Membrane curvature catalyzes actin nucleation through nano-scale condensation of N-WASP-FBP17.

Actin remodeling is spatiotemporally regulated by surface topographical cues on the membrane for signaling across diverse biological processes. Yet, the mechanism dynamic membrane curvature prompts quick actin cytoskeletal changes in signaling remain elusive. Leveraging the precision of nanolithography to control membrane curvature, we reconstructed catalytic reactions from the detection of nano-scale curvature by sensing molecules to the initiation of actin polymerization, which is challenging to study quantitatively in living cells. We show that this process occurs via topographical signal-triggered condensation and activation of the actin nucleation-promoting factor (NPF), Neuronal Wiskott-Aldrich Syndrome protein (N-WASP), which is orchestrated by curvature-sensing BAR-domain protein FBP17. Such N-WASP activation is fine-tuned by optimizing FBP17 to N-WASP stoichiometry over different curvature radii, allowing a curvature-guided macromolecular assembly pattern for polymerizing actin network locally. Our findings shed light on the intricate relationship between changes in curvature and actin remodeling via spatiotemporal regulation of NPF/BAR complex condensation.

Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

Aviptadil: A multifaceted approach to mitigating hypoxemia in acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is a severe and potentially life-threatening lung condition that often leads to Intensive Care Unit (ICU) admissions. Treating ARDS in the ICU involves providing essential support for proper oxygenation and ventilation, often requiring mechanical ventilation using high positive end-expiratory pressure (PEEP) to recruit alveoli. Strategies like prone positioning and extracorporeal membrane oxygenation (ECMO) may be necessary for stubbornly low oxygen levels. Addressing the underlying cause, if known, and employing additional therapies to prevent complications are also integral parts of the management. Despite advances in critical care, ARDS remains a formidable challenge with considerable risks of mortality and complications. Early recognition, immediate intervention, and comprehensive ICU care are pivotal in enhancing outcomes for ARDS patients. Ongoing research and clinical trials continue to explore innovative treatments and strategies to improve the prognosis of individuals with ARDS. In this series, we share our experience regarding the safe utilization of Aviptadil for treating ARDS arising from causes other than COVID-19.

Liquid-like condensates mediate competition between actin branching and bundling.

Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations, there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch or become bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells.

Liquid-like condensates mediate competition between actin branching and bundling.

Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch versus becoming bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells.

A new view of axon growth and guidance grounded in the stochastic dynamics of actin networks.

The mechanism of axon growth and guidance is a core, unsolved problem in neuroscience and cell biology. For nearly three decades, our view of this process has largely been based on deterministic models of motility derived from studies of neurons cultured in vitro on rigid substrates. Here, we suggest a fundamentally different, inherently probabilistic model of axon growth, one that is grounded in the stochastic dynamics of actin networks. This perspective is motivated and supported by a synthesis of results from live imaging of a specific axon growing in its native tissue in vivo, together with single-molecule computational simulations of actin dynamics. In particular, we show how axon growth arises from a small spatial bias in the intrinsic fluctuations of the axonal actin cytoskeleton, one that produces net translocation of the axonal actin network by differentially modulating local probabilities of network expansion versus compaction. We discuss the relationship between this model and current views of axon growth and guidance mechanism and demonstrate how it offers explanations for various longstanding puzzles in this field. We further point out the implications of the probabilistic nature of actin dynamics for many other processes of cell morphology and motility.

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Liquid-like VASP condensates drive actin polymerization and dynamic bundling.

The organization of actin filaments into bundles is required for cellular processes such as motility, morphogenesis, and cell division. Filament bundling is controlled by a network of actin-binding proteins. Recently, several proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might liquid-like condensates contribute to filament bundling? Here, we show that the processive actin polymerase and bundling protein, VASP, forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the droplet's surface tension, as predicted by a continuum-scale computational model. However, as actin polymerizes and the ring grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming into a linear bundle. The resulting bundles contain long, parallel actin filaments that grow from their tips. Significantly, the fluid nature of the droplets is critical for bundling, as more solid droplets resist deformation, preventing filaments from rearranging to form bundles. Once the parallel arrangement of filaments is created within a VASP droplet, it propagates through the addition of new actin monomers to achieve a length that is many times greater than the initial droplet. This droplet-based mechanism of bundling may be relevant to the assembly of cellular architectures rich in parallel actin filaments, such as filopodia, stress fibers, and focal adhesions.

Nucleation causes an actin network to fragment into multiple high-density domains.

Actin networks rely on nucleation mechanisms to generate new filaments because spontaneous nucleation is kinetically disfavored. Branching nucleation of actin filaments by actin-related protein (Arp2/3), in particular, is critical for actin self-organization. In this study, we use the simulation platform for active matter MEDYAN to generate 2000 s long stochastic trajectories of actin networks, under varying Arp2/3 concentrations, in reaction volumes of biologically meaningful size (>20 µm3). We find that the dynamics of Arp2/3 increase the abundance of short filaments and increases network treadmilling rate. By analyzing the density fields of F-actin, we find that at low Arp2/3 concentrations, F-actin is organized into a single connected and contractile domain, while at elevated Arp2/3 levels (10 nM and above), such high-density actin domains fragment into smaller domains spanning a wide range of volumes. These fragmented domains are extremely dynamic, continuously merging and splitting, owing to the high treadmilling rate of the underlying actin network. Treating the domain dynamics as a drift-diffusion process, we find that the fragmented state is stochastically favored, and the network state slowly drifts toward the fragmented state with considerable diffusion (variability) in the number of domains. We suggest that tuning the Arp2/3 concentration enables cells to transition from a globally coherent cytoskeleton, whose response involves the entire cytoplasmic network, to a fragmented cytoskeleton, where domains can respond independently to locally varying signals.

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones.

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multimicron-scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live imaging of actin in wild-type axons in vivo. Using a graph theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high-Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks.

Simulated actin reorganization mediated by motor proteins.

Cortical actin networks are highly dynamic and play critical roles in shaping the mechanical properties of cells. The actin cytoskeleton undergoes significant reorganization in many different contexts, including during directed cell migration and over the course of the cell cycle, when cortical actin can transition between different configurations such as open patched meshworks, homogeneous distributions, and aligned bundles. Several types of myosin motor proteins, characterized by different kinetic parameters, have been involved in this reorganization of actin filaments. Given the limitations in studying the interactions of actin with myosin in vivo, we propose stochastic agent-based models and develop a set of data analysis measures to assess how myosin motor proteins mediate various actin organizations. In particular, we identify individual motor parameters, such as motor binding rate and step size, that generate actin networks with different levels of contractility and different patterns of myosin motor localization, which have previously been observed experimentally. In simulations where two motor populations with distinct kinetic parameters interact with the same actin network, we find that motors may act in a complementary way, by tuning the actin network organization, or in an antagonistic way, where one motor emerges as dominant. This modeling and data analysis framework also uncovers parameter regimes where spatial segregation between motor populations is achieved. By allowing for changes in kinetic rates during the actin-myosin dynamic simulations, our work suggests that certain actin-myosin organizations may require additional regulation beyond mediation by motor proteins in order to reconfigure the cytoskeleton network on experimentally-observed timescales.

Optimizing Huddle Engagement Through Leadership and Problem Solving Within Primary Care: Results from a Cluster-Randomized Trial.

