The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we utilized two-color Quantum Dot tracking for visualization of erbB1 homodimerization and quantification of the dimer off rate (koff) on living cells. Kinetic parameters were extracted using a 3-state Hidden Markov Model to identify transition rates between free, co-confined, and dimerized states. We report that dimers composed of 2 ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have >4-fold faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases >6-fold when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.

The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcεRI motion and GFPtagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganisation of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time dependent. Multiple FcεRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcεRI signalling is activated by cross-linking with multivalent antigen. We show that receptors become immobilised within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilisation kinetics and increased diffusion of cross-linked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their longrange mobility, sequestration, and response to ligand binding.Signal transduction from the external environment to the cell interior is typically mediated by ligand-bound transmembrane receptors embedded in a lipid bilayer. In many systems, receptor activation is associated with changes in receptor dynamics and membrane topography1 -3 . Among these are the multi-chain immune recognition receptor family members that include the B-cell receptor (BCR) of B-cells, the T-cell receptor (TCR) of Tcells, and the high affinity IgE receptor (FcεRI) of mast cells and basophils, which are crucial to the execution of key events in the immune response. Cross-linking of these transmembrane receptors induces receptor oligomerisation, protein and lipid kinase activation and Ca 2+ mobilisation, leading in turn to cytoskeletal reorganisation, receptor trafficking and cell-specific responses including altered gene expression [4][5][6] . These signalling events have been well studied by biochemical techniques, but the precise mechanism by which oligomerisation initiates these events has remained elusive. Full understanding of these complex signalling cascades will require a more complete description of receptor movements in the membrane, including restrictions that might limit receptor diffusion and accessibility.Correspondence should be addressed to D.S.L. (dlidke@salud.unm.edu). 4 These authors contributed equally to this work. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2011 January 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA rich literature on single particle tracking (SPT) methods to follow the lateral diffusion of transmembrane and membrane-associated proteins7 -10 has revealed nanometer-scale "confinement zones" that restrict lateral diffusion and supports the general notion that plasma membrane organisation is more structured than originally postulated by the fluid mosaic model11. A membrane-skeleton fence (picket fence) model ...

Summary Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF – making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 milliseconds to follow three dimensional molecular motion. This method has substantial advantages over 3D molecular tracking methods based upon CCD cameras, including increased Z tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon anti-bunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatio-temporal dynamics of live cells, we follow individual QD-labeled IgE receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three dimensional, nano-scale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell. KeywordsQuantum Dot; Single Molecule; Fluorescence; Tracking; Microscopy; Cell The direct observation of individual biological molecules in motion can transform our view of important biophysical and cellular processes. 1 For example, single molecule tracking has shed significant light on cellular membrane dynamics 2-4 , motor protein kinetics 5,6 , and gene regulation 7 . Advantages of a single molecule approach include the ability to observe dynamic, stochastic behavior (such as compartmentalized diffusion 2, 4 ) that would be masked in ensemble measurements and the ability for localization of molecules with a precision well below the diffraction limit of light 5,6 . To date the field has primarily relied * Corresponding Author: jwerner@lanl.gov. 8,16 or follow the Z position with multiple cameras or image planes 9, 14 . While these camera-based techniques can capture 3D molecular motion, they are generally limited in their Z-tracking range in cells to approximately plus or minus one μm from a fixed focal plane 8,10,14,17 , limited by the shallow depth of field of high numerical aperture microscope objectives needed for single molecule work. We point out the obvious: many cells are substantially thicker than two microns and different methods and techniques are required to follow single molecules throughout entire three dimensional cell volumes. In addition to its quite limiting Z-tracking range, CCD-based tracking approaches are also bounded in temporal resolution by the CCD frame rate (~1 ms for fast EM-CCDs), and must illuminate an entire cell slice at relatively large excitation intensities (~40 W/cm 2 ).In contrast to ...

Distributions of ErbB receptors on membranes of SKBR3 breast cancer cells were mapped by immunoelectron microscopy. The most abundant receptor, ErbB2, is phosphorylated, clustered and active. Kinase inhibitors ablate ErbB2 phosphorylation without dispersing clusters. Modest co-clustering of ErbB2 and EGFR, even after EGF treatment, suggests that both are predominantly involved in homointeractions. Heregulin leads to dramatic clusters of ErbB3 that contain some ErbB2 and EGFR and abundant PI 3-kinase. Other docking proteins, such as Shc and STAT5, respond differently to receptor activation. Levels of Shc at the membrane increase two- to five-fold with EGF, whereas pre-associated STAT5 becomes strongly phosphorylated. These data suggest that the distinct topography of receptors and their docking partners modulates signaling activities.