Deciphering the role of curvature-sensitive BAR domain proteins for cell migration

Cell motility has a critical role in a range of biological processes including development, immunity and disease. Navigation through complex and ever-changing environments often relies on the activity of actin-rich protrusions at the leading edge, also referred to as lamellipodia. Lamellipodia are known to exhibit areas of continuously rearranging membrane curvature, and their dynamics determines motion persistence. One group of proteins interesting in the context of membrane curvature are BAR domain proteins. However, whether and how these curvature-sensitive proteins contribute to leading edge dynamics and function, remains poorly understood. Here, we use neutrophils as a vertebrate model system of a highly migratory cell type. By combining RNAseq with a localization screen we identify two BAR proteins that are relevant for cell surface organization during migration: SH3BP1 and Snx33. First, using fluorescent imaging and Atomic Force Microscopy, we show that SH3BP1 responds to changes in membrane mechanics and, vice-versa, modulates membrane tension. Using microfluidics, we further demonstrate that SH3BP1 is important for cell navigation through complex environments. Namely, its knockout displays increased cell speed and decision making during directed cell migration. Next, we used the above techniques complemented with machine learning-based segmentation for time-resolved TIRF microscopy to understand the role of Snx33. We show that motion persistence and directionality, in both freely moving and environmentally constrained cells, depends on Snx33 activity. Specifically, Snx33 has an inhibitory effect on the lamellipodia dynamics by regulating WAVE2-driven actin polymerization. Our work exposes a novel mechanism by which cells steer protrusions upon encountering obstacles that facilitates efficient migration. In summary, we discovered novel functions of the curvature-sensitive proteins SH3BP1 and Snx33 in regulating cell surface mechanics and efficiency of directed cell migration

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments

Load adaptation by endocytic actin networks

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feed back on assembly. By manipulating membrane tension and Arp2/3 complex activity, we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments

Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments

Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles

Abstract To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells’ capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment

Performance of language-coordinated collective systems: A study of wine recognition and description

Most of our perceptions of and engagements with the world are shaped by our immersion in socialinteractions, cultural traditions, tools and linguistic categories. In this study we experimentallyinvestigate the impact of two types of language-based coordination on the recognition anddescription of complex sensory stimuli: that of red wine. Participants were asked to taste,remember and successively recognize samples of wines within a larger set in a two-by-twoexperimental design: 1) either individually or in pairs, and 2) with or without the support of asommelier card – a cultural linguistic tool designed for wine description. Both effectiveness ofrecognition and the kinds of errors in the four conditions were analyzed. While our experimentalmanipulations did not impact recognition accuracy, bias-variance decomposition of error revealsnon-trivial differences in how participants solved the task. Pairs generally displayed reduced biasand increased variance compared to individuals, however the variance dropped significantly whenthey used the sommelier card. The effect of card reducing the variance was observed only inpairs, individuals did not seem to benefit from the cultural linguistic tool. Subsequent analysis ofdescriptions generated with the aid of card by individuals and pairs showed that they were moreconsistent and discriminative in the case of pairs. The findings are discussed in terms of globalproperties and dynamics of collective systems when constrained by different types of culturalpractices

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)