Conditional requirement for dimerization of the membrane-binding module for BTK signaling in lymphocyte cell lines.

Bruton's tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin homology and tec homology (PH-TH) domains of BTK are required for signaling. Dimerization of the PH-TH module strongly stimulates the kinase activity of BTK in vitro.

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab'-DNA ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs.

Discrete protein condensation events govern calcium signal dynamics in T cells.

Unlabelled: Calcium level variations, which occur downstream of T cell receptor (TCR) signaling, are an essential aspect of T cell antigen recognition. Although coordinated ion channel activities are known to drive calcium oscillations in other cell types, observations of nonperiodic and heterogeneous calcium patterns in T cells are inconsistent with this mechanism. Here, we track the complete ensemble of individual molecular peptide-major histocompatibility complex (pMHC) binding events to TCR, while simultaneously imaging LAT condensation events and calcium level.

Positive feedback in Ras activation by full-length SOS arises from autoinhibition release mechanism.

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism.

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without deterministic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays.

Differential Roles of Kinetic On- and Off-Rates in T-Cell Receptor Signal Integration Revealed with a Modified Fab'-DNA Ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly-binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide-MHC (pMHC) ligands bind their cognate TCRs.

Allosteric inhibition of the T cell receptor by a designed membrane ligand.

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model.

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability.

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays.

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2.

The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a 'Src module', an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP, or in solution by inositol hexakisphosphate (IP) (Wang et al., 2015, https://doi.

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins.

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed.

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events.

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event.

Kinetic frustration by limited bond availability controls the LAT protein condensation phase transition on membranes.

LAT is a membrane-linked scaffold protein that undergoes a phase transition to form a two-dimensional protein condensate on the membrane during T cell activation. Governed by tyrosine phosphorylation, LAT recruits various proteins that ultimately enable condensation through a percolation network of discrete and selective protein-protein interactions. Here, we describe detailed kinetic measurements of the phase transition, along with coarse-grained model simulations, that reveal that LAT condensation is kinetically frustrated by the availability of bonds to form the network.

Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P lipids and membrane-mediated dimerization of the kinase domain.

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane.

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation.

Competition for shared downstream signaling molecules establishes indirect negative feedback between EGFR and EphA2.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown.

Stochasticity and positive feedback enable enzyme kinetics at the membrane to sense reaction size.

Here, we present detailed kinetic analyses of a panel of soluble lipid kinases and phosphatases, as well as Ras activating proteins, acting on their respective membrane surface substrates. The results reveal that the mean catalytic rate of such interfacial enzymes can exhibit a strong dependence on the size of the reaction system-in this case membrane area. Experimental measurements and kinetic modeling reveal how stochastic effects stemming from low molecular copy numbers of the enzymes alter reaction kinetics based on mechanistic characteristics of the enzyme, such as positive feedback.

Relating cellular signaling timescales to single-molecule kinetics: A first-passage time analysis of Ras activation by SOS.

Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor (GEF) that plays a central role in numerous cellular signaling pathways. Like many other signaling molecules, SOS is autoinhibited in the cytosol and activates only after recruitment to the membrane. The mean activation time of individual SOS molecules has recently been measured to be ∼60 s, which is unexpectedly long and seemingly contradictory with cellular signaling timescales, which have been measured to be as fast as several seconds.

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.

Height, but not binding epitope, affects the potency of synthetic TCR agonists.

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab' antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor.

Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.

Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions.

The Realign-Resect Arthrodesis Technique.

During traditional fusion procedures surgeons initially perform a joint resection and then the structures are realigned for correction of deformity. The procedure described herein by the author reverses this traditional surgical approach by first realigning the joint to correct deformity, then after achieving a corrected alignment, joint resection is performed in parallel without wedging. Realigning deformity as an initial step creates the conditions for an in-situ fusion wherein the deformity is corrected simultaneously with parallel bone resection.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex.