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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 (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED 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.

Activating T cells reorganize their cortical actin to form a ramified transportation network beneath the immunological synapse., T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into “nano-domains” in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching non-activating surfaces probably don’t resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels.

We describe a means through which cells can be accurately counted in 3-D microscopy image data, using only weakly annotated images as input training material. We update an existing 2-D density kernel estimation approach into 3-D and we introduce novel 3-D features which encapsulate the 3-D neighbourhood surrounding each voxel. The proposed 3-D density kernel estimation (DKE-3-D) method, which utilises an ensemble of random decision trees, is computationally efficient and achieves state-of-the-art performance. DKE-3-D avoids the problem of discrete object identification and segmentation, common to many existing 3-D counting techniques, and we show that it outperforms other methods when quantification of densely packed and heterogeneous objects is desired. In this article we successfully apply the technique to two simulated and to two experimentally derived datasets and show that DKE-3-D has great potential in the biomedical sciences and any field where volumetric datasets are used.

The β-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, β-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, β-subunits affect the level of Ca(V)2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-Ca(V)2.2 containing a mutation (W391A), that prevents binding of β-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-Ca(V)2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-Ca(V)2.2(W391A) and CFP-Ca(V)2.2(WT) was lost in the absence of co-expressed β-subunits. Furthermore, the relative reduction of expression of Ca(V)2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface Ca(V)2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of Ca(V)2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of β-subunits on Ca(V)2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from which it buds out. Previous studies have shown that the HIV-1 envelope is an environment of very low mobility, with the diffusion of incorporated proteins two orders of magnitude slower than in the plasma membrane. One of the reasons for this difference is thought to be the HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing, which has, until now, been difficult to assess experimentally. To further refine the model of the molecular mobility on the HIV-1 surface, we herein investigated the relative importance of membrane composition and curvature in simplified model membrane systems, large unilamellar vesicles (LUVs) of different lipid compositions and sizes (0.1&ndash, 1 &micro, m), using super-resolution stimulated emission depletion (STED) microscopy-based fluorescence correlation spectroscopy (STED-FCS). Establishing an approach that is also applicable to measurements of molecule dynamics in virus-sized particles, we found, at least for the 0.1&ndash, m sized vesicles, that the lipid composition and thus membrane rigidity, but not the curvature, play an important role in the decreased molecular mobility on the vesicles&rsquo, surface. This observation suggests that the composition of the envelope rather than the particle geometry contributes to the previously described low mobility of proteins on the HIV-1 surface. Our vesicle-based study thus provides further insight into the dynamic properties of the surface of individual HIV-1 particles, as well as paves the methodological way towards better characterisation of the properties and function of viral lipid envelopes in general.

The role(s) of the novel stargazin-like γ-subunit proteins remain controversial. We have shown previously that the neuron-specific γ7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of γ7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of γ7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous γ7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed γ7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of γ7 is essential for all its effects, and we show that γ7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2IBa, and this enhancement is prevented by a concentration of γ7 that alone has no effect onIBa. The effect of γ7 is selective for certain mRNAs because it had no effect on α2δ-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that γ7 plays a role in stabilizing CaV2.2 mRNA.

Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-kappaB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IkappaBalpha), and that the phosphorylation of IkappaBalpha by IKK influences the amplitude of NF-kappaB oscillations. We have performed further analyses of the behavior of NF-kappaB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-kappaB translocation in 10,000 cells showed discernible oscillations in levels of nuclear NF-kappaB amongst cells when stimulated with interleukin (IL-1alpha), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IkappaBalpha by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IkappaBalpha were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-kappaB pathway resulted in significant differences in the oscillatory patterns of NF-kappaB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.

Cholesterol is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction and trafficking in cells, hence it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase separated giant unilamellar vesicles (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-FCS based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent cholesterol analogs in visualizing cellular cholesterol dynamics.

Motivation: Fluorescence Correlation Spectroscopy (FCS) is a popular tool for measuring molecular mobility and how mobility relates to molecular interaction dynamics and bioactivity in living cells. The FCS technique has been significantly advanced by its combination with super-resolution STED microscopy (STED-FCS). Specifically, the use of gated detection has shown great potential for enhancing STED-FCS, but has also created a demand for software which is efficient and also implements the latest algorithms. Prior to this study, no open software has been available which would allow practical time-gating and correlation of point data derived from STED-FCS experiments. Results: The product of this study is a piece of stand-alone software called FoCuS-point. FoCuS-point utilizes advanced time-correlated single-photon counting (TCSPC) correlation algorithms along with time-gated filtering and innovative data visualization. The software has been designed to be highly user-friendly and is tailored to handle batches of data with tools designed to process files in bulk. FoCuS-point also includes advanced fitting algorithms which allow the parameters of the correlation curves and thus the kinetics of diffusion to be established quickly and efficiently. Availability and implementation: FoCuS-point is written in python and is available through the github repository: https://github.com/dwaithe/FCS_point_correlator. Furthermore, compiled versions of the code are available as executables which can be run directly in Linux, Windows and Mac OSX operating systems. Contact: dominic.waithe@imm.ox.ac.uk

Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.

The role(s) of the newly discovered stargazin-like γ-subunit proteins remains unclear; although they are now widely accepted to be transmembrane AMPA receptor regulatory proteins (TARPs), rather than Ca2+ channel subunits, it is possible that they have more general roles in trafficking within neurons. We previously found that γ7 subunit is associated with vesicles when it is expressed in neurons and other cells. Here, we show that γ7 is present mainly in retrogradely transported organelles in sympathetic neurons, where it colocalises with TrkA–YFP, and with the early endosome marker EEA1, suggesting that γ7 localises to signalling endosomes. It was not found to colocalise with markers of the endoplasmic reticulum, mitochondria, lysosomes or late endosomes. Furthermore, knockdown of endogenous γ7 by short hairpin RNA transfection into sympathetic neurons reduced neurite outgrowth. The same was true in the PC12 neuronal cell line, where neurite outgrowth was restored by overexpression of human γ7. These findings open the possibility that γ7 has an essential trafficking role in relation to neurite outgrowth as a component of endosomes involved in neurite extension and growth cone remodelling.

Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-kappaB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IkappaBalpha), and that the phosphorylation of IkappaBalpha by IKK influences the amplitude of NF-kappaB oscillations. We have performed further analyses of the behavior of NF-kappaB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-kappaB translocation in 10,000 cells showed discernible oscillations in levels of nuclear NF-kappaB amongst cells when stimulated with interleukin (IL-1alpha), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IkappaBalpha by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IkappaBalpha were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-kappaB pathway resulted in significant differences in the oscillatory patterns of NF-kappaB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.

Background Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection. Materials and Methods PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs. Results PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker. Conclusion Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention., Author Summary Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection. In this study, we found that PAF was significantly increased in patients with DHF, and the PAF levels rose just before the onset of the critical phase of dengue, during which vascular leak is thought to occur. PAF in serum of dengue patients was associated with reduced expression of tight junction proteins (ZO-1) and reduction in trans-endothelial resistance (TEER) of human endothelial cells. Use of PAFR blockers significantly reduced the down regulation of ZO-1 by serum of dengue patients and also the reduction of TEER, suggesting that PAF plays a significant role in inducing vascular leak in acute dengue infections.

The three-dimensional (3D) organization of chromosomes can be probed using methods like Capture-C. However, it is unclear how such population-level data relate to the organization within a single cell, and the mechanisms leading to the observed interactions are still largely obscure. We present a polymer modeling scheme based on the assumption that chromosome architecture is maintained by protein bridges, which form chromatin loops. To test the model, we perform FISH experiments and compare with Capture-C data. Starting merely from the locations of protein binding sites, our model accurately predicts the experimentally observed chromatin interactions, revealing a population of 3D conformations. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0909-0) contains supplementary material, which is available to authorized users.