BACKGROUND: Leaders play a crucial role in implementing and sustaining changes in clinical practice, yet there is limited evidence on the strategies to engage them in team problem solving and communication. OBJECTIVE: Examine the impact of an intervention focused on facilitating leadership during daily huddles on optimizing team-based care and improving outcomes. DESIGN: Cluster-randomized trial using intention-to-treat analysis to measure the effects of the intervention (n = 13 teams) compared with routine practice (n = 16 teams). PARTICIPANTS: Twenty-nine primary care clinics affiliated with a large integrated health system in the upper Midwest; representing differing practice types and geographic settings. INTERVENTION: Full-day leadership training retreat for team leaders to facilitate of care team huddles. Biweekly coaching calls and two site visits with an assigned coach. MAIN MEASURES: Primary outcomes of team development and function were collected, pre- and post-intervention using surveys. Patient satisfaction and quality outcomes were compared pre- and post-intervention as secondary outcomes. Leadership engagement and adherence to the intervention were also assessed. KEY RESULTS: A total of 279 pre-intervention and 272 post-intervention surveys were completed. We found no impact on team development (- 0.98, 95% CI (- 3.18, 1.22)), improved team credibility (0.18, 95% CI (0.00, 0.35)), but worse psychological safety (- 0.19, 95% CI (- 0.38, 0.00)). No differences were observed in patient satisfaction; however, results were mixed among quality outcomes. Post hoc analysis within the intervention group showed higher adherence to the intervention was associated with improvement in team coordination (0.47, 95% CI (0.18, 0.76)), credibility (0.28, 95% CI (0.02, 0.53)), team learning (0.42, 95% CI (0.10, 0.74)), and knowledge creation (0.74, 95% CI (0.35, 1.13)) compared to teams that were less engaged. CONCLUSIONS: Results of this evaluation showed that leadership training and facilitation were not associated with better team functioning. Additional components to the intervention tested may be necessary to enhance team functioning. TRIAL REGISTRATION: Clinicaltrials.gov Identifier NCT03062670. Registration Date: February 23, 2017. URL: https://clinicaltrials.gov/ct2/show/NCT03062670.

Remarkable structural transformations of actin bundles are driven by their initial polarity, motor activity, crosslinking, and filament treadmilling.

Bundled actin structures play a key role in maintaining cellular shape, in aiding force transmission to and from extracellular substrates, and in affecting cellular motility. Recent studies have also brought to light new details on stress generation, force transmission and contractility of actin bundles. In this work, we are primarily interested in the question of what determines the stability of actin bundles and what network geometries do unstable bundles eventually transition to. To address this problem, we used the MEDYAN mechano-chemical force field, modeling several micron-long actin bundles in 3D, while accounting for a comprehensive set of chemical, mechanical and transport processes. We developed a hierarchical clustering algorithm for classification of the different long time scale morphologies in our study. Our main finding is that initially unipolar bundles are significantly more stable compared with an apolar initial configuration. Filaments within the latter bundles slide easily with respect to each other due to myosin activity, producing a loose network that can be subsequently severely distorted. At high myosin concentrations, a morphological transition to aster-like geometries was observed. We also investigated how actin treadmilling rates influence bundle dynamics, and found that enhanced treadmilling leads to network fragmentation and disintegration, while this process is opposed by myosin and crosslinking activities. Interestingly, treadmilling bundles with an initial apolar geometry eventually evolve to a whole gamut of network morphologies based on relative positions of filament ends, such as sarcomere-like organization. We found that apolar bundles show a remarkable sensitivity to environmental conditions, which may be important in enabling rapid cytoskeletal structural reorganization and adaptation in response to intracellular and extracellular cues.

Optimizing huddle engagement through leadership and problem-solving within primary care: A study protocol for a cluster randomized trial.

BACKGROUND: Team-based care has been identified as a key component in transforming primary care. An important factor in implementing team-based care is the requirement for teams to have daily huddles. During huddles, the care team, comprising physicians, nurses, and administrative staff, come together to discuss their daily schedules, track problems, and develop countermeasures to fix these problems. However, the impact of these huddles on staff burnout over time and patient outcomes are not clear. Further, there are challenges to implementing huddles in fast-paced primary care clinics. We will test whether the impact of a behavioral intervention of leadership training and problem-solving during the daily huddling process will result in higher consistent huddling in the intervention arm and result in higher team morale, reduced burnout, and improved patient outcomes. METHODS/DESIGN: We will conduct a care-team-level cluster randomized trial within primary care practices in two Midwestern states. The intervention will comprise a 1-day training retreat for leaders of primary care teams, biweekly sessions between huddle optimization coaches and members of the primary care teams, as well as coaching site visits at 30 and 100 days post intervention. This behavioral intervention will be compared to standard care, in which care teams have huddles without any support or training. Surveys of primary care team members will be administered at baseline (prior to training), 100 days (for the intervention arm only), and 180 days to assess team dynamics. The primary outcome of this trial will be team morale. Secondary outcomes will assess the impact of this intervention on clinician burnout, patient satisfaction, and quality of care. DISCUSSION: This trial will provide evidence on the impact of a behavioral intervention to implement huddles as a key component of team-based care models. Knowledge gained from this trial will be critical to broader deployment and successful implementation of team-based care models. TRIAL REGISTRATION: Clinicaltrials.gov , NCT03062670 . Registered on 23 February 2017.

