Patrick J. Cutler

ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (koff) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states. We report that dimers composed of two ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have a more than four times faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases more than six times 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.

Time-resolved three-dimensional molecular tracking in live cells.

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 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device 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 antibunching 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 spatiotemporal dynamics of live cells, we follow individual QD-labeled IgE-FcεRI receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three-dimensional, nanoscale 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.

Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope

Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.

Optimal Aggregation of FcεRI with a Structurally Defined Trivalent Ligand Overrides Negative Regulation Driven by Phosphatases

To investigate why responses of mast cells to antigen-induced IgE receptor (FcεRI) aggregation depend nonlinearly on antigen dose, we characterized a new artificial ligand, DF3, through complementary modeling and experimentation. This ligand is a stable trimer of peptides derived from bacteriophage T4 fibritin, each conjugated to a hapten (DNP). We found low and high doses of DF3 at which degranulation of mast cells sensitized with DNP-specific IgE is minimal, but ligand-induced receptor aggregation is comparable to aggregation at an intermediate dose, optimal for degranulation. This finding makes DF3 an ideal reagent for studying the balance of negative and positive signaling in the FcεRI pathway. We find that the lipid phosphatase SHIP and the protein tyrosine phosphatase SHP-1 negatively regulate mast cell degranulation over all doses considered. In contrast, SHP-2 promotes degranulation. With high DF3 doses, relatively rapid recruitment of SHIP to the plasma membrane may explain the reduced degranulation response. Our results demonstrate that optimal secretory responses of mast cells depend on the formation of receptor aggregates that promote sufficient positive signaling by Syk to override phosphatase-mediated negative regulatory signals.

Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties.

The movement of a particle described by Brownian motion is quantified by a single parameter, D, the diffusion constant. The estimation of D from a discrete sequence of noisy observations is a fundamental problem in biological single-particle tracking experiments since it can provide information on the environment and/or the state of the particle itself via the hydrodynamic radius. Here, we present a method to estimate D that takes into account several effects that occur in practice, important for the correct estimation of D, and that have hitherto not been combined together for an estimation of D. These effects are motion blur from the finite integration time of the camera, intermittent trajectories, and time-dependent localization uncertainty. Our estimation procedure, a maximum-likelihood estimation with an information-based confidence interval, follows directly from the likelihood expression for a discretely observed Brownian trajectory that explicitly includes these effects. We begin with the formulation of the likelihood expression and then present three methods to find the exact solution. Each method has its own advantages in either computational robustness, theoretical insight, or the estimation of hidden variables. The Fisher information for this likelihood distribution is calculated and analyzed to show that localization uncertainties impose a lower bound on the estimation of D. Confidence intervals are established and then used to evaluate our estimator on simulated data with experimentally relevant camera effects to demonstrate the benefit of incorporating variable localization errors.

Determining FcεRI Diffusional Dynamics via Single Quantum Dot Tracking

Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial-temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the high-affinity IgE Receptor. Here, we describe protocols for (1) coupling QDs to IgE, (2) tracking individual QD-bound receptors, and (3) analyzing one- and two-color tracking data.

Multi-Color Single Particle Tracking of QD-IgE-FcεRI: Directly Correlating Oligomer Size with Receptor Mobility and Signaling

IgE binds to its high affinity receptor, FceRI, expressed on mast cells and basophils. Crosslinking of IgE-FceRI complexes leads to intracellular signaling and eventual immune response. Although much is known about this receptor family, the precise mechanism of signal initiation remains elusive. Previous single particle tracking (SPT) experiments have revealed that small, mobile receptors are signaling competent. However, high-speed SPT had typically been limited to two-color tracking, such that aggregates larger than dimers could not be distinguished. In order to better understand the relationship between receptor mobility, aggregate size and signaling, we have developed a novel high-speed hyperspectral line-scanning microscope (HSM) to perform multi-color SPT (mcSPT) of up to 8 colors of quantum dot (QD)- labeled IgE-FceRI simultaneously on the surface of mast cells. The HSM is unique in its capability to acquire a 4D image (x,y,λ,t) of ∼30 microns⊥2 with 128 spectral channels at ∼30 fps. This combined with the advantageous characteristics of QDs (high quantum yield, single source excitation, narrow emission spectra) provides an exceptional platform for mcSPT. Using the HSM, we can determine receptor aggregate size from the spectral signature, and then correlate aggregate size with mobility. Comparison of aggregate mobility in the presence of the tyrosine kinase inhibitor, PP2, reveals the influence of adaptor protein recruitment on mobility. The HSM also allows for simultaneous measurement of calcium flux (Fluo-4) while performing mcSPT, providing a direct readout of signaling in response to receptor aggregate formation and mobility. These experiments not only address important questions in IgE-FceRI signal initiation, but also provide new biophysical insight into the long-standing debate over the relationship between receptor oligomer size and mobility in the plasma membrane.

