protein Gαq, which in turn activates phospholipase C, leading to Ca2+ release from the endoplasmic reticulum (
      • Quartara L.
      • Maggi C.A.
      The tachykinin NK1 receptor. Part I: ligands and mechanisms of cellular activation.
      ,
      • Khawaja A.M.
      • Rogers D.F.
      Tachykinins: receptor to effector.
      ). Thereafter, the NK1R is phosphorylated and binds β-arrestin to target the receptor to clathrin-coated pits (CCPs) and activate the CME machinery (
      • Valant C.
      • Robert Lane J.
      • Sexton P.M.
      • Christopoulos A.
      The best of both worlds? Bitopic orthosteric/allosteric ligands of g protein-coupled receptors.
      ,
      • Urban J.D.
      • Clarke W.P.
      • von Zastrow M.
      • Nichols D.E.
      • Kobilka B.
      • Weinstein H.
      • Javitch J.A.
      • Roth B.L.
      • Christopoulos A.
      • Sexton P.M.
      • Miller K.J.
      • Spedding M.
      • Mailman R.B.
      Functional selectivity and classical concepts of quantitative pharmacology.
      ,
      • Reiter E.
      • Ahn S.
      • Shukla A.K.
      • Lefkowitz R.J.
      Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors.
      ,
      • Barak L.S.
      • Warabi K.
      • Feng X.
      • Caron M.G.
      • Kwatra M.M.
      Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and β-arrestin 2 during homologous desensitization of the substance P receptor.
      ,
      • Luttrell L.M.
      • Lefkowitz R.J.
      The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals.
      ). Endocytosis of the β-arrestin·NK1R complex occurs within the 1st min of agonist exposition (
      • Takahashi K.
      • Tanaka A.
      • Hara M.
      • Nakanishi S.
      The primary structure and gene organization of human substance P and neuromedin K receptors.
      ,
      • McConalogue K.
      • Déry O.
      • Lovett M.
      • Wong H.
      • Walsh J.H.
      • Grady E.F.
      • Bunnett N.W.
      Substance P-induced trafficking of β-arrestins. The role of β-arrestins in endocytosis of the neurokinin-1 receptor.
      ). Internalization of the neurokinin-1 receptor is finalized about 10 min later (
      • Quartara L.
      • Maggi C.A.
      The tachykinin NK1 receptor. Part II: distribution and pathophysiological roles.
      ,
      • Garland A.M.
      • Grady E.F.
      • Payan D.G.
      • Vigna S.R.
      • Bunnett N.W.
      Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.
      ,
      • Grady E.F.
      • Garland A.M.
      • Gamp P.D.
      • Lovett M.
      • Payan D.G.
      • Bunnett N.W.
      Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.
      ). NK1R trafficking is influenced by SP; high SP concentration induces receptor internalization to perinuclear sorting endosomes, whereas low SP concentration induces receptor translocation to early endosomes followed by rapid recycling coinciding with the recovery of SP binding sites at the cell surface (
      • Steinhoff M.S.
      • von Mentzer B.
      • Geppetti P.
      • Pothoulakis C.
      • Bunnett N.W.
      Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.
      ,
      • Roosterman D.
      • Cottrell G.S.
      • Schmidlin F.
      • Steinhoff M.
      • Bunnett N.W.
      Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.
      ). Furthermore, an NK1R subpopulation can be desensitized and resensitized without leaving the vicinity of the plasma membrane (
      • Murphy J.E.
      • Roosterman D.
      • Cottrell G.S.
      • Padilla B.E.
      • Feld M.
      • Brand E.
      • Cedron W.J.
      • Bunnett N.W.
      • Steinhoff M.
      Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.
      ).
      Figure thumbnail gr1
      FIGURE 1Internalization and recycling of NK1R at the cell plasma membrane. The NK1R can be located in different regions of the plasma membrane and in intracellular endosomes: 1, nanometer-sized membrane domains; 2, clathrin-related region; 3, clathrin prepits; 4, clathrin-coated pits closed by dynamin; 5, clathrin-coated vesicles; 6, early, late, or perinuclear endosomes; 7, lysosomes; 8, recycling vesicles. NK1R can be recruited in the different regions described above. I, receptor exchanges between clathrin-related region and free membrane. II, after agonist binding, receptor is phosphorylated, leading to the recruitment of β-arrestin. The receptor is targeted to clathrin prepits. III, receptor·clathrin complex is bound to dynamin-dependent invaginating regions. IV, receptor is internalized in clathrin-coated vesicles, which are then transformed to early endosomes. V, SP is removed from NK1R in late endosomes where either in VI SP and a fraction of the receptors are degraded in lysosomes or in VII a fraction of the receptors is recycled to the cell membrane.
      In living 293T cells, individual NK1Rs show highly heterogeneous diffusion properties (
      • Prummer M.
      • Meyer B.H.
      • Franzini R.
      • Segura J.-M.
      • George N.
      • Johnsson K.
      • Vogel H.
      Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells.
      ). In general, the median diffusion coefficient of an NK1R population rapidly decreases after stimulation with SP (
      • Lill Y.
      • Martinez K.L.
      • Lill M.A.
      • Meyer B.H.
      • Vogel H.
      • Hecht B.
      Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.
      ). Immobile or confined receptors are already present in the basal state, i.e. in the absence of activating ligands, suggesting that SP influences the equilibrium between different receptor states (
      • Prummer M.
      • Meyer B.H.
      • Franzini R.
      • Segura J.-M.
      • George N.
      • Johnsson K.
      • Vogel H.
      Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells.
      ), which in turn would modulate the interaction between particular signaling proteins concentrated in membrane nano- or microdomains (
      • Meyer B.H.
      • Segura J.-M.
      • Martinez K.L.
      • Hovius R.
      • George N.
      • Johnsson K.
      • Vogel H.
      FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.
      ).
      The spatial organization and mobility of GPCRs in the cell membrane are of utmost importance to ensure correct signal transduction, fast desensitization, and endocytosis of the receptor. Here we addressed these issues by attaching a single quantum dot (Qdot) to individual NK1Rs, which enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane of the cell. By characterizing simultaneously the mobility and confinement of each individual receptor, it was possible to detect and distinguish different, highly dynamic receptor populations in the plasma membrane and correlate them with distinct steps of the GPCR-mediated transmembrane signaling cascade.

      Discussion

      Here we have investigated the mobility features of the neurokinin-1 receptor with an unprecedented level of mechanistic understanding. We used mobility patterns as a new, high content graphical representation of single molecule mobility. This representation is based on a two-dimensional density function of the short range diffusion coefficient D1–10 versus the mobility parameter SMSS, which is directly associated with the mode of motion of the receptor. This enabled us to visualize and analyze the complex information contained in a particular experiment within a single graph, substantially facilitating comparison of results obtained under different experimental conditions. Moreover, this method of analysis allowed us to easily define and classify the diffusing particles into different types according to their mobility regime.
      Our study revealed that, despite the very broad distribution of the mobility and sizes of membrane confinement, the overall NK1R mobility pattern remains highly reproducible between different days of experiment. This suggests the presence of a very distinct and stable network of functional interactions between the receptor and other cellular components. As presented under “Results,” NK1R can be classified into three major classes. Receptors assigned to type I are free to diffuse in the cellular membrane. Their general diffusion properties are accessible by other measurement techniques such as fluorescence recovery after photobleaching or fluorescence correlation spectroscopy. The overall features of type I receptors are comparable with those observed by single molecule tracking of other GPCRs (
      • Lill Y.
      • Martinez K.L.
      • Lill M.A.
      • Meyer B.H.
      • Vogel H.
      • Hecht B.
      Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.
      ,
      • Baker A.
      • Saulière A.
      • Dumas F.
      • Millot C.
      • Mazères S.
      • Lopez A.
      • Salomé L.
      Functional membrane diffusion of G-protein coupled receptors.
      ,
      • Calebiro D.
      • Rieken F.
      • Wagner J.
      • Sungkaworn T.
      • Zabel U.
      • Borzi A.
      • Cocucci E.
      • Zürn A.
      • Lohse M.J.
      Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.
      ,
      • Thurner P.
      • Gsandtner I.
      • Kudlacek O.
      • Choquet D.
      • Nanoff C.
