Membrane localization accelerates association under conditions relevant to cellular signaling.

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes. The measured rate constants show that for a 10-µm-radius spherical cell, association is 22- to 33-fold faster at the membrane than in the cytoplasm. The kinetic advantage depends on cell size and is essentially negligible for typical ~1 µm prokaryotic cells. The rate enhancement is attributable to a combination of higher encounter rates in two dimensions and a higher reaction probability per encounter.

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. 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.

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. 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.

Stochastic geometry sensing and polarization in a lipid kinase-phosphatase competitive reaction.

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.

Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions.

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.

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

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.

Proceedings of the fifth international RASopathies symposium: When development and cancer intersect.

This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer. The RAS pathway is a well-known oncogenic pathway that is commonly found to be activated in somatic malignancies. As in somatic cancers, the RASopathies can be caused by various pathogenetic mechanisms that ultimately impact or alter the normal function and regulation of the MAPK pathway. As such, the RASopathies represent an excellent model of study to explore the intersection of the effects of dysregulation and its consequence in both development and oncogenesis.

E-cadherin junction formation involves an active kinetic nucleation process.

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

Dynamic Scaling Analysis of Molecular Motion within the LAT:Grb2:SOS Protein Network on Membranes.

Biochemical signaling pathways often involve proteins with multiple, modular interaction domains. Signaling activates binding sites, such as by tyrosine phosphorylation, which enables protein recruitment and growth of networked protein assemblies. Although widely observed, the physical properties of the assemblies, as well as the mechanisms by which they function, remain largely unknown. Here we examine molecular mobility within LAT:Grb2:SOS assemblies on supported membranes by single-molecule tracking. Trajectory analysis reveals a discrete temporal transition to subdiffusive motion below a characteristic timescale, indicating that the LAT:Grb2:SOS assembly has the dynamical structure of a loosely entangled polymer. Such dynamical analysis is also applicable in living cells, where it offers another dimension on the characteristics of cellular signaling assemblies.

Allosteric Modulation of Grb2 Recruitment to the Intrinsically Disordered Scaffold Protein, LAT, by Remote Site Phosphorylation.

Tyrosine phosphorylation of membrane receptors and scaffold proteins followed by recruitment of SH2 domain-containing adaptor proteins constitutes a central mechanism of intracellular signal transduction. During early T-cell receptor (TCR) activation, phosphorylation of linker for activation of T cells (LAT) leading to recruitment of adaptor proteins, including Grb2, is one prototypical example. LAT contains multiple modifiable sites, and this multivalency may provide additional layers of regulation, although this is not well understood. Here, we quantitatively analyze the effects of multivalent phosphorylation of LAT by reconstituting the initial reactions of the TCR signaling pathway on supported membranes. Results from a series of LAT constructs with combinatorial mutations of tyrosine residues reveal a previously unidentified allosteric mechanism in which the binding affinity of LAT:Grb2 depends on the phosphorylation at remote tyrosine sites. Additionally, we find that LAT:Grb2 binding affinity is altered by membrane localization. This allostery mainly regulates the kinetic on-rate, not off-rate, of LAT:Grb2 interactions. LAT is an intrinsically disordered protein, and these data suggest that phosphorylation changes the overall ensemble of configurations to modulate the accessibility of other phosphorylated sites to Grb2. Using Grb2 as a phosphorylation reporter, we further monitored LAT phosphorylation by TCR ζ chain-recruited ZAP-70, which suggests a weakly processive catalysis on membranes. Taken together, these results suggest that signal transmission through LAT is strongly gated and requires multiple phosphorylation events before efficient signal transmission is achieved.

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

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 × 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.

Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts by Fluorescence Correlation Spectroscopy.

The cytoplasm is densely packed with macromolecules and organelles, displaying viscoelastic properties at various scales. How biochemical reactions function efficiently enough in a seemingly jammed environment remains elusive. Cell-free Xenopus laevis extracts represent a powerful system for investigating the biochemistry and biophysics of living systems. Here we present a protocol for characterizing macromolecular diffusion in self-organizing cytoplasmic extracts using fluorescence correlation spectroscopy (FCS), which measures the motions on a distance scale of ~200 nm. The method can also be used to characterize diffusion in the cytoplasm as it progresses through different phases of the cell cycle.

Robust trigger wave speed in Xenopus cytoplasmic extracts.

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we found that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derived a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

Cytoplasmic organization promotes protein diffusion in Xenopus extracts.

The cytoplasm is highly organized. However, the extent to which this organization influences the dynamics of cytoplasmic proteins is not well understood. Here, we use Xenopus laevis egg extracts as a model system to study diffusion dynamics in organized versus disorganized cytoplasm. Such extracts are initially homogenized and disorganized, and self-organize into cell-like units over the course of tens of minutes. Using fluorescence correlation spectroscopy, we observe that as the cytoplasm organizes, protein diffusion speeds up by about a factor of two over a length scale of a few hundred nanometers, eventually approaching the diffusion time measured in organelle-depleted cytosol. Even though the ordered cytoplasm contained organelles and cytoskeletal elements that might interfere with diffusion, the convergence of protein diffusion in the cytoplasm toward that in organelle-depleted cytosol suggests that subcellular organization maximizes protein diffusivity. The effect of organization on diffusion varies with molecular size, with the effects being largest for protein-sized molecules, and with the time scale of the measurement. These results show that cytoplasmic organization promotes the efficient diffusion of protein molecules in a densely packed environment.

Steric zipper formed by hydrophobic peptide fragment of Syrian hamster prion protein.

Steric zippers, where the residues of two neighboring ß-sheet layers are tightly interdigitated, have been proposed as fundamental structural units of amyloid fibrils by Eisenberg and co-workers. The steric zipper formed by polypeptides containing the palindromic sequence AGAAAAGA has a distinctive feature that the distance between two interdigitated ß-sheet layers is comparable to the interstrand distance of the individual ß-sheet. This structural motif is of great interest in the study of prion disease because the AGAAAAGA sequence is highly conserved in prion proteins of different species. In this work, the amyloid fibrils formed by the polypeptides of PrP(113-127), viz. Ac-AGAAAAGAVVGGLGG-NH(2), are taken as the model compound to investigate the biophysical principles governing the steric zipper formation. The target fibrils adopt the structural motif of class 7 steric zipper, which is formed by stacking of antiparallel ß-sheet layers with residue 117 + k forming backbone hydrogen bonds to residue 120 - k. Implication of our results in the infectivity of scrapie prion is briefly discussed.

Improving Student Confidence With Electronic Health Record Order Entry.

INTRODUCTION: Despite near-universal utilization of electronic health records (EHRs) by physicians in practice, medical students in most ambulatory settings gain limited experience with placing EHR orders. In this study, an individual preceptor site investigated the usefulness of a targeted curriculum in improving students' EHR confidence and clinical reasoning skills. METHODS: Family medicine clerkship students assigned to one community health center were invited to participate in this prospective, survey-based study. In their first week, students observed a preceptor performing EHR tasks. For the remainder of the 4-week clerkship, students utilized decision support tools, assigned a working diagnosis, entered unsigned orders in the EHR, proposed an assessment, and discussed a plan with a preceptor. Students completed weekly questionnaires to self-report confidence across several EHR domains while preceptors synchronously evaluated students' accuracy with entering orders correctly. RESULTS: From February 2017 to March 2020, all 49 eligible students completed the study. One hundred percent of students reported that placing EHR orders was beneficial to their medical education. The difference over time in learner confidence with placing EHR orders was statistically significant across every domain (eg, writing prescriptions, ordering labs and imaging). Preceptors' evaluations of students' accuracy with placing orders also showed significant improvement between each week. CONCLUSION: Clerkship-wide EHR training may be limited by multiple sites with multiple EHR products. This pilot study suggests that committed faculty at an individual preceptor site can offer a targeted curriculum to help students develop EHR confidence. We propose other preceptors similarly offer students this opportunity to maximize clerkship education.

Curriculum and evaluation results of a third-year medical student longitudinal pathway on underserved care.

BACKGROUND: There is a need to train compassionate and competent physicians to care for the growing underserved population in this country. DESCRIPTION: The authors developed the third-year Longitudinal Ambulatory Care Experience (LACE) Underserved Care pathway at Baylor College of Medicine in 2003 to help interested students prepare to be clinicians who care for the underserved. The pathway curriculum included seminar/journal clubs on relevant underserved care topics, clinical time with an underserved care preceptor, visits to community organizations, an assignment to help an uninsured patient obtain health care funding, and a group project. The authors report on the student evaluations of the first 4 years of the pathway, 2003 to 2007. The Institutional Review Board of Baylor College of Medicine granted this educational study exempt status. EVALUATION: Students highly rated each pathway component in enhancing their knowledge, skills and attitudes. For 2005 to 2007, students rated most knowledge, skills, and attitudes items more highly at the conclusion of the pathway compared to the beginning (p <.05). CONCLUSIONS: The pathway has been successful in enhancing knowledge, skills, and attitudes in underserved care for its participants. Further study is needed to evaluate long-term outcomes of participants in this pathway, including practice setting, knowledge, skills, attitudes, quality of care, and ability to help patients navigate through the health care system and overcome barriers.

