Stochastic geometry sensing and polarization in a lipid kinase-phosphatase competitive reaction.

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Single Proteoliposome High-Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs.

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (ß2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.

Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes.

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the ß2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.

H-Ras forms dimers on membrane surfaces via a protein-protein interface.

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to be ∼1 × 10(3) molecules/µm(2), and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes.

Interferometric Detection of Single Gold Nanoparticles Calibrated against TEM Size Distributions.

Single nanoparticle analysis: An interferometric optical approach calibrates sizes of gold nanoparticles (AuNPs) from the interference intensities by calibrating their interferometric signals against the corresponding transmission electron microscopy measurements. This method is used to investigate whether size affects the diffusion behavior of AuNPs conjugated to supported lipid bilayer membranes and to multiplex the simultaneous detection of three different AuNP labels.

Geometrical membrane curvature as an allosteric regulator of membrane protein structure and function.

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane ß-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ~1/40 nm(-1)) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (~40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ~50 mN/m and a curvature-imposed protein deformation energy of ~7 kBT. Such substantial energies have been shown to conformationally activate, or unfold, ß-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.

Investigating Lipase/Stain Interactions: Determining Interfacial Protein Conformation with Surface Spectroscopy.

Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion.

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity.

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca(2+) dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca(2+) accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca(2+) accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) ∼ 1 min and t(mix) ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.

Intermembrane docking reactions are regulated by membrane curvature.

The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle-vesicle pairs of different diameter (30-200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30-60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2-10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes.

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte.

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations in membrane thickness. We measured membrane thickness directly from electron micrographs of sections of fixed mucosal tissue. Indeed, mapping of the microvillar membrane revealed a biphasic distribution of membrane thickness. As a point of reference the thickness distribution of the basolateral membrane was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm(2). In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold labeling of alkaline phosphatase, a well documented raft marker. Strikingly, the alkaline phosphatase localized to distinct regions of the membrane in a pattern similar to the observed distribution of DITs. Although we were unable to measure membrane thickness directly on the immunogold labeled specimens, and thereby establish an unequivocal connection between DITs and rafts, we conclude that the brush border membrane of the enterocyte contains microdomains distinguishable both by membrane morphology and protein composition.

Single-Molecule Study of Thermomyces lanuginosus Lipase in a Detergency Application System Reveals Diffusion Pattern Remodeling by Surfactants and Calcium.

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

Membrane anchoring facilitates colocalization of enzymes in plant cytochrome P450 redox systems.

Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes.

Sensing-applications of surface-based single vesicle arrays.

A single lipid vesicle can be regarded as an autonomous ultra-miniaturised 3D biomimetic "scaffold" (Ø≥13 nm) ideally suited for reconstitution and interrogation of biochemical processes. The enclosing lipid bilayer membrane of a vesicle can be applied for studying binding (protein/lipid or receptor/ligand interactions) or transmembrane events (membrane permeability or ion channel activation) while the aqueous vesicle lumen can be used for confining few or single macromolecules and probe, e.g., protein folding, catalytic pathways of enzymes or more complex biochemical reactions, such as signal transduction cascades. Immobilisation (arraying) of single vesicles on a solid support is an extremely useful technique that allows detailed characterisation of vesicle preparations using surface sensitive techniques, in particular fluorescence microscopy. Surface-based single vesicle arrays allow a plethora of prototypic sensing applications in a high throughput format with high spatial and high temporal resolution. In this review we present a series of applications of single vesicle arrays for screening/sensing of: membrane curvature dependent protein-lipid interactions, bilayer tension, reactions triggered in the vesicle lumen, the activity of transmembrane protein channels and biological membrane fusion reactions.

Direct observation of Thermomyces lanuginosus lipase diffusional states by Single Particle Tracking and their remodeling by mutations and inhibition.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles.

We report a novel approach to quantitatively determine complete size distributions of surface-bound objects using fluorescence microscopy. We measure the integrated intensity of single particles and relate it to their size by taking into account the object geometry and the illumination profile of the microscope, here a confocal laser scanning microscope. Polydisperse (as well as monodisperse) size distributions containing objects both below and above the optical resolution of the microscope are recorded and analyzed. The data is collected online within minutes, which allows the user to correlate the size of an object with the response from any given fluorescence-based biochemical assay. We measured the mean diameter of extruded fluorescently labeled lipid vesicles using the proposed method, dynamic light scattering, and cryogenic transmission electron microscopy. The three techniques were in excellent agreement, measuring the same values within 7-9%. Furthermore we demonstrated here, for the first time that we know of, the ability to determine the full size distribution of polydisperse samples of nonextruded lipid vesicles. Knowledge of the vesicle size distribution before and after extrusion allowed us to propose an empirical model to account for the effect of extrusion on the complete size distribution of vesicle samples.

Surface-based lipid vesicle reactor systems: fabrication and applications.

Over the last ten years there has been a strong (bio)technological drive for the development of miniaturised reaction systems, motivated mainly by the need to reduce sample consumption and parallelise. Self-assembled soft-matter containers have naturally evolved to handle small volumes and could provide viable fluidic solutions especially in niche areas where ultra-miniaturisation, biocompatibility or cost are of critical importance. Here we focus on nanocontainers that are made of lipids and are immobilised on surfaces. We will highlight the most prominent contributions to date on the fabrication and the applications of surface-based vesicle systems as miniaturised reactors. Emphasis will be put on single-vesicle experiments.

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.