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Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000‐fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments. [ABSTRACT FROM AUTHOR]

The dynamic reorganization of plasma membrane nanodomains (lipid rafts) plays a key role in host cell infection by human pathogens. Bacteria can induce the reorganization of lipid rafts as a result of the interactions between bacterial lectins and lipid raft glycosphingolipids (GSLs). However, it is still unclear how such interactions trigger the initial stage of the infection mechanism. Here, we study the GSL globotriaosylceramide (Gb3), a key receptor involved in the cellular uptake of the Gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via a lipid zipper mechanism. We mimic the plasma membrane using supported lipid bilayers (SLBs), containing liquid-ordered and liquid-disordered lipid domains. We observe that the liquid-ordered domains in the SLBs disperse upon interaction with LecA, accompanied by a deformation of SLBs surface. Our results link the membrane reorganization triggered by LecA with the P. aeruginosa infections in host cells. Continuous, dynamic organisation of plasma membrane nanodomains, or lipid rafts, are closely related to membrane recognition and infection. The authors use supported lipid bilayers to study how the bacterial lectin LecA affects the nanometric organisation of membranes, with potential implications in regulating bacterial infection. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a Gram‐negative bacterium causing morbidity and mortality in immuno‐compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen‐specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE‐cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P. aeruginosa infection. Synopsis: Pseudomonas aeruginosa lectin B binds to endothelial cells and inflicts junctional and cytoskeletal changes. Lectin B restricts migration of dendritic cells from tissue to lymph nodes and thereby has negative repercussion on adaptive immune responses. Pseudomonas aeruginosa lectin B binds to the lymphatics.Endothelial cells undergo cell structural changes upon LecB binding.Leukocyte diapedesis is inhibited by LecB.LecB inhibits T cell priming by impeding dendritic cell migration to the lymph node paracortex. [ABSTRACT FROM AUTHOR]

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Bacterial adhesion, biofilm formation and host cell invasion of the ESKAPE pathogen Pseudomonas aeruginosa require the tetravalent lectins LecA and LecB, which are therefore drug targets to fight these infections. Recently, we have reported highly potent divalent galactosides as specific LecA inhibitors. However, they suffered from very low solubility and an intrinsic chemical instability due to two acylhydrazone motifs, which precluded further biological evaluation. Here, we isosterically substituted the acylhydrazones and systematically varied linker identity and length between the two galactosides necessary for LecA binding. The optimized divalent LecA ligands showed improved stability and were up to 1000‐fold more soluble. Importantly, these properties now enabled their biological characterization. The lead compound L2 potently inhibited LecA binding to lung epithelial cells, restored wound closure in a scratch assay and reduced the invasiveness of P. aeruginosa into host cells. [ABSTRACT FROM AUTHOR]

The present report assesses the capability of a soluble glycosyltransferase to modify glycolipids organized in two synthetic membrane systems that are attractive models to mimic cell membranes: giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs). The objective was to synthesize the Gb3 antigen (Galα1,4Galβ1,4Glcβ-Cer), a cancer biomarker, at the surface of these membrane models. A soluble form of LgtC that adds a galactose residue from UDP-Gal to lactose-containing acceptors was selected. Although less efficient than with lactose, the ability of LgtC to utilize lactosyl–ceramide as an acceptor was demonstrated on GUVs and SLBs. The reaction was monitored using the B-subunit of Shiga toxin as Gb3-binding lectin. Quartz crystal microbalance with dissipation analysis showed that transient binding of LgtC at the membrane surface was sufficient for a productive conversion of LacCer to Gb3. Molecular dynamics simulations provided structural elements to help rationalize experimental data. [ABSTRACT FROM AUTHOR]

