Tetraspanner-based nanodomains modulate BAR domain-induced membrane curvature.

The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR domain-induced PM furrows. Here we report a novel function for the Sur7 family of tetraspanner proteins in the regulation of local PM topography. Combining TIRF imaging, STED nanoscopy, freeze-fracture EM and membrane simulations we find that Sur7 tetraspanners form multimeric strands at the edges of PM furrows, where they modulate forces exerted by BAR domain proteins at the furrow base. Loss of Sur7 tetraspanners or Sur7 displacement due to altered PIP2 homeostasis leads to increased PM invagination and a distinct form of membrane tubulation. Physiological defects associated with PM tubulation are rescued by synthetic anchoring of Sur7 to furrows. Our findings suggest a key role for tetraspanner proteins in sculpting local membrane domains. The maintenance of stable PM furrows depends on a balance between negative curvature at the base which is generated by BAR domains and positive curvature at the furrows' edges which is stabilized by strands of Sur7 tetraspanners.

The impact of bilayer composition on the dimerization properties of the Slg1 stress sensor TMD from a multiscale analysis.

The impact of mutual interactions between the transmembrane domains of membrane proteins and lipids on bilayer properties has gained major attraction. Most simulation studies of membranes rely on the Martini force field, which has proven extremely helpful in providing molecular insights into realistic systems. Accordingly, an evaluation of the accuracy of the Martini force field is crucial to be able to correctly interpret the reported data. In this study, we combine atomistic and coarse-grained Martini simulations to investigate the properties of transmembrane domains (TMDs) in a model yeast membrane. The results show that the TMD binding state (monomeric and dimeric with positive or negative crossing angle) and the membrane composition significantly influence the properties around the TMDs and change TMD-TMD and TMD-lipid affinities. Furthermore, ergosterol (ERG) exhibits a strong affinity to TMD dimers. Importantly, the right-handed TMD dimer configuration is stabilized via TMD-TMD contacts by the addition of asymmetric anionic phosphatidylserine (PS). The coarse-grained simulations corroborate many of these findings, with two notable exceptions: a systematic overestimation of TMD-ERG interaction and lack of stabilization of the right-handed TMD dimers with the addition of PS.

Generation of a human iPSC line (MPIi008-A) from a patient with Denys-Drash syndrome.

An induced pluripotent stem cell (hiPSC) line (MPIi008-A) was generated from fibroblasts of a 1-year-old male patient with Denys-Drash syndrome using lentiviral delivery of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. The MPIi008-A iPSC line exhibited typical iPSC morphology and normal karyotype, expressed pluripotent stem cell markers, and showed developmental potential to differentiate into derivatives of all three germ layers in vivo. The hiPSC line harbours a heterozygous missense mutation (R394L) in exon 9 of the WT1 gene.

Lipid-Mediated Association of the Slg1 Transmembrane Domains in Yeast Plasma Membranes.

Clustering of transmembrane proteins underlies a multitude of fundamental biological processes at the plasma membrane (PM) such as receptor activation, lateral domain formation, and mechanotransduction. The self-association of the respective transmembrane domains (TMDs) has also been suggested to be responsible for the micron-scaled patterns seen for integral membrane proteins in the budding yeast PM. However, the underlying interplay between the local lipid composition and the TMD identity is still not mechanistically understood. In this work, we combined coarse-grained molecular dynamics simulations of simplified bilayer systems with high-resolution live-cell microscopy to analyze the distribution of a representative helical yeast TMD from the PM sensor Slg1 within different lipid environments. In our simulations, we specifically evaluated the effects of acyl chain saturation and anionic lipid head groups on the association of two TMDs. We found that weak lipid-protein interactions significantly affect the configuration of TMD dimers and the free energy of association. Increased amounts of unsaturated phospholipids (PLs) strongly reduced the helix-helix interaction, while the presence of anionic phosphatidylserine (PS) hardly affected the dimer formation. We could experimentally confirm this surprising lack of effect of PS using the network factor, a mesoscopic measure of PM pattern formation in yeast cells. Simulations also showed that the formation of TMD dimers in turn increased the order parameter of the surrounding lipids and induced long-range perturbations in lipid organization. In summary, our results shed new light on the mechanisms of lipid-mediated dimerization of TMDs in complex lipid mixtures.

