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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

Cell membranes exhibit multiple layers of complexity, ranging from their specific molecular content to their emergent mechanical properties and dynamic spatial organization. Both compositional and geometrical organizations of membrane components are known to play important roles in life processes, including signal transduction. Supported membranes, comprised of a bilayer assembly of phospholipids on the solid substrate, have been productively served as model systems to study wide range problems in cell biology. Because lateral mobility of membrane components is readily preserved, supported lipid membranes with signaling molecules can be utilized to effectively trigger various intercellular reactions. The spatial organization and mechanical deformation of supported membranes can also be manipulated by patterning underlying substrates with modern micro- and nano-fabrication techniques. This article focuses on various applications and methods to spatially patterned biomembranes by means of curvature modulations and spatial reorganizations, and utilizing them to interface with live cells. The integration of biological components into synthetic devices provides a unique approach to investigate molecular mechanisms in cell biology. [ABSTRACT FROM AUTHOR]

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. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient., Competing Interests: Competing interests statement:The authors declare no competing interest.

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. Individual LAT condensates induce a rapid and additive calcium response, which quickly attenuates upon condensate dissolution. No evidence of cooperativity between LAT condensates or oscillatory calcium response was detected. These results reveal stochastic LAT protein condensation events as a primary driver of calcium signal dynamics in T cells., One-Sentence Summary: Ca 2+ fluctuations in T cells reflect stochastic protein condensation events triggered by single molecular antigen-TCR binding.

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. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation., Competing Interests: Declaration of interests All authors declare they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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. The results reveal a bimodal Ras activation response, which is not a result of deterministic bistability but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts deterministic bistability and may be more resistant to pharmacological inhibition.

All organisms utilize S -adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S -adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt., Importance: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli , but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments., Competing Interests: The authors declare no conflict of interest.

Bovine hepacivirus (BoHV) is closely related to the hepatitis C virus (HCV) in humans and can cause both acute and chronic liver infections in cattle. BoHV was first identified in Ghana and Germany in 2015 and since then it has been detected and characterized in other countries around the world, but no strains have been sequenced from U.S. cattle. To date, BoHV has been classified into 2 genotypes (1 and 2), with genotype 1 being further divided into 11 subtypes (A-K). However, the true genetic diversity of BoHV is likely underestimated given limited surveillance and a lack of published genome sequences. Here, we sequenced 2 nearly complete BoHV genomes from serum samples collected in 2019 from beef cattle in Missouri. Sequence comparisons and phylogenetic analysis showed that isolate MARC/2019/60 had high sequence homology with genotype 1, subtype E isolates from China. In contrast, isolate MARC/2019/50 represented a novel BoHV subtype within genotype 2. Thus, we report the first genomic characterization of BoHV isolates from U.S. cattle, and the second complete BoHV2 genome worldwide. This work increases our knowledge of the global genetic diversity of BoHV and demonstrates the co-circulation of divergent BoHV strains in U.S. cattle., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Every year, perhaps as much as 800 million tons of hydrocarbons enters the environment; alkanes make up a large percentage of it. Most are transformed by organisms that utilize these molecules as sources of energy and carbon. Both aerobic and anaerobic alkane transformation chemistries exist, capitalizing on the presence of alkanes in both oxic and anoxic environments. Over the past 40 years, tremendous progress has been made in understanding the structure and mechanism of enzymes that catalyze the transformation of methane. By contrast, progress involving enzymes that transform liquid alkanes has been slower with the first structures of AlkB, the predominant aerobic alkane hydroxylase in the environment, appearing in 2023. Because of the fundamental importance of C-H bond activation chemistries, interest in understanding how biology activates and transforms alkanes is high.In this Account, we focus on steps we have taken to understand the mechanism and structure of alkane monooxygenase (AlkB), the metalloenzyme that dominates the transformation of liquid alkanes in the environment (not to be confused with another AlkB that is an α-ketogluturate-dependent enzyme involved in DNA repair). First, we briefly describe what is known about the prevalence of AlkB in the environment and its role in the carbon cycle. Then we review the key findings from our recent high-resolution cryoEM structure of AlkB and highlight important similarities and differences in the structures of members of class III diiron enzymes. Functional studies, which we summarize, from a number of single residue variants enable us to say a great deal about how the structure of AlkB facilitates its function. Next, we overview work from our laboratories using mechanistically diagnostic radical clock substrates to characterize the mechanism of AlkB and contextualize the results we have obtained on AlkB with results we have obtained on other alkane-oxidizing enzymes and explain these results in light of the enzyme's structure. Finally, we integrate recent work in our laboratories with information from prior studies of AlkB, and relevant model systems, to create a holistic picture of the enzyme. We end by pointing to critical questions that still need to be answered, questions about the electronic structure of the active site of the enzyme throughout the reaction cycle and about whether and to what extent the enzyme plays functional roles in biology beyond simply initiating the degradation of alkanes.

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. This model posits that binding of pMHC at the extracellular domain triggers a dynamic change in the transmembrane (TM) domain of the TCR subunits, which leads to signaling at the cytoplasmic side. We sought to test this hypothesis by creating a TM ligand for TCR. Previously we described a method to create a soluble peptide capable of inserting into membranes and binding to the TM domain of the receptor tyrosine kinase EphA2 (Alves et al., eLife, 2018). Here, we show that the approach is generalizable to complex membrane receptors, by designing a TM ligand for TCR. We observed that the designed peptide caused a reduction of Lck phosphorylation of TCR at the CD3ζ subunit in T cells. As a result, in the presence of this peptide inhibitor of TCR (PITCR), the proximal signaling cascade downstream of TCR activation was significantly dampened. Co-localization and co-immunoprecipitation in diisobutylene maleic acid (DIBMA) native nanodiscs confirmed that PITCR was able to bind to the TCR. AlphaFold-Multimer predicted that PITCR binds to the TM region of TCR, where it interacts with the two CD3ζ subunits. Our results additionally indicate that PITCR disrupts the allosteric changes in the compactness of the TM bundle that occur upon TCR activation, lending support to the allosteric TCR activation model. The TCR inhibition achieved by PITCR might be useful to treat inflammatory and autoimmune diseases and to prevent organ transplant rejection, as in these conditions aberrant activation of TCR contributes to disease., Competing Interests: YY, SM, JC, PB, KW, DD, JG, FB No competing interests declared, (© 2023, Ye et al.)

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.  The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system., (© 2023 Wiley-VCH GmbH.)

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 3 , or in solution by inositol hexakisphosphate (IP 6 ) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP 3 -bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP 3 -mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2., Competing Interests: LN, TE, AI, JG No competing interests declared, JK Co-founder of Nurix Therapeutics, (© 2023, Nocka et al.)

Bovine respiratory disease (BRD) is recognized as a complex multifactorial disease often resulting in significant economic losses for the stocker industry through reduced health and performance of feeder calves. Conventional approaches to manage BRD in stocker production systems can be challenged with a restricted view of the system, most importantly the structure, which drives the behavior of the system and fails to anticipate unintended consequences. The translation and implementation of systems thinking into veterinary medicine can offer an alternative method to problem-solving. Fundamental to the success of the systems thinker is the conceptualization of the Iceberg Diagram intended to identify root causes of complex problems such as BRD. Furthermore, veterinary and animal health professionals are well-positioned to serve as facilitators to establish creative tension, the positive energy necessary to identify high-leverage strategies. The interrelationships and interconnected behaviors of complex stocker systems warrant an understanding of various archetypes. Archetypes provide the systems thinker with a decision-making tool to explore tactics in a nonlinear fashion for the purpose of recognizing short- and long-term outcomes. Developing literacy in the discipline of systems thinking will further equip professionals with the skillset necessary to address the multitude of challenges ingrained in complex stocker cattle systems.

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. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a function for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells., (© 2022. The Author(s).)

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. Unlike typical miscibility transitions in which compact domains may coexist at equilibrium, the LAT condensates are dynamically arrested in extended states, kinetically trapped out of equilibrium. Modeling identifies the structural basis for this kinetic arrest as the formation of spindle arrangements, favored by limited multivalent binding interactions along the flexible, intrinsically disordered LAT protein. These results reveal how local factors controlling the kinetics of LAT condensation enable formation of different, stable condensates, which may ultimately coexist within the cell.

