Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome.

BACKGROUND: Scott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized. OBJECTIVES: Genetic and functional studies were undertaken in a consanguineous family with a history of excessive bleeding of unknown cause. METHODS: A targeted gene panel of known bleeding and platelet genes was used to identify possible genetic variants. Platelet phenotyping, flow adhesion, flow cytometry, whole blood and platelet-rich plasma thrombin generation, and specialized extracellular vesicle measurements were performed. RESULTS: We detected a novel homozygous frameshift variant, c.1943del (p.Arg648Hisfs∗23), in ANO6 encoding Anoctamin 6, in a patient with a bleeding history but interestingly with normal ANO6 expression. Phenotyping of the patient's platelets confirmed the absence of PS expression and procoagulant activity but also revealed other defects including reduced platelet δ granules, reduced ristocetin-mediated aggregation and secretion, and reduced P-selectin expression after stimulation. PS was absent on spread platelets, and thrombi formed over collagen at 1500/s. Reduced thrombin generation was observed in platelet-rich plasma and confirmed in whole blood using a new thrombin generation assay. CONCLUSION: We present a comprehensive report of a patient with Scott syndrome with a novel frameshift variant in AN06, which is associated with no platelet PS exposure and markedly reduced thrombin generation in whole blood, explaining the significant bleeding phenotype observed.

Imaging nanoscale nuclear structures with expansion microscopy.

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, limiting the numbers of three-dimensional (3D) nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy (ExM) solves many of these limitations, but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here, we demonstrate the conditions for isotropic expansion of the nucleus at a resolution equal to or better than 120-130 nm (pre-expansion). Using the DNA damage response proteins BRCA1, 53BP1 (also known as TP53BP1) and RAD51 as exemplars, we quantitatively describe the 3D nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin-regulated events and show the simultaneous assessment of four elements. This study thus demonstrates how ExM can contribute to the investigation of nanoscale nuclear processes.

Simple methods for quantifying super-resolved cortical actin.

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or 'corrals', in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

Analysis of preplatelets and their barbell platelet derivatives by imaging flow cytometry.

Circulating large "preplatelets" undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry and super-resolution microscopy. Immature platelets, preplatelets, and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia or undergoing chemotherapy. Preplatelets and barbells were 1.9% ± 0.18%/1.7% ± 0.48% (n = 6) and 3.3% ± 1.6%/0.5% ± 0.27% (n = 12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of major histocompatibility complex class I with high thiazole orange and Mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence on the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with immune thrombocytopenia had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent after chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.

Post-translational polymodification of ß1-tubulin regulates motor protein localisation in platelet production and function.

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which ß1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Super-Resolution Imaging Approaches for Quantifying F-Actin in Immune Cells.

Immune cells comprise a diverse set of cells that undergo a complex array of biological processes that must be tightly regulated. A key component of cellular machinery that achieves this is the cytoskeleton. Therefore, imaging and quantitatively describing the architecture and dynamics of the cytoskeleton is an important research goal. Optical microscopy is well suited to this task. Here, we review the latest in the state-of-the-art methodology for labeling the cytoskeleton, fluorescence microscopy hardware suitable for such imaging and quantitative statistical analysis software applicable to describing cytoskeletal structures. We also highlight ongoing challenges and areas for future development.

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function.

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

An adaptable analysis workflow for characterization of platelet spreading and morphology.

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimizing user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open-source software KNIME and ilastik.

A semi-automated finite difference mesh creation method for use with immersed boundary software IB2d and IBAMR.

Numerous fluid-structure interaction problems in biology have been investigated using the immersed boundary method. The advantage of this method is that complex geometries, e.g., internal or external morphology, can easily be handled without the need to generate matching grids for both the fluid and the structure. Consequently, the difficulty of modeling the structure lies often in discretizing the boundary of the complex geometry (morphology). Both commercial and open source mesh generators for finite element methods have long been established; however, the traditional immersed boundary method is based on a finite difference discretization of the structure. Here we present a software library for obtaining finite difference discretizations of boundaries for direct use in the 2D immersed boundary method. This library provides tools for extracting such boundaries as discrete mesh points from digital images. We give several examples of how the method can be applied that include passing flow through the veins of insect wings, within lymphatic capillaries, and around starfish using open-source immersed boundary software.

An introduction to the methodology of expansion microscopy.

Expansion microscopy is a novel, fluorescence imaging technique, which allows three-dimensional nanoscale imaging of specimens on a conventional fluorescence microscope. This is achieved through an innovative sample treatment, which culminates in approximately 4.5-fold expansion of specimens in each dimension. This allows 70 nm lateral and 200 nm axial resolution. To further develop application of the technique, there has been considerable focus on improving the methodology by i) extending the efficacy of labelling, ii) enabling multi-colour labelling of different biomolecules simultaneously, iii) further improving resolving power through alterations to sample preparation and iv) by combination of expansion microscopy with other well-established super resolution techniques. This review will highlight some of these recent advances and suggest ways that the technique could be developed further in the future.

Surface Chemistry-Dependent Evolution of the Nanomaterial Corona on TiO2 Nanomaterials Following Uptake and Sub-Cellular Localization.

Nanomaterial (NM) surface chemistry has an established and significant effect on interactions at the nano-bio interface, with important toxicological consequences for manufactured NMs, as well as potent effects on the pharmacokinetics and efficacy of nano-therapies. In this work, the effects of different surface modifications (PVP, Dispex AA4040, and Pluronic F127) on the uptake, cellular distribution, and degradation of titanium dioxide NMs (TiO2 NMs, ~10 nm core size) are assessed and correlated with the localization of fluorescently-labeled serum proteins forming their coronas. Imaging approaches with an increasing spatial resolution, including automated high throughput live cell imaging, correlative confocal fluorescence and reflectance microscopy, and dSTORM super-resolution microscopy, are used to explore the cellular fate of these NMs and their associated serum proteins. Uncoated TiO2 NMs demonstrate a rapid loss of corona proteins, while surface coating results in the retention of the corona signal after internalization for at least 24 h (varying with coating composition). Imaging with two-color super-resolution dSTORM revealed that the apparent TiO2 NM single agglomerates observed in diffraction-limited confocal microscopy are actually adjacent smaller agglomerates, and provides novel insights into the spatial arrangement of the initial and exchanged coronas adsorbed at the NM surfaces.

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

BACKGROUND: Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES: Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS: Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS: Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS: These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy.

MOTIVATION: Localization microscopy data is represented by a set of spatial coordinates, each corresponding to a single detection, that form a point cloud. This can be analyzed either by rendering an image from these coordinates, or by analyzing the point cloud directly. Analysis of this type has focused on clustering detections into distinct groups which produces measurements such as cluster area, but has limited capacity to quantify complex molecular organization and nano-structure. RESULTS: We present a segmentation protocol which, through the application of persistence-based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components, endocytic proteins and microtubule networks. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist-based analysis and batch processing of localization microscopy data. AVAILABILITY AND IMPLEMENTATION: RSMLM has been released under the GNU General Public License v3.0 and is available at https://github.com/JeremyPike/RSMLM. Tutorials for this library implemented as Binder ready Jupyter notebooks are available at https://github.com/JeremyPike/RSMLM-tutorials. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics.

The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.

Single-Molecule Localization and Structured Illumination Microscopy of Platelet Proteins.

Superresolution microscopy has become increasingly widespread over the past 5 years and allows users to image biological processes below the diffraction limit of traditional fluorescence microscopy where resolution is restricted to approximately 250 nm. Superresolution refers to a wide range of techniques which employ different approaches to circumvent the diffraction limit. Two of these approaches, structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM), which provide a doubling and tenfold increase in resolution respectively, are dominating the field. This is partly because of the insights into biology they offer and partly because of their commercialization by the main microscope manufacturers. This chapter provides background to the two techniques, practical considerations for their use, and protocols for their application to platelet biology.