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DNA breaks, in particular double-strand breaks (DSBs), produced by radiation and radiomimetic agents are amongst the most toxic forms of DNA damage, in part because they are associated with extensive oxidative chemical modification at the break termini. Prior to the completion of DSB repair, the chemically modified termini associated with radiation-induced damage must be removed. A number of DNA processing enzymes have been implicated in the processing of these ‘dirty ends’, but our knowledge of this process remains limited. Here, we demonstrate a role for the 5’-3’ exonuclease SNM1A in this process. Cells disrupted for SNM1A are sensitive to radiation and radiomimetic agents and show defects in the resolution of the lethal DSB damage they induce. SNM1A is recruited and retained to the sites of complex DNA damage via the concerted action of three highly conserved interaction domains the PBZ, UBZ and PIP box, mediating interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. Consistent with its unprecedented capacity to digest DNA containing a wide range of chemically modified nucleotides, we found that SNM1A can resect DNA containing oxidative lesions representative of those induced by radiation damage at break termini. Taken together, our work reveals a crucial role for SNM1A in the repair of toxic DNA breaks.

Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges., In Expansion Microscopy (ExM) subcellular structures are imaged in isotropically expanded fixed samples, consequently allowing to resolve enlarged subcellular structures, otherwise hidden to standard microscopy. Upon comparison of the expansion factors of different cellular compartments in cells within the same gel, we found significant differences in expansion factors of a factor of above 2.

The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.

Reperfusion-induced calcium overload profoundly affects the extent of myocardial injury following ischemia, impacting long-term morbidity and mortality. Reactive oxygen species play a crucial role in shaping the amplitude and spatiotemporal patterns of the intracellular calcium signal, but the mechanism governing this interplay remain unclear. Here we show that, in vivo, myocardial ischemia and reperfusion (I/R) potently induce formation of an intermolecular-disulfide within the type I regulatory subunits of protein kinase A (PKARIα), both in mice and in humans. This conformation does not increase intrinsic PKA catalytic activity, but rather promotes AKAP-mediated subcellular compartmentalization of PKARIα to the lysosome, where it inhibits calcium release from two-pore channels and prevents global calcium release from nearby ryanodine receptors. This regulatory mechanism is shown to be crucial for limiting I/R-induced cell death, as ‘redox dead’ Cys17Ser PKARIα knock-in mice, which are incapable of undergoing RIα disulfide formation, display substantially larger infarct sizes with concomitant reductions in left ventricular contractile recovery, both of which are prevented by inhibition of lysosomal calcium release at the time of reperfusion. These findings reveal a hitherto unknown role for PKARIα, in its disulfide-activated state, to regulate calcium homeostasis and, in this way, potently protect the myocardium from post-ischemic injury.

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

Supplemental Digital Content is available in the text., Background: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. Methods: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from “redox dead” (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo. Results: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P

Introduction: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, the regulatory Iα subunit of Protein Kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulfide bonds within the holoenzyme complex. However, the impact of disulfide formation on kinase function, and its influence on PKA signaling in the context of heart disease remains unknown. Methods & Results: Myocardial ischemia-reperfusion (I/R) was found to be a potent inducer of PKARIα disulfide formation in vivo , both in mice and in humans. Using imaging modalities with high spatial and temporal resolution, we found that this conformation did not increase intrinsic PKA catalytic activity, but rather facilitated enhanced AKAP-dependent compartmentation of PKARIα in the adult mouse left ventricular (LV) myocyte, with preferential localization to the lysosome under oxidized conditions (n=38-41 myocytes, N=3 animals, p Conclusions: Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKA oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics.

Significance MR1 is a monomorphic major histocompatibility complex (MHC) class I-like molecule that presents ligands to Mucosal Associated Invariant T cells. MR1 antigen presentation at the cell surface is tightly regulated by ligand availability. Although previously described MR1 ligands facilitate translocation of ER-resident MR1 to the cell surface, we describe nonmicrobial ligands, DB28 and its ester analogue NV18.1, which retain MR1 in the ER in an immature ligand-receptive form and competitively inhibit stimulatory ligands. We provide the molecular and functional basis underpinning the interactions of this class of ligands with MR1., The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A′-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.

Human immunodeficiency virus t ype 1 (H IV-1) assembles as immature particles, which require the proteolytic cleavage of structural polyprotein Gag and the clustering of envelope glycoprotein Env for infectivity. The details of mechanisms underlying Env clustering remain unknown. Here, we determine molecular dynamics of Env on the surface of individual HIV-1 particles using scanning fluorescence correlation spectroscopy on a super-resolution STED microscope. We find that Env undergoes a maturation-induced increase in mobility, highlighting diffusion as one cause for Env clustering. This mobility increase is dependent on Gag-interacting Env tail but not on changes in viral envelope lipid order. Diffusion of Env and other envelope incorporated proteins in mature HIV-1 is two orders of magnitude slower than in the plasma membrane, indicating that HIV-1 envelope is intrinsically a low mobility environment, mainly due to its general high lipid order. Our results provide insights into dynamic properties of proteins on the surface of individual virus particles., To become infectious, HIV-1 particles undergo a maturation process involving the clustering of envelope glycoprotein Env. Here, Chojnacki et al. employ super-resolution STED-FCS microscopy to study dynamics of Env molecules on HIV-1 particles and show that Env undergoes a maturation-induced increase in mobility.

Targeted inhibition of cytokine pathways provides opportunities to understand fundamental biology in vivo in humans. The IL-33 pathway has been implicated in the pathogenesis of atopy through genetic and functional associations. We investigated the role of IL-33 inhibition in a first-in-class phase 2a study of etokimab (ANB020), an IgG1 anti–IL-33 monoclonal antibody, in patients with atopic dermatitis (AD). Twelve adult patients with moderate to severe AD received a single systemic administration of etokimab. Rapid and sustained clinical benefit was observed, with 83% achieving Eczema Area and Severity Index 50 (EASI50), and 33% EASI75, with reduction in peripheral eosinophils at day 29 after administration. We noted significant reduction in skin neutrophil infiltration after etokimab compared with placebo upon skin challenge with house dust mite, reactivity to which has been implicated in the pathogenesis of AD. We showed that etokimab also inhibited neutrophil migration to skin interstitial fluid in vitro. Besides direct effects on neutrophil migration, etokimab revealed additional unexpected CXCR1-dependent effects on IL-8–induced neutrophil migration. These human in vivo findings confirm an IL-33 upstream role in modulating skin inflammatory cascades and define the therapeutic potential for IL-33 inhibition in human diseases, including AD.

The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

Background Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulphide bonds within the holoenzyme complex. However, the effect of PKARIα oxidation on downstream signalling in the heart, particularly under states of oxidative stress, remains unexplored. Purpose To determine the direct functional consequences of PKARIα oxidation in the heart and investigate their impact on ischaemia/reperfusion (I/R) injury. Methods and results Experiments using the AKAR3ev FRET biosensor in murine left ventricular (LV) myocytes and Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged wild-type (WT) and mutant RIα proteins expressed in RIα-null fibroblasts showed that PKARIα oxidation does not increase the kinases' catalytic activity, but enhances its binding to A-kinase anchoring proteins (AKAP; n=30–39/N=3, p Displacement of PKARIα from lysosomes resulted in spontaneous sarcoplasmic reticulum calcium release and dramatic calcium oscillations in KI LV myocytes (panel B), which were preventable by ryanodine receptor blockade (1 mM tetracaine; n=14, p I/R (secondary to cardiopulmonary bypass) was found to induce PKARIα oxidation in the myocardium of patients undergoing cardiac surgery (panel C; n=18, p Conclusions Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKARIα oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics. Acknowledgement/Funding British Heart Foundation CH/12/3/29609, RG/16/12/32451; Garfield-Weston Foundation MPS/IVIMS-11/12-4032; Wellcome Trust Fellowship 0998981Z/12/Z

Highlights • FoCuS-scan is software for processing and analysis of large-scale scanning FCS data. • FoCuS-scan can correlate data acquired on conventional turn-key confocal systems. • We show the precision of diffusion measurements depends on experimental duration. • This article gives specific acquisition, analysis and interpretation details., Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2–1 μm) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

Waithe et al. use high-performance, low-cost automated microscopy using object detection algorithms to identify and image cells in real time., Object detection networks are high-performance algorithms famously applied to the task of identifying and localizing objects in photography images. We demonstrate their application for the classification and localization of cells in fluorescence microscopy by benchmarking four leading object detection algorithms across multiple challenging 2D microscopy datasets. Furthermore we develop and demonstrate an algorithm that can localize and image cells in 3D, in close to real time, at the microscope using widely available and inexpensive hardware. Furthermore, we exploit the fast processing of these networks and develop a simple and effective augmented reality (AR) system for fluorescence microscopy systems using a display screen and back-projection onto the eyepiece. We show that it is possible to achieve very high classification accuracy using datasets with as few as 26 images present. Using our approach, it is possible for relatively nonskilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of fluorescence microscopy acquisition pipelines.

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.

In this paper we demonstrate the application of object detection networks for the classification and localization of cells in fluorescence microscopy. We benchmark two leading object detection algorithms across multiple challenging 2-D microscopy datasets as well as develop and demonstrate an algorithm which can localize and image cells in 3-D, in real-time. Furthermore, we exploit the fast processing of these algorithms and develop a simple and effective Augmented Reality (AR) system for fluorescence microscopy systems. Object detection networks are well-known high performance networks famously applied to the task of identifying and localizing objects in photography images. Here we show their application and efficiency for localizing cells in fluorescence microscopy images. Object detection algorithms are typically trained on many thousands of images, which can be prohibitive within the biological sciences due to the cost of imaging and annotating large amounts of data. Through taking different cell types and assays as an example, we show that with some careful considerations it is possible to achieve very high performance with datasets with as few as 26 images present. Using our approach, it is possible for relatively non-skilled users to automate detection of cell classes with a variety of appearances and enable new avenues for automation of conventionally manual fluorescence microscopy acquisition pipelines.

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes., Mechanisms that operate during embryonic development to restrict cell fate are currently under investigation. Here the authors characterise the role of SCL/TAL1 at the onset of blood specification in embryonic development using mouse EB differentiation culture as a model system.

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions., The Membrane-Proximal External Region (MPER) of the HIV Env gp41 subunit is a target for broadly neutralizing antibodies. Here, the authors apply super-resolution stimulated emission depletion (STED) microscopy on single virions and provide insights into how the MPER epitope is recognized.

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Through two superresolution microscopy techniques, STED and STORM, Spiess et al. visualize the organization of integrins in focal adhesions and show that active and inactive β1 integrins assemble into distinct nanoclusters within adhesions, suggesting the existence of a novel mechanism that locally coordinates integrin activity., Integrins are the core constituents of cell–matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation.

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse α-globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the α-globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA.

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D ‘point-and-click’ user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on such datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues., eLife digest There are around 200 billion cells in the human brain that are generated by a small pool of rapidly dividing stem cells. For the brain to develop correctly, these stem cells must produce an appropriate number of each type of cell in the right place, at the right time. However, it remains unclear how individual stem cells in the brain know when and where to divide. To answer this question, Hailstone et al. studied the larvae of fruit flies, which use similar genes and mechanisms as humans to control brain development. This involved devising a new method for extracting the brains of developing fruit flies and keeping the intact tissue alive for up to 24 hours while continuously imaging individual cells in three dimensions. Manually tracking the division of each cell across multiple frames of a time-lapse is extremely time consuming. To tackle this problem, Hailstone et al. created a tool called CytoCensus, which uses machine learning to automatically identify stem cells from three-dimensional images and track their rate of division over time. Using the CytoCensus tool, Hailstone et al. identified a gene that controls the diverse rates at whichstem cells divide in the brain. Earlier this year some of the same researchers also published a study showing that this gene regulates a well-known cancer-related protein using an unconventional mechanism. CytoCensus was also able to detect cells in other developing tissues, including the embryos of mice. In the future, this tool could aid research into diseases that affect complex tissues, such as neurodegenerative disorders and cancer.

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.