Publications by authors named "Josef Madl"

33 Publications

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

Thromb Haemost 2021 Feb 22. Epub 2021 Feb 22.

Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Objectives:  The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis.

Methods And Results:  Atherosclerotic plaques of apolipoprotein E-deficient ( ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCre-driven) deficiency of CD40 in mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs.

Conclusion:  Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.
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http://dx.doi.org/10.1055/a-1397-1858DOI Listing
February 2021

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa.

Cell Mol Life Sci 2021 Apr 8;78(7):3637-3656. Epub 2021 Feb 8.

Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104, Freiburg, Germany.

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 (PIP) 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, PIP and flotillin thereby facilitating efficient uptake of PAO1.
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http://dx.doi.org/10.1007/s00018-021-03766-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038999PMC
April 2021

Quantification of nanoscale forces in lectin-mediated bacterial attachment and uptake into giant liposomes.

Nanoscale 2021 Feb;13(7):4016-4028

Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany. and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany and Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT), University of Freiburg, Georges-Köhler-Allee 105, 79110 Freiburg, Germany.

Interactions of the bacterial lectin LecA with the host cells glycosphingolipid Gb3 have been shown to be crucial for the cellular uptake of the bacterium Pseudomonas aeruginosa. LecA-induced Gb3 clustering, referred to as lipid zipper mechanism, leads to full membrane engulfment of the bacterium. Here, we aim for a nanoscale force characterization of this mechanism using two complementary force probing techniques, atomic force microscopy (AFM) and optical tweezers (OT). The LecA-Gb3 interactions are reconstituted using giant unilamellar vesicles (GUVs), a well-controlled minimal system mimicking the plasma membrane and nanoscale forces between either bacteria (PAO1 wild-type and LecA-deletion mutant strains) or LecA-coated probes (as minimal, synthetic bacterial model) and vesicles are measured. LecA-Gb3 interactions strengthen the bacterial attachment to the membrane (1.5-8-fold) depending on the membrane tension and the applied technique. Moreover, significantly less energy (reduction up to 80%) is required for the full uptake of LecA-coated beads into Gb3-functionalized vesicles. This quantitative approach highlights that lectin-glycolipid interactions provide adequate forces and energies to drive bacterial attachment and uptake.
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http://dx.doi.org/10.1039/d0nr07726gDOI Listing
February 2021

Quantitative collagen assessment in right ventricular myectomies from patients with tetralogy of Fallot.

Europace 2021 Mar;23(Supplement_1):i38-i47

Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center- University of Freiburg, Faculty of Medicine, Elsässer Straße 2Q, 79110 Freiburg, Germany.

Aims: Patients with tetralogy of Fallot (TOF) are often affected by right ventricular fibrosis, which has been associated with arrhythmias. This study aimed to assess fibrosis distribution in right ventricular outflow tract (RVOT) myocardium of TOF patients to evaluate the utility of single histology-section analyses, and to explore the possibility of fibrosis quantification in unlabelled tissue by second harmonic generation imaging (SHGI) as an alternative to conventional histology-based assays.

Methods And Results: We quantified fibrosis in 11 TOF RVOT samples, using a tailor-made automated image analysis method on Picrosirius red-stained sections. In a subset of samples, histology- and SHGI-based fibrosis quantification approaches were compared. Fibrosis distribution was highly heterogeneous, with significant and comparable variability between and within samples. We found that, on average, 67.8 mm2 of 10 µm thick, histologically processed tissue per patient had to be analysed for accurate fibrosis quantification. SHGI provided data faster and on live tissue, additionally enabling quantification of collagen anisotropy.

Conclusion: Given the high intra-individual heterogeneity, fibrosis quantification should not be conducted on single sections of TOF RVOT myectomies. We provide an analysis algorithm for fibrosis quantification in histological images, which enables the required extended volume analyses in these patients.
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http://dx.doi.org/10.1093/europace/euaa389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943371PMC
March 2021

Achilles Subtendon Structure and Behavior as Evidenced From Tendon Imaging and Computational Modeling.

Front Sports Act Living 2020 23;2:70. Epub 2020 Jun 23.

Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.

The Achilles tendon is the largest and strongest tendon in the human body and is essential for storing elastic energy and positioning the foot for walking and running. Recent research into Achilles tendon anatomy and mechanics has revealed the importance of the Achilles subtendons, which are unique and semi-independent structures arising from each of the three muscular heads of the triceps surae. Of particular importance is the ability for the subtendons to slide, the role that this has in healthy tendons, and the alteration of this property in aging and disease. In this work, we discuss technical approaches that have led to the current understanding of Achilles subtendons, particularly imaging and computational modeling. We introduce a 3D geometrical model of the Achilles subtendons, built from dual-echo UTE MRI. We revisit and discuss computational models of Achilles subtendon twisting suggesting that optimal twist reduces both rupture loads and stress concentrations by distributing stresses. Second harmonic generation imaging shows collagenous subtendons within a rabbit Achilles tendon; a clear absence of signal between the subtendons indicates an inter-subtendon region on the order of 30 μm in our rabbit animal model. Entry of wheat germ agglutinin in both the inter-fascicular and the inter-subtendon regions suggests a glycoprotein-containing inter-subtendon matrix which may facilitate low friction sliding of the subtendons in healthy mammals. Lastly, we present a new computational model coupled with human exercise trials to demonstrate the magnitude of Achilles subtendon sliding which occurs during rehabilitation exercises for Achilles tendinopathy, and shows that specific exercise can maximize subtendon sliding and interface strains, without maximizing subtendon strains. This work demonstrates the value of imaging and computational modeling for probing tendon structure-function relationships and may serve to inform and develop treatments for Achilles tendinopathy.
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http://dx.doi.org/10.3389/fspor.2020.00070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7739789PMC
June 2020

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

Basic Res Cardiol 2020 12 9;115(6):78. Epub 2020 Dec 9.

Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, 55 Hugstetter St, 79106, Freiburg, Germany.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.
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http://dx.doi.org/10.1007/s00395-020-00838-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725697PMC
December 2020

Beat-by-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

Circ Res 2021 01 24;128(2):203-215. Epub 2020 Nov 24.

Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, and Faculty of Medicine, University of Freiburg, Germany (E.A.R.-Z., R.P., C.M.Z.-J., J.G., J.M., L.S., P.K.).

Rationale: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates.

Objective: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid.

Methods And Results: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics.

Conclusions: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834912PMC
January 2021

Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology.

Methods Mol Biol 2021 ;2191:287-307

Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center-University of Freiburg, Freiburg, Germany.

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.
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http://dx.doi.org/10.1007/978-1-0716-0830-2_17DOI Listing
March 2021

Sinoatrial Node Structure, Mechanics, Electrophysiology and the Chronotropic Response to Stretch in Rabbit and Mouse.

Front Physiol 2020 22;11:809. Epub 2020 Jul 22.

Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, Canada.

