Adam W. Smith

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Investigating Cell Surface Galectin-Mediated Cross-Linking on Glycoengineered Cells

The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin–ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs’ fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the “galectin lattice” hypothesis and to defining galectin ligand specificity in a physiologically relevant context.

Lipid-protein interactions in biological membranes: a dynamic perspective.

Though an increasing number of biological functions at the membrane are attributed to direct associations between lipid head groups and protein side chains or lipid protein hydrophobic attractive forces, surprisingly limited information is available about the dynamics of these interactions. The static in vitro representation provided by membrane protein structures, including very insightful lipid-protein binding geometries, still fails to recapitulate the dynamic behavior characteristic of lipid membranes. Experimental measures of the interaction time of lipid-protein association are very rare, and have only provided order-of-magnitude estimates in an extremely limited number of systems. In this review, a brief outline of the experimental approaches taken in this area to date is given. The bulk of the review will focus on two methods that are promising techniques for measuring lipid-protein interactions: time-resolved fluorescence microscopy, and two-dimensional infrared (2D IR) spectroscopy. Time-resolved fluorescence microscopy is the name given to a sophisticated toolbox of measurements taken using pulsed laser excitation and time-correlated single photon counting (TCSPC). With this technique the dynamics of interaction can be measured on the time scale of nanoseconds to milliseconds. 2D IR is a femtosecond nonlinear spectroscopy that can resolve vibrational coupling between lipids and proteins at molecular-scale distances and at time scales from femtoseconds to picoseconds. These two methods are poised to make significant advances in our understanding of the dynamic properties of biological membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.

Transient two-dimensional IR spectrometer for probing nanosecond temperature-jump kinetics

We have developed a Fourier transform two-dimensional infrared (2D IR) spectrometer to probe chemical reactions and biophysical processes triggered by a nanosecond temperature jump (T jump). The technical challenges for such a spectrometer involve (1) synchronization of a nanosecond T-jump laser and femtosecond laser system, (2) overcoming the decreased signal-to-noise ratio from low repetition rate data acquisition, and (3) performing an interferometric measurement through a sample with a density and index of refraction that varies with time delay after the T jump. The first challenge was overcome by synchronizing the two lasers to a clock derived from the Ti:sapphire oscillator, leading to timing accuracy of 2 ns for delays up to 50 ms. The data collection time is reduced by using undersampling with the improved signal-to-noise ratio obtained from a balanced detection scheme with a dual stripe array detector. Transient dispersed vibrational echo and 2D IR spectroscopy are applied to N-methylacetamide and ubiquitin, as examples, and the spectral responses by a temperature elevation and by structural changes of the protein are compared. The synchronization of 2D IR spectroscopy with a nanosecond temperature jump without losing its sensitivity at a low repetition rate opens a new applicability of the nonlinear spectroscopy to probe a variety of molecular structure changes induced by a nanosecond perturbation.

E-cadherin junction formation involves an active kinetic nucleation process

Significance Epithelial (E)-cadherin-based adherens junctions are the basis of epithelial tissue integrity in Metazoans. They are composed of E-cadherin molecules interacting with each other from apposed cells. Using artificial supported lipid bilayers functionalized with the full-length extracellular domain of E-cadherin and live cells, we show that E-cadherin junction formation involves a nucleation process mediated by active filopodia retraction and requiring reduced mobility of E-cadherin on supported lipid bilayers. These results underscore the importance of controlling physical aspects of the cellular microenvironment with synthetic materials for in vitro live cell applications. In this case, tuning the mobility of a viscous fluid display surface enabled functional reconstitution of a cadherin-mediated adhesion junction. Epithelial (E)-cadherin-mediated cell−cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

Monitoring Lipid Anchor Organization in Cell Membranes by PIE-FCCS

This study examines the dynamic co-localization of lipid-anchored fluorescent proteins in living cells using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and fluorescence lifetime analysis. Specifically, we look at the pairwise co-localization of anchors from lymphocyte cell kinase (LCK: myristoyl, palmitoyl, palmitoyl), RhoA (geranylgeranyl), and K-Ras (farnesyl) proteins in different cell types. In Jurkat cells, a density-dependent increase in cross-correlation among RhoA anchors is observed, while LCK anchors exhibit a more moderate increase and broader distribution. No correlation was detected among K-Ras anchors or between any of the different anchor types studied. Fluorescence lifetime data reveal no significant Förster resonance energy transfer in any of the data. In COS 7 cells, minimal correlation was detected among LCK or RhoA anchors. Taken together, these observations suggest that some lipid anchors take part in anchor-specific co-clustering with other existing clusters of native proteins and lipids in the membrane. Importantly, these observations do not support a simple interpretation of lipid anchor-mediated organization driven by partitioning based on binary lipid phase separation.

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells

T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell–nanopatterned supported lipid bilayer system to quantitatively probe the actin–TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if this is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.14107.001

Optical spectroscopy of tungsten carbide (WC)

Resonant two-photon ionization spectroscopy has been used to study the diatomic transition-metal carbide, WC. A low-resolution scan revealed a five-member vibrational progression beginning with the 0-0 band at 17 585 cm−1. Analysis of this progression yielded a vibrational frequency of ωe′(184W12C)=752.6(4.9) cm−1 and a bond length of re′(184W12C)=1.747(4) A. Several unassigned bands were also rotationally resolved and analyzed. All of the observed bands are Ω′=2←Ω″=1 transitions, confirming the predicted ground state of 3Δ1 arising from a 14σ28π415σ24δ116σ1 configuration. The measured line positions in these bands were simultaneously fitted to provide B0″=0.509 66(10) cm−1 for 184W12C, corresponding to r0″(184W12C)=1.713 5(2) A. These values are corrected for spin-uncoupling effects in the ground state and represent our best estimate of the true bond length of WC. Dispersed fluorescence studies provide the ground-state vibrational constants of ωe=983(4) cm−1 and ωexe=11(1) cm−1, and have also permitted t...

Time-Resolved Fluorescence Spectroscopy Measures Clustering and Mobility of a G Protein-Coupled Receptor Opsin in Live Cell Membranes

Determining membrane protein quaternary structure is extremely challenging, especially in live cell membranes. We measured the oligomerization of opsin, a prototypical G protein-coupled receptor with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Individual cell measurements revealed that opsin is predominantly organized into dimeric clusters. At low concentrations, we observed that the population of oligomers increased linearly with the square of the individual monomer populations. This finding supports a monomer–dimer equilibrium and provides an experimental measurement of the equilibrium constant.