      • Freissmuth M.
      • Zezula J.
      A two-state model for the diffusion of the A2A adenosine receptor in hippocampal neurons: agonist-induced switch to slow mobility is modified by synapse-associated protein 102 (SAP102).
      ). The low SMSS values measured here correlate with a restricted diffusion of the NK1R. A similar behavior was observed for other receptors in living cells and is often explained by multiple effects such as the rough, irregular shape of the plasma membrane; transient interactions with other membrane proteins; the heterogeneous composition of the plasma membrane; and the recruitment in caveolae (
      • Prummer M.
      • Meyer B.H.
      • Franzini R.
      • Segura J.-M.
      • George N.
      • Johnsson K.
      • Vogel H.
      Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells.
      ,
      • Kusumi A.
      • Suzuki K.G.
      • Kasai R.S.
      • Ritchie K.
      • Fujiwara T.K.
      Hierarchical mesoscale domain organization of the plasma membrane.
      ,
      • Nicolau Jr., D.V.
      • Hancock J.F.
      • Burrage K.
      Sources of anomalous diffusion on cell membranes: a Monte Carlo study.
      ,
      • Kubale V.
      • Abramović Z.
      • Pogacnik A.
      • Heding A.
      • Sentjurc M.
      • Vrecl M.
      Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2.
      ,
      • Jacquier V.
      • Prummer M.
      • Segura J.-M.
      • Pick H.
      • Vogel H.
      Visualizing odorant receptor trafficking in living cells down to the single-molecule level.
      ). The diffusion coefficients of GPCRs we and others have observed in the membranes of living cells are considerably lower than those of other membrane proteins of similar size or that of rhodopsin as an example of a class A GPCR in pure lipid bilayers (
      • Vaz W.L.
      • Criado M.
      • Madeira V.M.
      • Schoellmann G.
      • Jovin T.M.
      Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching.
      ,
      • Kusumi A.
      • Fujiwara T.K.
      • Chadda R.
      • Xie M.
      • Tsunoyama T.A.
      • Kalay Z.
      • Kasai R.S.
      • Suzuki K.G.
      Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model.
      ). The use of Qdots as a fluorescent label to track individual receptors allowed us to validate in an accurate and reliable manner previous single molecule diffusion measurements using organic dyes by Prummer et al. (
      • Prummer M.
      • Meyer B.H.
      • Franzini R.
      • Segura J.-M.
      • George N.
      • Johnsson K.
      • Vogel H.
      Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells.
      ). Consequently, the use of Qdots does not interfere with the diffusion properties of the receptor.
      Low diffusion coefficients are often explained by direct interactions of the membrane protein of interest with components of the cytoskeleton (
      • Jaqaman K.
      • Kuwata H.
      • Touret N.
      • Collins R.
      • Trimble W.S.
      • Danuser G.
      • Grinstein S.
      Cytoskeletal control of CD36 diffusion promotes its receptor and signaling function.
      ,
      • Andrews N.L.
      • Lidke K.A.
      • Pfeiffer J.R.
      • Burns A.R.
      • Wilson B.S.
      • Oliver J.M.
      • Lidke D.S.
      Actin restricts FcϵRI diffusion and facilitates antigen-induced receptor immobilization.
      ,
      • Bouzigues C.
      • Morel M.
      • Triller A.
      • Dahan M.
      Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging.
      ). According to the picket fence model (
      • Kusumi A.
      • Nakada C.
      • Ritchie K.
      • Murase K.
      • Suzuki K.
      • Murakoshi H.
      • Kasai R.S.
      • Kondo J.
      • Fujiwara T.
      Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules.
      ), drug treatment causing actin depolymerization should result in increased receptor diffusion due to the reduction of actin filament barriers. The NK1R does not follow this behavior. In the present case, the type I mobility receptor population did not increase after actin fiber or microtubule depolymerization, strongly suggesting a lack of direct interaction of type I NK1Rs with the cytoskeleton. The relative low mobility of type I receptors could be explained by the high propensity of NK1R to form diffusing membrane domains a few tens of nanometers in size with high receptor density (
      • Meyer B.H.
      • Segura J.-M.
      • Martinez K.L.
      • Hovius R.
      • George N.
      • Johnsson K.
      • Vogel H.
      FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.
      ).
      A high fraction of the NK1Rs exhibited a strictly restrained mobility and was therefore classified as type II. Indeed, more than one-third of the receptors in the basal state were found to be confined in submicrometer-sized domains. The low interchange rate between receptors of type II and other mobility regimes indicated that this restricted diffusion is not a consequence of the fast transient recruitment described in the picket fence model of the plasma membrane (
      • Kusumi A.
      • Fujiwara T.K.
      • Chadda R.
      • Xie M.
      • Tsunoyama T.A.
      • Kalay Z.
      • Kasai R.S.
      • Suzuki K.G.
      Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model.
      ) but is more likely due to the existence of very stable membrane structures in which the NK1R is integrated. The low diffusion coefficients measured for type II receptors did not depend on intact actin filaments or microtubule structures as no differences were observed in type II diffusion features upon treatment of cells with cytochalasin B or nocodazole. The low D1–10 values observed are very likely due to molecular crowding related to membrane regions of high protein content. The high frequency of our measurements (30 Hz) combined with the very high accuracy generated by the use of Qdots to localize individual receptors permitted us to exclude effects of domain size on the apparent diffusion coefficients observed elsewhere for other GPCRs with less photostable fluorophores (
      • Jacquier V.
      • Prummer M.
      • Segura J.-M.
      • Pick H.
      • Vogel H.
      Visualizing odorant receptor trafficking in living cells down to the single-molecule level.
      ,
      • Destainville N.
      • Salomé L.
      Quantification and correction of systematic errors due to detector time-averaging in single-molecule tracking experiments.
      ). A small part of the receptors in the basal state remained immobile; this population probably stems from constitutively internalized receptors.
      Although our results do not yield any indication of direct or mediated interactions by a simple protein assembly of the NK1R with the cytoskeleton of the cell, the NK1R is nevertheless tightly related to its surrounding. Indeed, disruption of the cytoskeleton had an indirect influence on the mobility pattern of the receptor through structural modifications of the membrane.
      Depolymerization of actin filaments is known to stimulate cell blebbing (
      • Albrecht-Buehler G.
      Autonomous movements of cytoplasmic fragments.
      ,
      • Pick H.
      • Schmid E.L.
      • Tairi A.-P.
      • Ilegems E.
      • Hovius R.
      • Vogel H.
      Investigating cellular signaling reactions in single attoliter vesicles.
      ). Under certain conditions, these blebs can be released as native microvesicles containing functional NK1R (
      • Grasso L.
      • Wyss R.
      • Piguet J.
      • Werner M.
      • Hassaïne G.
      • Hovius R.
      • Vogel H.
      Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.
      ). The high fraction of type III receptors after cytochalasin B treatment is related to membrane blebbing. The protein content of native vesicles is different from that of the plasma membrane of the cell; in particular, they lack cytoskeletal structure (
      • Bauer B.
      • Davidson M.
      • Orwar O.
      Proteomic analysis of plasma membrane vesicles.
      ). This explains the high D1–10 observed for this population. Furthermore, the submicrometer size of the confinement region is in total agreement with the results obtained elsewhere (
      • Pick H.
      • Schmid E.L.
      • Tairi A.-P.
      • Ilegems E.
      • Hovius R.
      • Vogel H.
      Investigating cellular signaling reactions in single attoliter vesicles.
      ,
      • Grasso L.
      • Wyss R.
      • Piguet J.
      • Werner M.
      • Hassaïne G.
      • Hovius R.
      • Vogel H.
      Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.
      ).
      SP is a potent natural agonist of the NK1R. It triggers multiple signaling pathways and receptor recycling. After activation, the NK1R is recycled via two distinct pathways: in a fast process, receptors are recruited in plasma membrane domains or in early endosomes in close proximity to the plasma membrane (
      • Ferguson S.S.
      Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling.
      ,
      • Murphy J.E.
      • Roosterman D.
      • Cottrell G.S.
      • Padilla B.E.
      • Feld M.
      • Brand E.
      • Cedron W.J.
      • Bunnett N.W.
      • Steinhoff M.
      Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.
      ); in a slow process, the receptors are transported to low pH perinuclear late endosomes (
      • Wang X.
      • Marvizón J.C.
      Time-course of the internalization and recycling of neurokinin 1 receptors in rat dorsal horn neurons.
      ,
      • Pelayo J.-C.
      • Poole D.P.
      • Steinhoff M.
      • Cottrell G.S.
      • Bunnett N.W.
      Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones.
      ). Both pathways are initiated by receptor phosphorylation and subsequent arrestin binding. Our SPT results in the presence of SP show a substantial decrease of NK1R mobility, consistent with an increased recruitment of the receptors in structures related to the recycling pathways. This effect correlates with a decrease of the overall diffusion coefficient mainly due to an increase of confinement as seen by an important shift from type I to type II receptors in the mobility patterns. Interestingly, only 30% of the receptors undergo a change of mobility after activation. It has been shown elsewhere that other cargo proteins associate transiently with CCPs during their formation and can dissociate before pit termination or internalization; the dwell times of this process display a very broad distribution from the second to hundred second regime (
      • Weigel A.V.
      • Tamkun M.M.
      • Krapf D.
      Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules.
      ). Taking this mechanism into consideration, our results can be explained by an increase of the receptor affinity for CCPs after agonist binding, thus increasing the dwell time and favoring internalization against release of the receptor in the plasma membrane.
      It is remarkable that this immobilization effect associated with receptor recruitment in the CME pathway depends on agonist concentration. The dose-response curve resulting from the measurement of the average Hurst parameter with increasing SP concentrations shows an EC50 value of about 100 pm, which is comparable with the EC50 of the intracellular Ca2+ response, indicating that the ligand-coordinated receptor immobilization might regulate the intracellular response. The NK1R mobility change induced by the presence of low concentrations of agonist in the environment would quickly regulate the cell response and therefore limit the intracellular Ca2+ release in the case of long term agonist exposition.
      An unexpected important result is that the decrease of CME functionality induced by the clathrin/dynamin inhibitors PitStop 2, Dyngo-4a, and Dynasore leads to a substantial increase of type II receptors confined in submicrometer membrane domains. Besides inhibition of endocytosis, these molecules induce an accumulation of clathrin at the plasma membrane, forming long lived clathrin structures (
      • Grove J.
      • Metcalf D.J.
      • Knight A.E.
      • Wavre-Shapton S.T.
      • Sun T.
      • Protonotarios E.D.
      • Griffin L.D.
      • Lippincott-Schwartz J.
      • Marsh M.
      Flat clathrin lattices: stable features of the plasma membrane.
      ), including membrane-attached vesicles (
      • Howes M.T.
      • Kirkham M.
      • Riches J.
      • Cortese K.
      • Walser P.J.
      • Simpson F.
      • Hill M.M.
      • Jones A.
      • Lundmark R.
      • Lindsay M.R.
      • Hernandez-Deviez D.J.
      • Hadzic G.
      • McCluskey A.
      • Bashir R.
      • Liu L.
      • Pilch P.
      • McMahon H.
      • Robinson P.J.
      • Hancock J.F.
      • Mayor S.
      • Parton R.G.
      Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells.
      ). Clathrin can also exist in large patches on the plasma membrane without forming functional pits or invagination (
      • Bellve K.D.
      • Leonard D.
      • Standley C.
      • Lifshitz L.M.
      • Tuft R.A.
      • Hayakawa A.
      • Corvera S.
      • Fogarty K.E.
      Plasma membrane domains specialized for clathrin-mediated endocytosis in primary cells.
      ,
      • Lin H.C.
      • Moore M.S.
      • Sanan D.A.
      • Anderson R.G.
      Reconstitution of clathrin-coated pit budding from plasma membranes.
      ,
      • Miller K.
      • Shipman M.
      • Trowbridge I.S.
      • Hopkins C.R.
      Transferrin receptors promote the formation of clathrin lattices.
      ). The correlated decrease of both the diffusion coefficient and the Hurst parameter after inhibition of clathrin or dynamin indicates a stable interaction of NK1R with these clathrin-related structures. Importantly, this was observed with all three CME inhibitors, indicating a specific clathrin effect. It is thus possible to exclude domain recruitment due to clathrin-independent membrane processes, which would be affected by dynamin inhibitors (
      • Sandvig K.
      • Pust S.
      • Skotland T.
      • van Deurs B.
      Clathrin-independent endocytosis: mechanisms and function.
      ). Receptors accumulate in a preinternalized state, that is in either clathrin lattices, prepits (
      • Cézanne L.
      • Lecat S.
      • Lagane B.
      • Millot C.
      • Vollmer J.-Y.
      • Matthes H.
      • Galzi J.-L.
      • Lopez A.
      Dynamic confinement of NK2 receptors in the plasma membrane. Improved FRAP analysis and biological relevance.
      ), coated pits (
      • Ehrlich M.
      • Boll W.
      • Van Oijen A.
      • Hariharan R.
      • Chandran K.
      • Nibert M.L.
      • Kirchhausen T.
      Endocytosis by random initiation and stabilization of clathrin-coated pits.
      ,
      • den Otter W.K.
      • Briels W.J.
      The generation of curved clathrin coats from flat plaques.
      ), or superficial early endosomes (
      • Roosterman D.
      • Cottrell G.S.
      • Schmidlin F.
      • Steinhoff M.
      • Bunnett N.W.
      Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist concentration and Rab GTPases.
      ). The fast accumulation of NK1R after CME inhibition strongly suggests a high association rate with these structures. Further support for the specific interaction of clathrin with the NK1R comes from the observation that the distinct membrane organization of the receptor is strongly affected after clathrin depletion.
      Interaction of NK1R with clathrin-dependent structures and immobilization of activated receptors are sequential events. In the absence of CME inhibitors, a large fraction of the receptors are localized in relatively stable domains in an intermediary mobility state between freely diffusing and internalized receptors. CME inhibitors promote this state by increasing the clathrin content at the membrane. In this state, receptors are diffusing in domains with lower diffusion coefficients and lower Hurst parameters. After activation with SP, the Hurst parameters remained unchanged, whereas the diffusion coefficients further decreased. The interactions involved in domain recruitment and in immobilization after activation are distinct. Non-activated receptors interact with clathrin-dependent structures, forming transient membrane domains, whereas activated receptors bind specifically to CCP through β-arrestin and AP2.
      CME inhibitors also strongly impaired receptor-mediated intracellular calcium signaling. This decrease of the NK1R canonical activity can arise from several, non-exclusive reasons. (i) The agonist binding site is not accessible due to the shape of the invagination as depicted in Fig. 1. (ii) G proteins cannot bind the intracellular region of the receptor due to the densely packed clathrin structures (
      • Cocucci E.
      • Aguet F.
      • Boulant S.
      • Kirchhausen T.
      The first five seconds in the life of a clathrin-coated pit.
      ). (iii) Receptor signaling is impaired by molecular crowding (
      • Zhou H.-X.
      Crowding effects of membrane proteins.
      ). Interestingly, disruption of microtubules, known to inhibit CME (
      • Subtil A.
      • Dautry-Varsat A.
      Microtubule depolymerization inhibits clathrin coated-pit internalization in non-adherent cell lines while interleukin 2 endocytosis is not affected.
      ), does not alter NK1R activity in our case.
      The correlation between the clathrin-dependent change of receptor mobility and the decrease of its activity, combined with the presence of a high fraction of type II receptors before activation, implies a clathrin-based mechanism for regulation of NK1R activity. Thereby, type II receptors could act as a non-activated receptor reservoir that is directly and quickly available at the cell membrane. This reservoir would have major implications in cell response to an agonist. In particular, it would allow responding to successive or long term agonist exposures. Indeed, for a short exposition time to an agonist, only a fraction of the receptors must respond. A gradual release of receptors from an inactive membrane reservoir could increase the response in the case of prolonged agonist exposition. Furthermore, such a mechanism would allow multiple intracellular Ca2+ responses to sequential agonist waves without the need of newly membrane-inserted receptors. This model is also compatible with the fast resensitization observed elsewhere (
      • Murphy J.E.
      • Roosterman D.
      • Cottrell G.S.
      • Padilla B.E.
      • Feld M.
      • Brand E.
      • Cedron W.J.
      • Bunnett N.W.
      • Steinhoff M.
      Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.
      ) albeit without the need of preliminary activation of NK1R.
      Cholesterol removal with mβCD affects NK1R mobility and activity in a similar manner as CME inhibition. Indeed, cholesterol depletion provokes a substantial decrease of the overall diffusion of the receptor and practically abolished the intracellular Ca2+ response. Thus, cholesterol, like clathrin, plays a major role in receptor mobility and is of critical importance for its activity, corroborating the close link between mobility and activity.
      The fast diffusing type III receptor population confined in circular domains, which appears 30 min after NK1R activation with SP, results from the presence of receptors in membrane blebs or microvesicles. Membrane blebs are balloon-like structures of the plasma membrane in which the cytoskeleton elements are generally absent, leading to enhanced molecular diffusion. Tank et al. (
      • Tank D.W.
      • Wu E.S.
      • Webb W.W.
      Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.
      ) found a considerable increase of the diffusion coefficients for both membrane proteins and lipids comparable with those found in liposomes (
      • Kusumi A.
      • Nakada C.
      • Ritchie K.
      • Murase K.
      • Suzuki K.
      • Murakoshi H.
      • Kasai R.S.
      • Kondo J.
      • Fujiwara T.
      Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules.
      ). Besides the canonical signaling pathway leading to Ca2+ release from the endoplasmic reticulum, SP induces cell membrane blebbing through the Rho/ROCK pathway by contraction of the actomyosin cell cortex (
      • Meshki J.
      • Douglas S.D.
      • Lai J.-P.
      • Schwartz L.
      • Kilpatrick L.E.
      • Tuluc F.
      Neurokinin 1 receptor mediates membrane blebbing in HEK293 cells through a Rho/Rho-associated coiled-coil kinase-dependent mechanism.
      ).
      Y-27632 is a highly specific and efficient cell-permeable ROCK inhibitor that prevents NK1R-induced blebbing without affecting the apoptotic state of the cells (
      • Meshki J.
      • Douglas S.D.
      • Lai J.-P.
      • Schwartz L.
      • Kilpatrick L.E.
      • Tuluc F.
      Neurokinin 1 receptor mediates membrane blebbing in HEK293 cells through a Rho/Rho-associated coiled-coil kinase-dependent mechanism.
      ). The mobility pattern resulting from NK1R trajectories measured in cells treated with this inhibitor and stimulated with SP is characterized by the absence of type III receptors. It demonstrates that the type III receptor population is directly dependent on the activation of the Rho/ROCK pathway and thus on the presence of membrane blebs.
      Membrane blebbing and excretion of microparticles are often associated with apoptosis. However, in our case, it has been shown that membrane blebbing is induced by activation of the NK1R by SP and is hence an apoptosis-independent phenomenon. This particular cellular mechanism may be of great importance for intercellular communication (
      • Chen P.
      • Douglas S.D.
      • Meshki J.
      • Tuluc F.
      Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.
      ).
      In summary, single particle tracking and multiparameter analysis allowed us to describe in detail the diffusional behavior of the neurokinin-1 receptor in the plasma membrane of living cells. The bimodal distribution of freely diffusing and confined receptors observed in the basal state is strongly shifted toward restricted mobility by receptor activation, whereas a new population of fast diffusing receptors in circular domains, corresponding to receptors in membrane blebs, resulted 30 min after activation of the Rho/ROCK pathway. Blocking of the CME pathway using different inhibitors leads to receptor confinement, which is correlated to a significant decrease of the receptor canonical pathway activity. Our results point to the central importance of clathrin, not only in receptor endocytosis and turnover but also in NK1R membrane homeostasis and fine regulation of its activity.

      Author Contributions

      H. V. initiated the project and was responsible for overall project management and strategy. L. V. and J. P. did the experiments and analyzed data. J. P. designed and implemented the computational methods. L. V., J. P., and H. V. designed the experiments, discussed the results, and contributed to the final manuscript.

      Acknowledgments

      We are very grateful to Shimon Weiss and Xavier Michalet from UCLA who generously hosted Luc Veya in the starting phase of this project. We appreciate many helpful discussions with Jean Gruenberg.

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