@article{15, abstract = {Although much is known about the physiological framework of T cell motility, and numerous rate-limiting molecules have been identified through loss-of-function approaches, an integrated functional concept of T cell motility is lacking. Here, we used in vivo precision morphometry together with analysis of cytoskeletal dynamics in vitro to deconstruct the basic mechanisms of T cell migration within lymphatic organs. We show that the contributions of the integrin LFA-1 and the chemokine receptor CCR7 are complementary rather than positioned in a linear pathway, as they are during leukocyte extravasation from the blood vasculature. Our data demonstrate that CCR7 controls cortical actin flows, whereas integrins mediate substrate friction that is sufficient to drive locomotion in the absence of considerable surface adhesions and plasma membrane flux.}, author = {Hons, Miroslav and Kopf, Aglaja and Hauschild, Robert and Leithner, Alexander F and Gärtner, Florian R and Abe, Jun and Renkawitz, Jörg and Stein, Jens and Sixt, Michael K}, journal = {Nature Immunology}, number = {6}, pages = {606 -- 616}, publisher = {Nature Publishing Group}, title = {{Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells}}, doi = {10.1038/s41590-018-0109-z}, volume = {19}, year = {2018}, } @article{569, abstract = {The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.}, author = {Spira, Felix and Cuylen Haering, Sara and Mehta, Shalin and Samwer, Matthias and Reversat, Anne and Verma, Amitabh and Oldenbourg, Rudolf and Sixt, Michael K and Gerlich, Daniel}, issn = {2050084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments}}, doi = {10.7554/eLife.30867}, volume = {6}, year = {2017}, } @article{571, abstract = {Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection. In addition to their role in thrombosis and hemostasis, platelets can also migrate to sites of infection to help trap bacteria and clear the vascular surface.}, author = {Gärtner, Florian R and Ahmad, Zerkah and Rosenberger, Gerhild and Fan, Shuxia and Nicolai, Leo and Busch, Benjamin and Yavuz, Gökce and Luckner, Manja and Ishikawa Ankerhold, Hellen and Hennel, Roman and Benechet, Alexandre and Lorenz, Michael and Chandraratne, Sue and Schubert, Irene and Helmer, Sebastian and Striednig, Bianca and Stark, Konstantin and Janko, Marek and Böttcher, Ralph and Verschoor, Admar and Leon, Catherine and Gachet, Christian and Gudermann, Thomas and Mederos Y Schnitzler, Michael and Pincus, Zachary and Iannacone, Matteo and Haas, Rainer and Wanner, Gerhard and Lauber, Kirsten and Sixt, Michael K and Massberg, Steffen}, issn = {00928674}, journal = {Cell Press}, number = {6}, pages = {1368 -- 1382}, publisher = {Cell Press}, title = {{Migrating platelets are mechano scavengers that collect and bundle bacteria}}, doi = {10.1016/j.cell.2017.11.001}, volume = {171}, year = {2017}, } @article{659, abstract = {Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.}, author = {Kage, Frieda and Winterhoff, Moritz and Dimchev, Vanessa and Müller, Jan and Thalheim, Tobias and Freise, Anika and Brühmann, Stefan and Kollasser, Jana and Block, Jennifer and Dimchev, Georgi A and Geyer, Matthias and Schnittler, Hams and Brakebusch, Cord and Stradal, Theresia and Carlier, Marie and Sixt, Michael K and Käs, Josef and Faix, Jan and Rottner, Klemens}, issn = {20411723}, journal = {Nature Communications}, publisher = {Nature Publishing Group}, title = {{FMNL formins boost lamellipodial force generation}}, doi = {10.1038/ncomms14832}, volume = {8}, year = {2017}, } @article{668, abstract = {Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.}, author = {Horsthemke, Markus and Bachg, Anne and Groll, Katharina and Moyzio, Sven and Müther, Barbara and Hemkemeyer, Sandra and Wedlich Söldner, Roland and Sixt, Michael K and Tacke, Sebastian and Bähler, Martin and Hanley, Peter}, issn = {00219258}, journal = {Journal of Biological Chemistry}, number = {17}, pages = {7258 -- 7273}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion}}, doi = {10.1074/jbc.M116.766923}, volume = {292}, year = {2017}, } @article{672, abstract = {Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration.}, author = {Vaahtomeri, Kari and Brown, Markus and Hauschild, Robert and De Vries, Ingrid and Leithner, Alexander F and Mehling, Matthias and Kaufmann, Walter and Sixt, Michael K}, issn = {22111247}, journal = {Cell Reports}, number = {5}, pages = {902 -- 909}, publisher = {Cell Press}, title = {{Locally triggered release of the chemokine CCL21 promotes dendritic cell transmigration across lymphatic endothelia}}, doi = {10.1016/j.celrep.2017.04.027}, volume = {19}, year = {2017}, } @article{674, abstract = {Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable “roads” ensure optimal guidance in vivo.}, author = {Schwarz, Jan and Bierbaum, Veronika and Vaahtomeri, Kari and Hauschild, Robert and Brown, Markus and De Vries, Ingrid and Leithner, Alexander F and Reversat, Anne and Merrin, Jack and Tarrant, Teresa and Bollenbach, Tobias and Sixt, Michael K}, issn = {09609822}, journal = {Current Biology}, number = {9}, pages = {1314 -- 1325}, publisher = {Cell Press}, title = {{Dendritic cells interpret haptotactic chemokine gradients in a manner governed by signal to noise ratio and dependent on GRK6}}, doi = {10.1016/j.cub.2017.04.004}, volume = {27}, year = {2017}, } @article{677, abstract = {The INO80 complex (INO80-C) is an evolutionarily conserved nucleosome remodeler that acts in transcription, replication, and genome stability. It is required for resistance against genotoxic agents and is involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). However, the causes of the HR defect in INO80-C mutant cells are controversial. Here, we unite previous findings using a system to study HR with high spatial resolution in budding yeast. We find that INO80-C has at least two distinct functions during HR—DNA end resection and presynaptic filament formation. Importantly, the second function is linked to the histone variant H2A.Z. In the absence of H2A.Z, presynaptic filament formation and HR are restored in INO80-C-deficient mutants, suggesting that presynaptic filament formation is the crucial INO80-C function during HR.}, author = {Lademann, Claudio and Renkawitz, Jörg and Pfander, Boris and Jentsch, Stefan}, issn = {22111247}, journal = {Cell Reports}, number = {7}, pages = {1294 -- 1303}, publisher = {Cell Press}, title = {{The INO80 complex removes H2A.Z to promote presynaptic filament formation during homologous recombination}}, doi = {10.1016/j.celrep.2017.04.051}, volume = {19}, year = {2017}, } @article{694, abstract = {A change regarding the extent of adhesion - hereafter referred to as adhesion plasticity - between adhesive and less-adhesive states of mammalian cells is important for their behavior. To investigate adhesion plasticity, we have selected a stable isogenic subpopulation of human MDA-MB-468 breast carcinoma cells growing in suspension. These suspension cells are unable to re-adhere to various matrices or to contract three-dimensional collagen lattices. By using transcriptome analysis, we identified the focal adhesion protein tensin3 (Tns3) as a determinant of adhesion plasticity. Tns3 is strongly reduced at mRNA and protein levels in suspension cells. Furthermore, by transiently challenging breast cancer cells to grow under non-adherent conditions markedly reduces Tns3 protein expression, which is regained upon re-adhesion. Stable knockdown of Tns3 in parental MDA-MB-468 cells results in defective adhesion, spreading and migration. Tns3-knockdown cells display impaired structure and dynamics of focal adhesion complexes as determined by immunostaining. Restoration of Tns3 protein expression in suspension cells partially rescues adhesion and focal contact composition. Our work identifies Tns3 as a crucial focal adhesion component regulated by, and functionally contributing to, the switch between adhesive and non-adhesive states in MDA-MB-468 cancer cells.}, author = {Veß, Astrid and Blache, Ulrich and Leitner, Laura and Kurz, Angela and Ehrenpfordt, Anja and Sixt, Michael K and Posern, Guido}, issn = {00219533}, journal = {Journal of Cell Science}, number = {13}, pages = {2172 -- 2184}, publisher = {Company of Biologists}, title = {{A dual phenotype of MDA MB 468 cancer cells reveals mutual regulation of tensin3 and adhesion plasticity}}, doi = {10.1242/jcs.200899}, volume = {130}, year = {2017}, } @article{1161, abstract = {Coordinated changes of cell shape are often the result of the excitable, wave-like dynamics of the actin cytoskeleton. New work shows that, in migrating cells, protrusion waves arise from mechanochemical crosstalk between adhesion sites, membrane tension and the actin protrusive machinery.}, author = {Müller, Jan and Sixt, Michael K}, issn = {09609822}, journal = {Current Biology}, number = {1}, pages = {R24 -- R25}, publisher = {Cell Press}, title = {{Cell migration: Making the waves}}, doi = {10.1016/j.cub.2016.11.035}, volume = {27}, year = {2017}, } @article{727, abstract = {Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.}, author = {Mueller, Jan and Szep, Gregory and Nemethova, Maria and De Vries, Ingrid and Lieber, Arnon and Winkler, Christoph and Kruse, Karsten and Small, John and Schmeiser, Christian and Keren, Kinneret and Hauschild, Robert and Sixt, Michael K}, issn = {00928674}, journal = {Cell}, number = {1}, pages = {188 -- 200}, publisher = {Cell Press}, title = {{Load adaptation of lamellipodial actin networks}}, doi = {10.1016/j.cell.2017.07.051}, volume = {171}, year = {2017}, } @misc{5567, abstract = {Immunological synapse DC-Tcells}, author = {Leithner, Alexander F}, keywords = {Immunological synapse}, publisher = {Institute of Science and Technology Austria}, title = {{Immunological synapse DC-Tcells}}, doi = {10.15479/AT:ISTA:71}, year = {2017}, } @article{664, abstract = {Immune cells communicate using cytokine signals, but the quantitative rules of this communication aren't clear. In this issue of Immunity, Oyler-Yaniv et al. (2017) suggest that the distribution of a cytokine within a lymphatic organ is primarily governed by the local density of cells consuming it.}, author = {Assen, Frank P and Sixt, Michael K}, issn = {10747613}, journal = {Immunity}, number = {4}, pages = {519 -- 520}, publisher = {Cell Press}, title = {{The dynamic cytokine niche}}, doi = {10.1016/j.immuni.2017.04.006}, volume = {46}, year = {2017}, } @article{679, abstract = {Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/ lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections.}, author = {Ebner, Florian and Sedlyarov, Vitaly and Tasciyan, Saren and Ivin, Masa and Kratochvill, Franz and Gratz, Nina and Kenner, Lukas and Villunger, Andreas and Sixt, Michael K and Kovarik, Pavel}, issn = {00219738}, journal = {The Journal of Clinical Investigation}, number = {6}, pages = {2051 -- 2065}, publisher = {American Society for Clinical Investigation}, title = {{The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection}}, doi = {10.1172/JCI80631}, volume = {127}, year = {2017}, } @article{1137, abstract = {RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.}, author = {Salzer, Elisabeth and Çaǧdaş, Deniz and Hons, Miroslav and Mace, Emily and Garncarz, Wojciech and Petronczki, Oezlem and Platzer, René and Pfajfer, Laurène and Bilic, Ivan and Ban, Sol and Willmann, Katharina and Mukherjee, Malini and Supper, Verena and Hsu, Hsiangting and Banerjee, Pinaki and Sinha, Papiya and Mcclanahan, Fabienne and Zlabinger, Gerhard and Pickl, Winfried and Gribben, John and Stockinger, Hannes and Bennett, Keiryn and Huppa, Johannes and Dupré, Loï̈C and Sanal, Özden and Jäger, Ulrich and Sixt, Michael K and Tezcan, Ilhan and Orange, Jordan and Boztug, Kaan}, journal = {Nature Immunology}, number = {12}, pages = {1352 -- 1360}, publisher = {Nature Publishing Group}, title = {{RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics}}, doi = {10.1038/ni.3575}, volume = {17}, year = {2016}, } @article{1142, abstract = {Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.}, author = {Martins, Rui and Maier, Julia and Gorki, Anna and Huber, Kilian and Sharif, Omar and Starkl, Philipp and Saluzzo, Simona and Quattrone, Federica and Gawish, Riem and Lakovits, Karin and Aichinger, Michael and Radic Sarikas, Branka and Lardeau, Charles and Hladik, Anastasiya and Korosec, Ana and Brown, Markus and Vaahtomeri, Kari and Duggan, Michelle and Kerjaschki, Dontscho and Esterbauer, Harald and Colinge, Jacques and Eisenbarth, Stephanie and Decker, Thomas and Bennett, Keiryn and Kubicek, Stefan and Sixt, Michael K and Superti Furga, Giulio and Knapp, Sylvia}, journal = {Nature Immunology}, number = {12}, pages = {1361 -- 1372}, publisher = {Nature Publishing Group}, title = {{Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions}}, doi = {10.1038/ni.3590}, volume = {17}, year = {2016}, } @article{1150, abstract = {When neutrophils infiltrate a site of inflammation, they have to stop at the right place to exert their effector function. In this issue of Developmental Cell, Wang et al. (2016) show that neutrophils sense reactive oxygen species via the TRPM2 channel to arrest migration at their target site. © 2016 Elsevier Inc.}, author = {Renkawitz, Jörg and Sixt, Michael K}, journal = {Developmental Cell}, number = {5}, pages = {448 -- 450}, publisher = {Cell Press}, title = {{A Radical Break Restraining Neutrophil Migration}}, doi = {10.1016/j.devcel.2016.08.017}, volume = {38}, year = {2016}, } @article{1154, abstract = {Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue. }, author = {Schwarz, Jan and Bierbaum, Veronika and Merrin, Jack and Frank, Tino and Hauschild, Robert and Bollenbach, Mark Tobias and Tay, Savaş and Sixt, Michael K and Mehling, Matthias}, journal = {Scientific Reports}, publisher = {Nature Publishing Group}, title = {{A microfluidic device for measuring cell migration towards substrate bound and soluble chemokine gradients}}, doi = {10.1038/srep36440}, volume = {6}, year = {2016}, } @article{1201, abstract = {In this issue of Cell, Skau et al. show that the formin FMN2 organizes a perinuclear actin cytoskeleton that protects the nucleus and its genomic content of migrating cells squeezing through small spaces.}, author = {Renkawitz, Jörg and Sixt, Michael K}, journal = {Cell}, number = {6}, pages = {1448 -- 1449}, publisher = {Cell Press}, title = {{Formin’ a nuclear protection}}, doi = {10.1016/j.cell.2016.11.024}, volume = {167}, year = {2016}, } @article{1217, abstract = {Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E 2 (PGE 2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE 2 during T-cell receptor stimulation. In addition, we show that autocrine PGE 2 signaling through EP receptors is essential for optimal CD4 + T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE 2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4 + Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE 2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE 2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.}, author = {Sreeramkumar, Vinatha and Hons, Miroslav and Punzón, Carmen and Stein, Jens and Sancho, David and Fresno Forcelledo, Manuel and Cuesta, Natalia}, journal = {Immunology and Cell Biology}, number = {1}, pages = {39 -- 51}, publisher = {Nature Publishing Group}, title = {{Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors}}, doi = {10.1038/icb.2015.62}, volume = {94}, year = {2016}, } @article{1285, abstract = {Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.}, author = {Paluch, Ewa and Aspalter, Irene and Sixt, Michael K}, journal = {Annual Review of Cell and Developmental Biology}, pages = {469 -- 490}, publisher = {Annual Reviews}, title = {{Focal adhesion-independent cell migration}}, doi = {10.1146/annurev-cellbio-111315-125341}, volume = {32}, year = {2016}, } @article{1490, abstract = {To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient.}, author = {Russo, Erica and Teijeira, Alvaro and Vaahtomeri, Kari and Willrodt, Ann and Bloch, Joël and Nitschké, Maximilian and Santambrogio, Laura and Kerjaschki, Dontscho and Sixt, Michael K and Halin, Cornelia}, journal = {Cell Reports}, number = {7}, pages = {1723 -- 1734}, publisher = {Cell Press}, title = {{Intralymphatic CCL21 promotes tissue egress of dendritic cells through afferent lymphatic vessels}}, doi = {10.1016/j.celrep.2016.01.048}, volume = {14}, year = {2016}, } @article{1599, abstract = {The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis. }, author = {Kiermaier, Eva and Moussion, Christine and Veldkamp, Christopher and Gerardy Schahn, Rita and De Vries, Ingrid and Williams, Larry and Chaffee, Gary and Phillips, Andrew and Freiberger, Friedrich and Imre, Richard and Taleski, Deni and Payne, Richard and Braun, Asolina and Förster, Reinhold and Mechtler, Karl and Mühlenhoff, Martina and Volkman, Brian and Sixt, Michael K}, journal = {Science}, number = {6269}, pages = {186 -- 190}, publisher = {American Association for the Advancement of Science}, title = {{Polysialylation controls dendritic cell trafficking by regulating chemokine recognition}}, doi = {10.1126/science.aad0512}, volume = {351}, year = {2016}, } @article{1597, abstract = {Chemokines are the main guidance cues directing leukocyte migration. Opposed to early assumptions, chemokines do not necessarily act as soluble cues but are often immobilized within tissues, e.g., dendritic cell migration toward lymphatic vessels is guided by a haptotactic gradient of the chemokine CCL21. Controlled assay systems to quantitatively study haptotaxis in vitro are still missing. In this chapter, we describe an in vitro haptotaxis assay optimized for the unique properties of dendritic cells. The chemokine CCL21 is immobilized in a bioactive state, using laser-assisted protein adsorption by photobleaching. The cells follow this immobilized CCL21 gradient in a haptotaxis chamber, which provides three dimensionally confined migration conditions.}, author = {Schwarz, Jan and Sixt, Michael K}, journal = {Methods in Enzymology}, pages = {567 -- 581}, publisher = {Elsevier}, title = {{Quantitative analysis of dendritic cell haptotaxis}}, doi = {10.1016/bs.mie.2015.11.004}, volume = {570}, year = {2016}, } @phdthesis{1129, abstract = {Directed cell migration is a hallmark feature, present in almost all multi-cellular organisms. Despite its importance, basic questions regarding force transduction or directional sensing are still heavily investigated. Directed migration of cells guided by immobilized guidance cues - haptotaxis - occurs in key-processes, such as embryonic development and immunity (Middleton et al., 1997; Nguyen et al., 2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues comprise adhesive ligands, such as collagen and fibronectin (Barczyk et al., 2009), or chemokines - the main guidance cues for migratory leukocytes (Middleton et al., 1997; Weber et al., 2013). While adhesive ligands serve as attachment sites guiding cell migration (Carter, 1965), chemokines instruct haptotactic migration by inducing adhesion to adhesive ligands and directional guidance (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis of the cellular response to immobilized guidance cues requires in vitro assays that foster cell migration, offer accurate control of the immobilized cues on a subcellular scale and in the ideal case closely reproduce in vivo conditions. The exploration of haptotactic cell migration through design and employment of such assays represents the main focus of this work. Dendritic cells (DCs) are leukocytes, which after encountering danger signals such as pathogens in peripheral organs instruct naïve T-cells and consequently the adaptive immune response in the lymph node (Mellman and Steinman, 2001). To reach the lymph node from the periphery, DCs follow haptotactic gradients of the chemokine CCL21 towards lymphatic vessels (Weber et al., 2013). Questions about how DCs interpret haptotactic CCL21 gradients have not yet been addressed. The main reason for this is the lack of an assay that offers diverse haptotactic environments, hence allowing the study of DC migration as a response to different signals of immobilized guidance cue. In this work, we developed an in vitro assay that enables us to quantitatively assess DC haptotaxis, by combining precisely controllable chemokine photo-patterning with physically confining migration conditions. With this tool at hand, we studied the influence of CCL21 gradient properties and concentration on DC haptotaxis. We found that haptotactic gradient sensing depends on the absolute CCL21 concentration in combination with the local steepness of the gradient. Our analysis suggests that the directionality of migrating DCs is governed by the signal-to-noise ratio of CCL21 binding to its receptor CCR7. Moreover, the haptotactic CCL21 gradient formed in vivo provides an optimal shape for DCs to recognize haptotactic guidance cue. By reconstitution of the CCL21 gradient in vitro we were also able to study the influence of CCR7 signal termination on DC haptotaxis. To this end, we used DCs lacking the G-protein coupled receptor kinase GRK6, which is responsible for CCL21 induced CCR7 receptor phosphorylation and desensitization (Zidar et al., 2009). We found that CCR7 desensitization by GRK6 is crucial for maintenance of haptotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. In the context of the organism, immobilized haptotactic guidance cues often coincide and compete with soluble chemotactic guidance cues. During wound healing, fibroblasts are exposed and influenced by adhesive cues and soluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly, migrating DCs are exposed to both, soluble chemokines (CCL19 and truncated CCL21) inducing chemotactic behavior as well as the immobilized CCL21. To quantitatively assess these complex coinciding immobilized and soluble guidance cues, we implemented our chemokine photo-patterning technique in a microfluidic system allowing for chemotactic gradient generation. To validate the assay, we observed DC migration in competing CCL19/CCL21 environments. Adhesiveness guided haptotaxis has been studied intensively over the last century. However, quantitative studies leading to conceptual models are largely missing, again due to the lack of a precisely controllable in vitro assay. A requirement for such an in vitro assay is that it must prevent any uncontrolled cell adhesion. This can be accomplished by stable passivation of the surface. In addition, controlled adhesion must be sustainable, quantifiable and dose dependent in order to create homogenous gradients. Therefore, we developed a novel covalent photo-patterning technique satisfying all these needs. In combination with a sustainable poly-vinyl alcohol (PVA) surface coating we were able to generate gradients of adhesive cue to direct cell migration. This approach allowed us to characterize the haptotactic migratory behavior of zebrafish keratocytes in vitro. Furthermore, defined patterns of adhesive cue allowed us to control for cell shape and growth on a subcellular scale.}, author = {Schwarz, Jan}, issn = {2663-337X}, pages = {178}, publisher = {Institute of Science and Technology Austria}, title = {{Quantitative analysis of haptotactic cell migration}}, year = {2016}, } @article{1321, abstract = {Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.}, author = {Leithner, Alexander F and Eichner, Alexander and Müller, Jan and Reversat, Anne and Brown, Markus and Schwarz, Jan and Merrin, Jack and De Gorter, David and Schur, Florian and Bayerl, Jonathan and De Vries, Ingrid and Wieser, Stefan and Hauschild, Robert and Lai, Frank and Moser, Markus and Kerjaschki, Dontscho and Rottner, Klemens and Small, Victor and Stradal, Theresia and Sixt, Michael K}, journal = {Nature Cell Biology}, pages = {1253 -- 1259}, publisher = {Nature Publishing Group}, title = {{Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes}}, doi = {10.1038/ncb3426}, volume = {18}, year = {2016}, } @article{1530, abstract = {In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.}, author = {Bierbaum, Veronika and Klumpp, Stefan}, journal = {Physical Biology}, number = {6}, publisher = {IOP Publishing Ltd.}, title = {{Impact of the cell division cycle on gene circuits}}, doi = {10.1088/1478-3975/12/6/066003}, volume = {12}, year = {2015}, } @article{1553, abstract = {Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.}, author = {Maiuri, Paolo and Rupprecht, Jean and Wieser, Stefan and Ruprecht, Verena and Bénichou, Olivier and Carpi, Nicolas and Coppey, Mathieu and De Beco, Simon and Gov, Nir and Heisenberg, Carl-Philipp J and Lage Crespo, Carolina and Lautenschlaeger, Franziska and Le Berre, Maël and Lennon Duménil, Ana and Raab, Matthew and Thiam, Hawa and Piel, Matthieu and Sixt, Michael K and Voituriez, Raphaël}, journal = {Cell}, number = {2}, pages = {374 -- 386}, publisher = {Cell Press}, title = {{Actin flows mediate a universal coupling between cell speed and cell persistence}}, doi = {10.1016/j.cell.2015.01.056}, volume = {161}, year = {2015}, } @article{1561, abstract = {Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1StopF (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.}, author = {Heger, Klaus and Kober, Maike and Rieß, David and Drees, Christoph and De Vries, Ingrid and Bertossi, Arianna and Roers, Axel and Sixt, Michael K and Schmidt Supprian, Marc}, journal = {European Journal of Immunology}, number = {6}, pages = {1614 -- 1620}, publisher = {Wiley}, title = {{A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors}}, doi = {10.1002/eji.201545457}, volume = {45}, year = {2015}, } @article{1560, abstract = {Stromal cells in the subcapsular sinus of the lymph node 'decide' which cells and molecules are allowed access to the deeper parenchyma. The glycoprotein PLVAP is a crucial component of this selector function.}, author = {Hons, Miroslav and Sixt, Michael K}, journal = {Nature Immunology}, number = {4}, pages = {338 -- 340}, publisher = {Nature Publishing Group}, title = {{The lymph node filter revealed}}, doi = {10.1038/ni.3126}, volume = {16}, year = {2015}, } @article{1575, abstract = {The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.}, author = {Chabaud, Mélanie and Heuzé, Mélina and Bretou, Marine and Vargas, Pablo and Maiuri, Paolo and Solanes, Paola and Maurin, Mathieu and Terriac, Emmanuel and Le Berre, Maël and Lankar, Danielle and Piolot, Tristan and Adelstein, Robert and Zhang, Yingfan and Sixt, Michael K and Jacobelli, Jordan and Bénichou, Olivier and Voituriez, Raphaël and Piel, Matthieu and Lennon Duménil, Ana}, journal = {Nature Communications}, publisher = {Nature Publishing Group}, title = {{Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells}}, doi = {10.1038/ncomms8526}, volume = {6}, year = {2015}, } @article{1676, author = {Sixt, Michael K and Raz, Erez}, journal = {Current Opinion in Cell Biology}, number = {10}, pages = {4 -- 6}, publisher = {Elsevier}, title = {{Editorial overview: Cell adhesion and migration}}, doi = {10.1016/j.ceb.2015.09.004}, volume = {36}, year = {2015}, } @article{1687, abstract = {Guided cell movement is essential for development and integrity of animals and crucially involved in cellular immune responses. Leukocytes are professional migratory cells that can navigate through most types of tissues and sense a wide range of directional cues. The responses of these cells to attractants have been mainly explored in tissue culture settings. How leukocytes make directional decisions in situ, within the challenging environment of a tissue maze, is less understood. Here we review recent advances in how leukocytes sense chemical cues in complex tissue settings and make links with paradigms of directed migration in development and Dictyostelium discoideum amoebae.}, author = {Sarris, Milka and Sixt, Michael K}, journal = {Current Opinion in Cell Biology}, number = {10}, pages = {93 -- 102}, publisher = {Elsevier}, title = {{Navigating in tissue mazes: Chemoattractant interpretation in complex environments}}, doi = {10.1016/j.ceb.2015.08.001}, volume = {36}, year = {2015}, } @article{1686, author = {Kiermaier, Eva and Sixt, Michael K}, journal = {Science}, number = {6252}, pages = {1055 -- 1056}, publisher = {American Association for the Advancement of Science}, title = {{Fragmented communication between immune cells: Neutrophils blaze a trail with migratory cues for T cells to follow to sites of infection}}, doi = {10.1126/science.aad0867}, volume = {349}, year = {2015}, } @article{477, abstract = {Dendritic cells are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of dendritic cell functions is poorly understood. Here, we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7TR) as well as its downstream effector Cdc42 is upregulated in dendritic cells upon maturation. Although dendritic cell maturation was independent of 5-HT7TR, receptor stimulation affected dendritic cell morphology through Cdc42-mediated signaling. In addition, basal activity of 5-HT7TR was required for the proper expression of the chemokine receptor CCR7, which is a key factor that controls dendritic cell migration. Consistent with this, we observed that 5-HT7TR enhances chemotactic motility of dendritic cells in vitro by modulating their directionality and migration velocity. Accordingly, migration of dendritic cells in murine colon explants was abolished after pharmacological receptor inhibition. Our results indicate that there is a crucial role for 5-HT7TR-Cdc42-mediated signaling in the regulation of dendritic cell morphology and motility, suggesting that 5-HT7TR could be a new target for treatment of a variety of inflammatory and immune disorders.}, author = {Holst, Katrin and Guseva, Daria and Schindler, Susann and Sixt, Michael K and Braun, Armin and Chopra, Himpriya and Pabst, Oliver and Ponimaskin, Evgeni}, journal = {Journal of Cell Science}, number = {15}, pages = {2866 -- 2880}, publisher = {Company of Biologists}, title = {{The serotonin receptor 5-HT7R regulates the morphology and migratory properties of dendritic cells}}, doi = {10.1242/jcs.167999}, volume = {128}, year = {2015}, } @article{1618, abstract = {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.}, author = {Veldkamp, Christopher and Kiermaier, Eva and Gabel Eissens, Skylar and Gillitzer, Miranda and Lippner, David and Disilvio, Frank and Mueller, Casey and Wantuch, Paeton and Chaffee, Gary and Famiglietti, Michael and Zgoba, Danielle and Bailey, Asha and Bah, Yaya and Engebretson, Samantha and Graupner, David and Lackner, Emily and Larosa, Vincent and Medeiros, Tysha and Olson, Michael and Phillips, Andrew and Pyles, Harley and Richard, Amanda and Schoeller, Scott and Touzeau, Boris and Williams, Larry and Sixt, Michael K and Peterson, Francis}, journal = {Biochemistry}, number = {27}, pages = {4163 -- 4166}, publisher = {American Chemical Society}, title = {{Solution structure of CCL19 and identification of overlapping CCR7 and PSGL-1 binding sites}}, doi = {10.1021/acs.biochem.5b00560}, volume = {54}, year = {2015}, } @article{1537, abstract = {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.}, author = {Ruprecht, Verena and Wieser, Stefan and Callan Jones, Andrew and Smutny, Michael and Morita, Hitoshi and Sako, Keisuke and Barone, Vanessa and Ritsch Marte, Monika and Sixt, Michael K and Voituriez, Raphaël and Heisenberg, Carl-Philipp J}, journal = {Cell}, number = {4}, pages = {673 -- 685}, publisher = {Cell Press}, title = {{Cortical contractility triggers a stochastic switch to fast amoeboid cell motility}}, doi = {10.1016/j.cell.2015.01.008}, volume = {160}, year = {2015}, } @article{1877, abstract = {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.}, author = {Sixt, Michael K and Vaahtomeri, Kari}, journal = {Nature}, number = {7523}, pages = {441 -- 442}, publisher = {Springer Nature}, title = {{Physiology: Relax and come in}}, doi = {10.1038/514441a}, volume = {514}, year = {2014}, } @article{1910, abstract = {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.}, author = {Konradi, Sabine and Yasmin, Nighat and Haslwanter, Denise and Weber, Michele and Gesslbauer, Bernd and Sixt, Michael K and Strobl, Herbert}, journal = {European Journal of Immunology}, number = {2}, pages = {553 -- 560}, publisher = {Wiley-Blackwell}, title = {{Langerhans cell maturation is accompanied by induction of N-cadherin and the transcriptional regulators of epithelial-mesenchymal transition ZEB1/2}}, doi = {10.1002/eji.201343681}, volume = {44}, year = {2014}, } @article{1925, abstract = {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.}, author = {Lamprecht, Constanze and Plochberger, Birgit and Ruprecht, Verena and Wieser, Stefan and Rankl, Christian and Heister, Elena and Unterauer, Barbara and Brameshuber, Mario and Danzberger, Jürgen and Lukanov, Petar and Flahaut, Emmanuel and Schütz, Gerhard and Hinterdorfer, Peter and Ebner, Andreas}, journal = {Nanotechnology}, number = {12}, publisher = {IOP Publishing}, title = {{A single-molecule approach to explore binding uptake and transport of cancer cell targeting nanotubes}}, doi = {10.1088/0957-4484/25/12/125704}, volume = {25}, year = {2014}, } @article{2158, abstract = {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.}, author = {Majumdar, Ritankar and Sixt, Michael K and Parent, Carole}, journal = {Current Opinion in Cell Biology}, number = {1}, pages = {33 -- 40}, publisher = {Elsevier}, title = {{New paradigms in the establishment and maintenance of gradients during directed cell migration}}, doi = {10.1016/j.ceb.2014.05.010}, volume = {30}, year = {2014}, } @article{2214, abstract = {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.}, author = {Stoler Barak, Liat and Moussion, Christine and Shezen, Elias and Hatzav, Miki and Sixt, Michael K and Alon, Ronen}, journal = {PLoS One}, number = {1}, publisher = {Public Library of Science}, title = {{Blood vessels pattern heparan sulfate gradients between their apical and basolateral aspects}}, doi = {10.1371/journal.pone.0085699}, volume = {9}, year = {2014}, } @article{2215, abstract = {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.}, author = {Renkawitz, Jörg and Lademann, Claudio and Jentsch, Stefan}, journal = {Nature Reviews Molecular Cell Biology}, number = {6}, pages = {369 -- 383}, publisher = {Nature Publishing Group}, title = {{Mechanisms and principles of homology search during recombination}}, doi = {10.1038/nrm3805}, volume = {15}, year = {2014}, } @article{2242, abstract = {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.}, author = {Dueck, Anne and Eichner, Alexander and Sixt, Michael K and Meister, Gunter}, issn = {00145793}, journal = {FEBS Letters}, number = {4}, pages = {632 -- 640}, publisher = {Elsevier}, title = {{A miR-155-dependent microRNA hierarchy in dendritic cell maturation and macrophage activation}}, doi = {10.1016/j.febslet.2014.01.009}, volume = {588}, year = {2014}, } @article{2830, author = {Moussion, Christine and Sixt, Michael K}, journal = {Immunity}, number = {5}, pages = {853 -- 854}, publisher = {Cell Press}, title = {{A conduit to amplify innate immunity}}, doi = {10.1016/j.immuni.2013.05.005}, volume = {38}, year = {2013}, } @article{2839, abstract = {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.}, author = {Weber, Michele and Hauschild, Robert and Schwarz, Jan and Moussion, Christine and De Vries, Ingrid and Legler, Daniel and Luther, Sanjiv and Bollenbach, Mark Tobias and Sixt, Michael K}, journal = {Science}, number = {6117}, pages = {328 -- 332}, publisher = {American Association for the Advancement of Science}, title = {{Interstitial dendritic cell guidance by haptotactic chemokine gradients}}, doi = {10.1126/science.1228456}, volume = {339}, year = {2013}, } @article{522, abstract = {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.}, author = {Fuertbauer, Elke and Zaujec, Jan and Uhrin, Pavel and Raab, Ingrid and Weber, Michele and Schachner, Helga and Bauer, Miroslav and Schütz, Gerhard and Binder, Bernd and Sixt, Michael K and Kerjaschki, Dontscho and Stockinger, Hannes}, journal = {Immunology Letters}, number = {1-2}, pages = {31 -- 41}, publisher = {Elsevier}, title = {{Thymic medullar conduits-associated podoplanin promotes natural regulatory T cells}}, doi = {10.1016/j.imlet.2013.07.007}, volume = {154}, year = {2013}, } @inbook{10900, abstract = {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.}, author = {Weber, Michele and Sixt, Michael K}, booktitle = {Chemokines}, editor = {Cardona, Astrid and Ubogu, Eroboghene}, isbn = {9781627034258}, issn = {1940-6029}, pages = {215--226}, publisher = {Humana Press}, title = {{Live Cell Imaging of Chemotactic Dendritic Cell Migration in Explanted Mouse Ear Preparations}}, doi = {10.1007/978-1-62703-426-5_14}, volume = {1013}, year = {2013}, } @article{2946, abstract = {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.}, author = {Dueck, Anne and Ziegler, Christian and Eichner, Alexander and Berezikov, Eugène and Meister, Gunter}, journal = {Nucleic Acids Research}, number = {19}, pages = {9850 -- 9862}, publisher = {Oxford University Press}, title = {{MicroRNAs associated with the different human Argonaute proteins}}, doi = {10.1093/nar/gks705}, volume = {40}, year = {2012}, } @article{2945, abstract = {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.}, author = {Girard, Jean and Moussion, Christine and Förster, Reinhold}, journal = {Nature Reviews Immunology}, number = {11}, pages = {762 -- 773}, publisher = {Nature Publishing Group}, title = {{HEVs, lymphatics and homeostatic immune cell trafficking in lymph nodes}}, doi = {10.1038/nri3298}, volume = {12}, year = {2012}, }