Quantifying colocalization: The case for discarding the Manders overlap coefficient.

Colocalization measurements aim to characterize the relative distribution of two molecules within a biologically relevant area. It is efficient to measure two distinct features, co-occurrence, the extent to which the molecules appear together, and correlation, how well variations in concentration of the two molecules match. The Manders overlap coefficient (MOC) appears in most colocalization software but the literature contains three interpretations of its measurements: (a) co-occurrence, (b) correlation, or (c) a combination of both. This is surprising given the simplicity of the underlying equation. Testing shows that the MOC responds both to changes in co-occurrence and to changes in correlation. Further testing reveals that different distributions of intensity (Gaussian, gamma, uniform, exponential) dramatically alter the balance between the contribution from co-occurrence and correlation. It follows that the MOC's ability to differentiate between different patterns of colocalization is very limited, since any value is compatible with widely differing combinations of co-occurrence, correlation, and intensity distribution. To characterize colocalization, we recommend reporting both co-occurrence and correlation, using coefficients specific for each attribute. Since the MOC has no clear role in the measurement of colocalization and causes considerable confusion, we conclude that it should be discarded.

Vγ9Vδ2 T cells proliferate in response to phosphoantigens released from erythrocytes infected with asexual and gametocyte stage Plasmodium falciparum.

Vγ9Vδ2 T cells, the dominant γδ T cell subset in human peripheral blood, are stimulated by phosphoantigens, of which (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, is produced in the apicoplast of malaria parasites. Cell-free media from synchronised Plasmodium falciparum asexual ring, trophozoite, and schizont stage-cultures of high purity as well as media from ruptured schizont cultures, all stimulated Vγ9Vδ2 T cell proliferation, as did media from pure gametocyte cultures, whereas media from uninfected erythrocytes cultures did not. The media from ruptured schizont cultures and all the asexual and gametocyte stage cultures contained only background iron levels, suggesting that all erythrocyte haemoglobin is consumed as the parasites develop and supporting that the phosphoantigens were released from intact parasitized erythrocytes. The Vγ9Vδ2 T cell-stimulating agent was not affected by freezing, thawing or heating but was sensitive to phosphatase treatment, confirming its phosphoantigen identity. In summary, phosphoantigens are released from parasitised erythrocytes at all developmental blood stages.

Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains.

The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerisation decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of 4-phosphate-inositides lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, lead to the formation of ordered domains. The increase in ordered domains was correlated with an increase in actin filaments just beneath the plasma membrane. In live cell plasma membrane blebs, which are detached from the underlying actin filaments, the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form when actin filaments attach to the plasma membrane. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

Membrane topography and the overestimation of protein clustering in single molecule localisation microscopy - identification and correction.

According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.

Optimised generalized polarisation analysis of C-laurdan reveals clear order differences between T cell membrane compartments.

Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (lo) phase to liquid-disordered (ld) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable.

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing.

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a ß-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

Potentiating Vγ9Vδ2†T cell proliferation and assessing their cytotoxicity towards adherent cancer cells at the single cell level.

Vγ9Vδ2 T cells is the dominant γδ T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for Vγ9Vδ2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise Vγ9Vδ2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly thawed human serum potentiated Vγ9Vδ2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and consistently enabled Vγ9Vδ2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallyl monophosphate stimulated Vγ9Vδ2 T cell proliferation. We developed a method where the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the Vγ9Vδ2 T cell cytotoxicity towards colon cancer cells. In summary, we have developed an easily interpretable method to assess the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells, found that Vγ9Vδ2 T cell proliferation can be potentiated by media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of Vγ9Vδ2 T cell immunotherapy.

Protective effects of statins on COVID-19 risk, severity and fatal outcome: a nationwide Swedish cohort study.

The impact of statins on COVID-19 remains unclear. This study aims to investigate whether statin exposure assessed both in the population and in well-defined cohorts of COVID-19 patients may affect the risk and severity of COVID-19 using nationwide Swedish population-based register data. A population ≥ 40 years was selected by age/sex-stratified random sampling from the Swedish population on 1 Jan 2020. COVID-19 outcomes were identified from the SmiNet database, the National Patient Register and/or Cause-of-Death Register and linked with the National Prescribed Drug Register and sociodemographic registers. Statin exposure was defined as any statin prescriptions in the year before index date. In Cox regressions, confounding was addressed using propensity score ATT (Average Treatment effect in the Treated) weighting. Of 572,695 individuals in the overall cohort, 22.3% had prior statin treatment. After ATT weighting, protective effects were observed among statin user for hospitalization and COVID-19 death in the overall cohort and onset cohort. In the hospitalized cohort, statin use was only associated with lower risk for death (HR = 0.86, 95% CI 0.79-0.95), but not ICU admission. Statin-treated individuals appear to have lower COVID-19 mortality than nonusers, whether assessed in the general population, from COVID-19 onset or from hospitalization.

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation.

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10-20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca(2+). Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD-cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.

The Pearson correlation coefficient (PCC) and the Mander's overlap coefficient (MOC) are used to quantify the degree of colocalization between fluorophores. The MOC was introduced to overcome perceived problems with the PCC. The two coefficients are mathematically similar, differing in the use of either the absolute intensities (MOC) or of the deviation from the mean (PCC). A range of correlated datasets, which extend to the limits of the PCC, only evoked a limited response from the MOC. The PCC is unaffected by changes to the offset while the MOC increases when the offset is positive. Both coefficients are independent of gain. The MOC is a confusing hybrid measurement, that combines correlation with a heavily weighted form of co-occurrence, favors high intensity combinations, downplays combinations in which either or both intensities are low and ignores blank pixels. The PCC only measures correlation. A surprising finding was that the addition of a second uncorrelated population can substantially increase the measured correlation, demonstrating the importance of excluding background pixels. Overall, since the MOC is unresponsive to substantial changes in the data and is hard to interpret, it is neither an alternative to nor a useful substitute for the PCC. The MOC is not suitable for making measurements of colocalization either by correlation or co-occurrence.

Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.

Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [3H]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

Conventional analysis of movement on non-flat surfaces like the plasma membrane makes Brownian motion appear anomalous.

Cells are neither flat nor smooth, which has serious implications for prevailing plasma membrane models and cellular processes like cell signalling, adhesion and molecular clustering. Using probability distributions from diffusion simulations, we demonstrate that 2D and 3D Euclidean distance measurements substantially underestimate diffusion on non-flat surfaces. Intuitively, the shortest within surface distance (SWSD), the geodesic distance, should reduce this problem. The SWSD is accurate for foldable surfaces but, although it outperforms 2D and 3D Euclidean measurements, it still underestimates movement on deformed surfaces. We demonstrate that the reason behind the underestimation is that topographical features themselves can produce both super- and subdiffusion, i.e. the appearance of anomalous diffusion. Differentiating between topography-induced and genuine anomalous diffusion requires characterising the surface by simulating Brownian motion on high-resolution cell surface images and a comparison with the experimental data.

Interleaflet Coupling, Pinning, and Leaflet Asymmetry-Major Players in Plasma Membrane Nanodomain Formation.

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

Methods applicable to membrane nanodomain studies?

Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

Cholesterol depletion using methyl-ß-cyclodextrin.

Cholesterol is an essential component of mammalian cells. It is the major lipid constituent of the plasma membrane and is also abundant in most other organelle membranes. In the plasma membrane cholesterol plays critical physical roles in the maintenance of membrane fluidity and membrane permeability. It is also important for membrane trafficking, cell signalling, and lipid as well as protein sorting. Cholesterol is essential for the formation of liquid ordered domains in model membranes, which in cells are known as lipid nanodomains or lipid rafts. Cholesterol depletion is widely used to study the role of cholesterol in cellular processes and can be performed over days using inhibitors of its synthesis or acutely over minutes using chemical reagents. Acute cholesterol depletion by methyl-ß-cyclodextrin (MBCD) is the most widely used method and here we describe how it should be performed to avoid the common side-effect cell death.

The T cell receptor resides in ordered plasma membrane nanodomains that aggregate upon patching of the receptor.

Two related models for T cell signalling initiation suggest either that T cell receptor (TCR) engagement leads to its recruitment to ordered membrane domains, often referred to as lipid rafts, where signalling molecules are enriched or that ordered TCR-containing membrane nanodomains coalesce upon TCR engagement. That ordered domains form upon TCR engagement, as they do upon lipid raft marker patching, has not been considered. The target of this study was to differentiate between those three options. Plasma membrane order was followed in live T cells at 37 °C using laurdan to report on lipid packing. Patching of the TCR that elicits a signalling response resulted in aggregation, not formation, of ordered plasma membrane domains in both Jurkat and primary T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement.