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* 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.
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.
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 (
). 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 (
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 (
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 (
) 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) (
). 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 (
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 (
). 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 (
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.
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 D1–10versus 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 (
). 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 (
). 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 (
). 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. (
), 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 (
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 (
) 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
) 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. (
). 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 (
). 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 (
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.
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.
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.