3D Molecular Tracking in Live Cells with Simultaneous Time-Resolved Spectroscopy

We report a confocal feedback method for tracking the motion of individual quantum dot labeled proteins as they move in three dimensions inside of cells that has sub-nanosecond temporal resolution. 3D tracking molecular is possible for tens of microns in X, Y, and Z, meaning tracking can occur throughout the entire cell volume volume for many cell lines. The sub-nanosecond temporal resolution enables time-resolved spectroscopies (e.g., fluorescence lifetime measurements or fluorescence correlation spectroscopy) to be made on the molecules as they are being tracked. In particular, recording of the arrival times of individual photons enabled, for the first time, photon pair correlation measurements showing fluorescence photon anti-bunching of individual QD labeled proteins in live cells. The power of this new technology is further illustrated through tracking of individual QD-labeled IgE receptor complexes on rat mast cells, revealing three-dimensional nano-scale topology of the cell membrane as individual receptors navigate hills and valleys of a dynamically changing plasma membrane landscape. In addition to mapping out cell surface topology, IgE-Fc{epsilon}RI signaling clusters were also captured in the act of ligand-mediated endocytosis and tracked during rapid (∼950 nm/s) vesicular transit through the cell.

Single Quantum Dot Tracking Coupled to a Three-State HMM Provides New Mechanistic Insight Into erbB1 Homodimerization

The erbB family of trans-membrane receptor tyrosine kinases serves as the prototypical model for dimerization-induced signal transduction. The current paradigm for signal initiation by erbB1 homo-dimers is based on ligand-induced conformational changes, which expose the dimerization arms of activated receptors. To track erbB1 dynamics at the molecular level, we used two-color single quantum dot (QD) tracking to determine diffusion and dimerization characteristics of resting, ligand-bound and kinase-inhibited receptors. Long-lived erbB1 homo-dimers were directly visualized between EGF-QD-bound receptors, characterized by a 50 nm separation. However, we also observed dimer interactions whose close approach was punctuated by periods of excursion up to hundreds of nanometers apart, suggesting a third domain-confined receptor state. For each condition, transition rates between free, domain-confined, and dimer states were extracted using a modified three-state Hidden Markov Model (HMM). This analytical model uses separation of pairwise QD trajectories to determine probabilities of state transitions, whose rates are fit over many candidate interactions. Diffusional behavior was determined by state and showed slowing upon dimer formation; EGF-bound monomeric receptors confined to the same domain showed two-fold slower diffusion than free receptors and showed a further three and a half-fold slowing upon dimerization. Resting or kinase inhibited receptors did not show this dramatic mobility change. While the small molecule tyrosine kinase inhibitor, PD153035 that blocks receptor phosphorylation altered receptor diffusion, it did not change the dimer off rate. Resting erbB1 dimers tracked via monovalent heavy-chain only antibody fragments (VhH) did not demonstrate correlated motion and had a >6-fold higher off-rate. Our studies provide new mechanistic insight into erbB1 dimerization, demonstrate a role for membrane domains in promoting protein-protein interactions, and suggest a reduction in receptor mobility as a feature of signaling competent erbB1 dimers.

Time-Resolved 3D Tracking of Individual Quantum Dot Labeled Proteins in Live Cells via Confocal Feedback

We have developed a microscope system that uses active feed-back to follow individual quantum dot labeled proteins moving in three dimensions in live cells at μm/s transport velocities with 100 picoseconds temporal resolution.