Experimental bovine respiratory syncytial virus (BRSV) infection can enhance Histophilus somni (Hs) disease in calves; we thus hypothesized that modified-live virus (MLV) vaccines containing BRSV may alter Hs carriage. Our objective was to determine the effects of an intranasal (IN) trivalent (infectious bovine rhinotracheitis virus [IBRV], parainfluenza-3 virus [PI3V], and BRSV) respiratory vaccine with parenteral (PT) bivalent bovine viral diarrhea virus (BVDV) type I + II vaccine, or a PT pentavalent (BVDV type I and II, IBRV, BRSV, and PI3V) respiratory vaccine, on health, growth, immunity, and nasal pathogen colonization in high-risk beef calves. Calves (n = 525) were received in five truckload blocks and stratified by body weight (213 ± 18.4 kg), sex, and presence of a pre-existing ear-tag. Pens were spatially arranged in sets of three within a block and randomly assigned to treatment with an empty pen between treatment groups consisting of: 1) no MLV respiratory vaccination (CON), 2) IN trivalent MLV respiratory vaccine with PT BVDV type I + II vaccine (INT), or 3) PT pentavalent, MLV respiratory vaccine (INJ). The pen was the experimental unit, with 15 pens/treatment and 11 to 12 calves/pen in this 70-d receiving study. Health, performance, and BRSV, Hs, Mycoplasma bovis (Mb), Mannheimia haemolytica (Mh), and Pasteurella multocida (Pm) level in nasal swabs via rtPCR was determined on days 0, 7, 14, and 28, and BRSV-specific serum neutralizing antibody titer, and serum IFN-γ concentration via ELISA, were evaluated on days 0, 14, 28, 42, 56, and 70. Morbidity (P = 0.83), mortality (P = 0.68) and average daily gain (P ≥ 0.82) did not differ. Serum antibodies against BRSV increased with time (P < 0.01). There was a treatment × time interaction (P < 0.01) for Hs detection; on days 14 and 28, INT (21.1% and 57.1%) were more frequently (P < 0.01) Hs positive than CON (3.6% and 25.3%) or INJ (3.4 % and 8.4%). Also, INT had reduced (P = 0.03) cycle time of Hs positive samples on day 28. No difference (P ≥ 0.17) was found for IFN-γ concentration and Mb, Mh, or Pm detection. The proportion of Mh positive culture from lung specimens differed (P < 0.01); INT had fewer (0.0%; 0 of 9) Mh positive lungs than INJ (45.5%; 6 of 13) or CON (74.0%; 14 of 19). Vaccination of high-risk calves with MLV did not clearly impact health or growth during the receiving period. However, INT was associated with an altered upper respiratory microbial community in cattle resulting in increased detection and level of Hs., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A cationic manganese porphyrin, Mn III -TDMImP, is an efficient, homogeneous, single-site water oxidation electrocatalyst at neutral pH. The measured turnover frequency for oxygen production is 32 s -1 . Mechanistic analyses indicate that Mn V (O)(OH 2 ), the protonated form of the corresponding trans -Mn V (O) 2 species, is generated from the Mn III (OH 2 ) 2 precursor in a 2-e - two-proton process and is responsible for O-O bond formation with a H 2 O molecule. Chloride ion is a competitive substrate with H 2 O for the Mn V (O)(OH 2 ) oxidant, forming hypochlorous acid with a rate constant that is 3 orders of magnitude larger than that of water oxidation. The data allow the construction of an experimental energy landscape for this water oxidation catalysis process.

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P 2 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 2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P 2 -containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P 2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P 2 and membrane-bound kinase., Competing Interests: SH, AL, BD, JG No competing interests declared, (© 2022, Hansen et al.)

Beef cattle veterinarians provide services to the increasingly complex beef industry system. Systems thinking offers pathways to better understand and communicate ranges of issues such as prevailing mental models, importance of match quality relative to clientele needs, and identification of leverage to better adapt and continually improve. Thinking in systems identifies and helps us to understand patterns or structures that are organized and interconnected that result in the outcomes observed and experienced in the practice of beef cattle veterinary medicine., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This chapter provides an introductory look into the practical application of the principals of systems thinking as a methodology to gain deeper understanding of the nature of bovine respiratory disease (BRD) in current North American beef production models. The "limits to success" archetype is used to explore the dynamic relationship between technological BRD mitigation improvements and the resultant adaptive changes made by the system. The chapter concludes, by using the tragedy of the common archetype, with an investigation into how the common shared resource of antimicrobials can be damaged and depleted over time., Competing Interests: Disclosure J.T. Groves has received honoraria to provide education on the topic of Systems Thinking to veterinarians. The other authors have nothing to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations., (Copyright © 2022. Published by Elsevier Inc.)