Effect of Cholesterol Versus Ergosterol on DPPC Bilayer Properties: Insights from Atomistic Simulations.

Sterols have been ascribed a major role in the organization of biological membranes, in particular for the formation of liquid ordered domains in complex lipid mixtures. Here, we employed molecular dynamics simulations to compare the effects of cholesterol and ergosterol as the major sterol of mammalian and fungal cells, respectively, on binary mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a proxy for saturated lipids. In agreement with previous work, we observe that the addition of sterol molecules modifies the order of DPPC both in the gel phase and in the liquid phase. When disentangling the overall tilt angle and the structure of the tail imposed by trans/gauche configurations of torsion angles in the tail, respectively, a more detailed picture of the impact of sterols can be formulated, revealing, for example, an approximate temperature-concentration superposition ranging from the liquid to the gel phase. Furthermore, a new quantitative measure to identify the presence of collective sterol effects is discussed. Moreover, when comparing both types of sterols, addition of cholesterol has a noticeably stronger impact on phospholipid properties than that of ergosterol. The observed differences can be attributed to higher planarity of the cholesterol ring system. This planarity combined with an inherent asymmetry in its molecular interactions leads to better alignment and hence stronger interaction with saturated acyl chains. Our results suggest that the high order demonstrated for ergosterol in fungal plasma membranes must therefore be generated via additional mechanisms.

Transcriptome analysis of primary podocytes reveals novel calcium regulated regulatory networks.

Podocytes are pivotal in establishing the selective permeability of the glomerular filtration barrier. Recently, we showed that an increase of the intracellular calcium ion concentration [Ca2+ ] causes a rapid and transient actin reset (CaAR) measurable through live imaging microscopy using lifeact-mCherry as an actin dye in different cell types including the podocyte. This and other studies show the critical role [Ca2+ ] and the actin cytoskeleton play in podocyte homeostasis. To further investigate the role of [Ca2+ ] and the actin cytoskeleton in podocytes, we used a double fluorescent reporter mouse model to establish a primary podocyte culture system. We treated these podocytes temporarily with a Calcium Ionophore and facultatively with Latrunculin A, an inhibitor of actin polymerization. Unbiased genome wide transcriptional analysis identified a transcriptional response in podocytes to elevated [Ca2+ ] levels, affecting mRNA levels of PDGF-BB, RICTOR, and MIR17HG as mediators of Ca2+ -signaling. Comparison of the ex vivo transcriptional response from the primary podocyte culture with glomerular transcripts across a wide spectrum of CKD disease confirmed co-regulation of transcript sets, establishing the disease relevance of the model system. Our findings demonstrate novel [Ca2+ ] regulated gene networks in podocytes deepening our understanding of podocyte biology and disease.

Dissecting the mechanism of signaling-triggered nuclear export of newly synthesized influenza virus ribonucleoprotein complexes.

Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.

A Deregulated Stress Response Underlies Distinct INF2-Associated Disease Profiles.

BACKGROUND: Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Inverted formin 2 (INF2), an actin regulator, mediates a stress response-calcium mediated actin reset, or CaAR-that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. It has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS), as well as to cases of the neurologic disorder Charcot-Marie-Tooth disease that are accompanied by nephropathy, mostly FSGS. METHODS: We used a combination of quantitative live cell imaging and validation in primary patient cells and Drosophila nephrocytes to systematically characterize a large panel of >50 autosomal dominant INF2 mutants that have been reported to cause either FSGS alone or with Charcot-Marie-Tooth disease. RESULTS: We found that INF2 mutations lead to deregulated activation of formin and a constitutive stress response in cultured cells, primary patient cells, and Drosophila nephrocytes. We were able to clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and Charcot-Marie-Tooth disease. Furthermore, we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. CONCLUSIONS: Our results suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of diseased-linked variants of formin. More broadly, these findings indicate that cellular profiling of disease-associated mutations has potential to contribute substantially to sequence-based phenotype predictions.

Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9-mediated genome editing events.

CRISPR-Cas9-mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knockout mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or nonhomologous end joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis, in most cases, failed to identify these multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential and offer practical solutions to correctly identify precisely edited chromosomes.

Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

The vascular endothelium acts as a selective barrier between the bloodstream and extravascular tissues. Intracellular [Ca2+]i signaling is essential for vasoactive agonist-induced stimulation of endothelial cells (ECs), typically including Ca2+ release from the endoplasmic reticulum (ER). Although it is known that interactions of Ca2+ and cAMP as ubiquitous messengers are involved in this process, the individual contribution of cAMP-generating adenylyl cyclases (ACs), including the only soluble AC (sAC; ADCY10), remains less clear. Using life-cell microscopy and plate reader-based [Ca2+]i measurements, we found that human immortalized ECs, primary aortic and cardiac microvascular ECs, and primary vascular smooth muscle cells treated with sAC-specific inhibitor KH7 or anti-sAC-small interfering RNA did not show endogenous or exogenous ATP-induced [Ca2+]i elevation. Of note, a transmembrane AC (tmAC) inhibitor did not prevent ATP-induced [Ca2+]i elevation in ECs. Moreover, l-phenylephrine-dependent constriction of ex vivo mouse aortic ring segments was also reduced by KH7. Analysis of the inositol-1,4,5-trisphosphate (IP3) pathway revealed reduced IP3 receptor phosphorylation after KH7 application, which also prevented [Ca2+]i elevation induced by IP3 receptor agonist adenophostin A. Our results suggest that sAC rather than tmAC controls the agonist-induced ER-dependent Ca2+ response in ECs and may represent a treatment target in arterial hypertension and heart failure.-Mewes, M., Lenders, M., Stappers, F., Scharnetzki, D., Nedele, J., Fels, J., Wedlich-Söldner, R., Brand, S.-M., Schmitz, B., Brand, E. Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

Integration Through Separation - The Role of Lateral Membrane Segregation in Nutrient Uptake.

Nutrient transporters are prominent and ubiquitous components of the plasma membrane in all cell types. Their expression and regulation are tightly linked to the cells' needs. Environmental factors such as nutrient starvation or osmotic stress prompt an acute remodeling of transporters and the plasma membrane to efficiently maintain homeostasis in cell metabolism. Lateral confinement of nutrient transporters through dynamic segregation within the plasma membrane has recently emerged as an important phenomenon that facilitates spatiotemporal control of nutrient uptake and metabolic regulation. Here, we review recent studies highlighting the mechanisms connecting the function of amino acid permeases with their endocytic turnover and lateral segregation within the plasma membrane. These findings indicate that actively controlled lateral compartmentalization of plasma membrane components constitutes an important level of regulation during acute cellular adaptations.

Nephrin Signaling Results in Integrin ß1 Activation.

BACKGROUND: Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. METHODS: To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. RESULTS: Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin ß1 in cultured podocytes. CONCLUSIONS: Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin ß1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

Actin dynamics during Ca2+-dependent exocytosis of endothelial Weibel-Palade bodies.

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Lateral plasma membrane compartmentalization links protein function and turnover.

Biological membranes organize their proteins and lipids into nano- and microscale patterns. In the yeast plasma membrane (PM), constituents segregate into a large number of distinct domains. However, whether and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, physiological function, and endocytic turnover. Using the methionine permease Mup1 as model system, we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner protein Nce102, and signaling through TORC2. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.

A two-phase response of endothelial cells to hydrostatic pressure.

