Publications
87 results found
Compte M, Lykke Harwood S, Munoz IG, et al., 2018, A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity, Nature Communications, Vol: 9, ISSN: 2041-1723
The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.
Schneider F, Waithe D, Galiani S, et al., 2018, Nanoscale spatiotemporal diffusion modes measured by simultaneous confocal and stimulated emission depletion nanoscopy imaging, Nano Letters, Vol: 18, Pages: 4233-4240, ISSN: 1530-6984
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.
Schneider F, Waithe D, Galiani S, et al., 2018, Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 18, Pages: 4233-4240, ISSN: 1530-6984
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.
Garcia E, Bernardino de la Serna J, 2018, Dissecting single-cell molecular spatiotemporal mobility and clustering at focal adhesions in polarised cells by fluorescence fluctuation spectroscopy methods, METHODS, Vol: 140, Pages: 85-96, ISSN: 1046-2023
Waithe D, Schneider F, Chojnacki J, et al., 2018, Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data, Methods, Vol: 140-141, Pages: 62-73, ISSN: 1046-2023
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.
Gutowska-Owsiak D, Bernardino de la Serna J, Fritzsche M, et al., 2018, Orchestrated control of filaggrin-actin scaffolds underpins cornification, Cell Death and Disease, Vol: 9, ISSN: 2041-4889
Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.
Santos AM, Ponjavic A, Fritzsche M, et al., 2018, Capturing resting T cells: the perils of PLL, NATURE IMMUNOLOGY, Vol: 19, Pages: 203-205, ISSN: 1529-2908
Garcia E, Bernardino de la Serna J, 2017, Dissecting single–cell molecular spatiotemporal mobility and clustering at Focal Adhesions in polarised cells by fluorescence fluctuation spectroscopy methods
<jats:title>Abstract</jats:title><jats:p>Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexibly and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine powerful sensitivity characteristics and some limitations of RICS and N&B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.</jats:p>
Sreedharan S, Gill MR, Garcia E, et al., 2017, Multimodal super-resolution optical microscopy using a transition-metal-based probe provides unprecedented capabilities for imaging both nuclear chromatin and mitochondria, Journal of the American Chemical Society, Vol: 139, Pages: 15907-15913, ISSN: 0002-7863
Detailed studies on the live cell uptake properties of a dinuclear membrane-permeable RuII cell probe show that, at low concentrations, the complex localizes and images mitochondria. At concentrations above ∼20 μM, the complex images nuclear DNA. Because the complex is extremely photostable, has a large Stokes shift, and displays intrinsic subcellular targeting, its compatibility with super-resolution techniques was investigated. It was found to be very well suited to image mitochondria and nuclear chromatin in two color, 2C-SIM, and STED and 3D-STED, both in fixed and live cells. In particular, due to its vastly improved photostability compared to that of conventional SR probes, it can provide images of nuclear DNA at unprecedented resolution.
Waithe D, Schneider F, Chojnacki J, et al., 2017, Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data, Publisher: bioRxiv
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.
Mao B, Calatayud DG, Mirabello V, et al., 2017, Fluorescence-lifetime imaging and super-resolution microscopies shed light on the directed- and self-assembly of functional porphyrins onto carbon nanotubes and flat surfaces, Chemistry - A European Journal, Vol: 23, Pages: 9772-9789, ISSN: 0947-6539
Functional porphyrins have attracted intense attention due to their remarkably high extinction coefficients in the visible region and potential for optical and energy‐related applications. Two new routes to functionalised SWNTs have been established using a bulky ZnII‐porphyrin featuring thiolate groups at the periphery. We probed the optical properties of this zinc(II)‐substituted, bulky aryl porphyrin and those of the corresponding new nano‐composites with single walled carbon nanotube (SWNTs) and coronene, as a model for graphene. We report hereby on: i) the supramolecular interactions between the pristine SWNTs and ZnII‐porphyrin by virtue of π–π stacking, and ii) a novel covalent binding strategy based on the Bingel reaction. The functional porphyrins used acted as dispersing agent for the SWNTs and the resulting nanohybrids showed improved dispersibility in common organic solvents. The synthesized hybrid materials were probed by various characterisation techniques, leading to the prediction that supramolecular polymerisation and host–guest functionalities control the fluorescence emission intensity and fluorescence lifetime properties. For the first time, XPS studies highlighted the differences in covalent versus non‐covalent attachments of functional metalloporphyrins to SWNTs. Gas‐phase DFT calculations indicated that the ZnII‐porphyrin interacts non‐covalently with SWNTs to form a donor–acceptor complex. The covalent attachment of the porphyrin chromophore to the surface of SWNTs affects the absorption and emission properties of the hybrid system to a greater extent than in the case of the supramolecular functionalisation of the SWNTs. This represents a synthetic challenge as well as an opportunity in the design of functional nanohybrids for future sensing and optoelectronic applications.
Fritzsche M, Fernandes RA, Chang VT, et al., 2017, Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation, Science Advances, Vol: 3, Pages: 1-18, ISSN: 2375-2548
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.
Aron M, Browning R, Carugo D, et al., 2017, Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order, BMC Bioinformatics, Vol: 18, ISSN: 1471-2105
Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and modelmembrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by thegeneralized polarization (GP) parameter. With the development of commercial microscopes equipped with spectraldetectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membraneproperties. The existing tools for spectral image processing, however, are insufficient for processing the large data setsafforded by this technological advancement, and are unsuitable for processing images acquired with rapidlyinternalized fluorescent probes.Results: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In additionto common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps,and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidlyinternalized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of datasets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution isdetermined, which can provide insight into the mechanisms underlying changes in membrane properties and isdesirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstratedfor cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitiveprobes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di-4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantificationof the local lateral density of lipids or lipid packing.Conclusions: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectralimaging datasets with a reliable method for membrane segmentation and no ability in programmin
Pereno V, Carugo D, Bau L, et al., 2017, Electroformation of giant unilamellar vesicles on stainless steel electrodes, ACS Omega, Vol: 2, Pages: 994-1002, ISSN: 2470-1343
Giant unilamellar vesicles (GUVs) are well-established model systems forstudying membrane structure and dynamics. Electroformation, also referred to aselectroswelling, is one of the most prevalent methods for producing GUVs, as it enablesmodulation of the lipid hydration process to form relatively monodisperse, defect-freevesicles. Currently, however, it is expensive and time-consuming compared with othermethods. In this study, we demonstrate that 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine GUVs can be prepared readily at a fraction of the cost on stainless steel electrodes,such as commercially available syringe needles, without any evidence of lipid oxidationor hydrolysis.
Polley A, Orlowski A, Danne R, et al., 2017, Glycosylation and lipids working in concert direct CD2 ectodomain orientation and presentation, Journal of Physical Chemistry Letters, Vol: 8, Pages: 1060-1066, ISSN: 1948-7185
Proteins embedded in the plasma membrane mediate interactions with the cell environment and play decisiveroles in many signaling events. For cell−cell recognition molecules, it is highly likely that their structures and behavior have beenoptimized in ways that overcome the limitations of membrane tethering. In particular, the ligand binding regions of theseproteins likely need to be maximally exposed. Here we show by means of atomistic simulations of membrane-bound CD2, a smallcell adhesion receptor expressed by human T-cells and natural killer cells, that the presentation of its ectodomain is highlydependent on membrane lipids and receptor glycosylation acting in apparent unison. Detailed analysis shows that the underlyingmechanism is based on electrostatic interactions complemented by steric interactions between glycans in the protein and themembrane surface. The findings are significant for understanding the factors that render membrane receptors accessible forbinding and signaling.
Shen C, Bernardino de la Serna J, Struth B, et al., 2017, Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 2. Membrane Structure, 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 319A-319A, ISSN: 0006-3495
Chan K-L, Gonzalez EG, Padilla-Parra S, et al., 2017, Well-Characterised Time-Gated Detector Photon Flux Resolves the Ultrastructure of DNA-Damage Nuclear Bodies with G-STED Nanoscopy, 61st Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 141A-141A, ISSN: 0006-3495
Carugo D, Aron M, Sezgin E, et al., 2016, Modulation of the molecular arrangement in artificial and biological membranes by phospholipid-shelled microbubbles, Biomaterials, Vol: 113, Pages: 105-117, ISSN: 1878-5905
The transfer of material from phospholipid-coated microbubbles to cell membranes has been hypothesized to play a role in ultrasound-mediated drug delivery. In this study, we employed quantitative fluorescence microscopy techniques to investigate this phenomenon in both artificial and biological membrane bilayers in an acoustofluidic system. The results of the present study provide strong evidence for the transfer of material from microbubble coatings into cell membranes. Our results indicate that transfer of phospholipids alters the organization of molecules in cell membranes, specifically the lipid ordering or packing, which is known to be a key determinant of membrane mechanical properties, protein dynamics, and permeability. We further show that polyethylene-glycol, used in many clinical microbubble formulations, also has a major impact on both membrane lipid ordering and the extent of lipid transfer, and that this occurs even in the absence of ultrasound exposure.
Jarrett R, Ogg G, Salio M, et al., 2016, Filaggrin inhibits house dust mite phospholipase generation of CD1a lipid antigens for recognition by T cells, 20th Anniversary Conference of the British-Skin-Foundation on Skin Deep - 20 Years of Research, Publisher: WILEY-BLACKWELL, Pages: 50-50, ISSN: 0007-0963
Bernardino de la Serna J, Schuetz GJ, Eggeling C, et al., 2016, There is no simple model of the plasma membrane organizationyy, Frontiers in Cell and Developmental Biology, Vol: 4, ISSN: 2296-634X
Ever since technologies enabled the characterization of eukaryotic plasma membranes,heterogeneities in the distributions of its constituents were observed. Over the years thisled to the proposal of various models describing the plasma membrane organizationsuch as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed innumerous publications and reviews. Instead of emphasizing on one model we in thisreview give a brief overview over current models and highlight how current experimentalwork in one or the other way do not support the existence of a single overarching model.Instead, we highlight the vast variety of membrane properties and components, theirinfluences and impacts. We believe that highlighting such controversial discoveries willstimulate unbiased research on plasma membrane organization and functionality, leadingto a better understanding of this essential cellular structure.
Stanly TA, Fritzsche M, Banerji S, et al., 2016, Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters, Biology Open, Vol: 5, Pages: 1343-1350, ISSN: 2046-6390
Receptor clustering is known to trigger signalling events that contributeto critical changes in cellular functions. Faithful imaging of suchclusters by means of fluorescence microscopy relies on the applicationof adequate cell fixation methods prior to immunolabelling in order toavoid artefactual redistribution by the antibodies themselves. Previouswork has highlighted the inadequacy of fixation with paraformaldehyde(PFA) alone for efficient immobilisation of membrane-associatedmolecules, and the advantages of fixation with PFA in combinationwith glutaraldehyde (GA). Using fluorescence microscopy, we herehighlight how inadequate fixation can lead to the formation ofartefactual clustering of receptors in lymphatic endothelial cells,focussing on the transmembrane hyaluronan receptors LYVE-1 andCD44, and the homotypic adhesion molecule CD31, each of whichdisplays their native diffuse surface distribution pattern only whenvisualised with the right fixation techniques, i.e. PFA/GA incombination. Fluorescence recovery after photobleaching (FRAP)confirms that the artefactual receptor clusters are indeed introduced byresidual mobility. In contrast, we observed full immobilisation ofmembrane proteins in cells that were fixed and then subsequentlypermeabilised, irrespective of whether the fixative was PFA or PFA/GAin combination. Our study underlines the importance of choosingappropriate sample preparation protocols for preserving authenticreceptor organisation in advanced fluorescence microscopy
Jarrett R, Salio M, Lloyd-Lavery A, et al., 2016, Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase, Annual Meeting of the British-Society-for-Investigative-Dermatology (BSID), Publisher: WILEY-BLACKWELL, Pages: E49-E50, ISSN: 0007-0963
Garcia E, Ragazzini C, Yu X, et al., 2016, WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion, Scientific Reports, Vol: 6, ISSN: 2045-2322
Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), whichallows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches,we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles incancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodiumassembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturationand degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulatesinvadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution ofNck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate withhigh invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP asa hub for signaling molecule recruitment during invadopodium generation and cancer progression, aswell as a potential diagnostic biomarker and an optimal target for therapeutic approaches.
Bernardino de la Serna J, Chang VT, Waithe D, et al., 2016, T-Cells in Suspension Do Not Show Pre-Clustered LCK, 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 570A-570A, ISSN: 0006-3495
Jarrett R, Salio M, Lloyd-Lavery A, et al., 2016, Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase, Science Translational Medicine, Vol: 8, ISSN: 1946-6234
Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.
Clausen MP, Sezgin E, Bernardino de la Serna J, et al., 2015, A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics, Methods, Vol: 88, Pages: 67-75, ISSN: 1046-2023
Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.
de la Serna JB, Fritzsche M, Chang V, et al., 2015, Advanced microscopy studies of the true resting state of T-cells, 10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Publisher: SPRINGER, Pages: S74-S74, ISSN: 0175-7571
Stanly TA, de la Serna JB, Banerji S, et al., 2015, A STED microscopy study: Cell surface distribution of the lymphatic endothelial cell receptor LYVE-1, 10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Publisher: SPRINGER, Pages: S63-S63, ISSN: 0175-7571
Sezgin E, Waithe D, Bernardino de la Serna J, et al., 2015, Spectral imaging to measure heterogeneity in membrane lipid packing, ChemPhysChem, Vol: 16, Pages: 1387-1394, ISSN: 1439-7641
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.
Shen C, de la Serna JB, Struth B, et al., 2015, DPPC Monolayers Exhibit an Additional Phase Transition at High Surface Pressure, Publisher: CELL PRESS, Pages: 85A-85A, ISSN: 0006-3495
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