Phagocytic particle uptake is crucial for the fate of both living cells and pathogens. Invading particles have to overcome fluctuating lipid membranes as the first physical barrier. However, the energy and the role of the fluctuation-based particle-membrane interactions during particle uptake are not understood. We tackle this problem by indenting the membrane of differently composed Giant Unilamellar Vesicles (GUVs) with optically trapped particles until particle uptake. By continuous 1 MHz tracking and autocorrelating the particle's positions within 30µs delays for different indentations, the fluctuations' amplitude, the damping, the mean forces, and the energy profiles were obtained. Remarkably, the uptake energy into a GUV becomes predictable since it increases for smaller fluctuation amplitudes and longer relaxation time. Our observations could be explained by a mathematical model based on continuous suppression of fluctuation modes. Hence, the reduced particle uptake energy for protein-ligand interactions LecA-Gb3 or Biotin-Streptavidin results also from pronounced, low-friction membrane fluctuations. Particulates, bacteria and viruses wrap into cell membranes. Here the authors use optical tweezers, particle tracking and mathematical modelling to show that the uptake process into giant vesicles is influenced by thermal membrane fluctuations. [ABSTRACT FROM AUTHOR]

Receptor-mediated transcytosis is an elegant and promising strategy for drug delivery across biological barriers. Here, we describe a novel ligand–receptor pair based on a dimeric, engineered derivative of the Pseudomonas aeruginosa lectin LecA, here termed Di-LecA, and the host cell glycosphingolipid Gb3. We characterized the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells using mainly immunofluorescence in combination with confocal microscopy. To evaluate the delivery capacity of dimeric LecA chimeras, EGFP was chosen as a fluorescent model protein representing macromolecules, such as antibody fragments, and fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology. Both LecA/EGFP fusion proteins crossed cellular monolayers in vitro. Of note, the conjugate with EGFP at the N-terminus of LecA (EGFP-LecA) showed a higher release rate than the conjugate with EGFP at the C-terminus (LecA-EGFP). Based on molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles, we speculate that EGFP-LecA tends to be a dimer while LecA-EGFP forms a tetramer. Overall, we confidently propose the dimeric LecA chimeras as transcytotic drug delivery tools through Gb3-positive cellular barriers for future in vivo tests. [ABSTRACT FROM AUTHOR]

Background: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. Methods: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. Results: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis—up to 93%—of Gb3+ tumor cells in vitro. Conclusions: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes. [ABSTRACT FROM AUTHOR]

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours. [ABSTRACT FROM AUTHOR]

Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a β-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 β-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both β-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation. A curated lectin database, structural characterization, and in vitro assays show that choanoflagellate lectin SaroL-1 binds to cancer-related α-galactosylated epitopes and can be toxic to cancer cells through a carbohydrate-dependent pore-formation mechanism. [ABSTRACT FROM AUTHOR]

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell. [ABSTRACT FROM AUTHOR]

Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions. In live-cell microscopy, motion blur limits resolution and contrast. Here the authors use 100 Hz super-resolving Rotating Coherent Scattering (ROCS) microscopy on various dynamic biological systems, and time-window analysis to understand biological effects. [ABSTRACT FROM AUTHOR]

B cell superantigens crosslink conserved domains of B cell receptors (BCRs) and cause dysregulated, polyclonal B cell activation irrespective of normal BCR-antigen complementarity. The cells typically succumb to activation-induced cell death, which can impede the adaptive immune response and favor infection. In the present study, we demonstrate that the fucose-binding lectin of Burkholderia ambifaria, BambL, bears functional resemblance to B cell superantigens. By engaging surface glycans, the bacterial lectin activated human peripheral blood B cells, which manifested in the surface expression of CD69, CD54 and CD86 but became increasingly cytotoxic at higher concentrations. The effects were sensitive to BCR pathway inhibitors and excess fucose, which corroborates a glycan-driven mode of action. Interactome analyses in a model cell line suggest BambL binds directly to glycans of the BCR and regulatory coreceptors. In vitro, BambL triggered BCR signaling and induced CD19 internalization and degradation. Owing to the lectin's six binding sites, we propose a BCR activation model in which BambL functions as a clustering hub for receptor glycans, modulates normal BCR regulation, and induces cell death through exhaustive activation. [ABSTRACT FROM AUTHOR]

Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal. [ABSTRACT FROM AUTHOR]

Photorhabdus luminescens Tc toxins are large tripartite ABC‐type toxin complexes, composed of TcA, TcB and TcC proteins. Tc toxins are widespread and have shown a tropism for a variety of targets including insect, mammalian and human cells. However, their receptors and the specific mechanisms of uptake into target cells remain unknown. Here, we show that the TcA protein TcdA1 interacts with N‐glycans, particularly Lewis X/Y antigens. This is confirmed using N‐acetylglucosamine transferase I (Mgat1 gene product)‐deficient Chinese hamster ovary (CHO) Lec1 cells, which are highly resistant to intoxication by the Tc toxin complex most likely due to the absence of complex N‐glycans. Restoring Mgat1 gene activity, and hence complex N‐glycan biosynthesis, recapitulated the sensitivity of these cells to the toxin. Exogenous addition of Lewis X trisaccharide partially inhibits intoxication in wild‐type cells. Additionally, sialic acid also largely reduced binding of the Tc toxin. Moreover, proteolytic activation of TcdA1 alters glycan‐binding and uptake into target cells. The data suggest that TcdA1‐binding is most likely multivalent, and carbohydrates probably work cooperatively to facilitate binding and intoxication. [ABSTRACT FROM AUTHOR]

The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1. [ABSTRACT FROM AUTHOR]

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. We hypothesized that the orientation of the carbohydrate head group of Gb3 embedded in the lipid bilayer differentially influences LecA and StxB binding. To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and were indeed able to rebuild LecA and StxB segregation. Both, the Gb3 fatty acyl chain structure and the local membrane environment, modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes compared to LecA. Based on our findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify this approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB recognized Gb3 at the primary cilium and the periciliary membrane, whereas LecA only bound periciliary Gb3. This suggests that the ciliary membrane is of higher order than the surrounding periciliary membrane. [ABSTRACT FROM AUTHOR]

The healthy heart adapts continuously to a complex set of dynamically changing mechanical conditions. The mechanical environment is altered by, and contributes to, multiple cardiac diseases. Mechanical stimuli are detected and transduced by cellular mechano-sensors, including stretch-activated ion channels (SAC). The precise role of SAC in the heart is unclear, in part because there are few SAC-specific pharmacological modulators. That said, most SAC can be activated by inducers of membrane curvature. The lectin LecA is a virulence factor of Pseudomonas aeruginosa and essential for P. aeruginosa -induced membrane curvature, resulting in formation of endocytic structures and bacterial cell invasion. We investigate whether LecA modulates SAC activity. TREK-1 and Piezo1 have been selected, as they are widely expressed in the body, including cardiac tissue, and they are "canonical representatives" for the potassium selective and the cation non-selective SAC families, respectively. Live cell confocal microscopy and electron tomographic imaging were used to follow binding dynamics of LecA, and to track changes in cell morphology and membrane topology in human embryonic kidney (HEK) cells and in giant unilamellar vesicles (GUV). HEK cells were further transfected with human TREK-1 or Piezo1 constructs, and ion channel activity was recorded using the patch-clamp technique. Finally, freshly isolated cardiac cells were used for studies into cell type dependency of LecA binding. LecA (500 nM) binds within seconds to the surface of HEK cells, with highest concentration at cell-cell contact sites. Local membrane invaginations are detected in the presence of LecA, both in the plasma membrane of cells (by 17 min of LecA exposure) as well as in GUV. In HEK cells, LecA sensitizes TREK-1, but not Piezo1, to voltage and mechanical stimulation. In freshly isolated cardiac cells, LecA binds to non-myocytes, but not to ventricular or atrial cardiomyocytes. This cell type specific lack of binding is observed across cardiomyocytes from mouse, rabbit, pig, and human. Our results suggest that LecA may serve as a pharmacological tool to study SAC in a cell type-preferential manner. This could aid tissue-based research into the roles of SAC in cardiac non-myocytes. [ABSTRACT FROM AUTHOR]

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane. [ABSTRACT FROM AUTHOR]

The plasmamembrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears--globally speaking--homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations. This article is part of the theme issue 'Self-organization in cell biology'. [ABSTRACT FROM AUTHOR]

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell-surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA-fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand-protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700-fold compared to l-fucose. [ABSTRACT FROM AUTHOR]

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell-surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA-fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand-protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700-fold compared to l-fucose. [ABSTRACT FROM AUTHOR]

Motivation Giant Unilamellar Vesicles (GUVs) are widely used synthetic membrane systems that mimic native membranes and cellular processes. Various fluorescence imaging techniques can be employed for their characterization. In order to guarantee a fast and unbiased analysis of imaging data, the development of automated recognition and processing steps is required. Results We developed a fast and versatile Fiji-based macro for the analysis of digital microscopy images of GUVs. This macro was designed to investigate membrane dye incorporation and protein binding to membranes. Moreover, we propose a fluorescence intensity-based method to quantitatively assess protein binding. Availability and implementation The ImageJ distribution package FIJI is freely available online: https://imagej.net/Fiji. The macro file GUV-AP.ijm is available at https://github.com/AG-Roemer/GUV-AP. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Septin GTPases polymerize into higher-ordered structures as a part of the cytoskeleton and are involved in interactions of the host with a wide spectrum of pathogens. Many pathogens foster an ambiguous relationship with septins. They exploit septins for uptake, but septins also prevent their intracellular replication and target them for autophagy. We demonstrate that septins are involved in a defense mechanism against the pathogen Pseudomonas aeruginosa , which enters cells via a lipid zippering mechanism relying on interaction of the lectin LecA with the glycosphingolipid Gb3 on the host membrane. LecA-dependent invagination of the plasma membrane triggers septin recruitment to the site of bacterial attachment. We also find a septin-dependent reinforcement of cortical actin at attachment sites. Atomic force microscopy reveals formation of a septin-dependent rigid barrier below the membrane, preventing bacterial penetration. Our data suggest that septin barriers represent a cellular defense against bacteria inducing membrane curvature for invasion. [Display omitted] • Host cell attachment of P. aeruginosa triggers localized septin recruitment • Septins form a local barrier by increasing cellular rigidity • Septin recruitment inhibits LecA-dependent bacterial uptake into host cells Septins are involved in host-pathogen interactions. Aigal et al. show that septins are recruited to the attachment site of P. aeruginosa , which utilizes a surface lectin to bind to the host membrane, resulting in attachment, membrane deformation, and entry. Septins can prevent this invasion process by locally increasing membrane rigidity. [ABSTRACT FROM AUTHOR]

Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5), a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex) to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs) in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type. [ABSTRACT FROM AUTHOR]

Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains ("lipid rafts"), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium-a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a "lipid zipper," which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA-Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes. [ABSTRACT FROM AUTHOR]

Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites. [ABSTRACT FROM AUTHOR]

Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites. [ABSTRACT FROM AUTHOR]

Lectin LecA is a virulence factor of Pseudomonas aeruginosa involved in lung injury, mortality, and cellular invasion. Ligands competing with human glycoconjugates for LecA binding are thus promising candidates to counteract P. aeruginosa infections. We have identified a novel divalent ligand from a focused galactoside(Gal)-conjugate array which binds to LecA with very high affinity ( Kd=82 n M). Crystal structures of LecA complexed with the ligand together with modeling studies confirmed its ability to chelate two binding sites of LecA. The ligand lowers cellular invasiveness of P. aeruginosa up to 90 % when applied in the range of 0.05-5 μ M. Hence, this ligand might lead to the development of drugs against P. aeruginosa infection. [ABSTRACT FROM AUTHOR]

Lectin LecA is a virulence factor of Pseudomonas aeruginosa involved in lung injury, mortality, and cellular invasion. Ligands competing with human glycoconjugates for LecA binding are thus promising candidates to counteract P. aeruginosa infections. We have identified a novel divalent ligand from a focused galactoside(Gal)-conjugate array which binds to LecA with very high affinity ( Kd=82 n M). Crystal structures of LecA complexed with the ligand together with modeling studies confirmed its ability to chelate two binding sites of LecA. The ligand lowers cellular invasiveness of P. aeruginosa up to 90 % when applied in the range of 0.05-5 μ M. Hence, this ligand might lead to the development of drugs against P. aeruginosa infection. [ABSTRACT FROM AUTHOR]

RATIONALE A rapid and precise analytical method for the investigation of natural products is required for pathway monitoring of the biosynthesis of secondary metabolites. Phenalinolactones, used in antibiotic research, are produced by Streptomyces sp. Tü6071. For the analysis of those compounds, prior to mass spectrometric analysis, an efficient separation technique is required. METHODS For the identification of phenalinolactones from liquid cultures of Streptomyces sp. Tü6071, a new method comprising the combination of solid-phase extraction (SPE) prior to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was established. MS/MS product ion scans were applied for phenalinolactone detection and structure elucidation, performed in negative mode and optimized for sensitivity and specificity. For the discovery of new intermediates, a MS/MS precursor ion scan was applied. RESULTS Analysis of the extracts revealed that the Oasis® MAX cartridge, containing a quaternary amine functionality, is the most efficient SPE material for purification of phenalinolactones, since it allowed sufficient enrichment and detection of intermediates from the biosynthetic pathway by LC/ESI-MS/MS. Using the precursor ion scan technique, two new secondary metabolites, PL IM1 with m/z 672.6 and PL IM2 with m/z 433.3, have been detected. The structures of the new intermediates are postulated and arranged into the biosynthetic pathway of phenalinolactones. CONCLUSIONS A precise analytical method was established for the identification of phenalinolactones by combining purification from Streptomyces using SPE prior to LC/ESI-MS/MS. By optimising LC/ESI-MS/MS settings, this method has been successfully applied for pathway monitoring of secondary metabolites. Application of a precursor ion scan allowed for the identification of unknown intermediates in biosynthetic pathways. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. [ABSTRACT FROM AUTHOR]

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin-independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture ( SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin-induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP-Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor-binding B-subunit of Shiga toxin reaching the trans-Golgi/ TGN membranes was decreased in Rab12-depleted cells, and that cells were partially protected against intoxication by Shiga-like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady-state localizations of TGN46 and cation-independent mannose-6-phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route. [ABSTRACT FROM AUTHOR]

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane. [ABSTRACT FROM AUTHOR]

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein-mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli is an emergent pathogen that can induce haemolytic uraemic syndrome. The toxin has received considerable attention not only from microbiologists but also in the field of cell biology, where it has become a powerful tool to study intracellular trafficking. In this Review, we summarize the Shiga toxin family members and their structures, receptors, trafficking pathways and cellular targets. We discuss how Shiga toxin affects cells not only by inhibiting protein biosynthesis but also through the induction of signalling cascades that lead to apoptosis. Finally, we discuss how Shiga toxins might be exploited in cancer therapy and immunotherapy. [ABSTRACT FROM AUTHOR]

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells. [ABSTRACT FROM AUTHOR]

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb3 in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb3 lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-snglycero- 3-phosphocholine (DOPC)/cholesterol/Gb3 (65:30:5) bilayers. In contrast, membranes composed of DOPC/ cholesterol/sphingomyelin/Gb3 (40:35:20:5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb3, STxB-Gb3 complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations. [ABSTRACT FROM AUTHOR]

Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit–Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes. [ABSTRACT FROM AUTHOR]