Podcasts about CDC42

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550329v1?rss=1 Authors: Tong, C. S., Su, M., Sun, H., Chua, X. L., Guo, S., Ramaraj, R. S., Lee, A. G., Ong, N. W. P., Miao, Y., Wu, M. Abstract: The coordination between actin and microtubule network is crucial, yet our understanding of the underlying mechanisms remains limited. In this study, we used travelling waves in the cell cortex to characterize the collective dynamics of cytoskeletal networks. Our findings show that Cdc42 and F-BAR-dependent actin waves in mast cells are mainly driven by formin-mediated actin polymerization, with the microtubule-binding formin FH2 domain-containing protein 1 (FHDC1) identified as an early regulator. The depolymerization of microtubules coincides with the nucleation of actin waves, and the concurrent release of FHDC1 from microtubule is required for actin waves. Lastly, we show the importance of the actin-microtubule linkage mediated by FHDC1 in crucial cellular processes such as cell division and migration. Our data provided molecular insights into the nucleation mechanisms of actin waves and uncover an antagonistic interplay between microtubule and actin polymerization in their collective dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550042v1?rss=1 Authors: Rutkowski, D. M., Vincenzetti, V., Vavylonis, D., Martin, S. G. Abstract: Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeast Schizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537540v1?rss=1 Authors: Khan, S. T., Ahuja, N., Taib, S., Vohra, S., Cleaver, O., Nunes, S. S. Abstract: The pancreatic islet vasculature displays tissue-specific physiological and functional adaptations that support rapid glucose sensing and insulin response by beta-cells. To uncover the transcriptomic basis of this specialization, we performed a meta-analysis of multi-organ single cell RNA sequencing atlases employing a unique strategy to avoid transcriptomic contamination. We identified biologically relevant genes involved in sphingosine-1-phosphate-mediated insulin secretion (PLPP1, RDX, CDC42), islet basement membrane formation (SPARC, COL15A1), endothelial cell (EC) permeability (PLVAP, EHD4), membrane transporters (CD320, SLCO2A1) and developmental transcription factors (NKX2-3, AHR). These were validated in silico in an independent dataset. We further established the first integrated transcriptomic atlas of human pancreatic ECs and described two unique capillary subpopulations: exocrine and endocrine pancreas ECs. We validated the spatial localization of key markers using RNAscope and immunofluorescence staining on mouse pancreatic tissue cross-sections. Our findings provide novel insights into pancreatic EC heterogeneity and islet EC function with potential implications in therapeutic strategies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535690v1?rss=1 Authors: Kirchenwitz, M., Halfen, J., von Peinen, K., Prettin, S., Kollasser, J., Brakebusch, C. H., Rottner, K., Steffen, A., Stradal, T. Abstract: Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and -E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535147v1?rss=1 Authors: Hladyshau, S., Stoop, J. P., Kamada, K., Nie, S., Tsygankov, D. V. Abstract: Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new research paper was published in Oncotarget's Volume 14 on March 24, 2023, entitled, “Polyisoprenylated cysteinyl amide inhibitors deplete singly polyisoprenylated monomeric G-proteins in lung and breast cancer cell lines.” Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. In their new study, researchers Nada Tawfeeq, Jassy Mary S. Lazarte, Yonghao Jin, Matthew D. Gregory, and Nazarius S. Lamango from Florida A&M University College of Pharmacy Pharmaceutical Sciences and Imam Abdulrahman bin Faisal University tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. “To investigate the hypothesized anticancer mechanisms of the PCAIs through disruption of G-protein function, we checked the effects of the PCAIs on the G-protein levels in lung cancer (A549 and NCI-H1299) and breast cancer (MDA-MB-231 and MDA-MB-468) cell lines.” Following 48 hours of exposure, they found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20–66% after NSL-YHJ-2-27 (5 μM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. Their results show that PCAIs deplete the protein levels of some significant G-proteins which are known to be involved in the migration and invasion of cells (i.e., metastasis) such as RAC1, RHOA, and CDC42. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins. “The initial findings presented here indicate how PCAIs can be used as potent agents in developing new anticancer therapeutics, therefore, more extensive studies need to be done to elucidate on its potency. Although we cannot conclusively explain the exact mechanism of action of PCAIs on how they affect the levels of some G-proteins yet, but we can say that these PCAIs have the ability to affect the progression of cancer.” Research paper: DOI: https://doi.org/10.18632/oncotarget.28390 Correspondence to: Nazarius S. Lamango - nazarius.lamango@famu.edu Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords: PCAIs, G-proteins, KRAS, RHOA, RAC1 About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.528078v1?rss=1 Authors: Ravichandran, Y., Hanisch, J., Murray, K., Roca, V., Dingli, F., Loew, D., Sabatet, V., Boeda, B., Etienne-Manneville, S. Abstract: The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of numerous polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins; the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), whose specific role are not fully elucidated. The two isoforms only differ in their carboxy-terminal sequence, which includes the CAAX motif essential for CDC42 interaction with membrane. Here we show that these divergent sequences do not directly affect CDC42 ability to interact with its binding partners, but rather indirectly influence CDC42-mediated signaling by controlling the distinct subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u is mainly cytosolic and associates with the leading edge plasma membrane of migrating cells, whereas CDC42b mainly localizes to intracellular membrane compartments. During directed persistent migration CDC42u alone fulfills the polarity function while CDC42b embodies the major isoform regulating endocytosis. Both CDC42 isoforms act in concert by contributing their specific functions to promote chemotaxis of neural precursors, demonstrating that the expression pattern of the two isoforms is decisive for the tissue specific behavior of cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.24.529907v1?rss=1 Authors: Basant, A., Way, M. Abstract: Vaccinia virus exiting from host cells activates Src/Abl kinases to phosphorylate A36, an integral membrane viral protein. Phosphorylated A36 binds the adaptors Nck and Grb2 which recruit N-WASP to activate Arp2/3-driven actin polymerisation to promote viral spread. A36 also recruits intersectin, which enhances actin polymerization via AP-2/clathrin and Cdc42. To obtain a better quantitative understanding of this signalling network we still need to know the absolute numbers of the key molecules. To achieve this goal, we have now used fluorescent molecule counting approaches in live cells. There are 1156{+/-}120 A36 molecules on virus particles inducing actin polymerization in HeLa cells. This number, however, is over 2000 in mouse embryonic fibroblasts (MEFs), suggesting that A36 levels on the virion are not fixed. In MEFs, viruses recruit 1032{+/-}200 Nck and 434{+/-}10 N-WASP molecules, suggesting a ratio of 4:2:1 for the A36:Nck:N-WASP signalling network. Loss of A36 binding to either Grb2 or intersectin results in a 1.3- and 2.5-fold reduction in Nck respectively. Despite recruiting comparable numbers of the Arp2/3 activator, N-WASP (245{+/-}26 and 276{+/-}66), these mutant viruses move at different speeds that inversely correlate with the number of Nck molecules. Our analysis has uncovered two unexpected new aspects of Vaccinia egress, A36 levels can vary in the viral membrane and the velocity of virus movement depends on Nck. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.20.529203v1?rss=1 Authors: Nanda, S., Calderon, A., Duong, T.-T., Koch, J., Sachan, A., Xin, X., Solouk, D., Wu, Y.-W., Nalbant, P., Dehmelt, L. Abstract: Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by an increase in migration directionality and reduced migration velocity. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.31.522400v1?rss=1 Authors: Eid, L., Lokmane, L., Raju, P. K., Tene Tadoum, S. B., Jiang, X., Toulouse, K., Lupien-Meilleur, A., Charron-Ligez, F., Toumi, A., Backer, S., Lachance, M., Lavertu-Jolin, M., Montseny, M., Lacaille, J.-C., Bloch-Gallego, E., Rossignol, E. Abstract: Recessive mutations in the TRIO gene are associated with intellectual deficiency (ID), autism spectrum disorder (ASD) and developmental epileptic encephalopathies (DEE). TRIO is a dual guanine nucleotide exchange factor (GEF) that activates Rac1, Cdc42 and RhoA. Trio has been extensively studied in excitatory neurons, and has recently been found to regulate the switch from tangential to radial migration in GABAergic interneurons (INs), through GEFD1-Rac1-dependent SDF1/CXCR4 signalling. Given the central role of Rho-GTPases during neuronal migration and the implication of IN pathologies in ASD and DEE, we investigated the relative roles of both Trio's GEF domains in regulating the dynamics of INs tangential migration. In Trio-/- mice, we observed reduced numbers of tangentially migrating INs, with intact progenitor proliferation. Further, we noted increased growth cone collapse in developing INs, suggesting altered cytoskeleton dynamics. To bypass the embryonic mortality of Trio-/- mice, we generated Dlx5/6Cre;Trioc/c conditional mutant mice, which develop spontaneous seizures and behavioral deficits reminiscent of ASD and ID. These phenotypes are associated with reduced cortical IN density and functional cortical inhibition. Mechanistically, this reduction of cortical IN numbers reflects a premature switch to radial migration, with an aberrant early entry in the cortical plate, as well as major deficits in cytoskeletal dynamics, including enhanced leading neurite branching and slower nucleokinesis reflecting reduced actin filament condensation and turnover. Further, we show that both Trio GEFD1 and GEFD2 domains are required for proper IN migration, with a dominant role of the RhoA-activating GEFD2 domain. Altogether, our data show a critical role of the DEE/ASD-associated Trio gene in the establishment of cortical inhibition and the requirement of both GEF domains in regulating IN migration dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.10.515940v1?rss=1 Authors: Borgqvist, J. G., Gerlee, P., Lundholm, C. Abstract: The formation of buds on the cell membrane of budding yeast cells is thought to be driven by reactions and diffusion involving the protein Cdc42. These processes can be described by a coupled system of partial differential equations known as the Schnakenberg system. The Schnakenberg system is known to exhibit diffusion-driven pattern formation, thus providing a mechanism for bud formation. However, it is not known how the accumulation of bud scars on the cell membrane affect the ability of the Schnakenberg system to form patterns. We have approached this problem by modelling a bud scar on the cell membrane with a hole on the sphere. We have studied how the spectrum of the Laplace-Beltrami operator, which determines the resulting pattern, is affected by the size of the hole, and by numerically solving the Schnakenberg system on a sphere with a hole using the finite element method. Both theoretical predictions and numerical solutions show that pattern formation is robust to the introduction of a bud scar of considerable size, which lends credence to the hypothesis that bud formation is driven by diffusion-driven instability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.09.515863v1?rss=1 Authors: Shouib, R., Eitzen, G. Abstract: Airway epithelial cells can respond to incoming pathogens, allergens and stimulants through the secretion of cytokines and chemokines. These pro-inflammatory mediators activate inflammatory signaling cascades that allow a robust immune response to be mounted. However, uncontrolled production and release of cytokines and chemokines can result in chronic inflammation and appears to be an underlying mechanism for the pathogenesis of pulmonary disorders such as asthma and COPD. The Rho GTPase, Cdc42, is an important signaling molecule that we hypothesize can regulate cytokine production and release from epithelial cells. We treated BEAS-2B lung epithelial cells with a set of stimulants to activate inflammatory pathways and cytokine release. The production, trafficking and secretion of cytokines were assessed when Cdc42 was pharmacologically inhibited with ML141 drug or silenced with lentiviral-mediated shRNA knockdown. We found that Cdc42 inhibition with ML141 differentially affected gene expression of a subset of cytokines; transcription of IL-6 and IL-8 were increased while MCP-1 was decreased. However, Cdc42 inhibition or depletion disrupted IL-8 trafficking and reduced its secretion even though transcription was increased. Cytokines transiting through the Golgi were particularly affected by Cdc42 disruption. Our results define a role for Cdc42 in the regulation of cytokine production and release in airway epithelial cells. This underscores the role of Cdc42 in coupling receptor activation to downstream gene expression and also as a regulator of cytokine secretory pathways. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.514246v1?rss=1 Authors: Bea-Mascato, B., Giudice, G., Pinheiro-de-Sousa, I., Petsalaki, E., Valverde, D. Abstract: BACKGROUND: The primary cilium is a sensory organelle that extends from the plasma membrane. It plays a vital role in physiological and developmental processes by controlling different signalling pathways such as WNT, Sonic hedgehog (SHh), and transforming growth factor {beta} (TGF-{beta}). Ciliary dysfunction has been related to different pathologies such as Alstrom (ALMS) or Bardet-Biedl (BBS) syndrome. The leading cause of death in adults with these syndromes is chronic kidney disease (CKD), which is characterised by fibrotic and inflammatory processes often involving the TGF-{beta} pathway. METHODS: Using genomic editing with CRISPR-CAS9 and phosphoproteomics we have studied the TGF- {beta} signalling pathway in knockout (KO) models for ALMS1 and BBS1 genes. We have developed a network diffusion-based analysis pipeline to expand the data initially obtained and to be able to determinate which processes were deregulated in TGF-{beta} pathway. Finally, we have analysed protein-protein interactions to prioritise candidate genes in the regulation of the TGF-{beta} pathway in Alstrom and Bardet-Biedl syndrome. RESULTS: Analysis of differentially phosphorylated proteins identified 10 candidate proteins in the ALMS1 KO model and 41 in the BBS1 KO model. After network expansion using a random walk with a restart model, we were able to obtain processes related to TGF-{beta} signalling such as endocytosis in the case of ALMS1 or extracellular matrix regulation in BBS1. Protein interaction analyses demonstrated the involvement of CDC42 as a central protein in the interactome in ALMS1 and CDK2 in the case of BBS1. CONCLUSION: In conclusion, the depletion of ALMS1 and BBS1 affects the TGF-{beta} signalling pathway, conditioning the phosphorylation and activation of several proteins, including CDC42 in the case of ALMS1 and CDK2 in the case of BBS1. KEYWORDS: ALMS1, BBS1, ciliopathies, TGF-{beta}, phosphoproteomics, Alstrom syndrome, Bardet-Biedl syndrome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.30.514432v1?rss=1 Authors: Fradet, A., Fitzgerald, J. Abstract: Mutations in INPPL1, the gene coding for SH2 Domain-Containing Inositol 5'-Phosphatase 2 (SHIP2), cause Opsismodysplasia, a severe chondrodysplasia characterized by delayed bone maturation. The mechanism by which the loss of an inositol phosphatase causes a major skeletal developmental defect is unclear. To investigate the role of SHIP2 in mineralization, the INPPL1 gene was deleted in vitro in chondrocyte and osteoblast differentiation models and the effect of the loss of SHIP2 on cell differentiation, subsequent mineralization, and on actin cytoskeleton formation was investigated. The loss of SHIP2 does not impact differentiation but, consistent with the disease phenotype, induces a significant reduction in extracellular matrix mineralization in both cell types. Absence of SHIP2 also altered the actin cytoskeleton to increase cell adhesion and focal adhesion formation. Furthermore, inhibition of actin polymerization in SHIP2-deficient cells rescued the mineralization phenotype. RhoA/ROCK, Cdc42 and Rac1 are the three main RhoGTPases responsible for actin cytoskeleton regulation in bone cells. Specific inhibitors of these RhoGTPases were used to determine the pathways involved in SHIP2-mediated mineralization. Since only the ROCK pathway inhibitor rescued the mineralization phenotype, it is concluded that SHIP2 regulates actin cytoskeleton remodeling and consequently extracellular matrix mineralization by inhibiting the RhoA/ROCK pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512957v1?rss=1 Authors: Jiu, Y., Zhang, Y., Zhang, X., Li, Z., Yang, H., Tang, D., Zhao, S., Zhang, Q., Li, B., Lappalainen, P., Cui, Z., Liu, H., Li, H., Zhao, W. Abstract: Emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great threat to human health and economics. Although SARS-CoV-2 entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the host cell remodeling to facilitate virus invasion process. Here we unveil that SARS-CoV-2 boosts and repurposes filopodia for entry to the target cells. Using SARS-CoV-2 virus-like particle (VLP), real-time live-cell imaging and simulation of active gel model, we reveal that VLP-induced Cdc42 activation leads to the formation of filopodia, which reinforce the viral entry to host cells. By single-particle tracking and sparse deconvolution algorithm, we uncover that VLP particles utilize filopodia to reach the entry site in two patterns, surfing and grabbing, which are more efficient and faster than entry via flat plasma membrane regions. Furthermore, the entry process via filopodia is dependent on the actin cytoskeleton and actin-associated proteins fascin, formin, and Arp2/3. Importantly, either inhibition the actin cross-linking protein fascin or the active level of Cdc42 could significantly hinders both the VLP and the authentic SARS-CoV-2 entry. Together, our results highlight that the spatial-temporal regulation of the actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry, which emerges as an antiviral target. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512253v1?rss=1 Authors: Mahlandt, E. K., Palacios Martinez, S., Arts, J. J. G., Tol, S., van Buul, J. D., Goedhart, J. Abstract: The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated to allow the passage of essential molecules like oxygen, carbon-dioxide, water, ions, and nutrients. The vascular endothelial barrier is defined by cell-cell contacts through adherens and tight junctions. To further investigate the signaling in the endothelium that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. Rho GTPase signaling is confined in space and time. To manipulate the signaling in a temporal and spatial manner we applied optogenetics. Guanine exchange factor (GEF) domains from ITSN1, TIAM1 and p63RhoGEF, activating Cdc42, Rac and Rho respectively, were integrated into the optogenetic recruitment tool iLID. This tool allows for activation at the subcellular level in a reversible and non-invasive manner and thereby to recruit a GEF to local areas at the plasma membrane, enabling the local activation of specific Rho GTPases. The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting Opto-RhoGEFs were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell-size, -roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool i.e. Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511969v1?rss=1 Authors: Bogucka-Janczi, K., Harms, G., May-Coissieux, M., Bentires-Alj, M., Thiede, B., Rajalingam, K. Abstract: The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.19.508514v1?rss=1 Authors: Kamiyama, D., Nishida, Y., Kamiyama, R., Fitch, M. A., Chihara, T. Abstract: The formation of primary dendrites (dendritogenesis) significantly affects the overall orientation and coverage of dendritic arborization, limiting the number and types of inputs a neuron can receive. Previously we reported how a Drosophila motoneuron spatially controls the positioning of dendritogenesis through the Dscam1/Dock/Pak1 pathway; however, how the neuron defines the timing of this process remains elusive. Here we show that the Eph receptor tyrosine kinase provides a temporal cue. We find that, at the onset of dendritogenesis, the Eph receptor recruits the Rho Family GEF Vav to the intracellular domain of Eph, which transiently activates the Cdc42 family of small GTPase. We also show that vap33 (vesicle-associated membrane protein-associated protein) mutants exhibit defects in Cdc42 activation and dendritic outgrowth, indicating Vap33 may play an upstream role in Eph signaling. Together, our result and previous studies argue that the formation of primary dendrites requires the proximity of active Cdc42 and membrane-anchored Pak1 driven by collaborative action between two distinct signaling complexes, Vap33/Eph/Vav and Dscam1/Dock. Signal integration from multiple input pathways would represent a general mechanism for the spatiotemporal precision of dendrite branch formation. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.20.344556v1?rss=1 Authors: Osmak, G., Kiselev, I., Baulina, N., Favorova, O. Abstract: MicroRNAs (miRNAs) are short single-stranded non-coding RNA molecules, which are involved in regulation of main biological processes, such as apoptosis, cell proliferation and differentiation, through sequence-specific interaction with target mRNAs. In this study we propose a workflow for predicting miRNAs function by analyzing the structure of the network of their target genes. This workflow was applied to study the functional role of miR-375 in the heart muscle (myocardium), since this miRNA was previously shown to be associated with heart diseases and data on its function in myocardium are mostly unclear. We identified PIK3CA, RHOA, MAPK3, PAFAH1B1, CTNNB1, MYC, PRKCA, ERBB2, and CDC42 as key genes in the miR-375 regulated network and predicted the possible function of miR-375 in the heart muscle, consisting mainly in the regulation of the Rho-GTPases-dependent signalling pathways. We implemented our algorithm for miRNA function prediction into Python module, which is available at GitHub (https://github.com/GJOsmak/miRNET). Copy rights belong to original authors. Visit the link for more info

This week’s episode includes author Charlotte Andersson and Associate Editor Naveed Sattar as they discuss familial clustering of aortic size, aneurysms, and dissections in the community. TRANSCRIPT: Dr Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast, summary, and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, Associate Editor from the National Heart Center and Duke national University of Singapore. Dr Greg Hundley: And I'm Greg Hundley, director of the Pauley Heart Center at VCU health in Richmond, Virginia. Well, Carolyn, our feature this week has to do with aortic size, aneurysms, and predilection to dissection. But before we get to that, how about if we grab a cup of coffee and go through some of the other articles in the issue? Dr Carolyn Lam: I got my coffee, Greg, and you know what? I'm going to start with quiz for you. Dr Greg Hundley: All right. Dr Carolyn Lam:  True or false, in the setting of obesity and/or diabetes, cardiac substrate metabolism shifts towards increased fatty acid oxidation, while lipid accumulates in the heart? True or false? Of course, you're right. Oh, but there's a part two. Can you guess, by increasing fatty acid oxidation, will we induce or prevent obesity-induced lipotoxic cardiomyopathy? Dr Greg Hundley: I'm going to say, because you asked it in the way you asked it, prevent. Dr Carolyn Lam: Wow. All right. Well, the truth is we didn't really know before today's paper. The specific link between cardiac metabolism and lipotoxic cardiomyopathy was elusive and there was no specific therapy available for this condition. And these authors, Dr Rong Tian from University of Washington and colleagues, hypothesized that cardiac pathology-associated obesity would be attributable to the imbalance of fatty acid supply and oxidation. So using a diet-induced obesity model in the current study, they demonstrated that enhancing fatty acid oxidation through deletion of acetyl-CoA carboxylase 2, was sufficient to prevent obesity-induced cardiomyopathy. So, increasing cardiac fatty acid oxidation alone does not cause cardiac dysfunction, but instead protects against cardiomyopathy in chronically obese mice. The cardiac-protective effect of increasing fatty acid oxidation and obese mice is through maintenance of Parkin-mediated mitophagy, and thus preventing mitochondrial dysfunction. These findings indicate that impaired mitophagy contributes to mitochondrial dysfunction in obese mice, and that targeting the Parkin-dependent pathway is a viable therapeutic intervention for obesity-induced cardiomyopathy. Dr Greg Hundley: Very nice. Carolyn. Dr Carolyn Lam: I'm going to be greedy and go on to my next paper. So Greg, do you think cardiac regeneration is possible? Dr Greg Hundley: Well, Carolyn, I would have said, several years ago, no, but that trip that we took to China with Joe Hill and Hesham Sadek, our Associate Editor and our Chief Editor, convinced me otherwise. So I'm going to definitely answer yes on this one. Dr Carolyn Lam: Oh, Greg, you're just too smart. And speaking of China, this next paper is from there, from co-corresponding authors, Dr Nie and Hu, from Fuwai Hospital National Center for Cardiovascular Disease and Chinese Academy of Medical Sciences and Peking Union Medical College. So, using seven genetic mouse lines, they identify that Oncostatin M is the top upregulated cytokine during neonatal heart regeneration. Oncostatin M is a pleiotropic secretory protein that belongs to the interleukin 6 family, and associates with the pathological process of dilated cardiomyopathy. And these authors found that macrophages promote heart regeneration by secreting Oncostatin M, which promotes cardiomyocyte proliferation via a co-receptor, gp130. Employing RNA-seq and functional screening, they further found that Src-mediated gp130 triggered cardiomyocyte proliferation by activating the downstream signaling pathway involving Yap, with Y357 phosphorylation independent of the Hippo pathway. So the last thing that they did was show that gene therapy with adenovirus-associated virus and coding this activated gp130 improved heart regeneration and pumping function, thus serving as a potential therapeutic target. An amazing paper. Dr Greg Hundley: Very nice, Carolyn. What a great summary and so much detail. Well, Carolyn, I'm going to turn our attention to catecholaminergic polymorphic ventricular tachycardia. And this article comes to us from Dr Jason Roberts, from the Western University. Carolyn, genetic variants in calsequestrin 2 can cause an autosomal recessive form of catecholaminergic polymorphic ventricular tachycardia, though isolated reports have identified arrhythmogenic phenotypes among heterozygotes. So in this study, a total of 112 individuals, including 36 catecholaminergic polymorphic ventricular tachycardia probands, 24 were homozygotes for compound heterozygotes, and 12 were pure heterozygotes, against 76 family members possessing at least one presumed pathogenic calsequestrin 2 variant. These were all identified. Dr Carolyn Lam: Wow, a very precious cohort. So what did they find, Greg? Dr Greg Hundley: This international multicenter study of calsequestrin 2 catecholaminergic polymorphic ventricular tachycardia really redefined its heritability and confirmed that pathogenic heterozygous calsequestrin 2 variants may manifest with a catecholaminergic polymorphic ventricular tachycardia phenotype, indicating a need to clinically screen these individuals. Among individuals heterozygous for a pathogenic calsequestrin 2 rare variant, medical therapy and exercise restriction are likely not necessary in the absence of the catecholaminergic polymorphic ventricular tachycardia phenotype. Though, you have to be certain over time, an intermittent clinical screening to ensure they remain phenotype-negative should be obtained. Dr Carolyn Lam: Wow, Greg, clinically important study there. Well, I'm going to go back to the basic science world and talk about calcineurin. Now, calcineurin has long been implicated in the induction of pathological cardiac remodeling but has not been therapeutically targetable for the prevention of heart failure because of its pleiotropy and our lack of understanding of its specific protein-protein interactions and compartmentation within the cardiomyocyte. Dr Greg Hundley: Okay. Carolyn, do you want me to give background on calcineurin? Dr Carolyn Lam: No, Greg, you're off the hook. I'm going to give you some background on calcineurin. So, calcineurin is the calcium-calmodulin-dependent phosphatase that exists as a heterodimer, consisting of a catalytic subunit and a regulatory subunit. Now, of the three catalytic subunit isoforms, alpha, beta, gamma, it's the beta isoform that appears to be the most important for the development of cardiac hypertrophy. Binding of calcium to the calcineurin regulatory subunit enables binding of the calcium-calmodulin complex, thereby releasing auto-inhibition and freeing the enzyme to dephosphorylate downstream substrates. That's the background. Now, in today's issue, we have this great paper from co-corresponding authors, Dr Kapiloff from Stanford University, and Dr Nikolaev from University Medical Center Hamburg. And, with their colleagues, they described the discovery of a calcineurin catalytic subunit beta binding protein Cdc42-interacting proteins 4, and I'm going to call that CIP4, which functions as a scaffold to sequester the pool of calcineurin near the sarcolemma of cardiomyocytes, where it regulates pro-hypertrophic signaling. These findings have really important implications for understanding how cardiac calcineurin is selectively activated by stress signals, as opposed to the pleiotropic second messenger, calcium, that really floods the cardiomyocytes during each contractile cycle. Furthermore, the data provide proof of concept for an innovative therapeutic approach, whereby CIP4-anchoring activity is selectively inhibited to block the action of a small pathogenic pool of calcineurin as a means of treating heart failure. How about that? This is really discussed in an elegant editorial by doctors, Woulfe, Travers, and McKinsey. Dr Greg Hundley: Very interesting, Carolyn. Sounds like another possibility for treating and managing heart failure. Well, let me share with you some of the other findings in our mailbag this week. First, I've got, from Professor Lang Li and Stephen Wiviott, they swap research correspondence regarding the prior publication entitled, Effect of Dapagliflozin on Atrial Fibrillation in Patients with Type 2 Diabetes Mellitus, Insights from the DECLARE-TIMI 58 Trial. And then Professor Laszlo Littmann has a nice ECG challenge for us related to a high-risk ECG that exposed some downstream worrisome vital signs. Dr Carolyn Lam: In addition, there's a perspective piece by Dr Nambi discussing the fact that a zero-calcium score is desirable, but isn't enough to defer therapy, given that up to one-third of events will occur in this group. There's also an In Depth paper by Dr Borlaug, entitled, “Altered Hemodynamics and End Organ Damage in Heart Failure, The Impact on the Lung and Kidney,” and oh boy, this one is so beautifully illustrated. Just a must read for the understanding of the hemodynamics in the lung and kidney and heart failure. Next is a research letter by Dr Loeys on enrichment of rare variants in the Loeys-Dietz syndrome genes in spontaneous coronary artery dissection, and not in severe fibromuscular dysplasia. And finally, another research letter by Dr Arora on racial differences in serial NT-proBNP levels in heart failure management with insights from the GUIDE-IT Trial. What a rich issue, but let's move on to our future discussion, shall we? Dr Greg Hundley: You bet, Carolyn. Well, listeners, we're now getting to our feature discussion and it's very interesting this week where we're going to evaluate aortic aneurysms. And we have with us one of the lead authors of this paper, Dr Charlotte Andersson from Boston Medical Center, and our own Associate Editor, Naveed Sattar from Glasgow, Scotland. Charlotte, welcome to our feature discussion. Could you tell us a little bit about the background and the hypothesis that you put forward with this study? Dr Charlotte Andersson: The background for this study was based on clinical work and what we observed in clinic. We had a few patients where we had been stricken by the fact that they came in with an acute aortic syndrome and they had a first-degree relative themself with the condition, but they did not look syndromic at all. And we started to wonder, what is the actual risk in the community, in people without obvious syndromic features of suffering from an aortic event itself. And although there are quite a few studies out there that have, to some degree, focused on the familial clustering of aortopathies, there is not a lot of information based on communities and whole entire populations. So we wanted to, frankly, estimate what is the incidence rates of aortic dissections and aortic aneurism in the community if you have a first-degree relative that has suffered from the disease themselves. Dr Greg Hundley: How you organize your study and what was your population and what was your design? Dr Charlotte Andersson: This study was actually based on two independent samples. First, we used the Framingham Heart study population that is very densely phenotypes over many years of spanning three generations of participants, where we looked at people who had at least one parent who had an aortic size in the upper quartile index to body-surface area and adjusted for age and sex. And we saw what's the risk of you, as a child, having an aortic size in the same upper quartile. And second, we looked in the general Danish population, the Danish healthcare system is, as you probably know, governmental funded and we have very good registries of all hospitalizations, all outpatient visits, and so we were able to link more hard clinical events in people with and without a first-degree relative. What we did was we started time when people had an aortic dissection, we identified all the first-degree relatives in these people, and we matched them with up to 10 sex and age match controls from the general population without a first-degree relative with the disease. Dr Greg Hundley: What did you find? Dr Charlotte Andersson: We found that in the Framingham sample, if you had at least one parent who belongs into the upper quartile of aortic size, you had an odds ratio of two to three, adjusted for various clinical risk factors, such as hypertension and smoking yourself. And in Danish population, we found that if you had a first-degree relative with an aortopathy, the hazard rates for you developing the disease yourself was almost a tenfold-increase compared to age and sex match controls. And importantly, seemed like hazard ratios use were, more or less, unchanged when we start adjusting various known risk factors, such as bicuspid aortic valve, Marfan syndrome, and Ehlers-Danlos syndrome, normally those kinds of things. And we also found that the younger your proband were at the time of an acute event, the higher was your relative risk yourself. So among people who were below the age of 50 when they suffered an event, the hazard ratios were up to a 50-fold increase. Dr Greg Hundley: Very nice. Naveed, what attracted you to this article as it was coming through the editorial process? And then second, how do we take the information that Charlotte's just conveyed and will be published here today, how do we take this in the context of what we already know about aortic aneurysms? Dr Naveed Sattar: I think it's a beautiful study, so well done, Charlotte. I think it's a beautiful fusion. As Charlotte said, an in-depth cohort study, which has got very well-measured parameters of systematic points and a fantastic population-based data set from Denmark, which Sweden shares and Scotland shares and relatively small countries like us share. So small countries like Denmark punch above their weight in these kinds of studies, which is fantastic. But there's a rich seam of research that comes from these, and this is one of them. So I think that fusion of two data sets with different strengths and limitations combined giving off same signals is good. I think, as Charlotte said, this is the first major population study to look at this question. So there's been people around the world who have got this sense that the aortic aneurism may well be familial, this provides, probably, some of the best data to suggest, yes, it definitely is. Now the questions going forward is, okay, at what point do you screen everybody's got a family history with a proband, or do you screen those who've got a family history of younger probands? And I think what Charlotte and the team and other people around the world thar are going to look at this say, "Okay, we now think, in addition to screening, for example, in the UK and the US we probably screen just men above 65, where most of the disease is, do we also then implement screening in younger people with family histories? And who do we screen, and when and how? And do we need to develop some kind of risk score?" And then when we do that screening, what do we do about it? Is going to be the questions and I'm sure Charlotte and her colleagues have thought about these things and it'd be interesting to see what her view is on those things. But I think it was a beautiful study in every sense. Dr Greg Hundley: So Charlotte, he's really set you up nicely, what study do we need to perform next in this area? What are you and your group thinking about? Dr Charlotte Andersson: Yeah, I think there are two implications of this study. First, clinical, as Naveed says. They already had a sense that aortic diseases were heritable, and I think these data definitely support that we should probably screen first-degree relatives. And I think, at some extent, this is what the guidelines already encourage us to do. So I'm not sure it would be feasible to randomize people or do a clinical trial where we screen some but not others. I'm not sure that would be ethical. I think the evidence is too strong for familial clustering and that we should probably screen these people. But I think also, our estimates, they are so strong that I suspect that there are likely more genetic variants associated with non-syndromic aortopathies that we are not aware of just yet. So I think the next step would be to try to disentangle the genetics a little bit more. I have seen some preliminary analysis based on the UK Biobank, for instance, and I think there are more genetic variants to come up with also, more common genetic variants, at least, that we are not aware of just yet. So that would be the next step as I see it. Dr Naveed Sattar: And that might particularity in younger probands. Dr Charlotte Andersson: Right. Dr Naveed Sattar: Those with the younger probands, because it looks like, as you said, the hazard ratio, the risks, are so high, it could also potentially be monogenic, but anyway. Dr Charlotte Andersson: I agree. Dr Greg Hundley: Well, Charlotte, Naveed, we really appreciate your time and taking this opportunity to discuss these really interesting findings and helping us understand that, truly, there may be a familial component to understanding this disease process, particularly in patients with aortic aneurysms that may go on to develop aortic dissections. Well listeners, we hope you have a great week and on behalf of Carolyn and myself, catch you on The Run next week. This program is copyright, the American Heart Association, 2020.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.16.155523v1?rss=1 Authors: Benitez-Burraco, A., Fernandez-Urquiza, M., Jimenez-Romero, M. S. Abstract: Copy-number variations of the distal region of the short arm of chromosome 9 are associated with learning disabilities and behavioral disturbances. Deletions of the 9p are more frequent than duplications. We report in detail on the cognitive and language features of a child with a duplication in the 9p24.3 region (arr[hg19] 9p24.3(266,045-459,076)x3). He exhibits marked expressive and receptive problems, which affect to both structural aspects of language (notably, inflectional morphology, complex syntax, and sentence semantics), and to functional aspects (pragmatics). These problems might result from a severe underlying deficit in working memory. Regarding the molecular causes of the observed symptoms, they might result from the altered expression of selected genes involved in procedural learning, particularly, some of components of the SLIT/ROBO/FOXP2 network, strongly related to the development and evolution of language. Dysregulation of specific components of this network can result in turn from an altered interaction between DOCK8, affected by the microduplication in 9p24.3 borne by our proband, and CDC42, acting as the hub component of the network encompassing language-related genes. Still, some genes found strongly upregulated in the subject and not related to these genes, particularly NRCAM, can contribute to the observed problems in the language domain, as well as to specific features of the proband, particularly, his impulsivity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.14.095117v1?rss=1 Authors: Andrews, M. G., Subramanian, L., Kriegstein, A. R. Abstract: Outer radial glial (oRG) cells are a population of neural stem cells prevalent in the developing human cortex that contribute to its cellular diversity and evolutionary expansion. The mammalian Target of Rapamycin (mTOR) signaling pathway is active in human oRG cells. Mutations in mTOR pathway genes are linked to a variety of neurodevelopmental disorders and malformations of cortical development. We find that dysregulation of mTOR signaling specifically affects oRG cells, but not other progenitor types, by changing the actin cytoskeleton through the activity of the GTPase, CDC42. These effects change oRG cellular morphology, migration, and mitotic behavior. Thus, mTOR signaling can regulate the architecture of the developing human cortex by maintaining the cytoskeletal organization of oRG cells and the radial glia scaffold. Our study provides insight into how mTOR dysregulation may contribute to neurodevelopmental disease. Copy rights belong to original authors. Visit the link for more info

