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Register a new userIdentification and super-resolution imaging of ligand-activated receptor dimers in live cells
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Authors
Pascale Winckler
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Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Forster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.
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Rebinding kinetics of molecular ligands plays a critical role in biomachinery, from regulatory networks to protein transcription, and is also a key factor for designing drugs and high-precision biosensors.In this study, we investigate initial release and rebinding of ligands to their binding sites grafted on a planar surface, a situation commonly observed in single molecule experiments and which occurs during exocytosis in vivo. Via scaling arguments and molecular dynamic simulations, we analyze the dependence of non-equilibrium rebinding kinetics on two intrinsic length scales: average separation distance between the binding sites and dimensions of diffusion volume (e.g., height of the experimental reservoir in which diffusion takes place or average distance between receptor-bearing surfaces). We obtain time-dependent scaling laws for on rates and for the cumulative number of rebinding events for various regimes. Our analyses reveal that, for diffusion-limited cases, the on rate decreases via multiple power law regimes prior to the terminal steady-state regime, in which the on rate becomes constant. At intermediate times, at which particle density has not yet become uniform throughout the reservoir, the number of rebindings exhibits a distinct plateau regime due to the three dimensional escape process of ligands from their binding sites. The duration of this regime depends on the average separation distance between binding sites. Following the three-dimensional diffusive escape process, a one-dimensional diffusive regime describes on rates. In the reaction-limited scenario, ligands with higher affinity to their binding sites delay the power laws. Our results can be useful for extracting hidden time scales in experiments where kinetic rates for ligand-receptor interactions are measured in microchannels, as well as for cell signaling via diffusing molecules.
In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are cell-type- or species-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.
We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of 1.22 pm 0.12, such that the wall is significantly stiffer in intact cells (E = 23 pm 8 MPa and 49 pm 20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29 pm 3 kPa.
Ligand-receptor binding and unbinding are fundamental biomolecular processes and particularly essential to drug efficacy. Environmental water fluctuations, however, impact the corresponding thermodynamics and kinetics and thereby challenge theoretical descriptions. Here, we devise a holistic, implicit-solvent, multi-method approach to predict the (un)binding kinetics for a generic ligand-pocket model. We use the variational implicit-solvent model (VISM) to calculate the solute-solvent interfacial structures and the corresponding free energies, and combine the VISM with the string method to obtain the minimum energy paths and transition states between the various metastable (dry and wet) hydration states. The resulting dry-wet transition rates are then used in a spatially-dependent multi-state continuous-time Markov chain Brownian dynamics simulations, and the related Fokker-Planck equation calculations, of the ligand stochastic motion, providing the mean first-passage times for binding and unbinding. We find the hydration transitions to significantly slow down the binding process, in semi-quantitative agreement with existing explicit-water simulations, but significantly accelerate the unbinding process. Moreover, our methods allow the characterization of non-equilibrium hydration states of pocket and ligand during the ligand movement, for which we find substantial memory and hysteresis effects for binding versus unbinding. Our study thus provides a significant step forward towards efficient, physics-based interpretation and predictions of the complex kinetics in realistic ligand-receptor systems.
Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.
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