The rhythmic electrical activity of the heart's natural pacemaker, the sinoatrial node (SAN), determines cardiac beating rate (BR). SAN electrical activity is tightly controlled by multiple factors, including tissue stretch, which may contribute to adaptation of BR to changes in venous return. In most animals, including human, there is a robust increase in BR when the SAN is stretched. However, the chronotropic response to sustained stretch differs in mouse SAN, where it causes variable responses, including decreased BR. The reasons for this species difference are unclear. They are thought to relate to dissimilarities in SAN electrophysiology (particularly action potential morphology) between mouse and other species and to how these interact with subcellular stretch-activated mechanisms. Furthermore, species-related differences in structural and mechanical properties of the SAN may influence the chronotropic response to SAN stretch. Here we assess (i) how the BR response to sustained stretch of rabbit and mouse isolated SAN relates to tissue stiffness, (ii) whether structural differences could account for observed differences in BR responsiveness to stretch, and (iii) whether pharmacological modification of mouse SAN electrophysiology alters stretch-induced chronotropy. We found disparities in the relationship between SAN stiffness and the of the chronotropic response to stretch between rabbit and mouse along with differences in SAN collagen structure, alignment, and changes with stretch. We further observed that pharmacological modification to prolong mouse SAN action potential plateau duration rectified the of BR changes during sustained stretch, resulting in a positive chronotropic response akin to that of other species. Overall, our results suggest that structural, mechanical, and background electrophysiological properties of the SAN influence the chronotropic response to stretch. Improved insight into the biophysical determinants of stretch effects on SAN pacemaking is essential for a comprehensive understanding of SAN regulation with important implications for studies of SAN physiology and its dysfunction, such as in the aging and fibrotic heart.
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http://dx.doi.org/10.3389/fphys.2020.00809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388775PMC
July 2020

Differential recognition of lipid domains by two Gb3-binding lectins.

Sci Rep 2020 06 16;10(1):9752. Epub 2020 Jun 16.

Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

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.
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http://dx.doi.org/10.1038/s41598-020-66522-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297801PMC
June 2020

The Lectin LecA Sensitizes the Human Stretch-Activated Channel TREK-1 but Not Piezo1 and Binds Selectively to Cardiac Non-myocytes.

Front Physiol 2020 15;11:457. Epub 2020 May 15.

Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, Medical Center-University of Freiburg, Freiburg, Germany.

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 and essential for -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.
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http://dx.doi.org/10.3389/fphys.2020.00457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243936PMC
May 2020

Dissection of heterocellular cross-talk in vascularized cardiac tissue mimetics.

J Mol Cell Cardiol 2020 01 19;138:269-282. Epub 2019 Dec 19.

Institute for Cardiovascular Regeneration, Goethe University Frankfurt, Germany; Cardiopulmonary Institute, Frankfurt, Germany; Genome Biologics, Frankfurt, Germany. Electronic address:

Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular cross-talk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/β-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon β-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF β2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology - and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.
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http://dx.doi.org/10.1016/j.yjmcc.2019.12.005DOI Listing
January 2020

Phytochrome-Based Extracellular Matrix with Reversibly Tunable Mechanical Properties.

Adv Mater 2019 Mar 27;31(12):e1806727. Epub 2019 Jan 27.

Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104, Freiburg, Germany.

Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.
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http://dx.doi.org/10.1002/adma.201806727DOI Listing
March 2019

GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles.

Bioinformatics 2019 07;35(13):2340-2342

Faculty of Biology, Albert Ludwigs University Freiburg, Schänzlestraße 1, Freiburg im Breisgau, Germany.

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.
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http://dx.doi.org/10.1093/bioinformatics/bty962DOI Listing
July 2019

A microfluidic biochip for locally confined stimulation of cells within an epithelial monolayer.

RSC Adv 2018 Feb;8(14):7839-7846

Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany. Email: Email:

A key factor determining the fate of individual cells within an epithelium is the unique microenvironment that surrounds each cell. It regulates location-dependent differentiation into specific cellular sub-types, but, on the other hand, a disturbed microenvironment can promote malignant transformation of epithelial cells leading to cancer formation. Here, we present a tool based on a microfluidic biochip that enables novel research approaches by providing a means to control the basolateral microenvironment of a confined number of neighbouring cells within an epithelial monolayer. Through isolated single pores in a thin membrane carrying the epithelial cell layer only cells above the pores are stimulated by solutes. The very thin design of the biochip (<75 μm) enabled us to apply a high-resolution inverted confocal fluorescence microscope to show by live cell imaging that such a manipulation of the microenvironment remained locally restricted to cells located above the pores. In addition, the biochip allows access for the force probe of an atomic force microscope (AFM) from the apical side to determine the topography and mechanical properties of individual cells, which we demonstrated by combined AFM and fluorescence microscopy imaging experiments. Taken together, the presented microfluidic biochip is a powerful tool that will enable studying the initial steps of malignant transformation of epithelial cells by directly manipulating their microenvironment and by real-time monitoring of affected cells with fluorescence microscopy and AFM.
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http://dx.doi.org/10.1039/c7ra11943gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830875PMC
February 2018

Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol.