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. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an obligate intracellular pathogen that lives within the phagosome of macrophages. Here we demonstrate that the siderophore mycobactin J, produced by the closely related intracellular pathogen Mycobacterium paratuberculosis, is toxic to murine macrophage cells. Its median lethal dose, 10 μM, is lower than that of the iron chelators desferrioxamine B and TrenCAM, an enterobactin analog. To determine the source of this toxicity, we conducted microarray, ELISA, and metabolite profiling experiments. The primary response is hypoxia-like, which implies iron starvation as the underlying cause of the toxicity. This observation is consistent with our recent finding that mycobactin J is a stronger iron chelator than had been inferred from previous studies. Mycobactin J is known to partition into cell membranes and hydrophobic organelles indicating that enhanced membrane penetration is also a likely factor. Thus, mycobactin J is shown to be toxic, eliciting a hypoxia-like response under physiological conditions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Photodriven oxidations of alkanes in trifluoroacetic acid using commercial and synthesized Fe(III) sources as catalyst precursors and dioxygen (O 2 ) as the terminal oxidant are reported. The reactions produce alkyl esters and occur at ambient temperature in the presence of air, and catalytic turnover is observed for the oxidation of methane in a pure O 2 atmosphere. Under optimized conditions, approximately 17% conversion of methane to methyl trifluoroacetate at more than 50% selectivity is observed. It is demonstrated that methyl trifluoroacetate is stable under catalytic conditions, and thus overoxidized products are not formed through secondary oxidation of methyl trifluoroacetate.

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. For the competitive enzymatic cycles studied here, the final product-consisting of a specific lipid composition or Ras activity state-depends on the size of the reaction system. Furthermore, we demonstrate how these reaction size dependencies can be controlled by engineering feedback mechanisms into the enzymes., Competing Interests: The authors declare no competing interest.

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. Here, we rectify this discrepancy using a first-passage time analysis to reconstruct the effective signaling timescale of multiple SOS molecules from their single-molecule activation kinetics. Along with corresponding experimental measurements, this analysis reveals how the functional response time, comprised of many slowly activating molecules, can become substantially faster than the average molecular kinetics. This consequence stems from the enzymatic processivity of SOS in a highly out-of-equilibrium reaction cycle during receptor triggering. Ultimately, rare, early activation events dominate the macroscopic reaction dynamics., Competing Interests: The authors declare no competing interest.

A modular synthesis of mycobactin T and its N -acetyl analogue is reported in a route that facilitates permutation of the lipid tails. A key feature is the generation of N(α)-Cbz-N(ε)-benzyloxy-N(ε)-Boc-lysine ( A4 ) with methyl(trifluoromethyl)dioxirane in 59% yield. Selective hydroxamate N -acylation was achieved with acyl fluorides, enabling installation of lipids tails in the final step. O -Benzyl-dehydrocobactin T ( B4 ) was prepared by modifying a known five-step sequence with an overall yield of 49%. 2-Hydroxyphenyl-4-carboxyloxazoline ( C3 ) was prepared from 2-hydroxybenzoic acid and l-serine methyl ester in three steps with an overall yield of 55%. Ester coupling of A4 and B4 with EDCI afforded MbI-1 in 73% yield. Catalytic hydrogenation with Pd/BaSO 4 and 50 psi of H 2 simultaneously effected alkene reduction and debenzylation to afford MbI-2 in 96% yield. Fragment C3 was converted into acyl fluoride C4 , which coupled with MbI-2 to afford MbI-3 in 51% yield. Finally, Boc-removal with HCl/EtOAc and treatment of the resultant hydroxylamine with stearyl fluoride furnished mycobactin T in 65% yield. Overall, the yield is 4% over 14 steps. The gallium mycobactin T- N -acetyl derivative (GaMbT-NAc) structure was determined by 1 H NMR. The structure shows an octahedral Ga and two internal hydrogen bonds between peptidic N-Hs and two of the oxygen atoms coordinating Ga.

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., (© 2021. The Author(s).)

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. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation., Competing Interests: ZC, DO, KB, RZ, JG No competing interests declared, (© 2021, Chen et al.)