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr. Carolyn Lam, associate editor from the National Heart Centre and Duke National University of Singapore. Our featured paper this week is an in-depth paper on the cardiovascular and metabolic heterogeneity of obesity, and we will have a discussion with the authors on the clinical challenges, implications for management, and much more coming right up after these summaries.                                                 How does MRI quantification compare with standard Doppler echo approach to identify organic mitral regurgitation and predict adverse outcomes? Well, our first paper this week addresses this question, led by first and corresponding author, Dr. Penicka from the Cardiovascular Center OLV Clinic in Belgium. These authors studied 258 asymptomatic patients with preserved left ventricular ejection fraction and chronic moderate and severe organic mitral regurgitation by echo. All patients underwent MRI to quantify regurgitant volume of this organic mitral regurgitation by subtracting aortic flow volume from the total left ventricular stroke volume. Severe organic mitral regurgitation was defined as a regurgitant volume of greater or equal to 60 milliliters.                                                 The authors found that mean echo-derived regurgitant volume was an average 17 milliliters larger than the MRI-derived regurgitant volume. Concordant grading of organic mitral regurgitation severity with both techniques was observed in 76% of individuals. In the remaining 24% of individuals with discordant findings between the two techniques, this was mainly observed in patients with late systolic, eccentric, or multiple jets.                                                 The MRI-derived regurgitant volume showed the highest discriminative power among all the imaging parameters to predict all cause mortality or its combination with development of indication for mitral valve surgery. Thus, this study demonstrates that MRI-derived assessments of organic mitral regurgitation are clinically accurate to identify asymptomatic patients with severe organic mitral regurgitation and at first outcomes. This may be particularly so when the mitral regurgitation is late systolic, eccentric, or multiple in jets where misclassification may occur with echo-derived approach.                                                 The next study is the first large population-based study to analyze the association between low-dose ionizing radiation from cardiac procedures and incident cancer in adults with congenital heart disease. First author Dr. Cohen, corresponding author Dr. Marelli from McGill University, studied the population from the Quebec Congenital Heart Disease Database and performed a nested case control study comparing cancer cases with controls matched on sex, congenital heart disease severity, birth year, and age. They found that the cumulative incidence of cancer in adults with congenital heart disease between the ages of 18 and 64 years was 15%. The cumulative low-dose ionizing radiation exposure from cardiac procedures was independently associated with incident cancer after adjusting for age, sex, year of birth, congenital heart disease severity and comorbidities.                                                 Results were similar using either the number of procedures or estimates of the effective doses with a possible dose-related response relationship between the low-dose ionizing radiation exposure level and cancer risk. Thus, increasing exposure to low-dose ionizing radiation from cardiac imaging in adults with congenital heart disease raises concerns about life-long risk of malignancy.  Confirmation of these findings by prospective studies is needed to reinforce policy recommendations for radiation surveillance in patients with congenital heart disease.                                                 The next study characterizes the long-term dynamics of potassium in heart failure and its associated risk of mortality. First and corresponding author, Dr. Nunez from Hospital Clinic University of Valencia in Spain, evaluated the prognostic implications of long-term longitudinal monitoring and dynamics of serum potassium in a prospective and consecutive cohort of patients following a hospitalization for acute heart failure. In these patients, serum potassium was measured at every physician-patient encounter, including hospital admissions and ambulatory settings.                                                 The authors found that on a continuous scale, the followup trajectory of serum potassium levels independently predicted mortality through a U-shaped association with higher risk at both ends of the distribution, and the same was true using potassium categories. Furthermore, dynamic changes in potassium were independently associated with substantial differences in mortality risk. Persistence of normal potassium levels was linked to a higher risk of death compared to patients who maintained or returned to normal values. Conversely, potassium normalization was independently associated with a lower mortality risk.                                                 These findings support the need for close monitoring of serum potassium after an episode of acute decompensated heart failure and suggest that maintaining serum potassium levels within normal range may be considered a therapeutic target.                                                 The next study gives us an example of how functional metabolomics can translate into metabolomics derived biomarkers of disease mechanisms. Co-first authors, Dr. Zhang, Wei, and Li; co-corresponding authors, Dr. Zhu, Li, and Qi from Nanjing, China, studied a cohort of 2324 patients who underwent coronary angiography from four independent centers. They used a combination of ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry in the negative ion mode for untargeted analysis of metabolites in the plasma.                                                 The authors identified a total of 36 differential metabolites related to coronary artery disease progression. In particular, N-Acetyl-neuraminic acid, a metabolic marker highly elevated during coronary artery disease progression, acted as a signaling molecule to trigger RhoA and Cdc42 dependent myocardial injury via activation of the Rho-RACK signaling pathway.                                                 Silencing neuraminidase-1, which is the enzyme that regulates N-Acetyl-neuraminic acid generation, ameliorated myocardial injury in vitro and in vivo. Pharmacologic inhibition of neuraminidase by anti-influenza drugs protected cardiomyocytes and the heart from myocardial injury.                                                 Thus, in summary, functional metabolomics identified a key role for N-Acetyl-neuraminic acid in acute myocardial injury, and targeting neuraminidase-1 may represent an unrecognized therapeutic intervention for coronary artery disease.                                                 The final study addresses the controversy of whether high density lipoprotein, or HDL cholesterol, plays a causal role in cardioprotection. First and corresponding author, Dr. Jensen from Harvard T.H. Chan School of Public Health and colleagues, hypothesized that subspecies of HDL defined by apolipoprotein C3, a key regulator of lipoprotein metabolism, may contribute new information to prediction of cardiovascular risk.                                                 They used immunoaffinity chromatography to measure the apo A1 concentrations of HDL that contained or lacked apolipoprotein C3, or apo C3, in two prospective studies of adults free of coronary heart disease, the Multiethnic Study of Atherosclerosis and the Danish Diet, Cancer and Health Study. They then conducted a meta-analysis that combined these results with the previously published findings from two cohort studies that used similar laboratory methodology to measure lipoproteins.                                                 The authors identified a subspecies of HDL that contained apo C3. HDL that contained apo C3 comprised 5 to 6% of apo A1 or 10 to 15% of HDL cholesterol. In the four prospective studies, HDL containing apo C3 was associated with a greater risk of coronary heart disease, whereas HDL that lacked apo C3 was inversely associated with risk more strongly than the total HDL.                                                 These findings support the hypothesis that apo C3 may mark a subfraction of HDL cholesterol that is associated with higher risk of coronary heart disease. These findings therefore provide novel insights for cardiovascular risk that extend beyond traditional plasma HDL cholesterol concentrations. And that brings us to a close for the summaries. Now for our feature discussion.                                                 For today's featured discussion, we are talking about obesity, a universal issue, or is it? And when we talk about obesity, are we talking about one thing or many things? Today's in-depth review is just such a great paper. I highly recommend it to everyone. So pleased to be discussing it with Dr. Ian Neeland today from UT Southwestern Medical Center.                                                 Ian, first of all, congratulations. A beautiful paper. I learned so much reading it, and I've got so many questions. You started off pointing out that we talk about obesity. We've always defined it by body mass index, but that may not be the ideal biomarker. I love the way you said that. So, tell us a bit more about the reason for this review. Dr Ian Neeland:                Obesity, like you said, we define it by body mass index, but body mass index is such a crude marker. It's great to use for the clinic. It's easy to implement, but it doesn't really tell us a lot of information about the person. And so you can just look at a third of the population in the US right now is thought to be obese. And if you take a third of the population, clearly not everyone has diabetes and heart disease.                                                 So, obesity in and of itself, defined by the body mass index really is very heterogeneous, and it's not possible to use that alone to tell an individual if they're really at risk for disease. And so this review is really about getting deeper under the skin, no pun intended, to really get a sense of what it means to be obese, how the body fat plays a role in disease, and really getting to the different aspects of obesity and how we can understand it a little bit better. Dr Carolyn Lam:                Yeah. You know, Ian, you had me at hello if I could say when I read your paper because I'm from Asia, and here, the World Health Organization actually even suggests that we use lower body mass index cutoffs to define obesity, simply because there's a different relationship as well with cardiometabolic disease. So, so true, but before we get there, to maybe ethnic differences, I want to ask you something. I heard the term, obesity paradox, thrown around a lot, and sometimes I think we don't really know what we're talking about when we say obesity paradox.                                                 I love the way, in your paper, you broke it down into four types. There are four paradoxes. Do you want to just clarify this for the audience? I think it's important. Dr Ian Neeland:                So, the obesity paradox, what we mean by that is we think that obesity causes disease and gives someone an increased risk for disease and mortality and death, but the obesity paradox means that some people who are obese we see actually have better outcomes than those who are not obese. And how to describe that paradox and why that exists is really the subject of lots and lots of research and discussion.                                                 And so when we talk about the obesity paradox, really it's important to understand that most of the time we're talking about people who already have established disease. Let's say, for example, heart disease. So people with heart disease who are obese tend to have better outcomes than those who are not, and there are a few ways to understand that.                                                 So people who have obesity with established disease who may have better outcomes; that's the classic obesity paradox. Then there's a paradox really about fitness and being fat and fit, and that concept that you can be fat, but if you're fit, if you're able to do exercise and you have good cardiorespiratory fitness, that you actually may be protected from disease as well. And then there's also the obesity paradox of basically the pre-obesity paradox, so that overweight, right, where you haven't yet met the threshold for obesity can also be protective in people who don't have disease. And so being a little bit plump may be protective for different diseases down the road. And then the final one is that the metabolically health obesity. When we say that, it means that the person who is obese by body mass index but doesn't really have any hypertension or diabetes or lipid abnormalities. So, that's the metabolically healthy obese person.                                                 Those are the four types of individuals we see who may be obese but actually have better outcomes long term, and the question is why that exists. So there's a lot of thinking about it. Maybe it has to do with the fact that being normal weight nowadays, often we have older folks that are normal weight. Well, they tend to be more deconditioned. They may be frail. They may have undiagnosed disease like cancer. And that might be why those people are the worst. And there are the naysayers out there who think that it's all just about what we call confounding, so things we can't account for when we look at that. People who smoke tend to be lower weight, and obviously they have worse outcomes, and then also people who are older.  So it's kind of a conundrum, this obesity paradox, but there's lots and lots of data out there coming out all the time that we keep seeing it again and again and again.                                                 One of the areas in the paper that I wanted to address was this concept of obesity heterogeneity in the obesity paradox, meaning to say is it potentially where the body fat is that may be playing a role in which obese person gets disease, and which obese person may be protected from disease. So it could be that it's not how much fat you have but where that fat is that is really telling about what someone's risk is, and that might help to describe the obesity paradox and get us a little bit more understanding. Dr Carolyn Lam:                Yeah, now, I thought that bit was just so key and important. Not how much fat, not weight per se, but where that fat is. Do you want to elaborate on that a bit? Dr Ian Neeland:                Sure. For, I don't know, 50, 60 years we've had this concept of the apple and the pear. Right? Fat in the belly being the apple shape and fat in the pear being fat in the hips and buttocks and that being two different body types of body fat. So we have a lot of technology nowadays, and we can actually directly image body fat and where it is in the body. So we can do MRI, we can do CT, and we can actually see where the body fat is distributed and how much body fat in one area may be related to disease compared with another area.                                                 So we've gone away from the apple and pear and really getting down to what we call body fat depots or adipose tissue depots where we deposit fat. And the area that we deposit fat that has the most risk for cardiometabolic diseases is this visceral adipose tissue or VAT.  VAT is fat that's around the intra-abdominal organs, also near the kidneys, and you can't actually tell how much visceral fat someone has just by BMI or waist circumference or just looking at them. You really have to do this dedicated imaging to find out. And the reason for that is that in the belly there's two types of fat. There's the visceral fat, and there's the subcutaneous, which is the fat under the skin. Both those fat areas make up the belly fat, but they're very different. And part of the review is really going into depth about why these are different and how they're different.                                                 They have completely different metabolic profiles, so if you would take blood, lipids, inflammatory markers, they would look completely different even in a single individual. And then if you look at the genetics of where the fat is, they're different. If you look at what these fat areas secrete, they're completely different. So it's really important to know where the fat is, and that's why I think this concept of sick fat versus healthy fat comes into play.                                                 So, sick fat is fat that's usually in this visceral fat depot, and that is really the three central tenets we talk about are visceral fat or ectopic fat. Ectopic means fat where it doesn't belong. Then inflammation and cytokines, so secretion of abnormal factors in the blood from this fat, and then insulin resistance. So those are the three kind of tenets of this sick fat.  So that's why we think that the sick fat plays a role in disease, and then there's a concept of less sick fat or healthy fat, which is maybe a sink. It actually buffers some of these cytokines and inflammation from causing disease in the body. Dr Carolyn Lam:                Yeah. I found that concept so fascinating, and just to bring it back to the obesity paradox. So, some larger people may enjoy better outcomes because they actually have a predisposition to put the fat subcutaneously perhaps, rather than viscerally. Would that be correct? You worded it so eloquently in your paper. There are some ethnicities or some genetic predispositions that could make one lose that inability to put it peripherally, and therefore it all goes viscerally, is what I got from it. And that's the stuff that puts people at risk. Dr Ian Neeland:                Yeah. We find that fat in the lower body, the hips and the buttocks, is actually in epidemiology, protective against heart disease, protective against cancer. And the problem is we don't know why some people put fat in the belly and some people put it in the hips and buttocks. There's very interesting twin-twin studies that show if someone has a predisposition for obesity, so twins may be both obese, but there is some difference in where they actually put the fat. So I think genetics certainly plays a role, but environment also plays a role. And environments, things like appropriate nutrition and physical activity can really alter genetics and help someone to put fat where it should be and prevent disease.                                                 So this obesity paradox, this concept of putting fat where it should be, is really the next frontier for this type of research. How can we modulate it? How can we fix it? Dr Carolyn Lam:                Exactly, and I love the way you ended your review when you said, "Therefore, maybe in all our complaints and so on, saying that we want weight loss, we should actually be focusing on waist loss. You could redistribute the fat to healthy areas, not change your weight, and still become healthier."  That was the concept, right? Dr Ian Neeland:                That's right. Yeah. It really is amazing, and it's been shown again and again that people can stay the same weight, but their body fat really is very plastic. It can change, and it's modifiable. And that really makes a difference with health outcomes. So whether we can do that with lifestyle changes, so there's some data to support that. There's also some data to support pharmacology, so medications may be able to move fat from one area to another. And then certainly surgery, which is now getting a lot of popularity for people who are really high risk for cardiometabolic disease. Bariatric surgery has been shown to decrease visceral fat significantly, and that may be one of the reasons why it works so well. Dr Carolyn Lam:                Exactly, Ian. Fascinating, fascinating. I tell you what. Could I just ask you to give us some take-home messages? Dr Ian Neeland:                Sure. So one take-home message I think is that we can move beyond the BMI, beyond the body mass index. Obesity is no longer just a number. It's really about the entire individual, biologic systems, what's going on, and there's just remarkable heterogeneity in the structure of obesity, where body fat is, the activity of body fat, the physiology of it, and also how it relates to diseases, either causing disease and potentially being protective for harmful outcomes.                                                 I think it's also a key message to understand that there's sick fat and there is healthy fat and they're very different. And we can get to the bottom of those using specialized tools like imaging and special testing, but they're really very different, and not all body fat is created equal.                                                 And then lastly, I think it's important to consider, like you mentioned earlier, that really public health and lifestyle going forward is going to be so important, and focusing on those areas that will have the biggest impact for people such as trying to promote waist loss, like you said, as opposed to weight loss. Really focusing and using our knowledge of body fat and obesity and how it's so different across individuals and populations, that it's really important to use that knowledge for our future goals and to have that mind when we recommend weight-modifying therapies for our patients.                                                 It's really going to be a new frontier in weight. We're really moving beyond this concept of just check your weight and your height, and we can tell you what your risk is. No, it's really much more complex and complicated and much more interesting than that. Dr Carolyn Lam:                Oh, Ian, that's just so wonderful. I cannot help this last question. Who knows whether we'll put it in, but I just have to ask you. So how do you monitor your own status or your patients' status? Do you really get them DEXAs, all of them? Or PETs, FDGs? Or do you take your own weight? Dr Ian Neeland:                Yeah. I do. One thing I have noticed, I actually started an exercise and diet program for myself to improve my health about a year and a half ago. I took the research, and I said, "Okay, I'm really going to use this and apply this to my life." So, what's interesting is what I found and actually what other colleagues of mine in research are finding is that you can actually melt away visceral fat just with exercise alone, even if you don't actually go on a diet. And they've done studies like this where they do DEXA scans, and they give people high-intensity interval training. They don't give them a special diet. They just say maintain your current diet, and the visceral fat goes away.                                                 It's really remarkable how lifestyle can be so important and make such a change. And you can see people who have diabetes who can cure their diabetes with a lifestyle program by really decreasing the visceral fat. Even if their weight doesn't change or only changes by a small amount, but their weight may change by, I don't know, five, 10 pounds, but their visceral fat may go away by 50%. And that really makes the difference.                                                 It's obviously hard to monitor. We don't really have these tools clinically every day. Not everyone can do a DEXA and has the software to measure the visceral fat. Certainly could be coming in the future, but right now we should use the tools we do have and use the biomarkers we have and the clinical use, the waist circumference, triglycerides. These things are all surrogates for visceral fat but can be very useful to monitor for change. And it's not just about the scale. It's really about more than that with a person's metabolic status. Dr Carolyn Lam:                That is so helpful. Thank you so much, and I'm so glad you said that it was exercise, and you don't jump into a ice pool or something to try and convert the fat to brown fat or something. That's really, really encouraging to me. Thank you, Ian. This was so enjoyable. I'm sure all our listeners are thanking you as well.                                                 Listeners, you've been listening to Circulation on the Run. Please tune in again next week.  

P-cadherin provides the driving force for collective cell migration Collective cell migration is an important process in normal development, wound repair, and tumor metastasis. Plutoni et al. reveal that the cell adhesion molecule P-cadherin promotes collective cell migration via the small GTPase Cdc42, inducing cell polarization and increasing the strength and orientation of mechanical forces. This biosights episode presents the paper by Plutoni et al. from the January 18th, 2016, issue of The Journal of Cell Biology and includes an interview with the paper's senior author, Cécile Gauthier-Rouvière (Centre de Recherche de Biochimie Macromoléculaire, Centre National de la Recherche Scientifique, Montpellier, France). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Cdc42 prompts a change of heart The apical surfaces of polarized epithelia are covered by short, actin-rich protrusions called microvilli, but what happens to these structures when cells detach from a monolayer during development or disease is unclear. Klingner et al. reveal that non-confluent epithelial cells form longer, more dynamic microvilli on their apical surface that connect to a cortical actomyosin network. This biosights episode presents the paper by Klingner et al. from the October 13, 2014, issue of The Journal of Cell Biology and includes an interview with the paper's senior author, Roland Wedlich-Söldner (University of Münster, Germany). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Cdc42 prompts a change of heart The Drosophila heart is a simple tubular structure with a central lumen. Vogler et al. reveal that the small GTPase Cdc42 and actin-nucleating formin proteins promote lumen formation by regulating the dynamics of non-muscle myosin in cardioblasts during heart morphogenesis. This biosights episode presents the paper by Vogler et al. from the September 29, 2014, issue of The Journal of Cell Biology and includes an interview with two of the paper's authors, Georg Vogler and Rolf Bodmer (Sanford-Burnham Medical Research Institute, La Jolla, CA). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Wed, 6 Nov 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18855/ https://edoc.ub.uni-muenchen.de/18855/1/Schulz_Anna.pdf Schulz, Anna ddc:610, ddc:600,

Astrocytes present a major population of glial cells in the adult mammalian brain. The heterogeneity of astrocytes in different regions of the healthy central nervous system (CNS) and their physiological functions are well understood. In contrast, rather little is known about the diversity of astrocyte reactions under pathological conditions. After CNS injury the reaction of astrocytes, also termed ‘reactive astrogliosis’, is characterized by morphological and molecular changes such as hypertrophy, polarization, migration and up-regulation of intermediate filaments. So far, it was unknown whether all astrocytes undergo these changes, or whether only specific subpopulations of reactive astrocytes possess special plasticity. Since some quiescent, postmitotic astrocytes in the cortical gray matter apparently de-differentiate and re-enter the cell cycle upon injury, reactive astrocytes have the ability to acquire restrictive stem cell potential. However, the mechanisms leading to increased astrocyte numbers after acute injury, e.g. proliferation and migration, had not been investigated live in vivo. For the first time, recently established in vivo imaging using 2-photon laser scanning microscopy (2pLSM) allowed to follow single GFP-labeled astrocytes for days and weeks after cortical stab wound injury. Tracing morphological changes during the transition from a quiescent to reactive state, these live observations revealed a heterogeneous behavior of reactive astrocytes depending on the lesion size. Different subsets of astrocytes either became hypertrophic, polarized and/ or divided, but never migrated towards the injury. Intriguingly, the lack of astrocyte migration was not only contradictory to what had been predicted based on in vitro and in situ studies, but was also in stark contrast to the motility of other glial cells. Additionally, live imaging provided first evidence that only a small subset of reactive astrocytes in juxtavascular positions re-gains proliferative capacity after injury. While astrocyte proliferation was affected by conditional deletion of RhoGTPase Cdc42 – a key regulator of cell polarity –, the vascular niche was preserved, indicating that juxtavascular astrocytes are uniquely suited for proliferation after injury. Following the behavior of cdc42-deficient astrocytes by live imaging using an in vitro scratch wound assay, cell-autonomous effects including disturbed polarity and impaired directional migration confirmed a crucial role of Cdc42 signaling in reactive astrocytes after acute injury in vitro and in vivo. These novel insights revise current concepts of reactive astrocytes involved in glial scar formation by assigning regenerative potential to a minor pool of proliferative, juxtavascular astrocytes, and suggesting specific functions of different astrocyte subsets after CNS trauma.

Thu, 14 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16018/ https://edoc.ub.uni-muenchen.de/16018/1/Freisinger_Tina.pdf Freisinger, Tina ddc:570, ddc:500, Fakultät für Biologie

Fibroblasts moving across two-dimensional surfaces form lamellipodial protrusions at their leading edge, but how cells move through three-dimensional environments is less well understood. Petrie et al. reveal that, depending on the strength of intracellular RhoA signaling and on the elastic properties of the extracellular matrix, fibroblasts can migrate through 3D environments using either lamellipodia or blunt, cylindrical protrusions called lobopodia. This biosights episode presents the paper by Petrie et al. from the April 30, 2012, issue of The Journal of Cell Biology and includes an interview with first author Ryan Petrie (NIH, Bethesda, MD). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research. Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Insights into the developmental processes during which the brain forms from the neuroepithelium may provide a deeper understanding how the brain works. The Rho family of small GTPases is known for its many cell biological functions such as regulation of the cytoskeleton, gene expression, cell migration, adhesion, cell polarity and the cell cycle. All of these functions are of importance during the formation of the cerebral neocortex, which consists of the generation of its different cell types, their migration to their destination and their maturation to a functional network. These roles have been mostly established in vitro using dominant negative or constitutively active constructs. Since these approaches are often not entirely specific for single pathways, this work used the Cre/loxP system to genetically delete an individual member of the Rho family, RhoA, to examine its role following a loss-of-function approach. Specifically, we examined a mouse line where part of the RhoA gene has been deleted by means of the Emx1::Cre mouse line. This idea is based on previous experiences with the deletion of Cdc42 in the developing neocortex, which leads to a loss of apical progenitors. RhoA often works as a functional antagonist to Cdc42. Using immunofluorescence, we could detect a loss of RhoA at embryonic day 12 (E12) in Emx1::Cre-positive offspring carrying the floxed RhoA-construct in both alleles (cKO). At E14, we detected an increase in mitotic cells to 160% (±25%, p

Most migrating cells place their centrosome in front of the nucleus, facing the direction in which the cell is moving. Manneville et al. identify a signaling pathway that moves the centrosome into position by recruiting the motor protein dynein to leading edge microtubules. This biosights episode presents the paper by Manneville et al. from the November 1, 2010 issue of The Journal of Cell Biology, and includes an interview with senior author Sandrine Etienne-Manneville (Institut Pasteur, Paris, France). Produced by Caitlin Sedwick and Ben Short.   Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Astrocytes perform many functions in the adult brain and even act as neural stem cells after brain injury (Buffo et al., 2008) or in regions where neurogenesis persists, e.g. in the subependymal zone of the lateral ventricle. The stem cell astrocytes possess an apicobasal polarity as they are coupled by adherens junctions to neighbouring ependymal cells and possess an apical membrane domain with CD133 and Par complex proteins and a basolateral membrane domain including contact of processes to the basement membrane (BM). This is notably different from parenchymal astrocytes that only have contacts to the BM under physiological conditions. The major underlying question is how differences between neural stem cells and 'normal' astrocytes are generated and how polarity mechanisms may be involved in generating this difference. Here, I set out to determine the role of BM contact and the Par complex for astrocyte function in the normal brain parenchyma as well as in the neurogenic niche. First, I examined the influence of BM-mediated signaling by conditional deletion of β1-integrin, one of the major BM receptors in the CNS. The use of specific Cre lines resulted in a loss of β1-integrin protein only at postnatal stages either in both glia and neurons or specifically in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and upregulation of the intermediate filaments GFAP and Vimentin as well as pericellular components, such Tenascin-C and the 473HD proteoglycan. This reaction to the loss of β1-integrin was further accompanied by non-cell autonomous activation of microglial cells. However, neither reactive astrocytes nor microglia divided, suggesting that the loss of β1-integrin-mediated signaling is not sufficient to elicit proliferation of these cells. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood-brain barrier disturbances. As these effects did not appear after neuron-specific deletion of β1-integrin, we conclude that β1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, non-reactive state. Interestingly, neural stem cell astrocytes in the SEZ were not affected in their proliferation and fate, suggesting that β1-integrins are not involved in the regulation of these stem cell properties. However, loss of β1-integrins interfered with the normal dedifferentiation of astrocytes into stem cells after brain injury. Next, I examined the role of Cdc42, a key activator of the Par complex, but also a mediator of β1-integrin signalling in adult stem cell astrocytes. Therefore, I genetically deleted this small RhoGTPase in astroglia at adult stages. In contrast to what has been observed during development, loss of Cdc42 had no influence on proliferation or fate of subependymal zone astrocytes. These effects on adult astroglia-like stem cells differ profoundly from effects on parenchymal astrocytes upon injury. Here, deletion of Cdc42 resulted in severe defects of astrocyte polarity as measured by centrosome reorientation and oriented process extension in the scratch assay in vitro. In vivo, astrocytes could still orient towards the injury site suggesting the existence of compensating signaling pathways. However, the increase of astrocyte numbers around the injury site was reduced. Impaired proliferation certainly contributes to this phenotype. Most importantly, loss of Cdc42 resulted in a significantly increased size of brain injury enlightening the importance of this pathway in the wound reaction towards brain injury. Conversely, no effects were seen by Cdc42 deletion in astrocytes in the absence of injury, suggesting that integrin-mediated signaling from the BM maintains the hallmarks of mature non-reactive astrocytes while Cdc42, most likely via activation of the Par complex, regulates polarity and dedifferentiation after injury. Taken together, this work elucidated for the first time specific signaling pathways regulating the role of astrocytes as stem cells during wound reaction of the injured brain.

Discover how a well-known polarity protein keeps intestinal cells facing the right way by controlling their division direction. This biosights episode presents a paper by Jaffe et al. in The Journal of Cell Biology, and includes excerpts from an interview with senior author Alan Hall. Produced by Justin Paul and Ruth Williams.

Inhalational anthrax is an acute infectious disease caused by exposure of the lungs to B. anthracis spores. Alveolar macrophages engulf spores causing them to germinate to the vegetative form of B. anthracis, which secretes edema toxin (ET)and lethal toxin (LT). The pathogenesis of inhalational anthrax is characterized by flu-like symptoms, respiratory distress, meningitis and shock, which is fatal in almost all cases. The mechanism behind the respiratory distress is not well understood. Therefore, our goal was to determine the effects of lethal toxin in the human lung epithelium. To study alterations in a more physiological setting, we developed a differentiated, polarized lung epithelial system. Lethal toxin exposure disrupted the lung barrier function and wound healing. Assembly defects of junction proteins and additional multicellular junction sites resulted in a higher permeability. Pretreatment with keratinocyte growth factor (KGF) and dexamethasone increased the viability, resulting in the rescue of the permeability changes. Upon LT treatment, a more rigid cytoskeleton was observed, evidenced by enhanced actin stress fiber formations and tubulin stabilization. Cytoskeleton and adhesion alterations prevented the epithelial cells from polarization, directed migration, and wound healing. The MAPK pathway and Cdc42 activity might be partially responsible for these motility defects. Lethal toxin is known to induce rapid cell death in murine macrophages. In contrast, human epithelial cells are more resistant to the cytotoxic effect of LT. By following the growth of epithelial cells after LT treatment, we observed inhibited cell proliferation due to a cell cycle arrest in the G1 phase. Surprisingly, biotinylated lethal factor did not induce cytotoxicity in murine macrophages. This is not due to an internalization or proteolytic activity defect; instead changes in the mitochondrial potential and proteasome activity were observed. Biotinylated LT did not reduce proteasome activity as seen in LT treated cells and caused hypopolarization of the mitochondria. However, it is possible that biotinylation of lethal toxin could prevent interaction of LT with proteins that induce cell death. The major challenge for anthrax treatment is to find a treatment, which can act faster, is easy to use and can bring patient out of the dangerous physiological state in late pathogenesis. Our study has implications in saving the viability and barrier function of lung epithelial cells. One can devise better dosage and delivery of KGF and dexamethasone as treatment modality for post anthrax exposure to reduce respiratory distress. Furthermore, overcoming the cell cycle arrest by the development of a drug would reduce the damage of lung epithelial cells and induce proliferation. The discovery that biotinylated LT is non-toxic to murine macrophages could revolutionize treatment of anthrax infection. Exploring the types of posttranslational modifications of LT that decrease toxicity and finding the mechanism behind it might, lead to therapies that directly counteract the effects of the lethal toxin in vivo.

Cell migration plays a central role in the development and maintenance of multicellular organisms. It involves regulated cell adhesion, mediated by integrins, and polarized changes of the cytoskeleton, controlled by Rho GTPases such as Cdc42. Aim of this study was to investigate the role of integrins and Cdc42 in cell migration and in particular the cross-talk between these molecules. In addition, the structure–function relationship of beta1 integrin in mediating migration associated events was studied. To test whether Cdc42 is essential for directed cell migration in mammalian cells and to investigate the cross-talks between integrin and Cdc42 mediated signalling, fibroblastoid cell lines lacking a functional Cdc42 gene were established and analyzed in wound closure assays. Contrary to the expectations, we could show that Cdc42 is neither required for integrin activation nor for integrin mediated protrusion formation. Moreover, Cdc42 has no significant influence on the speed of directed migration. However, it contributes to the directionality of migration and to the re-orientation of the Golgi apparatus into the direction of migration by a mechanism independent of Gsk3beta phosphorylation. Furthermore, we demonstrated that Cdc42 controls cell morphology, quite likely by regulating Rac1 activity. Expression of dominant negative Cdc42 (dnCdc42) in Cdc42-null cells revealed that dnCdc42 non-specifically inhibits other Rho GTPases besides Cdc42, since it aggravates the impairments observed in Cdc42-null cells, resulting in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation towards the wound. Beta1 integrins were previously shown to activate Cdc42 in response to wounding and thus to regulate the directionality of migration. We demonstrated now, that fourfold reduction of beta1 integrin expression in keratinocytes in vivo did not impair wound healing. However, keratinocyte stem cells with normal levels of beta1 integrin had a competitive advantage over the hypomorphic cells and expanded steadily in the skin of mice harbouring both cell types in the epidermis. Finally, we analysed the importance of specific amino acids of the intracellular domain of beta1 integrin in keratinocytes in vivo by generating 8 mice strains which in skin express only point or deletion mutants of beta1 integrin. Our data are for the most part strikingly different from previous results obtained in vitro and significantly revise proposed models for the role of serine and tyrosine phosphorylation and the function of a salt bridge between the integrin beta subunits and the integrin alpha tails.

Podosomen sind ein prominenter Teil des Aktinzytoskelettes primärer humaner Makrophagen und wahrscheinlich essentiell für Adhäsion, Matrixverdau und gerichtete Migration. In der vorliegenden Arbeit wurde die Regulation dieser Strukturen untersucht. Es konnte zunächst gezeigt werden, dass Monozyten Podosomen nicht nur auf starren, künstlichen Oberflächen wie Glas-Deckgläschen ausbilden, sondern auch auf einem Monolayer aus Endothelzellen. Dies unterscheidet sie klar von anderen Adhäsionsstrukturen wie z.B. focal adhesions. Auch in verschiedenen Zelllinien, unter anderem in Krebszellen, ließen sich podosomale Strukturen nachweisen bzw. induzieren. Diese Befunde sind Hinweis einerseits auf die physiologische Relevanz von Podosomen und andererseits auf eine wahrscheinlich weite Verbreitung dieser Strukturen in verschiedenen Zelltypen. Podosomen sind hochdynamische Strukturen mit einer Halbwertszeit von 2-12 Minuten, das heißt, es werden permanent Podosomen abgebaut und neu gebildet. Dazu ist die Polymerisation und Depolymerisation von filamentösem (F-)Aktin notwendig. Regulationsmechanismen F-Aktin-aufbauender Wege sind gut untersucht und bekannt, weshalb in der vorliegenden Arbeit F-Aktin-abbauende Wege untersucht wurden. Ein wichtiger Regulator des Aktinzytoskelettes ist Cofilin, das die Depolymerisierung von Aktinfilamenten beschleunigt und unter anderem durch Phosphorylierung am Serin-3 inaktiviert werden kann. Folgende Ergebnisse sprechen für eine wichtige Rolle von Cofilin in der Podosomen-Regulation: Es konnte eine spezifische Lokalisation von Cofilin und phosphoryliertem Cofilin in der Aktin-reichen Podosomen-Kernstruktur nachgewiesen werden. Im Western Blot zeigte sich eine Korrelation des Grades der Cofilin-Phosphorylierung mit der Podosomenanzahl. Durch Mikroinjektion eines kurzen Peptids, welches die Cofilin-Phosphorylierung inhibiert, sowie durch Transfektion von Cofilin-siRNA konnte die Podosomen-Bildung reduziert werden. Die am besten untersuchten Cofilin-Kinasen sind die LIM-Kinasen 1 und 2. Mittels RT-PCR war in unserer Arbeitsgruppe bereits die Expression von LIMK1 in Makrophagen nachgewiesen worden. Auch Ergebnisse im Western Blot sowie in DNA-Arrays weisen auf LIMK1 als dominante Isoform in Makrophagen hin. In fixierten Präparaten konnte allerdings weder mit kommerziell erhältlichen noch mit einem selbst hergestellten, gegen die LIM-Domänen von LIMK1 gerichteten Antikörper eine spezifische Lokalisation von LIMK1 an Podosomen nachgewiesen werden. Mittels Nucleofection wurden deshalb verschiedene LIM-Kinase-Konstrukte transfiziert und überexprimiert. Dabei bestätigten sich die Ergebnisse der Antikörperfärbungen, keines der Konstrukte war in Podosomen zu finden. Alle Konstrukte mit Kinase-Aktivität führten zum raschen Krampfen und Ablösen der Zellen, wobei die Adhäsionsfläche bis zuletzt mit Podosomen bedeckt war. Im Gegensatz zu den Befunden aus der Transfektion war durch Mikroinjektion der konstitutiv aktiven Kinase-Domäne von LIMK1 eine deutliche Reduktion der Podosomen-Bildung zu erzielen. Hier können konzentrationsabhängige Effekte eine Rolle spielen. Als Gegenspieler der LIM-Kinasen wurden die Phosphatasen PP1 und PP2A beschrieben. Eine spezifische Lokalisation von PP2A an Podosomen war jedoch nicht nachzuweisen, zudem hatte eine Inhibition der beiden Phosphatasen keinen Effekt auf die Podosomenbildung oder den Podosomenabbau. Dies spricht gegen eine Beteiligung von PP1 oder PP2A an der Podosomenregulation. LIM-Kinasen selbst können durch Effektoren der Rho-GTPasen Rho, Rac und Cdc42 reguliert werden. So aktiviert der Rho-Effektor ROCK LIMK1 und LIMK2. Der ROCK-Inhibitor Y?27632 führte zu einer Störung der Podosomen-Verteilung, auch die Podosomen-Neubildung wurde stark inhibiert. Dies spricht für eine Beteiligung von ROCK an der Podosomenregulation. Auch Rac und Cdc42 können durch die gemeinsamen Effektoren der PAK-Familie eine Aktivierung von LIMK1 bewirken, dabei sind PAK1 und PAK4 die am besten untersuchten Isoformen. Die Transfektion verschiedener PAK1- und PAK4-Konstrukte führte jeweils zu einer Reduktion der Podosomen-Anzahl, unabhängig von der Kinase-Aktivität des Konstruktes. Die Kinase-inaktive PAK4-Mutante führte zu einer Reduktion des F-Aktin mit kleinen Podosomen, während die konstitutiv-aktive PAK4-Mutante große Podosomen mit vermehrtem F-Aktin bewirkte. Weitere Arbeiten zur Untersuchung vor allem von PAK4 in unserer Arbeitsgruppe konnten diese Ergebnisse bestätigen und quantifizieren sowie weitere Interaktionspartner nachweisen. Eine weitere Regulationsmöglichkeit von Cofilin ist die Bindung des second messengers PIP2, welcher unter anderem durch Isoformen der Phospholipase C (PLC) hydrolysiert werden kann. Die Mikroinjektion zweier Peptide, die laut Literatur zu einer PIP2-Inhibition bzw. einer Steigerung des PIP2-Abbaus führen, hatte keinen Einfluss auf Podosomen. Durch Transfektion der PH-Domäne von PLCd1, welche als PIP2-Sensor eingesetzt werden kann, konnte jedoch eine teilweise Lokalisation von PIP2 an Podosomen gefunden werden. Mit spezifischen Antikörpern konnte zudem eine Lokalisation von PLCb1 im Aktin-reichen Podosomenkern und von PLCb2 in der podosomalen Ringstruktur nachgewiesen werden, PLCb3 zeigte keine spezifische Lokalisation. Auch ein PLCb2-Konstrukt reicherte sich nach Transfektion in der podosomalen Ringstruktur an. Der PLC-Inhibitor U-73122 führte zu einem kompletten Verschwinden der Podosomen mit nachfolgender Ablösung der Zellen. Aufgrund dieses Befundes und der spezifischen Lokalisation ist von einer Beteiligung der PLCb1 und PLCb2 in der Podosomen-Regulation auszugehen. Im Rahmen der vorliegenden Arbeit konnten somit wichtige Effektoren der podosomalen Aktinregulation identifiziert werden: Cofilin als direkter Interaktionspartner von Aktin, LIMK1 als Cofilin-Regulator sowie ROCK und PAK als upstream-Regulatoren in der Signalkaskade. Darüber hinaus scheinen PLCb1 und PLCb2, möglicherweise über PIP2, ebenfalls an der Podosomen-Regulation beteiligt zu sein. Dies legt die Grundlage für weitere Untersuchungen über die molekularen Mechanismen der podosomalen Aktinregulation.

In dieser Arbeit sollte mittels eines in vitro-Systems die Wirkung des Yersinia-Effektorproteins YopT, einer Cysteinprotease, auf zelluläre Signalwege untersucht werden. Hierzu wurde rekombinant exprimiertes YopT, welches dieselbe biologische Aktivität aufwies wie Yersinia-transloziertes YopT, über die Coexpression mit seinem spezifischen Chaperon SycT in löslicher Form gereinigt. Mittels Interaktionsstudien wurde die Wirkung von YopT auf die RhoGTPasen RhoA, Rac1 und Cdc42 analysiert. Es konnte gezeigt werden, dass RhoGTPasen, die im Komplex mit ihren zytosolischen Inhibitor RhoGDI vorliegen, als Target für YopT dienen. Dabei wird RhoA von YopT gebunden, proteolytisch modifiziert und anschließend entlassen. Als Folge der Modifikation wird der Komplex zwischen RhoA und RhoGDI getrennt. Rac1 und Cdc42 interagieren ebenfalls mit YopT. Es konnte jedoch keine Modifikation beider GTPasen durch YopT dargestellt werden. Stattdessen binden Cdc42 und Rac1 nach der Wechselwirkung mit YopT in erhöhtem Maße an RhoGDI. Zusätzlich werden die biochemischen Eigenschaften von RhoGDI durch die Wirkung von YopT verändert. RhoA wird durch den Einfluss der Cysteinprotease YopT inaktiviert und kann nicht mehr mit nachgeschalteten Effektoren interagieren. Rac1 und Cdc42 dagegen binden nach Interaktion mit YopT weiterhin in ihrem aktivierten Zustand an ihre Effektoren. Neben YopT ist ein weiteres Yersinia-Effektorprotein bekannt, das mit den RhoGTPasen RhoA und Rac1 interagiert: die Serin/Threonin-Kinase YopO. Hier konnte die Bindung beider GTPasen an YopO unabhängig von der Anwesenheit des Prenylrestes am CTerminus der GTPasen dargestellt werden. Auch als Komplex mit RhoGDI interagierten Rac1 und RhoA mit der Kinase YopO. Zusätzlich konnte gezeigt werden, dass durch die Translokation von YopO im Gegensatz zur Translokation von YopTC139S die drei GTPasen RhoA, Rac1 und Cdc42 zumindest vorübergehend aktiviert werden. In dieser Arbeit konnte außerdem die Kristallstruktur des Chaperons SycT aufgelöst werden. SycT bildet ein Dimer und weist im allgemeinen Ähnlichkeiten zur Struktur anderer Chaperone des Typ III Sekretions- und Translokationsapparates (TTSS) auf. Jedoch unterscheidet sich der Dimerisierungsbereich von SycT strukturell von dem der anderen TTSS-Chaperone. Außerdem konnte gezeigt werden, dass weniger SycT an das biologisch inaktive YopTC139S bindet, was auf eine Interaktion des Chaperons mit dem katalytischen Zentrum der Cysteinprotease YopT hinweist.

Bakterielle Toxine aktivieren spezifische Signalwege in humanen Zellen und modulieren so deren Funktion und Morpholgie. Ein besseres Verständnis der Effekte von Toxinen auf humane Zellen könnte zur Aufklärung der Pathogenese bakterieller Erkrankungen beitragen. In dieser Arbeit wurde der Effekt des Exotoxins CNF-1 aus Escherichia coli auf die Morphologie humaner Endothelzellen (HUVEC) und die beteiligten Signalwege untersucht. CNF-1 führt in HUVEC zeitabhängig sowohl zur Bildung von Aktinfasern als auch zur Ausbildung von "membrane ruffles" und Filopodien. Diese Aktinstrukturen werden durch Aktivierung der GTPasen Rho, Rac und CDC42 induziert. Rho führt in Endothelzellen über Rho-Kinase zu einer Myosinleichtketten (MLC)- Phosphorylierung und dadurch zur Hemmung von MLC-Phosphatase. Stimulation der Endothelzellen mit CNF-1 führt hingegen abhängig von Rho und Rho-Kinase ohne Hemmung der MLC-Phosphataseaktivität zu einem Anstieg der MLC-Phosphorylierung und einer Zellkontraktion. Es konnte gezeigt werden, dass zwar Rac und CDC42 in den ersten Stunden durch CNF aktiviert werden, diese aber nicht für die MLC-Phosphorylierung verantwortlich sind. 24h nach CNF-Stimulation zeigt sich immer noch eine Aktivierung von RhoA, nicht aber von CDC42 und Rac. Trotzdem kommt es zu einem Anstieg der MLC-Phosphatase und dadurch zu einem Abfall der MLC-Phosphorylierung und Zellausbreitung. Diese Ergebnisse zeigen, dass CNF zu einer Entkopplung des Rho, Rho-Kinase, Myosinleichtketten-Phosphatase-Signalweges führt. Diese Entkopplung könnte eine Rolle bei der pathologischen Wirkung des Toxins spielen.

Pathogene Yersinien injizieren während einer Infektion über ihr TypIII-Sekretionssystem bakterielle Moduline, sogenannte Yops, in Immunzellen, um das Immunsystem zu stören. Zu Beginn dieser Arbeit war bekannt, dass die injizierten Yersinia enterocolitica-Moduline YopE und YopT das Aktinzytoskelett angreifen. Mit dem Ziel der näheren Charakterisierung der zugrunde liegenden Mechanismen wurde insbesondere der Effekt von Y. enterocolitica-YopE auf aktinregulierende Signaltransduktionswege in menschlichen Endothelzellen (HUVEC) untersucht. Zu diesem Zweck wurde ein Yersinia-Stamm hergestellt, welcher YopE als einzigen Effektor transloziert. Mit diesem Stamm infizierte ruhende HUVEC zeigten eine Veränderung des Aktinzytoskeletts ähnlich wie nach Mikroinjektion von dominant negativem N17Rac, was auf eine Inaktivierung von Rac hindeutete. Zur weiteren Untersuchung wurden in infizierten Endothelzellen durch extrazelluläre Stimuli einzelne Rho-GTPasen aktiviert und die dabei ausgebildeten Aktinstrukturen beobachtet. Dabei ergab sich keine Beeinträchtigung der Neubildung CDC42- und Rho-vermittelter Aktinstrukturen (Filopodien und Stressfasern) durch YopE, jedoch eine spezifische Hemmung Rac-induzierter Lamellipodien. Frühere Untersuchungen hatten demonstriert, dass es sich bei YopE um ein sogenanntes GAP („GTPase activating protein“) handelt, welches in vitro die Proteine Rho, Rac und CDC42 hemmt. Die Ergebnisse der vorliegenden Arbeit weisen darauf hin, dass in Endothelzellen transloziertes YopE höchst selektiv auf die Ras-ähnliche GTPase Rac wirkt, jedoch keinen Effekt auf CDC42 oder Rho ausübt. Diese Ergebnisse zeigen, dass YopE von Yersinia Rho- GTPase-abhängige Signaltransduktionswege mit einer bemerkenswerten Spezifität in primären Zielzellen beeinflussen kann. Überdies wurde die genannte Spezifität auch in primären Makrophagen nachgewiesen. Weiterhin zeigte sich im HUVEC-Infektionsversuch, dass die Hemmung der typischerweise Rho-vermittelten Aktin-Stressfasern YopT-abhängig ist. Morphologische Veränderungen von Aktinstrukturen, wie sie typischerweise bei der Unterbrechung von CDC42- oder Racvermittelten Signalen vorkommen, wurden nicht beobachtet. In Zusammenhang damit konnte gezeigt werden, dass genannter Effekt auf eine chemische Modifikation und folgliche Inaktivierung von RhoA zurückzuführen ist (Zumbihl et al., 1999). Damit unterscheiden sich YopT und YopE in ihrem Wirkmechanismus und spezifischen Zielmolekül, greifen andererseits jedoch beide direkt an Rho-GTPasen an. Sie könnten deshalb synergistisch bei der Pathogenität von Y. enterocolitica wirken. Darüber hinaus könnten YopE und YopT aufgrund ihrer Spezifität zukünftig als wertvolle Hilfsmittel zur Untersuchung zellulärer Regulationsvorgänge dienen

Podosomen sind aktinreiche Strukturen des Zytoskeletts primärer humaner Makrophagen. Für Adhäsion, Polarisation und Chemotaxis sind diese Strukturen von essentieller Bedeutung. Ihr ständiger Umbau und ihre Regulation unterliegt einer fein abgestimmten Balance der Rho GTPasen Rho, Rac und Cdc42. Pathogene Yersinien spp. haben Aktinzytoskelett von Wirtszellen durch Modulation von Rho GTPasen als Angriffsobjekt gewählt. Mit ihrem plasmidkodierten Typ III Sekretions- und Translokationsapparat werden wichtige Immunfunktionen paralysiert. In dieser Arbeit wurde in primären humanen Makrophagen der Einfluss von Yersinien-Effektoren auf Podosomen untersucht. Konkret interessierte die Frage, welchen Effekt YopE auf diese Strukturen hat. Hierzu wurden in einem standardisierten Verfahren gewonnene und gereinigte Makrophagen gesunder Spender mit unterschiedlichen Mutanten der Spezies Yersinia enterocolitica für verschiedene Zeiten infiziert. Nach Färbung der Zellen mit Rhodamin-Phalloidin wurde die Anzahl der verbliebenen Zellen mit Podosomen im konfokalen Mikroskop ermittelt und statistisch ausgewertet. Es konnte erstens gezeigt werden, daß ein voll virulenter Yersinien Stamm in der Lage ist, nach einer Infektion von bereits 30 min die podosomalen Strukturen der Makrophagen vollkommen zu zerstören. Zweitens sind an diesem Effekt verschiedene Yersinien-Effektoren und zusätzlich der Typ III Sekretions- und Translokationsapparat beteiligt. Drittens reicht YopE für die Zerstörung von Podosomen alleine aus. Viertens ist die GAP-Aktivität von YopE für die Destruktion von Podosomen nicht notwendig und lässt auf GAP-unabhängige Mechanismen von YopE schliessen. Zusammenfassend lassen die Ergebnisse dieser Arbeit vermuten, daß YopE ein wichtiger aber nicht der alleinige Effektor der Yersinien bei der Paralyse von menschlichen Makrophagen und insbesondere der Zerstörung podosomaler Adhäsionsstrukturen ist.

Während der Abspaltung von der E. coli-Linie wurde durch die Aufnahme der Salmonella-Pathogenitätsinsel 1 der Grundstein für die Entwicklung vom Kommensalen zum Pathogen gelegt. Die hier kodierten Effektoren vermitteln ebenso wie einige außerhalb von SPI1 gelegene Effektoren (z. B. SopE2) zentrale Virulenzeigenschaften. Zusätzlich zu diesen konservierten Genen gibt es einige variable Effektoren wie SopE, die erst in jüngerer Zeit erworben wurden und auch heute noch durch horizontalen Transfer zwischen verschiedenen Salmonella-Stämmen weitergegeben werden. Sie dienen wahrscheinlich der Optimierung der Wechselwirkung mit dem Wirt. In dieser Arbeit konnte gezeigt werden, dass die G-Nukleotidaustauschfaktoren SopE und das zu 69 % homologe SopE2 eine differenzielle Substratspezifität gegenüber den RhoGTPasen Cdc42 und Rac1 besitzen. Während SopE und SopE2 vergleichbar gut an Cdc42 binden, wird Rac1 von SopE deutlich stärker gebunden als von SopE2. Die schwächere Bindung von Rac1 an SopE2 führt in der Folge zu einer deutlich schwächeren Aktivierung von Rac1. Zellkulturversuche haben gezeigt, dass diese biochemischen Unterschiede zumindest in einigen Zelltypen zu unterschiedlichen Antworten (z. B. Morphologie des Aktinzytoskeletts) führen. Es ist zu vermuten, dass die Unterschiede der molekularen Eigenschaften von SopE und SopE2 einen Selektionsvorteil für S. typhimurium-Stämme darstellen, die beide SopE-Homologe besitzen. Bei der Analyse der Kristallstruktur des SopE ⋅Cdc42-Komplexes zeigte sich, dass SopE eine von der Struktur zellulärer GEFs unabhängige Lösung gefunden hat, den Nukleotidaustausch an RhoGTPasen zu katalysieren. In einer Mutagenesestudie konnten diejenigen Aminosäuren identifiziert werden, die das katalytische Zentrum von SopE bilden. Die Ergebnisse dieser Arbeit sprechen dafür, dass das katalytische Zentrum von SopE durch das 166 GAGA 169 -Motiv repräsentiert ist. Dieses Motiv weist keine Ähnlichkeiten zur katalytischen DH-Domäne eukaryontischer Rho-GEFs auf. Das deutet darauf hin, dass SopE durch konvergente Evolution entstanden sein muss. In ähnlicher Weise wie in dieser Arbeit könnten auch in Eukaryonten durch funktionelle Analysen noch weitere, bisher unbekannte GEF-Familien identifiziert werden, deren Untersuchung das Verständnis der zellulären Signaltransduktionswege weiter fördern könnte.

Die enterpathogenen Yersinia-Spezies unterlaufen die primären Abwehrmechanismen des Wirtes durch Einflußnahme auf Signaltransduktionskaskaden. Diese Subversion wird von einem 70 kDa Virulenzplasmid vermittelt, welches für ein Typ III Sekretion/Translokationsssystem und einige Virulenzfaktoren, sogenannte Yops (Yersinia outer proteins) kodiert. Die sechs bisher bekannten Effektor-Yops gelangen über das Sekretionssystem ins Zytoplasma von Makrophagen und Granulozyten, was in diesen zu einer Inhibition von Phagozytose, oxidative burst und Zytokinfreisetzung sowie zur Induktion der Apoptose führt. Ziel dieser Arbeit war es, die zellulären Angriffsproteine der Effektor-Yops YopM und YopO zu identifizieren. Als Methode diente das Yeast Two- Hybrid System. Es stellte sich heraus, dass YopM für diese Methode aufgrund seiner transskriptionsaktivierenden Eigenschaft nicht verwendbar ist. Dies und die Beobachtung, dass YopM in den Zellkern lokalisiert wird (77) deuten darauf hin, dass YopM möglicherweise als Transkriptionsfaktor in der Wirtszelle wirken könnte. Für YopO erbrachte die Two-Hybrid Untersuchung 31 positiv interagierende Klone, die als Rac1 (17 Klone, davon 4 unabhängige), Snk i.p. (11 Klone, davon 6 unabhängige) und Mus musculus spindlin (3 Klone) identifiziert werden konnten. Rac1 gehört zur Familie der Rho-GTPasen zu denen u.a. auch die Proteine RhoA und Cdc42 gehören. Diese kleinen G-Proteine sind in komplexer und in noch unvollkommen verstandener Weise an der Regulation vielfältiger Zellfunktionen beteiligt. Es konnte gezeigt werden, dass YopO neben Rac1 auch mit RhoA, nicht jedoch mit Cdc42 im Yeast Two-Hybrid System interagiert. Für YpkA, dem zu YopO homologen Protein aus Y. pseudotuberculosis zeigte sich das gleiche Interaktionsverhalten. Durch Koimmunopräzipitation konnten die Ergebnisse der Yeast Two-Hybrid Untersuchung mit einer zweiten, unabhängigen Methode bestätigt werden. Aus dem Zellysat von mit Yersinien infizierten humanen COS-Zellen, ließen sich RhoA und Rac1, nicht aber Cdc42 mit Hilfe von Anti-YpkA-Antikörpern koimmunopräzipitieren. In dieser Arbeit konnte mit zwei unabhängigen Methoden eine bis dahin unbekannte Affinität zwischen YpkA/YopO mit RhoA und Rac1 gezeigt werden. Es kann somit davon ausgegangen werden, dass RhoA und Rac1 die intrazellulären Angriffspunkte der Effektor- Yops YpkA bzw. YopO darstellen.