Cell Stress Chaperones 2018 07 5;23(4):673-683. Epub 2018 Feb 5.

Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.

Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.
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http://dx.doi.org/10.1007/s12192-018-0879-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045550PMC
July 2018

Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion.

Sci Rep 2018 01 31;8(1):1932. Epub 2018 Jan 31.

Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, 79104, Freiburg, Germany.

Cell adhesion is a crucial feature of all multicellular organisms, as it allows cells to organise themselves into tissues to carry out specific functions. Here, we present a mimetic approach that uses multivalent lectins with opposing binding sites to crosslink glycan-functionalised giant unilamellar vesicles. The crosslinking process drives the progression from contact puncta into elongated protocellular junctions, which form the vesicles into polygonal clusters resembling tissues. Due to their carbohydrate specificity, different lectins can be engaged in parallel with both natural and synthetic glycoconjugates to generate complex interfaces with distinct lectin domains. In addition, the formation of protocellular junctions can be combined with adhesion to a functionalised support by other ligand-receptor interactions to render increased stability against fluid flow. Furthermore, we consider that adhesion is a complex process of attraction and repulsion by doping the vesicles with a PEG-modified lipid, and demonstrate a dose-dependent decrease of lectin binding and formation of protocellular junctions. We suggest that the engineering of prototissues through lectin-glycan interactions is an important step towards synthetic minimal tissues and in designing artificial systems to reconstruct the fundamental functions of biology.
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http://dx.doi.org/10.1038/s41598-018-20230-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792463PMC
January 2018

HDL particles incorporate into lipid bilayers - a combined AFM and single molecule fluorescence microscopy study.

Sci Rep 2017 Nov 21;7(1):15886. Epub 2017 Nov 21.

TU Wien, Institute of Applied Physics, Vienna, 1040, Austria.

The process, how lipids are removed from the circulation and transferred from high density lipoprotein (HDL) - a main carrier of cholesterol in the blood stream - to cells, is highly complex. HDL particles are captured from the blood stream by the scavenger receptor, class B, type I (SR-BI), the so-called HDL receptor. The details in subsequent lipid-transfer process, however, have not yet been completely understood. The transfer has been proposed to occur directly at the cell surface across an unstirred water layer, via a hydrophobic channel in the receptor, or after HDL endocytosis. The role of the target lipid membrane for the transfer process, however, has largely been overlooked. Here, we studied at the single molecule level how HDL particles interact with synthetic lipid membranes. Using (high-speed) atomic force microscopy and fluorescence correlation spectroscopy (FCS) we found out that, upon contact with the membrane, HDL becomes integrated into the lipid bilayer. Combined force and single molecule fluorescence microscopy allowed us to directly monitor the transfer process of fluorescently labelled amphiphilic lipid probe from HDL particles to the lipid bilayer upon contact.
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http://dx.doi.org/10.1038/s41598-017-15949-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698431PMC
November 2017

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

Genes Dev 2017 09;31(17):1754-1769

Institute of Medical Microbiology and Hygiene, Faculty of Medicine, Medical Center-University of Freiburg, 79104 Freiburg, Germany.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.
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http://dx.doi.org/10.1101/gad.302497.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666674PMC
September 2017

Biglycan expression in the melanoma microenvironment promotes invasiveness via increased tissue stiffness inducing integrin-β1 expression.

Oncotarget 2017 Jun;8(26):42901-42916

Department of Hematology and Oncology, University Medical Center, Faculty of Medicine, Freiburg, Germany.

Novel targeted and immunotherapeutic approaches have revolutionized the treatment of metastatic melanoma. A better understanding of the melanoma-microenvironment, in particular the interaction of cells with extracellular matrix molecules, may help to further improve these new therapeutic strategies.We observed that the extracellular matrix molecule biglycan (Bgn) was expressed in certain human melanoma cells and primary fibroblasts when evaluated by microarray-based gene expression analysis. Bgn expression in the melanoma tissues correlated with low overall-survival and low progression-free-survival in patients. To understand the functional role of Bgn we used gene-targeted mice lacking functional Bgn. Here we observed that melanoma growth, metastasis-formation and tumor-related death were reduced in Bgn-/- mice compared to Bgn+/+ mice. In vitro invasion of melanoma cells into organotypic-matrices derived from Bgn-/- fibroblasts was reduced compared to melanoma invasion into Bgn-proficient matrices. Tissue stiffness as determined by atomic-force-microscopy was reduced in Bgn-/- matrices. Isolation of melanoma cells and fibroblasts from the stiffer Bgn+/+ matrices revealed an increase in integrin-β1 expression compared to the Bgn-/- fibroblast matrices. Overexpression of integrin-β1 in B16-melanoma cells abolished the survival benefit seen in Bgn-/- mice. Consistent with the studies performed in mice, the abundance of Bgn-expression in human melanoma samples positively correlated with the expression of integrin-β1, which is in agreement with results from the organotypic invasion-assay and the in vivo mouse studies.This study describes a novel role for Bgn-related tissue stiffness in the melanoma-microenvironment via regulation of integrin-β1 expression by melanoma cells in both mice and humans.
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http://dx.doi.org/10.18632/oncotarget.17160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522114PMC
June 2017

Gb3-binding lectins as potential carriers for transcellular drug delivery.

Expert Opin Drug Deliv 2017 Feb 16;14(2):141-153. Epub 2016 Dec 16.

a Faculty of Biology , Albert-Ludwigs-University Freiburg , Freiburg , Germany.

Objectives: Epithelial cell layers as well as endothelia forming the blood-brain barrier can drastically reduce the efficiency of drug targeting. Our goal was to investigate lectins recognizing the glycosphingolipid globotriaosylceramide (Gb3) for their potential as carriers for transcytotic drug delivery.

Methods: We utilized an in vitro model based on Madin-Darby canine kidney cells transfected with Gb3 synthase to characterize transcytosis of the Gb3-binding lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin (StxB).

Results: Both lectins were rapidly transcytosed from the apical to the basolateral plasma membrane and vice versa. Whereas StxB proceeded on retrograde and transcytotic routes, LecA avoided retrograde transport. This differential trafficking could be explained by our observation that LecA and StxB segregated into different domains during endocytosis. Furthermore, inhibiting the small GTPase Rab11a, which organizes trafficking through apical recycling endosomes, blocked basolateral to apical transcytosis of both lectins.

Conclusions: Gb3-binding lectins are promising candidates for transcytotic drug delivery. Our findings highlight that LecA and StxB, which both bind Gb3 but exhibit dissimilar valence and molecular structures of their carbohydrate binding sites and can take divergent intracellular trafficking routes. This opens up the possibility of developing tailor-made glycosphingolipid-binding carrier lectins, which take optimized trafficking pathways.
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http://dx.doi.org/10.1080/17425247.2017.1266327DOI Listing
February 2017

Synthesis of Cholesterol-Substituted Glycopeptides for Tailor-Made Glycocalyxification of Artificial Membrane Systems.

Chembiochem 2016 08 17;17(15):1403-6. Epub 2016 Jun 17.

Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.

Synthetic minimal membrane systems are extremely useful for better understanding of complex cellular structures and cell surface processes. We have developed a facile method for synthesis of cholesterylated peptides, each bearing a carbohydrate moiety and a fluorescent tag. The position of the cholesterol moiety on the peptide can be controlled by using a new Fmoc-protected cholesterol-triazole-lysine group, which we constructed by means of solid-phase peptide synthesis. We succeeded in integrating the glyco modules into giant unilamellar vesicles by electroformation or infusion in buffer solution. The glyco-decorated liposomes were recognized by a lectin and had unique topological membrane features. In conclusion, this work is a proof of principle for the functionalization of artificial membranes with a primitive synthetic glycocalyx useful for studying carbohydrate-protein interactions on a simplified cell-like membrane surface.
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http://dx.doi.org/10.1002/cbic.201600258DOI Listing
August 2016

Pseudomonas aeruginosa lectin LecB inhibits tissue repair processes by triggering β-catenin degradation.

Biochim Biophys Acta 2016 Jun 6;1863(6 Pt A):1106-18. Epub 2016 Feb 6.

Faculty of Biology, Schänzlestraße 1, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, Schänzlestraße 18, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Albertstraße 19, 79104 Freiburg, Germany. Electronic address:

Pseudomonas aeruginosa is an opportunistic pathogen that induces severe lung infections such as ventilator-associated pneumonia and acute lung injury. Under these conditions, the bacterium diminishes epithelial integrity and inhibits tissue repair mechanisms, leading to persistent infections. Understanding the involved bacterial virulence factors and their mode of action is essential for the development of new therapeutic approaches. In our study we discovered a so far unknown effect of the P. aeruginosa lectin LecB on host cell physiology. LecB alone was sufficient to attenuate migration and proliferation of human lung epithelial cells and to induce transcriptional activity of NF-κB. These effects are characteristic of impaired tissue repair. Moreover, we found a strong degradation of β-catenin, which was partially recovered by the proteasome inhibitor lactacystin. In addition, LecB induced loss of cell-cell contacts and reduced expression of the β-catenin targets c-myc and cyclin D1. Blocking of LecB binding to host cell plasma membrane receptors by soluble l-fucose prevented these changes in host cell behavior and signaling, and thereby provides a powerful strategy to suppress LecB function. Our findings suggest that P. aeruginosa employs LecB as a virulence factor to induce β-catenin degradation, which then represses processes that are directly linked to tissue recovery.
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http://dx.doi.org/10.1016/j.bbamcr.2016.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859328PMC
June 2016

Injury-Driven Stiffening of the Dermis Expedites Skin Carcinoma Progression.

Cancer Res 2016 Feb 16;76(4):940-51. Epub 2015 Dec 16.

Department of Dermatology, University Medical Center Freiburg, Freiburg, Germany. BIOSS Centre for Biological Signalling Studies, Albert Ludwigs University of Freiburg, Freiburg, Germany.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility disorder characterized by injury-driven blister formation, progressive soft-tissue fibrosis, and a highly elevated risk of early-onset aggressive cutaneous squamous cell carcinoma (cSCC). However, the mechanisms underlying the unusually rapid progression of RDEB to cSCC are unknown. In this study, we investigated the contribution of injury-induced skin alterations to cSCC development by using a genetic model of RDEB and organotypic skin cultures. Analysis of RDEB patient samples suggested that premalignant changes to the dermal microenvironment drive tumor progression, which led us to subject a collagen VII hypomorphic mouse model of RDEB to chemical carcinogenesis. Carcinogen-treated RDEB mice developed invasive tumors phenocopying human RDEB-cSCC, whereas wild-type mice formed papillomas, indicating that the aggressiveness of RDEB-cSCC is mutation-independent. The inherent structural instability of the RDEB dermis, combined with repeated injury, increased the bioavailability of TGFβ, which promoted extracellular matrix production, cross-linking, thickening of dermal fibrils, and tissue stiffening. The biophysically altered dermis increased myofibroblast activity and integrin β1/pFAK/pAKT mechanosignaling in tumor cells, further demonstrating that cSCC progression is governed by pre-existing injury-driven changes in the RDEB tissue microenvironment. Treatment of three-dimensional organotypic RDEB skin cultures with inhibitors of TGFβ signaling, lysyl oxidase, or integrin β1-mediated mechanosignaling reduced or bypassed tissue stiffness and limited tumor cell invasion. Collectively, these findings provide a new mechanism by which RDEB tissue becomes malignant and offer new druggable therapeutic targets to prevent cSCC onset.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1348DOI Listing
February 2016

Signalling to the nucleus under the control of light and small molecules.

Mol Biosyst 2016 Feb;12(2):345-9

Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany and Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, D-79104 Freiburg, Germany.

One major regulatory mechanism in cell signalling is the spatio-temporal control of the localization of signalling molecules. We synthetically designed an entire cell signalling pathway, which allows controlling the transport of signalling molecules from the plasma membrane to the nucleus, by using light and small molecules.
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http://dx.doi.org/10.1039/c5mb00763aDOI Listing
February 2016

Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion.

Biochim Biophys Acta 2016 Feb 10;1860(2):392-401. Epub 2015 Nov 10.

Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, D-79104 Freiburg, Germany; BIOSS-Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Schänzlestraße 18, D-79104 Freiburg, Germany. Electronic address:

Background: Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family.

Methods: Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line.

Results: In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of β1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments.

Conclusion: Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability.

General Significance: After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity.
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http://dx.doi.org/10.1016/j.bbagen.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717121PMC
February 2016

A lipid zipper triggers bacterial invasion.

Proc Natl Acad Sci U S A 2014 Sep 18;111(35):12895-900. Epub 2014 Aug 18.

Faculty of Biology, BIOSS Centre for Biological Signalling Studies, Spemann Graduate School of Biology and Medicine, Albert Ludwigs University Freiburg, 79104 Freiburg, Germany;

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.
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http://dx.doi.org/10.1073/pnas.1402637111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156737PMC
September 2014

Sphingosine-1-phosphate receptors control B-cell migration through signaling components associated with primary immunodeficiencies, chronic lymphocytic leukemia, and multiple sclerosis.

J Allergy Clin Immunol 2014 Aug 26;134(2):420-8. Epub 2014 Mar 26.

Center of Chronic Immunodeficiency, University Medical Center, Freiburg, Germany. Electronic address:

Background: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells.

Objective: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies.

Methods: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy.

Results: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, β-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1.

Conclusion: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.
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http://dx.doi.org/10.1016/j.jaci.2014.01.037DOI Listing
August 2014

Structure, regulation and biophysics of I(CRAC), STIM/Orai1.

Adv Exp Med Biol 2012 ;740:383-410

Institute of Biophysics, University of Linz, Austria.

Ca(2+) release activated Ca(2+) (CRAC) channels mediate robust Ca(2+) influx when the endoplasmic reticulum Ca(2+) stores are depleted. This essential process for T-cell activation as well as degranulation of mast cells involves the Ca(2+) sensor STIM1, located in the endoplasmic reticulum and the Ca(2+) selective Orai1 channel in the plasma membrane. Our review describes the CRAC signaling pathway, the activation of which is initiated by a drop in the endoplasmic Ca(2+) level sensed by STIM1. This in term induces multimerisation and puncta-formation of STIM1 proteins is followed by their coupling to and activation of Orai channels. Consequently Ca(2+) entry is triggered through the Orai pore into the cytosol with subsequent closure of the channel by Ca(2+)-dependent inactivation. We will portray a mechanistic view of the events coupling STIM1 to Orai activation based on their structure and biophysics.
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http://dx.doi.org/10.1007/978-94-007-2888-2_16DOI Listing
September 2012

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells.

Biochim Biophys Acta 2011 Oct 28;1808(10):2581-90. Epub 2011 Jun 28.

Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr 69, A-4040 Linz, Austria.

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.
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http://dx.doi.org/10.1016/j.bbamem.2011.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161180PMC
October 2011
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