Pulsed-interleaved Excitation Fluorescence Cross-correlation Research Articles

Resolving the hierarchical network of receptor tyrosine kinase interactions and the effect of oncogenic mutations.

Cells have an array of protein receptors on the plasma membrane that help process environmental cues. The spatial and temporal arrangement of these receptors is critical to function, but the chemical forces driving this organization are not well understood. This talk will focus on receptor tyrosine kinases (RTKs), which are transmembrane proteins that regulate cell growth, proliferation, and differentiation. Several RTKs are oncogenic and are targeted in next generation anti-cancer drug development. The degree to which these oncogenic mutations affect the local network of interactions between RTKs has not been explored beyond a few high-profile examples. We use pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to resolve membrane protein mobility, concentration, and monomer/dimer/oligomer distributions. I will describe ongoing work in my group to resolve the network of interactions between EGFR, HER2, HER3 and HER4, and how this network responds to oncogenic mutations.

Resolving Membrane Protein-Protein Interactions in Live Cells with Pulsed Interleaved Excitation Fluorescence Cross-Correlation Spectroscopy.

The cell plasma membrane (PM) contains thousands of proteins that sense and respond to the outside environment. These proteins have evolved sensitivity to a wide variety of physical and chemical signals and act as a delivery system across the PM. Membrane proteins are critical for information flow and decision making in the cell and thus are important targets in drug development. A critical aspect of membrane protein function is the way they interact with other proteins, often through the formation of dimers or small oligomers that regulate function at the protein, cell, and organism levels. Resolving membrane protein interactions in a live cell environment is challenging because of the chemical diversity and spatial heterogeneity of the PM. In this Account, we describe a fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) that is ideally suited to quantify membrane associations in live cells. PIE-FCCS is a two-color fluorescence fluctuation method that can simultaneously measure the concentration, mobility, proximity, and oligomerization state of membrane proteins in situ. It has several advantages over two related approaches, single-molecule tracking (SMT) and Förster resonance energy transfer (FRET), including that it measures all of the properties listed above in a single measurement. Another advantage is that PIE-FCCS is most sensitive at the physiological expression levels for many membrane proteins rather than the very low or high levels typical in other techniques. Here, we review the history of FCCS as it has been applied to study membrane protein interactions in cells. We also describe PIE-FCCS and the advantages it has over biochemical approaches like coimmunoprecipitation (co-IP) and proximity ligation assays (PLA). Finally, we review two classes of membrane proteins that have been studied with FCCS and PIE-FCCS: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). For RTKs, ligand induced dimerization directly regulates the catalytic activity of the kinase, but higher order oligomerization and ligand-independent dimerization can complicate this historically simple paradigm. PIE-FCCS data have resolved a low population of EGFR dimers under basal conditions and assembly into multimers when stimulated with ligand. While GPCRs function primarily as monomers, dimerization has been hypothesized to regulate function for some receptors. PIE-FCCS data have established the dimerization potential of rhodopsin at low densities and were critical for the discovery of a novel dimerization interface in human cone opsins. This Account describes the how FCCS and PIE-FCCS can reveal the details of quaternary interactions in each of these receptor systems.

Single Molecule Diffusion of Phosphatidylinositol Bisphosphate (PIP2) Lipids on Asymmetric Lipid Bilayers under the Influence of Polycationic Macromolecules

Proteins binding with PIP2 lipids in the plasma membrane initiate and regulate many cell signaling events. Previous research suggests that proteins interact with PIP2 lipids through electrostatic interactions between polycationic protein motifs and anionic lipid head groups, leading to potential sequestration of multiple PIP2 lipids. The sequestration of PIP2 lipids will cause a change in the lipid mobility and the spatial distribution of PIP2 in the plasma membrane. We set out to understand the molecular structure and dynamics of the protein-PIP2 interface at single molecule level. In this study, a polycationic polymer, quaternized polyvinylpyridine (QPVP), and Myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were chosen to mimic the membrane binding proteins. The behavior of PIP2 lipids in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid.

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Tuning the Mobility Coupling of Quaternized Polyvinylpyridine and Anionic Phospholipids in Supported Lipid Bilayers

Binding of biomacromolecules to anionic lipids in the plasma membrane is a common motif in many cell signaling pathways. Previous work has shown that macromolecules with cationic sequences can form nanodomains with sequestered anionic lipids, which alters the lateral distribution and mobility of the membrane lipids. Such sequestration is believed to result from the formation of a lipid-macromolecule complex. To date, however, the molecular structure and dynamics of the lipid-polymer interface are poorly understood. We have investigated the behavior of polycationic quaternized polyvinylpyridine (QPVP) on supported lipid bilayers doped with phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids using time-resolved fluorescence microscopy, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and colocalization. By labeling the polymer and lipids, we investigated the adsorption-induced translational mobility of lipids and systematically studied the influence of lipid charge density and solution ionic strength. Our results show that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid headgroup and is modulated by the ionic strength of the solution, indicating that electrostatic interactions drive the decrease in lateral mobility of anionic lipids by adsorbed QPVP. At physiological salt concentration we observe that the lipid lateral mobility is weakly influenced by QPVP and that there is no evidence of stable lipid-polymer complexes.

Decoding the Role of Receptor Dimerization in Plexin-Semaphorin Signaling

Plexin-semaphorin signaling plays an important role in the formation of neural networks by acting as the guidance cue for axon growth or retraction. The plexin receptor is a single-pass transmembrane protein with a sema domain at the extracellular N-terminus and a GTP-ase activating protein domain on the cytoplasmic C-terminus. Binding of semaphorin is known to activate the plexin receptor, but the mechanism of how that binding event leads to activation is not understood. We will present our recent work using pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to measure plexin protein-protein interactions in live cells. PIE-FCCS transforms fluctuations in fluorescence intensity (arising mainly from diffusion) into information about a protein's mobility and concentration. By cross-correlating the fluorescence fluctuations in two color channels, PIE-FCCS also quantifies co-localization and co-diffusion with high accuracy. This method has been used to resolve the mechanism of epidermal growth factor receptor (EGFR) activation and inhibition, as well as the dimerization constant of the opsin G protein-coupled receptor. In this work we measure the ligand-free dimerization of plexin receptors in live cell membranes and the response of the receptor to semaphorin-induced activation.

PIE-FCCS Study of the Effects of Polycationic Macromolecules on Phosphatidylserine and Phosphatidylinositol Phosphate Lipid Mobility

The interaction between proteins and anionic lipids in the plasma membrane is a common motif in cell communication at the plasma membrane. Such interactions can gate membrane protein function and have also been proposed to sequester membrane lipids during quiescent phases of signaling. To date, however, the molecular structure and dynamics of the lipid-protein interface is poorly understood. To isolate these interactions in a biophysical assay, we have investigated the behavior of a polycationic polymer, quaternized polyvinylpyridine (QPVP), on supported lipid bilayers doped with tail-labeled phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids. To measure the mobility and association of the lipid and adsorbed polymer, we use a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and co-localization. With PIE-FCCS we have investigated the polymer adsorption-dependent translational mobility of the lipids and systematically studied the influence of lipid head-group charge and solvent ionic strength. Our results indicate that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid head group and is significantly screened by the ionic strength of the solution. At physiological salt concentration we observe that the lipid lateral mobility is nearly unaffected by mobile, adsorbed polymers and that there is no evidence of stable lipid-polymer complexes.