Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes

Rupture of Stochastically Occurring Vesicle Clusters Limits Bilayer Formation on Alkane-PEG-Type Supports:Uncoupling Clustering from Surface Coverage

Polymer-supported bilayers (PSBs) are a recognized tool for drug discovery through function-interaction analysis of membrane proteins. While silica-supported bilayers (SSBs) spontaneously form from surface-adsorbed vesicles, successful PSB formation via a similar method has thus far been limited by an insufficient understanding of the underlying vesicle-remodelling processes. Here, we generated a polymer support through the incubation of poly-l-lysine conjugated to alkyl-chain-terminated poly­(ethylene)­glycol on silica. This polymer-coated silica substrate yielded efficient vesicle adsorption and spontaneous bilayer formation, thereby providing a rare opportunity to address the mechanism of PSB formation and compare it to that of SSB. The combined use of super-resolution imaging, kinetics, and simulations indicates that the rupture of stochastically formed vesicle clusters is the rate-limiting step, which is an order of magnitude higher for silica than for polymer-coated silica. This was confirmed by directly demonstrating increased rupture rates for surface adsorbed multivesicle assemblies formed by vesicle cross-linking in solution. On the basis of this key insight we surmised that a low propensity of cluster rupture can be compensated for by an increase in the number density of clusters: the deposition of a mixture of oppositely charged vesicles resulted in bilayer formation on another alkane–PEG type of interface, which despite efficient vesicle adsorption otherwise fails to support spontaneous bilayer formation. This potentially provides a universal strategy for promoting bilayer formation on resistant surfaces without resorting to modifying the surface itself. Therefore, multivesicle assemblies with tailored geometries not only could facilitate bilayer formation on polymers with interesting functional properties but also could instigate the exploration of vesicle architecture for other processes involving vesicle remodelling such as drug delivery

Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requiresSNAREproteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, theRAB7GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1-Ccz1. We and others recently identified Ykt6 as the autophagosomalSNAREprotein. However, it has not been resolved when and how lipid-anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)-resident Dsl1 complex andCOPII-coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event

Receptor dimer stabilization By hierarchical plasma membrane microcompartments regulates cytokine signaling

The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane

Structural insights into IL-11-mediated signalling and human IL6ST variant-associated immunodeficiency

IL-11 and IL-6 activate signalling via assembly of the cell surface receptor gp130; however, it is unclear how signals are transmitted across the membrane to instruct cellular responses. Here we solve the cryoEM structure of the IL-11 receptor recognition complex to discover how differences in gp130-binding interfaces may drive signalling outcomes. We explore how mutations in the IL6ST gene encoding for gp130, which cause severe immune deficiencies in humans, impair signalling without blocking cytokine binding. We use cryoEM to solve structures of both IL-11 and IL-6 complexes with a mutant form of gp130 associated with human disease. Together with molecular dynamics simulations, we show that the disease-associated variant led to an increase in flexibility including motion within the cytokine-binding core and increased distance between extracellular domains. However, these distances are minimized as the transmembrane helix exits the membrane, suggesting a stringency in geometry for signalling and dimmer switch mode of action.</p

Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy

Quantitative protein sensing with germanium THz-antennas manufactured using CMOS processes

The development of a CMOS manufactured THz sensing platform could enable the integration of state-of-the-art sensing principles with the mixed signal electronics ecosystem in small footprint, low-cost devices. To this aim, in this work we demonstrate a label-free protein sensing platform using highly doped germanium plasmonic antennas realized on Si and SOI substrates and operating in the THz range of the electromagnetic spectrum. The antenna response to different concentrations of BSA shows in both cases a linear response with saturation above 20 mg/mL. Ge antennas on SOI substrates feature a two-fold sensitivity as compared to conventional Si substrates, reaching a value of 6 GHz/(mg/mL), which is four-fold what reported using metal-based metamaterials. We believe that this result could pave the way to a low-cost lab-on-a-chip biosensing platform

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells