Biology subjects

Sezgin, E.

Publications and source records attributed to Sezgin, E..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Discovery of a novel inhibitor of macropinocytosis with antiviral activity

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a new molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using High-Throughput Microscopy, where we identified new chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as Monkeypox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present a new molecule that inhibits viral entry via the endocytic route, offering a new alternative to prevent viral infection.

pharmacology and toxicology↗

bioRxiv2023

Quantifying fluorescence lifetime responsiveness of environment sensitive probes for membrane fluidity measurements

The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and to avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as degree of lipid saturation, cholesterol content, double bond position and configuration and phospholipid headgroup. As comparison, we investigated lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime, but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g. cholesterol). While the lifetime of Flipper is particularly sensitive to membrane cholesterol content, NR12S and NR12A lifetime is moderately sensitive to both cholesterol content and lipid acyl chains. Moreover, all probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission-wavelength dependency results in varying lifetime resolution at different spectral regions, highly relevant for FLIM data acquisition. Our data provides valuable insights on how to perform FLIM with these probes and highlights both their potential and limitations.

biophysics↗

bioRxiv2023

Quantification of membrane fluidity in bacteria using TIR-FCS

Cell membrane fluidity is an important phenotypic feature that regulates the diffusion, function and folding of transmembrane and membrane-associated proteins. It is particularly interesting to study it in bacteria as variations in membrane fluidity are known to affect fundamental cellular processes such as respiration, transport and antibiotic resistance. As such key parameter, membrane fluidity is regulated to adapt to environmental variations and stresses like temperature fluctuations or osmotic shocks. Membrane fluidity has been however scarcely studied quantitatively in bacterial cells, mostly because of the lack of available tools. Here, we developed an assay based on total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. We used this assay to quantify the fluidity of the cytoplasmic membrane of the Gram-positive model bacterium Bacillus subtilis in response to a cold shock, caused by a shift from 37{degrees}C to 20{degrees}C. In our experimental conditions, steady-state fluidity was recovered within 30 mins, and the steady-state fluidity at 20{degrees}C was about half of that at 37{degrees}C. Our minimally invasive assay opens up exciting perspectives and could be used to study a wide range of phenomena affecting the bacterial membrane, from disruption by antibiotics, antimicrobial peptides, or osmotic shocks. SignificanceUsing fluorescence correlation spectroscopy (FCS) with total internal reflection fluorescence (TIRF) illumination, we measured the diffusion speed of fluorescent membrane markers as a readout for membrane fluidity of growing B. subtilis cells. Quantification of the effect of cold shock provided unique information about the dynamics of the plasma membrane of B. subtilis. The unprecedented capability of TIR-FCS to quantify membrane fluidity in living bacteria opens the door to a whole set of new studies that will shed light on the bacterial plasma membrane and its interactions with the environment.

biophysics↗

bioRxiv2023

Estrogen receptor activation remodels TEAD1 gene expression to alleviate nonalcoholic fatty liver disease

IntroductionThe occurrence of obesity-related hepatic malignancies differs between sexes, suggesting the involvement of sex hormones. Female sex hormones maintain cell homeostasis through estrogen receptor (ER) signaling and protect from developing nonalcoholic fatty liver disease (NAFLD) in mice and humans. RationaleTo understand recovery from high-fat diet (HFD)-induced liver disease in males upon estrogen treatment, we comprehensively characterized molecular changes in the liver upon selective activation of estrogen receptors (ERs) to identify novel therapeutic targets downstream of estrogen signaling. MethodsTo dissect hepatic ER isoform-driven responses, we integrated liver transcriptomes from female and male HFD mice treated with or without four different estrogen agonists, along with multiomics data, including bulk, single-cell and spatial transcriptomics, chromatin profiling, machine learning models and advanced microscopy. Patient cohorts and primary human hepatocyte spheroids datasets were included. ResultsOnly males developed liver steatosis. We found that selective activation of either ER or ER{beta} reduced HFD-induced hepatic steatosis in male mice. Systemic ER activation restored HFD-induced aberrant gene expression of cellular processes across liver cell types, including hepatocytes. Profiling of marked histones revealed that ER activation modulated promoter and enhancer sites and identified 68 estrogen-sensitive enhancer-gene pairs. Most of these genes were similarly deregulated in human nonalcoholic fatty liver disease (NAFLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in NAFLD patients, and inhibiting TEAD ameliorated steatosis in spheroids by suppressing lipogenic pathways. ConclusionsSystemic activation of ER or ER{beta} modulates molecular pathways in the liver to counteract NAFLD. Our study identified TEAD1 as a key ER-sensitive gene and suggested that its inhibition poses a therapeutic strategy to combat NAFLD without the undesired side effects elicited by estrogen signaling. Clinical research relevanceWe identified drug targets downstream of estrogen signaling, including TEAD1, and demonstrate that TEAD inhibition improves steatosis by suppressing lipogenic pathways. Basic research relevanceThe targeted activation of nuclear ERs recovers high-fat diet-induced molecular and physiological liver phenotypes by remodeling core pathways beyond lipid metabolism. ER-responsive enhancers regulate central metabolic genes of clinical significance in NAFLD patients, highlighting the potential impact of this research on understanding liver cell plasticity. HIGHLIGHTSO_LIsteatosis in livers of high-fat diet (HFD) male mice was effectively reduced by selective activation of estrogen receptors (ER and ER{beta}) with four different agonists. C_LIO_LIER agonist treatments successfully reversed HFD-induced changes in gene regulation and expression, revealing new treatment targets involving previously unconnected molecular pathways. C_LIO_LIestrogen-sensitive enhancers regulated important genes, including TEAD1, emerging as pivotal NAFLD regulators significantly impacting metabolic processes. C_LIO_LIhigh TEAD1 gene expression in NAFLD patients correlated with disease severity, underscoring its clinical significance in disease progression. C_LIO_LIinhibiting TEAD with small molecules alleviated steatosis by suppressing lipogenic pathways, resembling some of the same beneficial effects as estrogen treatment. C_LI

genomics↗

bioRxiv2023

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that act as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings reveal a novel glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

cell biology↗

bioRxiv2023

Cell Membranes Sustain Phospholipid Imbalance Via Cholesterol Asymmetry

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function often include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PM). Using quantitative lipidomics, we discovered that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared to exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization. These largely overlooked aspects of membrane asymmetry represent an evolution of classic paradigms of biomembrane structure and physiology.

biophysics↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

MIGH-T: A Multi-Parametric and High-Throughput Platform for Host-Virus Binding Screens

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to the pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here we describe a multi-parametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform was validated by blocking SARS-CoV-2 spike particles with nanobodies and IgGs from human serum samples. TeaserA fast screening platform tackling host-pathogen interactions.

synthetic biology↗

bioRxiv2022

Single particle profiler for measuring properties of nano-sized bioparticles

It is technically challenging to study the content and properties of nanoscale bioparticles in a high-throughput and single-molecule manner. We developed a high-throughput analysis method, called single particle profiler (SPP) that provides single-particle information on content and biophysical properties of thousands of particles. We applied SPP to measure the mRNA encapsulation efficiency of lipid nanoparticles, viral binding efficiency of different nanobodies and biophysical heterogeneity of liposomes, lipoproteins, exosomes and viruses.

biophysics↗

bioRxiv2022

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties

The plasma membrane serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades several environment-sensitive probes have been popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we studied the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration, phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

biophysics↗

bioRxiv2022

PCYT2 controls muscle health and muscle aging

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive muscle weakness. Pcyt2 mutant zebrafish recapitulate the patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss, accelerated ageing, and reduced lifespan. Mechanistically, Pcyt2 deficiency affects mitochondrial bioenergetics and physicochemical properties of the myofiber membrane lipid bilayer, in particular under exercise strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice. AAV-based delivery of PCYT2 rescued muscle weakness in Pcyt2 knock-out mice and, importantly, improved muscle strength in old mice, offering a novel therapeutic avenue for rare disease patients and muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.

genetics↗

bioRxiv2021

The influence of the extracellular domains on the dynamic behavior of membrane proteins

The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how the ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data shows that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconciles different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane. SIGNIFICANCE STATEMENTWe studied how the size and glycosylation of the proteins influences their dynamic behavior in a lipid bilayer by reconstituting the ECDs of different sizes or glycosylation in model membrane systems and analyzing their sorting into lipid domains as well as their mobility. We observe that increasing the ECD apparent mass leads to a decrease in membrane ordered domain partitioning and diffusivity. Our data reconciles multiple mechanisms proposed for the initiation of cellular signaling by linking the ECD properties of membrane proteins with their localization and diffusion dynamics in the plasma membrane.

biophysics↗

bioRxiv2021

STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution, permitting for long acquisition times, and that allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

biophysics↗

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

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.

biophysics↗

bioRxiv2021

A FOXN1 mutation competitively displaces wild-type FOXN1 from higher order nuclear condensates to cause immunodeficiency

The transcription factor FOXN1 is a master regulator of thymic epithelial cell development and function. Here we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multi-molecular nuclear condensates essential for the factors transcriptional activity. FOXN1s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are significantly altered in a patient with a mutant FOXN1 which is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog, selectively impairs mouse thymic epithelial cell (TEC) differentiation revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

immunology↗

bioRxiv2021

The glycosphingolipid inhibitor eliglustat inhibits autophagy in osteoclasts to increase bone mass and reduce myeloma bone disease

Multiple myeloma (MM) is a fatal hematological malignancy, where the majority of patients are diagnosed with, or develop, destructive and debilitating osteolytic bone lesions. Current treatments for MM bone disease such as the bisphosphonate zoledronic acid can result in deleterious side effects at high doses. In this study, eliglustat, an FDA approved glycosphingolipid inhibitor, was shown to reduce MM bone disease in preclinical models of MM. Mechanistically, eliglustat alters the lipid composition and plasma membrane fluidity and acts as an autophagy flux inhibitor in bone-resorbing osteoclasts (OC). Autophagic degradation of the signaling molecule TRAF3 is key step in OC differentiation; this was prevented by eliglustat in OC precursors. In addition, eliglustat works depend on TRAF3 in vivo. Furthermore, the combination of eliglustat and zoledronic acid was found to have an additive effect to reduce MM bone disease, suggesting the potential for combination therapies that would allow for drug dose reductions. Taken together, this project identifies a novel mechanism in which glycosphingolipid inhibition reduces osteoclastogenesis via autophagy and highlights the translational potential of eliglustat for the treatment of bone loss disorders such as MM. One Sentence SummaryTranslational use of eliglustat as an autophagy inhibitor to limit bone lesions in multiple myeloma.

cell biology↗

bioRxiv2020

Aggregation and immobilisation of membrane proteins interplay with local lipid order in the plasma membrane of T cells

The quest for understanding of numerous vital membrane-associated cellular processes, such as signalling, has largely focussed on the spatiotemporal orchestration and reorganisation of the identified key proteins, including their binding and aggregation. Despite strong indications of the involvement of membrane lipid heterogeneities, historically often termed lipid rafts, their roles in many processes remain controversial and mechanisms elusive. Taking activation of T lymphocytes as an example, we here investigate membrane properties around the key proteins - in particular the T cell receptor (TCR), its main kinase Lck, and phosphatase CD45. We determine their partitioning and co-localisation in passive cell-derived model membranes (i.e. giant plasma-membrane vesicles, GPMVs), and explore their mobility and local lipid order in live Jurkat T cells using fluorescence correlation spectroscopy and spectral imaging with polarity-sensitive membrane probes. We find that upon aggregation and partial immobilisation, the TCR changes its preference towards more ordered lipid environments, which can in turn passively recruit Lck. We observe similar aggregation-induced local membrane ordering and recruitment of Lck also by CD45, as well as by a membrane protein of antigen-presenting cells, CD86, which is not supposed to interact with Lck directly. This highlights the involvement of lipid-mediated interactions and suggests that the cellular membrane is poised to modulate the frequency of protein encounters according to their aggregation state and alterations of their mobility, e.g. upon ligand binding.

biophysics↗