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.

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.signal transduction | protein assembly | kinetic proofreading | membrane dwell time | single molecule

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 K d 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.Ras signaling | Ras assay

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications.

A versatile platform for photodynamic therapy (PDT), mesoporous silica nanoparticles functionalized with protoporphyrin IX (PpIX‐MSNs), has been developed. In vitro studies on HeLa cells show high uptake efficiency. Phototoxicity results give both irradiation time‐ and dosage‐dependent cell death events. Because of the ease of incorporating other biomedical functional groups, we believe MSNs would be an ideal platform for biomedical applications.

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.

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.

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 are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis.