TY - JOUR AB - CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1's enhancement of resting T-cell recruitment are discussed. AU - Veldkamp, Christopher AU - Kiermaier, Eva AU - Gabel Eissens, Skylar AU - Gillitzer, Miranda AU - Lippner, David AU - Disilvio, Frank AU - Mueller, Casey AU - Wantuch, Paeton AU - Chaffee, Gary AU - Famiglietti, Michael AU - Zgoba, Danielle AU - Bailey, Asha AU - Bah, Yaya AU - Engebretson, Samantha AU - Graupner, David AU - Lackner, Emily AU - Larosa, Vincent AU - Medeiros, Tysha AU - Olson, Michael AU - Phillips, Andrew AU - Pyles, Harley AU - Richard, Amanda AU - Schoeller, Scott AU - Touzeau, Boris AU - Williams, Larry AU - Sixt, Michael K AU - Peterson, Francis ID - 1618 IS - 27 JF - Biochemistry TI - Solution structure of CCL19 and identification of overlapping CCR7 and PSGL-1 binding sites VL - 54 ER - TY - JOUR AB - 3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype. AU - Ruprecht, Verena AU - Wieser, Stefan AU - Callan Jones, Andrew AU - Smutny, Michael AU - Morita, Hitoshi AU - Sako, Keisuke AU - Barone, Vanessa AU - Ritsch Marte, Monika AU - Sixt, Michael K AU - Voituriez, Raphaël AU - Heisenberg, Carl-Philipp J ID - 1537 IS - 4 JF - Cell TI - Cortical contractility triggers a stochastic switch to fast amoeboid cell motility VL - 160 ER - TY - JOUR AB - During inflammation, lymph nodes swell with an influx of immune cells. New findings identify a signalling pathway that induces relaxation in the contractile cells that give structure to these organs. AU - Sixt, Michael K AU - Vaahtomeri, Kari ID - 1877 IS - 7523 JF - Nature TI - Physiology: Relax and come in VL - 514 ER - TY - JOUR AB - angerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene-expression changes during LC maturation. LCs down-regulate a set of epithelial genes including E-cadherin, while they upregulate the mesenchymal marker N-cadherin known to facilitate cell migration. In addition, N-cadherin is constitutively expressed by monocyte-derived DCs known to exhibit characteristics of both inflammatory-type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc-finger E-box-binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial-to-mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N-cadherin plays a role during DC migration. AU - Konradi, Sabine AU - Yasmin, Nighat AU - Haslwanter, Denise AU - Weber, Michele AU - Gesslbauer, Bernd AU - Sixt, Michael K AU - Strobl, Herbert ID - 1910 IS - 2 JF - European Journal of Immunology TI - Langerhans cell maturation is accompanied by induction of N-cadherin and the transcriptional regulators of epithelial-mesenchymal transition ZEB1/2 VL - 44 ER - TY - JOUR AB - In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity. AU - Lamprecht, Constanze AU - Plochberger, Birgit AU - Ruprecht, Verena AU - Wieser, Stefan AU - Rankl, Christian AU - Heister, Elena AU - Unterauer, Barbara AU - Brameshuber, Mario AU - Danzberger, Jürgen AU - Lukanov, Petar AU - Flahaut, Emmanuel AU - Schütz, Gerhard AU - Hinterdorfer, Peter AU - Ebner, Andreas ID - 1925 IS - 12 JF - Nanotechnology TI - A single-molecule approach to explore binding uptake and transport of cancer cell targeting nanotubes VL - 25 ER - TY - JOUR AB - Directional guidance of migrating cells is relatively well explored in the reductionist setting of cell culture experiments. Here spatial gradients of chemical cues as well as gradients of mechanical substrate characteristics prove sufficient to attract single cells as well as their collectives. How such gradients present and act in the context of an organism is far less clear. Here we review recent advances in understanding how guidance cues emerge and operate in the physiological context. AU - Majumdar, Ritankar AU - Sixt, Michael K AU - Parent, Carole ID - 2158 IS - 1 JF - Current Opinion in Cell Biology TI - New paradigms in the establishment and maintenance of gradients during directed cell migration VL - 30 ER - TY - JOUR AB - A hallmark of immune cell trafficking is directional guidance via gradients of soluble or surface bound chemokines. Vascular endothelial cells produce, transport and deposit either their own chemokines or chemokines produced by the underlying stroma. Endothelial heparan sulfate (HS) was suggested to be a critical scaffold for these chemokine pools, but it is unclear how steep chemokine gradients are sustained between the lumenal and ablumenal aspects of blood vessels. Addressing this question by semi-quantitative immunostaining of HS moieties around blood vessels with a pan anti-HS IgM mAb, we found a striking HS enrichment in the basal lamina of resting and inflamed post capillary skin venules, as well as in high endothelial venules (HEVs) of lymph nodes. Staining of skin vessels with a glycocalyx probe further suggested that their lumenal glycocalyx contains much lower HS density than their basolateral extracellular matrix (ECM). This polarized HS pattern was observed also in isolated resting and inflamed microvascular dermal cells. Notably, progressive skin inflammation resulted in massive ECM deposition and in further HS enrichment around skin post capillary venules and their associated pericytes. Inflammation-dependent HS enrichment was not compromised in mice deficient in the main HS degrading enzyme, heparanase. Our results suggest that the blood vasculature patterns steep gradients of HS scaffolds between their lumenal and basolateral endothelial aspects, and that inflammatory processes can further enrich the HS content nearby inflamed vessels. We propose that chemokine gradients between the lumenal and ablumenal sides of vessels could be favored by these sharp HS scaffold gradients. AU - Stoler Barak, Liat AU - Moussion, Christine AU - Shezen, Elias AU - Hatzav, Miki AU - Sixt, Michael K AU - Alon, Ronen ID - 2214 IS - 1 JF - PLoS One TI - Blood vessels pattern heparan sulfate gradients between their apical and basolateral aspects VL - 9 ER - TY - JOUR AB - Homologous recombination is crucial for genome stability and for genetic exchange. Although our knowledge of the principle steps in recombination and its machinery is well advanced, homology search, the critical step of exploring the genome for homologous sequences to enable recombination, has remained mostly enigmatic. However, recent methodological advances have provided considerable new insights into this fundamental step in recombination that can be integrated into a mechanistic model. These advances emphasize the importance of genomic proximity and nuclear organization for homology search and the critical role of homology search mediators in this process. They also aid our understanding of how homology search might lead to unwanted and potentially disease-promoting recombination events. AU - Renkawitz, Jörg AU - Lademann, Claudio AU - Jentsch, Stefan ID - 2215 IS - 6 JF - Nature Reviews Molecular Cell Biology TI - Mechanisms and principles of homology search during recombination VL - 15 ER - TY - JOUR AB - MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in many cellular pathways. MiRNAs associate with members of the Argonaute protein family and bind to partially complementary sequences on mRNAs and induce translational repression or mRNA decay. Using deep sequencing and Northern blotting, we characterized miRNA expression in wild type and miR-155-deficient dendritic cells (DCs) and macrophages. Analysis of different stimuli (LPS, LDL, eLDL, oxLDL) reveals a direct influence of miR-155 on the expression levels of other miRNAs. For example, miR-455 is negatively regulated in miR-155-deficient cells possibly due to inhibition of the transcription factor C/EBPbeta by miR-155. Based on our comprehensive data sets, we propose a model of hierarchical miRNA expression dominated by miR-155 in DCs and macrophages. AU - Dueck, Anne AU - Eichner, Alexander AU - Sixt, Michael K AU - Meister, Gunter ID - 2242 IS - 4 JF - FEBS Letters SN - 00145793 TI - A miR-155-dependent microRNA hierarchy in dendritic cell maturation and macrophage activation VL - 588 ER - TY - JOUR AU - Moussion, Christine AU - Sixt, Michael K ID - 2830 IS - 5 JF - Immunity TI - A conduit to amplify innate immunity VL - 38 ER - TY - JOUR AB - Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues. AU - Weber, Michele AU - Hauschild, Robert AU - Schwarz, Jan AU - Moussion, Christine AU - De Vries, Ingrid AU - Legler, Daniel AU - Luther, Sanjiv AU - Bollenbach, Mark Tobias AU - Sixt, Michael K ID - 2839 IS - 6117 JF - Science TI - Interstitial dendritic cell guidance by haptotactic chemokine gradients VL - 339 ER - TY - JOUR AB - Podoplanin, a mucin-like plasma membrane protein, is expressed by lymphatic endothelial cells and responsible for separation of blood and lymphatic circulation through activation of platelets. Here we show that podoplanin is also expressed by thymic fibroblastic reticular cells (tFRC), a novel thymic medulla stroma cell type associated with thymic conduits, and involved in development of natural regulatory T cells (nTreg). Young mice deficient in podoplanin lack nTreg owing to retardation of CD4+CD25+ thymocytes in the cortex and missing differentiation of Foxp3+ thymocytes in the medulla. This might be due to CCL21 that delocalizes upon deletion of the CCL21-binding podoplanin from medullar tFRC to cortex areas. The animals do not remain devoid of nTreg but generate them delayed within the first month resulting in Th2-biased hypergammaglobulinemia but not in the death-causing autoimmune phenotype of Foxp3-deficient Scurfy mice. AU - Fuertbauer, Elke AU - Zaujec, Jan AU - Uhrin, Pavel AU - Raab, Ingrid AU - Weber, Michele AU - Schachner, Helga AU - Bauer, Miroslav AU - Schütz, Gerhard AU - Binder, Bernd AU - Sixt, Michael K AU - Kerjaschki, Dontscho AU - Stockinger, Hannes ID - 522 IS - 1-2 JF - Immunology Letters TI - Thymic medullar conduits-associated podoplanin promotes natural regulatory T cells VL - 154 ER - TY - CHAP AB - Leukocyte migration through the interstitial space is crucial for the maintenance of tolerance and immunity. The main cues for leukocyte trafficking are chemokines thought to directionally guide these cells towards their targets. However, model systems that facilitate quantification of chemokine-guided leukocyte migration in vivo are uncommon. Here we describe an ex vivo crawl-in assay using explanted mouse ears that allows the visualization of chemokine-dependent dendritic cell (DC) motility in the dermal interstitium in real time. We present methods for the preparation of mouse ear sheets and their use in multidimensional confocal imaging experiments to monitor and analyze the directional migration of fluorescently labelled DCs through the dermis and into afferent lymphatic vessels. The assay provides a more physiological approach to study leukocyte migration than in vitro three-dimensional (3D) or 2-dimensional (2D) migration assays such as collagen gels and transwell assays. AU - Weber, Michele AU - Sixt, Michael K ED - Cardona, Astrid ED - Ubogu, Eroboghene ID - 10900 SN - 1064-3745 T2 - Chemokines TI - Live Cell Imaging of Chemotactic Dendritic Cell Migration in Explanted Mouse Ear Preparations VL - 1013 ER - TY - JOUR AB - MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3′-untranslated region (3′-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1-3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected. AU - Dueck, Anne AU - Ziegler, Christian AU - Eichner, Alexander AU - Berezikov, Eugène AU - Meister, Gunter ID - 2946 IS - 19 JF - Nucleic Acids Research TI - MicroRNAs associated with the different human Argonaute proteins VL - 40 ER - TY - JOUR AB - In search of foreign antigens, lymphocytes recirculate from the blood, through lymph nodes, into lymphatics and back to the blood. Dendritic cells also migrate to lymph nodes for optimal interaction with lymphocytes. This continuous trafficking of immune cells into and out of lymph nodes is essential for immune surveillance of foreign invaders. In this article, we review our current understanding of the functions of high endothelial venules (HEVs), stroma and lymphatics in the entry, positioning and exit of immune cells in lymph nodes during homeostasis, and we highlight the unexpected role of dendritic cells in the control of lymphocyte homing through HEVs. AU - Girard, Jean AU - Moussion, Christine AU - Förster, Reinhold ID - 2945 IS - 11 JF - Nature Reviews Immunology TI - HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes VL - 12 ER - TY - JOUR AU - Weber, Michele ID - 3167 IS - 6077 JF - Science TI - NextGen speaks 13 VL - 336 ER - TY - JOUR AB - We describe here the development and characterization of a conditionally inducible mouse model expressing Lifeact-GFP, a peptide that reports the dynamics of filamentous actin. We have used this model to study platelets, megakaryocytes and melanoblasts and we provide evidence that Lifeact-GFP is a useful reporter in these cell types ex vivo. In the case of platelets and megakaryocytes, these cells are not transfectable by traditional methods, so conditional activation of Lifeact allows the study of actin dynamics in these cells live. We studied melanoblasts in native skin explants from embryos, allowing the visualization of live actin dynamics during cytokinesis and migration. Our study revealed that melanoblasts lacking the small GTPase Rac1 show a delay in the formation of new pseudopodia following cytokinesis that accounts for the previously reported cytokinesis delay in these cells. Thus, through use of this mouse model, we were able to gain insights into the actin dynamics of cells that could only previously be studied using fixed specimens or following isolation from their native tissue environment. AU - Schachtner, Hannah AU - Li, Ang AU - Stevenson, David AU - Calaminus, Simon AU - Thomas, Steven AU - Watson, Steve AU - Sixt, Michael K AU - Wedlich Söldner, Roland AU - Strathdee, Douglas AU - Machesky, Laura ID - 3158 IS - 11-12 JF - European Journal of Cell Biology TI - Tissue inducible Lifeact expression allows visualization of actin dynamics in vivo and ex vivo VL - 91 ER - TY - JOUR AU - Sixt, Michael K ID - 506 IS - 3 JF - Journal of Cell Biology TI - Cell migration: Fibroblasts find a new way to get ahead VL - 197 ER - TY - JOUR AB - Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion. AU - Ruprecht, Verena AU - Axmann, Markus AU - Wieser, Stefan AU - Schuetz, Gerhard ID - 3287 IS - 8 JF - Current Protein & Peptide Science TI - What can we learn from single molecule trajectories? VL - 12 ER - TY - JOUR AB - The Minisymposium “Cell Migration and Motility” was attended by approximately 500 visitors and covered a broad range of questions in the field using diverse model systems. Topics comprised actin dynamics, cell polarity, force transduction, signal transduction, bar- rier transmigration, and chemotactic guidance. AU - Sixt, Michael K AU - Parent, Carole ID - 3371 IS - 6 JF - Molecular Biology and Evolution TI - Cells on the move in Philadelphia VL - 22 ER -