The vascular endothelium is exposed to three types of mechanical forces: blood flow-mediated shear stress, vessel diameter-dependent wall tension and hydrostatic pressure. Despite considerable variations of blood pressure during normal and pathological physiology, little is known about the acute molecular and cellular effects of hydrostatic pressure on endothelial cells. Here, we used a combination of quantitative fluorescence microscopy, atomic force microscopy and molecular perturbations to characterize the specific response of endothelial cells to application of pressure. We identified a two-phase response of endothelial cells with an initial response to acute (1 h) application of pressure (100 mmHg) followed by a different response to chronic (24 h) application. While both regimes induce cortical stiffening, the acute response is linked to Ca2+-mediated myosin activation, whereas the chronic cell response is dominated by increased cortical actin density and a loss in endothelial barrier function. GsMTx-4 and amiloride inhibit the acute pressure response, which suggests that the ENaC Na+ channel is a key player in endothelial pressure sensing. The described two-phase pressure response may participate in the differential effects of transient changes in blood pressure and hypertension.

Self-organization: the fundament of cell biology.

Self-organization refers to the emergence of an overall order in time and space of a given system that results from the collective interactions of its individual components. This concept has been widely recognized as a core principle in pattern formation for multi-component systems of the physical, chemical and biological world. It can be distinguished from self-assembly by the constant input of energy required to maintain order-and self-organization therefore typically occurs in non-equilibrium or dissipative systems. Cells, with their constant energy consumption and myriads of local interactions between distinct proteins, lipids, carbohydrates and nucleic acids, represent the perfect playground for self-organization. It therefore comes as no surprise that many properties and features of self-organized systems, such as spontaneous formation of patterns, nonlinear coupling of reactions, bi-stable switches, waves and oscillations, are found in all aspects of modern cell biology. Ultimately, self-organization lies at the heart of the robustness and adaptability found in cellular and organismal organization, and hence constitutes a fundamental basis for natural selection and evolution.This article is part of the theme issue 'Self-organization in cell biology'.

Intracellular APOL1 Risk Variants Cause Cytotoxicity Accompanied by Energy Depletion.

Population genetic approaches have uncovered a strong association between kidney diseases and two sequence variants of the APOL1 gene, called APOL1 risk variant G1 and variant G2, compared with the nonrisk G0 allele. However, the mechanism whereby these variants lead to disease manifestation and, in particular, whether this involves an intracellular or extracellular pool of APOL1 remains unclear. Herein, we show a predominantly intracellular localization of APOL1 G0 and the renal risk variants, which localized to membranes of the endoplasmic reticulum in podocyte cell lines. This localization did not depend on the N-terminal signal peptide that mediates APOL1 secretion into the circulation. Additionally, a fraction of these proteins localized to structures surrounding mitochondria. In vitro overexpression of G1 or G2 lacking the signal peptide inhibited cell viability, triggered phosphorylation of stress-induced kinases, increased the phosphorylation of AMP-activated protein kinase, reduced intracellular potassium levels, and reduced mitochondrial respiration rates. These findings indicate that functions at intracellular membranes, specifically those of the endoplasmic reticulum and mitochondria, are crucial factors in APOL1 renal risk variant-mediated cell injury.

Apical and basal epitheliomuscular F-actin dynamics during Hydra bud evagination.

Bending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analyzed actin-based processes during asexual bud evagination in the simple metazoan Hydra We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells. We then combined live imaging with conventional phalloidin staining to directly follow actin reorganization. Bending of the Hydra epithelial double layer is initiated by a group of epitheliomuscular cells in the endodermal layer. These cells shorten their apical-basal axis and arrange their basal muscle processes in a circular configuration. We propose that this rearrangement generates the initial forces to bend the endoderm towards the ectoderm. Convergent tissue movement in both epithelial layers towards the centre of evagination then leads to elongation and extension of the bud along its new body axis. Tissue movement into the bud is associated with lateral intercalation of epithelial cells, remodelling of apical septate junctions, and rearrangement of basal muscle processes. The work presented here extends the analysis of morphogenetic mechanisms beyond embryonic tissues of model bilaterians.

Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion.

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations.

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.