@article{7009, abstract = {Cell migration is essential for physiological processes as diverse as development, immune defence and wound healing. It is also a hallmark of cancer malignancy. Thousands of publications have elucidated detailed molecular and biophysical mechanisms of cultured cells migrating on flat, 2D substrates of glass and plastic. However, much less is known about how cells successfully navigate the complex 3D environments of living tissues. In these more complex, native environments, cells use multiple modes of migration, including mesenchymal, amoeboid, lobopodial and collective, and these are governed by the local extracellular microenvironment, specific modalities of Rho GTPase signalling and non- muscle myosin contractility. Migration through 3D environments is challenging because it requires the cell to squeeze through complex or dense extracellular structures. Doing so requires specific cellular adaptations to mechanical features of the extracellular matrix (ECM) or its remodelling. In addition, besides navigating through diverse ECM environments and overcoming extracellular barriers, cells often interact with neighbouring cells and tissues through physical and signalling interactions. Accordingly, cells need to call on an impressively wide diversity of mechanisms to meet these challenges. This Review examines how cells use both classical and novel mechanisms of locomotion as they traverse challenging 3D matrices and cellular environments. It focuses on principles rather than details of migratory mechanisms and draws comparisons between 1D, 2D and 3D migration.}, author = {Yamada, KM and Sixt, Michael K}, issn = {1471-0080}, journal = {Nature Reviews Molecular Cell Biology}, number = {12}, pages = {738–752}, publisher = {Springer Nature}, title = {{Mechanisms of 3D cell migration}}, doi = {10.1038/s41580-019-0172-9}, volume = {20}, year = {2019}, } @article{6988, abstract = {Platelets are central players in thrombosis and hemostasis but are increasingly recognized as key components of the immune system. They shape ensuing immune responses by recruiting leukocytes, and support the development of adaptive immunity. Recent data shed new light on the complex role of platelets in immunity. Here, we summarize experimental and clinical data on the role of platelets in host defense against bacteria. Platelets bind, contain, and kill bacteria directly; however, platelet proinflammatory effector functions and cross-talk with the coagulation system, can also result in damage to the host (e.g., acute lung injury and sepsis). Novel clinical insights support this dichotomy: platelet inhibition/thrombocytopenia can be either harmful or protective, depending on pathophysiological context. Clinical studies are currently addressing this aspect in greater depth.}, author = {Nicolai, Leo and Gärtner, Florian R and Massberg, Steffen}, issn = {1471-4906}, journal = {Trends in Immunology}, number = {10}, pages = {922--938}, publisher = {Cell Press}, title = {{Platelets in host defense: Experimental and clinical insights}}, doi = {10.1016/j.it.2019.08.004}, volume = {40}, year = {2019}, } @article{6979, author = {Kopf, Aglaja and Sixt, Michael K}, issn = {1879-0445}, journal = {Current Biology}, number = {20}, pages = {R1091--R1093}, publisher = {Cell Press}, title = {{Gut homeostasis: Active migration of intestinal epithelial cells in tissue renewal}}, doi = {10.1016/j.cub.2019.08.068}, volume = {29}, year = {2019}, } @article{7105, abstract = {Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.}, author = {Yolland, Lawrence and Burki, Mubarik and Marcotti, Stefania and Luchici, Andrei and Kenny, Fiona N. and Davis, John Robert and Serna-Morales, Eduardo and Müller, Jan and Sixt, Michael K and Davidson, Andrew and Wood, Will and Schumacher, Linus J. and Endres, Robert G. and Miodownik, Mark and Stramer, Brian M.}, issn = {1476-4679}, journal = {Nature Cell Biology}, number = {11}, pages = {1370--1381}, publisher = {Springer Nature}, title = {{Persistent and polarized global actin flow is essential for directionality during cell migration}}, doi = {10.1038/s41556-019-0411-5}, volume = {21}, year = {2019}, } @article{7420, abstract = {β1-integrins mediate cell–matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes, including cancer cell invasion. Here, we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with β1-integrin, and its role in recycling of active but not inactive β1-integrin receptors. Silencing of GGA2 limits active β1-integrin levels in focal adhesions and decreases cancer cell migration and invasion, which is in agreement with its ability to regulate the dynamics of active integrins. By using the proximity-dependent biotin identification (BioID) method, we identified two RAB family small GTPases, i.e. RAB13 and RAB10, as novel interactors of GGA2. Functionally, RAB13 silencing triggers the intracellular accumulation of active β1-integrin, and reduces integrin activity in focal adhesions and cell migration similarly to GGA2 depletion, indicating that both facilitate active β1-integrin recycling to the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.}, author = {Sahgal, Pranshu and Alanko, Jonna H and Icha, Jaroslav and Paatero, Ilkka and Hamidi, Hellyeh and Arjonen, Antti and Pietilä, Mika and Rokka, Anne and Ivaska, Johanna}, issn = {1477-9137}, journal = {Journal of Cell Science}, number = {11}, publisher = {The Company of Biologists}, title = {{GGA2 and RAB13 promote activity-dependent β1-integrin recycling}}, doi = {10.1242/jcs.233387}, volume = {132}, year = {2019}, } @article{7404, abstract = {The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved in vivo through a dynamic process of de novo branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through in vivo time-lapse imaging of the subcellular localization of actin during the process of branching of Drosophila larva sensory neurons, combined with genetic analysis and electron tomography, we have identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation in vivo. These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.}, author = {Stürner, Tomke and Tatarnikova, Anastasia and Müller, Jan and Schaffran, Barbara and Cuntz, Hermann and Zhang, Yun and Nemethova, Maria and Bogdan, Sven and Small, Vic and Tavosanis, Gaia}, issn = {1477-9129}, journal = {Development}, number = {7}, publisher = {The Company of Biologists}, title = {{Transient localization of the Arp2/3 complex initiates neuronal dendrite branching in vivo}}, doi = {10.1242/dev.171397}, volume = {146}, year = {2019}, } @phdthesis{6947, abstract = {Lymph nodes are es s ential organs of the immune s ys tem where adaptive immune responses originate, and consist of various leukocyte populations and a stromal backbone. Fibroblastic reticular cells (FRCs) are the main stromal cells and form a sponge-like extracellular matrix network, called conduits , which they thems elves enwrap and contract. Lymph, containing s oluble antigens , arrive in lymph nodes via afferent lymphatic vessels that connect to the s ubcaps ular s inus and conduit network. According to the current paradigm, the conduit network dis tributes afferent lymph through lymph nodes and thus provides acces s for immune cells to lymph-borne antigens. An elas tic caps ule s urrounds the organ and confines the immune cells and FRC network. Lymph nodes are completely packed with lymphocytes and lymphocyte numbers directly dictates the size of the organ. Although lymphocytes cons tantly enter and leave the lymph node, its s ize remains remarkedly s table under homeostatic conditions. It is only partly known how the cellularity and s ize of the lymph node is regulated and how the lymph node is able to swell in inflammation. The role of the FRC network in lymph node s welling and trans fer of fluids are inves tigated in this thes is. Furthermore, we s tudied what trafficking routes are us ed by cancer cells in lymph nodes to form distal metastases.We examined the role of a mechanical feedback in regulation of lymph node swelling. Using parallel plate compression and UV-las er cutting experiments we dis s ected the mechanical force dynamics of the whole lymph node, and individually for FRCs and the caps ule. Physical forces generated by packed lymphocytes directly affect the tens ion on the FRC network and capsule, which increases its resistance to swelling. This implies a feedback mechanism between tis s ue pres s ure and ability of lymphocytes to enter the organ. Following inflammation, the lymph node swells ∼10 fold in two weeks . Yet, what is the role for tens ion on the FRC network and caps ule, and how are lymphocytes able to enter in conditions that resist swelling remain open ques tions . We s how that tens ion on the FRC network is important to limit the swelling rate of the organ so that the FRC network can grow in a coordinated fashion. This is illustrated by interfering with FRC contractility, which leads to faster swelling rates and a dis organized FRC network in the inflamed lymph node. Growth of the FRC network in turn is expected to releas e tens ion on thes e s tructures and lowers the res is tance to swelling, thereby allowing more lymphocytes to enter the organ and drive more swelling. Halt of swelling coincides with a thickening of the caps ule, which forms a thick res is tant band around the organ and lowers tens ion on the FRC network to form a new force equilibrium.The FRC and conduit network are further believed to be a privileged s ite of s oluble information within the lymph node, although many details remain uns olved. We s how by 3D ultra-recons truction that FRCs and antigen pres enting cells cover the s urface of conduit s ys tem for more than 99% and we dis cus s the implications for s oluble information exchangeat the conduit level.Finally, there is an ongoing debate in the cancer field whether and how cancer cells in lymph nodes s eed dis tal metas tas es . We s how that cancer cells infus ed into the lymph node can utilize trafficking routes of immune cells and rapidly migrate to blood vessels. Once in the blood circulation, these cells are able to form metastases in distal tissues.}, author = {Assen, Frank P}, issn = {2663-337X}, pages = {142}, publisher = {Institute of Science and Technology Austria}, title = {{Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking}}, doi = {10.15479/AT:ISTA:6947}, year = {2019}, } @phdthesis{6891, abstract = {While cells of mesenchymal or epithelial origin perform their effector functions in a purely anchorage dependent manner, cells derived from the hematopoietic lineage are not committed to operate only within a specific niche. Instead, these cells are able to function autonomously of the molecular composition in a broad range of tissue compartments. By this means, cells of the hematopoietic lineage retain the capacity to disseminate into connective tissue and recirculate between organs, building the foundation for essential processes such as tissue regeneration or immune surveillance. Cells of the immune system, specifically leukocytes, are extraordinarily good at performing this task. These cells are able to flexibly shift their mode of migration between an adhesion-mediated and an adhesion-independent manner, instantaneously accommodating for any changes in molecular composition of the external scaffold. The key component driving directed leukocyte migration is the chemokine receptor 7, which guides the cell along gradients of chemokine ligand. Therefore, the physical destination of migrating leukocytes is purely deterministic, i.e. given by global directional cues such as chemokine gradients. Nevertheless, these cells typically reside in three-dimensional scaffolds of inhomogeneous complexity, raising the question whether cells are able to locally discriminate between multiple optional migration routes. Current literature provides evidence that leukocytes, specifically dendritic cells, do indeed probe their surrounding by virtue of multiple explorative protrusions. However, it remains enigmatic how these cells decide which one is the more favorable route to follow and what are the key players involved in performing this task. Due to the heterogeneous environment of most tissues, and the vast adaptability of migrating leukocytes, at this time it is not clear to what extent leukocytes are able to optimize their migratory strategy by adapting their level of adhesiveness. And, given the fact that leukocyte migration is characterized by branched cell shapes in combination with high migration velocities, it is reasonable to assume that these cells require fine tuned shape maintenance mechanisms that tightly coordinate protrusion and adhesion dynamics in a spatiotemporal manner. Therefore, this study aimed to elucidate how rapidly migrating leukocytes opt for an ideal migratory path while maintaining a continuous cell shape and balancing adhesive forces to efficiently navigate through complex microenvironments. The results of this study unraveled a role for the microtubule cytoskeleton in promoting the decision making process during path finding and for the first time point towards a microtubule-mediated function in cell shape maintenance of highly ramified cells such as dendritic cells. Furthermore, we found that migrating low-adhesive leukocytes are able to instantaneously adapt to increased tensile load by engaging adhesion receptors. This response was only occurring tangential to the substrate while adhesive properties in the vertical direction were not increased. As leukocytes are primed for rapid migration velocities, these results demonstrate that leukocyte integrins are able to confer a high level of traction forces parallel to the cell membrane along the direction of migration without wasting energy in gluing the cell to the substrate. Thus, the data in the here presented thesis provide new insights into the pivotal role of cytoskeletal dynamics and the mechanisms of force transduction during leukocyte migration. Thereby the here presented results help to further define fundamental principles underlying leukocyte migration and open up potential therapeutic avenues of clinical relevance. }, author = {Kopf, Aglaja}, isbn = {978-3-99078-002-2}, issn = {2663-337X}, keywords = {cell biology, immunology, leukocyte, migration, microfluidics}, pages = {171}, publisher = {Institute of Science and Technology Austria}, title = {{The implication of cytoskeletal dynamics on leukocyte migration}}, doi = {10.15479/AT:ISTA:6891}, year = {2019}, } @article{6328, abstract = {During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1,2,3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some—but not all—cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion7, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.