Protein Dynamics and Contact Topology Reveal Protein-DNA Binding Orientation.

Structure-encoded conformational dynamics are crucial for biomolecular functions. However, there is insufficient evidence to support the notion that dynamics play a role in guiding protein-nucleic acid interactions. Here, we show that protein-DNA docking orientation is a function of protein intrinsic dynamics, but the binding site itself does not display unique patterns in the examined spectrum of motions. This revelation is made possible by a novel technique that locates "dynamics interfaces" in proteins across which protein parts are anticorrelated in their slowest dynamics. A striking statistic is that such interfaces intersect the DNA in 97% of the 104 examined cases. These findings were then used to screen decoys generated by rigid-body docking of DNA molecules onto DNA-binding proteins. Using our method, the chance to discern near-native poses from non-native decoys increased by 2.5- and 1.6-fold, as compared to a random guess and methods based on surface complementarity, respectively. Hence, dynamically allowed protein-DNA docking orientations can work as new filters to cull and rerank docking poses and therefore enhance the predictability of DNA-binding sites that themselves do not have distinct dynamics features. Computer software implementing the method can be accessed via http://dyn.life.nthu.edu.tw/IDD/DNA.htm .

Role of in-hospital care quality in reducing anxiety and readmissions of kidney transplant recipients.

BACKGROUND: A total of 17,000 patients receive kidney transplants each year in the United States. The 30-day readmission rate for kidney transplant recipients is over 30%. Our research focuses on the relationship between the quality of care delivered during the patient's hospital stay for a kidney transplant, and the patient health outcomes and readmissions related to the transplant. METHODS: We interviewed 20 kidney transplant recipients at a major transplant center in the United States. Findings from these interviews were used to inform the data collection using structured surveys, which were administered to an additional 77 kidney transplant recipients. We used ordinary least squares regression to predict the effects of two dimensions of in-hospital care quality-information consistency and empathetic care delivery-on level of patient anxiety 1 week following discharge. Further, we estimated a logistic regression to predict the effect of anxiety, combined with the two dimensions of in-hospital care quality, on occurrence of 30-day readmissions. RESULTS: Patient anxiety levels 1 wk after discharge are significantly associated with information consistency and empathetic delivery of care. Patient anxiety 1 wk after discharge is associated with occurrence of 30-d readmissions. The logistic regression model indicates that the risk of getting readmitted is 110% higher for a one unit increase in patient anxiety level 1 wk after discharge. Finally, patient anxiety fully mediates the effects of consistency of information and empathetic care delivery on occurrence of 30-d readmissions (50.96% of the effect is mediated). CONCLUSIONS: Our study suggests two ways of preventing readmissions through reduction of postdischarge anxiety: (1) standardizing in-hospital care, so that information received by patients is consistent, and (2) by training caregivers to be more empathetic toward patients during the delivery of this information.

Molecular binding sites are located near the interface of intrinsic dynamics domains (IDDs).

We provide evidence supporting that protein-protein and protein-ligand docking poses are functions of protein shape and intrinsic dynamics. Over sets of 68 protein-protein complexes and 240 nonhomologous enzymes, we recognize common predispositions for binding sites to have minimal vibrations and angular momenta, while two interacting proteins orient so as to maximize the angle between their rotation/bending axes (>65°). The findings are then used to define quantitative criteria to filter out docking decoys less likely to be the near-native poses; hence, the chances to find near-native hits can be doubled. With the novel approach to partition a protein into "domains" of robust but disparate intrinsic dynamics, 90% of catalytic residues in enzymes can be found within the first 50% of the residues closest to the interface of these dynamics domains. The results suggest an anisotropic rather than isotropic distribution of catalytic residues near the mass centers of enzymes.