Hsiung-Lin Tu

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

Significance Ras is a key signaling molecule in living cells, and mutations in Ras are involved in 30% of human cancers. It is becoming progressively more clear that the spatial arrangement of proteins within a cell, not just their chemical structure, is an important aspect of their function. In this work, we use a series of quantitative physical techniques to map out the tendency of two Ras molecules to bind together to form a dimer on membrane surfaces. Insights from this work, as well as the technical assays developed, may help to discover new therapeutic drugs capable of modulating the errant behavior of Ras in cancer. 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 × 103 molecules/µm2, 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.

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

Outliers dominate signaling at cell membrane SOS enzymes act at cell membranes to activate Ras, a regulatory protein often overactive in cancer cells. Iversen et al. devised a system where they could observe the activity of individual enzymes at work. The single SOS molecules occupied stable states that varied greatly in their catalytic activity. Regulation appeared to occur by altering the time spent in active states. The overall activity of SOS was determined by just a few molecules that achieved the highest catalytic activity. The methods described should allow further detailed kinetic analysis of this and other signaling events that occur at the cell membrane — properties that it is not possible to discern from bulk biochemical measurements. Science, this issue p. 50 Single-molecule measurements reveal insights into regulation of the small GTPase Ras. Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

Single molecule tracking on supported membranes with arrays of optical nanoantennas.

Coupling of the localized surface plasmons between two closely apposed gold nanoparticles (nanoantenna) can cause strong enhancements of fluorescence or Raman signal intensity from molecules in the plasmonic “hot-spot”. Harnessing these properties for practical applications is challenging due to the need to fabricate gold particle arrays with well-defined nanometer spacing and a means of delivering functional molecules to the hot-spot. We report fabrication of billions of plasmon-coupled nanostructures on a single substrate by a combination of colloid lithography and plasma processing. Controlled spacing of the nanoantenna gaps is achieved by taking advantage of the fact that polystyrene particles melt together at their contact point during plasma processing. The resulting polymer thread shadows a gap of well-defined spacing between each pair of gold triangles in the final array. Confocal surface-enhanced Raman spectroscopy imaging confirms the array is functionally uniform. Furthermore, a fully intact supported membrane can be formed on the intervening substrate by vesicle fusion. Trajectories of freely diffusing individual proteins are traced as they sequentially pass through, and are enhanced by, multiple gaps. The nanoantenna array thus enables enhanced observation of a fluid membrane system without static entrapment of the molecules.

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

Significance The assembly of receptors and downstream signaling molecules into extended networks is a commonly observed phenomenon in signal transduction. However, little is known about how such assemblies physically modulate signal propagation. Here, based on single-molecule kinetics studies, we report that phosphotyrosine-mediated assembly of adaptor protein LAT networks yields two distinct kinetic species of the Ras activator SOS. This system regulates the transmission of signal from activated T-cell receptor to Ras. We propose and evaluate how the emergence of a long-dwelling SOS species may serve as a kinetic proofreading mechanism to discriminate stochastic noise from genuinely activated receptors. 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.

Nonionic fluorescent oligomeric surfactant for ordered mesoporous silica structure

A fluorescent oligomeric surfactant prepared by multi-step synthesis was used as a template to obtain ordered mesostructured silica, the fluorescent surfactant completely fills the nano-channels and the emission spectrum shows that the composite is red-shifted 32 nm when compared with the surfactant in aqueous solution.

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.

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.

One-step synthesis of ordered mesostructural organic/silica nanocomposites with tunable fluorescence surfactants

Mesostructured nanocomposites with tunable fluorescence were prepared by one-step synthesis using fluorescent surfactants with 1, 2 and 3 units of thiophene as templates. The XRD data showed that the resulting nanocomposites exhibited ordered mesostructures, which could be further confirmed by TEM observation. Moreover, the surfactants with longer hydrophobic chain lengths result in mesostructured nanocomposites with larger lattice constants. Fluorescent properties of these nanocomposites, compared with the same fluorescent surfactants in other environments, showed fluorescence bands that were red-shifted in the fluorescence spectra. N2 adsorption–desorption experiments indicated that the rearrangement of PEO chains and the conjugated part of surfactants occurs upon hydrothermal treatment, thus enlarging the pore diameter, surface area and pore volume of the resulting mesostructured nanocomposites.

The Residence Time and Processivity Study of the Ras/Sos Interaction

Ras is a membrane-bound small GTPase protein that plays a central role in the signal transduction pathways that control cell proliferation, differentiation, and apoptosis. Its deregulation is a hallmark of many cancers and developmental defects. Son of Sevenless (SOS) is a guanine nucleotide exchange factor (GEF) enzyme that activates Ras by catalyzing the conversion of Ras from the GDP- to the GTP-bound state.SOS has two binding sites for Ras, a catalytic site and an allosteric site, which can both be occupied simultaneously by membrane-bound Ras. Previous studies have shown that binding to the allosteric site by Ras-GTP will localize SOS to the membrane and therefore stimulate the nucleotide exchange activity of the catalytic site (positive-feedback), raising the question of whether SOS is processive, capable of remaining surface bound while catalyzing the nucleotide exchange of multiple Ras. In this study we employ fluorescence microscopy on Ras functionalized supported lipid bilayers to demonstrate that the catalytic core of SOS (SOScat) is processive. In the absence of GTP, SOScat remains surface bound via Ras in a non-processive state for ∼hours. Addition of GTP triggers processive turnover of multiple Ras by surface bound SOScat. Using single molecule TIRF microscopy, the result indicates that most of the initial surface bound SOScat rapidly desorbs when GTP is added, and that most of the Ras turnover is catalyzed by a small but processive fraction of the initial SOScat population.