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway., Competing Interests: The authors declare no competing interest.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation., Competing Interests: The authors declare no competing interest.

Paramyxovirus membrane fusion requires an attachment protein that binds to a host cell receptor and a fusion protein that merges the viral and host membranes. For Nipah virus (NiV), the G attachment protein binds ephrinB2/B3 receptors and activates F-mediated fusion. To visualize dynamic events of these proteins at the membrane interface, we reconstituted NiV fusion activation by overlaying F- and G-expressing cells onto ephrinB2-functionalized supported lipid bilayers and used TIRF microscopy to follow F, G, and ephrinB2. We found that G and ephrinB2 form clusters and that oligomerization of ephrinB2 is necessary for F activation. Single-molecule tracking of F particles revealed accumulation of an immobilized intermediate upon activation. We found no evidence for stable F-G protein complexes before or after activation. These observations lead to a revised model for NiV fusion activation and provide a foundation for investigating other multicomponent viral fusion systems., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

T cells exhibit remarkable sensitivity and selectivity in detecting and responding to agonist peptides (p) bound to MHC molecules in a sea of self pMHC molecules. Despite much work, understanding of the underlying mechanisms of distinguishing such ligands remains incomplete. Here, we quantify T cell discriminatory capacity using channel capacity, a direct measure of the signaling network's ability to discriminate between antigen-presenting cells (APCs) displaying either self ligands or a mixture of self and agonist ligands. This metric shows how differences in information content between these two types of peptidomes are decoded by the topology and rates of kinetic proofreading signaling steps inside T cells. Using channel capacity, we constructed numerically substantiated hypotheses to explain the discriminatory role of a recently identified slow LAT Y132 phosphorylation step. Our results revealed that in addition to the number and kinetics of sequential signaling steps, a key determinant of discriminatory capability is spatial localization of a minimum number of these steps to the engaged TCR. Biochemical and imaging experiments support these findings. Our results also reveal the discriminatory role of early negative feedback and necessary amplification conferred by late positive feedback., Competing Interests: The authors declare no competing interest.

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is an oligomeric enzyme with crucial roles in neuronal signaling and cardiac function. Previously, we showed that activation of CaMKII triggers the exchange of subunits between holoenzymes, potentially increasing the spread of the active state (Stratton et al., 2014; Bhattacharyya et al., 2016). Using mass spectrometry, we show now that unphosphorylated and phosphorylated peptides derived from the CaMKII-α regulatory segment bind to the CaMKII-α hub and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of CaMKII-α expressed in mammalian cells shows that activation of CaMKII-α results in the destabilization of the holoenzyme. Our results suggest that release of the regulatory segment by activation and phosphorylation allows it to destabilize the hub, producing smaller assemblies that might reassemble to form new holoenzymes., Competing Interests: DK, MB, ZX, YL, SM, DM, EM, BQ, JG, EW No competing interests declared, JK Senior editor, eLife, (© 2020, Karandur et al.)

The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.

This article provides insights into the management of bovine respiratory disease in high-risk cattle populations. Biocontainment strategies, records, procurement, transport, arrival/receiving management, vaccination, and treatment protocols are discussed from practical and systems-thinking perspectives regarding their impact on health in high-risk cattle. Arrival management considerations, such as facilities, nutritional management, metaphylaxis, bovine viral diarrhea virus persistent infection testing, parasite control, and castration, are also addressed. Caretaker morale and job satisfaction are suggested as important factors to consider when managing high-risk cattle. The inter-relationships of variables within the system are explored as contributing causative factors to bovine respiratory disease in high-risk cattle., Competing Interests: Disclosure The author has nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRβ H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation., (Copyright © 2020. Published by Elsevier Inc.)

The many variants of human Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers., Competing Interests: MB, YL, SM, BQ, PN, HS, JG No competing interests declared, JK Senior editor, eLife, (© 2020, Bhattacharyya et al.)

Tracking inorganic photochemistry with high resolution poses considerable challenges. Here, sub-picosecond electronic and structural motions and MLCT/d-d intersystem crossing in a cationic iron-porphyrazine are probed using ultrafast transient absorption, stimulated Raman spectroscopy, and quantum calculations. By delineating photoinduced energy relaxation, strategies for extending the lifetime of MLCT state are discussed.