Family Medicine Clerkship Directors' Handling of Student Mistreatment: Results From a CERA Survey.

BACKGROUND AND OBJECTIVES: Little is known about how family medicine clerkship directors (FMCDs) handle reports of student mistreatment. We investigated FMCDs' involvement in handling and resolving these reports. METHODS: We collected data as part of the 2019 CERA survey of FMCDs. FMCDs provided responses on how they handled student mistreatment reports and their comfort level in resolving these reports. RESULTS: Ninety-nine out of 142 FMCDs (69.7%) responded to the survey. Regarding mistreatment reports, 24.2% of FMCDs had received at least one report of student mistreatment about full-time faculty in the past 3 years, compared to 64.6% of FMCDs receiving at least one report about community preceptors (P<.001). Regarding who determined the response to the mistreatment, 13.1% of FMCDs were the highest level of leadership responsible for stopping use of a full-time faculty member for mistreatment concerns, while 42.4% of FMCDs were the highest level of leadership responsible for stopping use of a community preceptor. Regarding their comfort level in resolving mistreatment reports, 59.1% of FMCDs were either somewhat or very comfortable resolving a mistreatment report about a community preceptor, while only 48.9% reported those comfort levels for full-time faculty. FMCDs who had previously stopped using full-time faculty and/or community preceptors due to mistreatment reports were less likely to feel comfortable with resolving reports about full-time faculty compared to those who had no such experience (P=.03). CONCLUSIONS: FMCDs more frequently receive mistreatment reports about community preceptors than full-time faculty and are more likely to be the highest decision maker to stop using a community preceptor for mistreatment concerns. Further study is needed to elucidate factors that affect FMCDs' comfort in handling student mistreatment reports.

How to Teach Laboratory Stewardship in the Undergraduate Medical Curriculum?

OBJECTIVES: Promotion of high-quality care at a lower cost requires educational initiatives across the continuum of medical education. A needs assessment was performed to inform the design of an educational tool with the goal of teaching laboratory stewardship to medical students. METHODS: The needs assessment consisted of semistructured interviews with core clerkship directors and residency program directors at our institution, a national survey to the Undergraduate Medical Educators Section (UMEDS) of the Association of Pathology Chairs, and a review of existing online resources that teach high-value care. RESULTS: Two major themes emerged regarding opportunities to enhance laboratory stewardship education: appropriate ordering (knowledge of test indications, pretest/posttest probability, appropriateness criteria, recognition of unnecessary testing) and correct interpretation (understanding test specifications, factors that affect the test result, recognizing inaccurate results). CONCLUSIONS: The online educational tool will focus on the curricular needs identified, using a multidisciplinary approach for development and implementation.

Predictors of student use of an electronic record.

BACKGROUND: Little is known on ambulatory clerkship students' use of an electronic medical record (EMR). We investigated students' use of recommended EMR tasks across different types of sites and studied the predictors of these recommended tasks. METHODS: Students documented how often they performed recommended EMR tasks and suggested improvements to enhance EMR use. We compared student performance of recommended tasks across different types of sites using χ2 tests and the Fisher's exact test. We performed regression analyses to investigate factors predicting students' performance of EMR tasks. Two faculty members read all of the suggested improvements and agreed on themes. RESULTS: From January 2014 to June 2015, 263 of 295 Family and Community Medicine Clerkship (FCMC) students (89.2%) were at sites that used an EMR. Of the 263 students, 68.4% typed their own note into the EMR, but only 31.2% entered orders and 27.8% entered prescriptions for their teacher to sign. Students' rating of the orientation to the EMR predicted their use of all EMR tasks. The number of years that the teaching site used an EMR predicted the students' use of some tasks. Suggested improvements included a better orientation to the EMR, more use of the EMR, and access to a computer and the EMR. Little is known on ambulatory clerkship students' use of an electronic medical record DISCUSSION: Many students did not perform recommended EMR tasks. To help more students learn EMR tasks, clinical teachers can offer students a detailed orientation to their EMR, provide them with access to a computer and the EMR, and give them the opportunity to perform recommended EMR tasks, including typing their own note and entering orders and prescriptions.