Violin SuperPlots: visualizing replicate heterogeneity in large data sets.

A recent article in MBoC (Goedhart, 2021) presented a web interface for the creation of ‘SuperPlots’. SuperPlots were introduced by Lord and colleagues last year (Lord et al., 2020) to visualise both cell-level variability within replicates as well as the experimental reproducibility between replicates in one single plot. Simple bar charts or boxplots of mean or median values from experimental replicates mask the contribution of underlying cell-to-cell variations in individual experiments, whereas pooling cell-level data across replicates overemphasises statistical differences. The SuperPlot put forward by Lord et al. uses a beeswarm plot to display the cell-level data color-coded according to the individual replicates, and overlays the mean (or median) and error bars (standard deviation or confidence intervals) of each replicate (Figure 1a). The new web interface (Goedhart, 2021) offers an online option for researchers to generate beeswarm SuperPlots, as well as RainCloud plots (Allen et al., 2021), using their own data. We welcome the transparency brought by SuperPlots and would like to introduce an augmentation, the Violin SuperPlot, to further simplify visual inspection of raw data containing large sample sizes

A handshake between platelets and neutrophils might fuel deep vein thrombosis

Deep vein thrombosis (DVT) describes the clogging of veins in extremities, leading to pain, swelling, and potentially life-threatening pulmonary embolism. Although several known factors can provoke DVT, it is not entirely clear how they contribute to thrombus formation. Long-term immobilization of hospitalized patients is thought to provoke DVT through reducing blood flow in the legs. While the associated ischemia activates endothelial cells, two mechanisms drive thrombosis: hypercoagulability caused by the dysregulation of clotting factors, or inflammation caused by the recruitment and activation of immune cells, especially neutrophils. Platelets play an important role in DVT, but how they help to attract neutrophils is incompletely understood. Two independent studies published in the Journal of Thrombosis and Hemostasis and in eLife now provide a fresh perspective on how this might work.</p

Platelet mechanosensing as key to understanding platelet function

Purpose of review: This review highlights how the perception of platelet function is evolving based on recent insights into platelet mechanobiology. Recent findings: The mechanosensitive ion channel Piezo1 mediates activation of free-flowing platelets under conditions of flow acceleration through mechanisms independent of adhesion receptors and classical activation pathways. Interference with the initiation of platelet migration or with the phenotypic switch of migrating platelets to a procoagulant state aggravates inflammatory bleeding. Mechanosensing of biochemical and biophysical microenvironmental cues during thrombus formation feed into platelet contractile force generation. Measurements of single platelet contraction and bulk clot retraction show promise to identify individuals at risk for hemorrhage. Summary: New findings unravel novel mechanotransduction pathways and effector functions in platelets, establishing mechanobiology as a pivotal component of platelet function. These insights highlight limitations of existing treatments and offer new potential therapeutic approaches and diagnostic avenues based on mechanobiological principles. Further extensive research is required to distinguish between core hemostatic and pathological mechanisms influenced by platelet mechanosensing.</p

PIP2-induced membrane binding of the Vinculin tail competes with its other binding partners.

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its Tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-Vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, where unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond long all-atom simulations. This allowed us to map the interaction of the Vinculin Tail with PIP2-enriched membranes at atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between Vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the Basic Collar and the Basic Ladder, as the main PIP2 interaction sites. By introducing a valency disaggregated binding network analysis, we were able to map the protein lipid interactions at unprecedented detail. In contrast to the Basic Collar where PIP2 is specifically recognized by an up to hexavalent binding pocket, the Basic Ladder forms a series of low valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining Vinculin in a closed, auto-inhibited conformation. These findings led us to propose a molecular mechanism for the coupling between Vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-Actin binding that disfavors simultaneous interaction with both ligands despite non-overlapping binding sites

A comprehensive program in academic health professions to enhance faculty recruitment, mentoring, productivity, and retention: the case of RCSI's StAR program

Top faculty talent recruitment, mentoring, productivity, and retention are paramount for organizational success among institutions of higher learning. Programs would do well to treat these various aspects of faculty management/development as inextricably linked to one another, rather than viewing recruitment or retention in a vacuum. The Strategic Academic Recruitment (StAR) program at the Royal College of Surgeons in Ireland (RCSI) University of Medicine and Health Sciences in Dublin was founded to bear these things, along with best practices in faculty development, in mind to enhance organizational effectiveness. This paper provides some background, description, and outcomes of the program thus far, revealing positive trends in scholarly productivity, teaching, program faculty commitment, and the development of future leaders for the institution, even while further evaluation and continued quality improvement for the StAR initiative are called for. It is hoped that the details provided here can be helpful for other academic organizations as they consider any of various initiatives aimed to attract high-quality labor capital, position those faculty for success, and enhance organizational effectiveness and reputation.</p

Nanofiber topographies enhance platelet-fibrinogen scaffold interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. In this study, we fabricate planar and nanofiber scaffolds from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine (PS) exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy (SEM). Different fibrinogen topographies equally support the spreading and granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. Our data show that the enhanced interactions of platelets with nanofibers results from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets towards robust adhesion formation and α-granule secretion while minimizing their pro-coagulant activity. Similar results on fibrinogen-coated PDMS substrates with micron-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.</p

Phosphorylated fibronectin enhances cell attachment and upregulates mechanical cell functions

A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers

RhoGAP6 interacts with COPI to regulate protein transport

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets

O-glycan determinants regulate VWF trafficking to Weibel-Palade bodies.

Von Willebrand factor (VWF) undergoes complex post-translational modification within endothelial cells (EC) prior to secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLG) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within EC. We demonstrate that alterations in OLG (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB following EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted following EC activation, but also affected its hemostatic efficacy. Notably, VWF secreted following WPB exocytosis consisted predominantly of low molecular weight multimers and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly impact VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T antigen expression) in patients with von Willebrand disease

Real-time 3D single-molecule localization using experimental point spread functions.

We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves minimal uncertainty in 3D on any microscope and is compatible with any PSF engineering approach. We used this method to image cellular structures and attained unprecedented image quality for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D super-resolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.</p