}, author = {Renkawitz, Jörg and Kopf, Aglaja and Stopp, Julian A and de Vries, Ingrid and Driscoll, Meghan K. and Merrin, Jack and Hauschild, Robert and Welf, Erik S. and Danuser, Gaudenz and Fiolka, Reto and Sixt, Michael K}, journal = {Nature}, pages = {546--550}, publisher = {Springer Nature}, title = {{Nuclear positioning facilitates amoeboid migration along the path of least resistance}}, doi = {10.1038/s41586-019-1087-5}, volume = {568}, year = {2019}, } @article{6877, author = {Kopf, Aglaja and Sixt, Michael K}, issn = {1097-4172}, journal = {Cell}, number = {1}, pages = {51--53}, publisher = {Elsevier}, title = {{The neural crest pitches in to remove apoptotic debris}}, doi = {10.1016/j.cell.2019.08.047}, volume = {179}, year = {2019}, } @article{6354, abstract = {Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow.}, author = {Fan, Shuxia and Lorenz, Michael and Massberg, Steffen and Gärtner, Florian R}, issn = {2331-8325}, journal = {Bio-Protocol}, keywords = {Platelets, Cell migration, Bacteria, Shear flow, Fibrinogen, E. coli}, number = {18}, publisher = {Bio-Protocol}, title = {{Platelet migration and bacterial trapping assay under flow}}, doi = {10.21769/bioprotoc.3018}, volume = {8}, year = {2018}, } @article{318, abstract = {The insect’s fat body combines metabolic and immunological functions. In this issue of Developmental Cell, Franz et al. (2018) show that in Drosophila, cells of the fat body are not static, but can actively “swim” toward sites of epithelial injury, where they physically clog the wound and locally secrete antimicrobial peptides.}, author = {Casano, Alessandra M and Sixt, Michael K}, journal = {Developmental Cell}, number = {4}, pages = {405 -- 406}, publisher = {Cell Press}, title = {{A fat lot of good for wound healing}}, doi = {10.1016/j.devcel.2018.02.009}, volume = {44}, year = {2018}, } @article{308, abstract = {Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.}, author = {Ratheesh, Aparna and Biebl, Julia and Smutny, Michael and Veselá, Jana and Papusheva, Ekaterina and Krens, Gabriel and Kaufmann, Walter and György, Attila and Casano, Alessandra M and Siekhaus, Daria E}, journal = {Developmental Cell}, number = {3}, pages = {331 -- 346}, publisher = {Elsevier}, title = {{Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration}}, doi = {10.1016/j.devcel.2018.04.002}, volume = {45}, year = {2018}, } @article{437, abstract = {Dendritic cells (DCs) are sentinels of the adaptive immune system that reside in peripheral organs of mammals. Upon pathogen encounter, they undergo maturation and up-regulate the chemokine receptor CCR7 that guides them along gradients of its chemokine ligands CCL19 and 21 to the next draining lymph node. There, DCs present peripherally acquired antigen to naïve T cells, thereby triggering adaptive immunity.}, author = {Leithner, Alexander F and Renkawitz, Jörg and De Vries, Ingrid and Hauschild, Robert and Haecker, Hans and Sixt, Michael K}, journal = {European Journal of Immunology}, number = {6}, pages = {1074 -- 1077}, publisher = {Wiley-Blackwell}, title = {{Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration}}, doi = {10.1002/eji.201747358}, volume = {48}, year = {2018}, } @article{5672, abstract = {The release of IgM is the first line of an antibody response and precedes the generation of high affinity IgG in germinal centers. Once secreted by freshly activated plasmablasts, IgM is released into the efferent lymph of reactive lymph nodes as early as 3 d after immunization. As pentameric IgM has an enormous size of 1,000 kD, its diffusibility is low, and one might wonder how it can pass through the densely lymphocyte-packed environment of a lymph node parenchyma in order to reach its exit. In this issue of JEM, Thierry et al. show that, in order to reach the blood stream, IgM molecules take a specific micro-anatomical route via lymph node conduits.}, author = {Reversat, Anne and Sixt, Michael K}, issn = {00221007}, journal = {Journal of Experimental Medicine}, number = {12}, pages = {2959--2961}, publisher = {Rockefeller University Press}, title = {{IgM's exit route}}, doi = {10.1084/jem.20181934}, volume = {215}, year = {2018}, } @article{275, abstract = {Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified > 1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.}, author = {Brown, Markus and Johnson, Louise and Leone, Dario and Májek, Peter and Vaahtomeri, Kari and Senfter, Daniel and Bukosza, Nora and Schachner, Helga and Asfour, Gabriele and Langer, Brigitte and Hauschild, Robert and Parapatics, Katja and Hong, Young and Bennett, Keiryn and Kain, Renate and Detmar, Michael and Sixt, Michael K and Jackson, David and Kerjaschki, Dontscho}, journal = {Journal of Cell Biology}, number = {6}, pages = {2205 -- 2221}, publisher = {Rockefeller University Press}, title = {{Lymphatic exosomes promote dendritic cell migration along guidance cues}}, doi = {10.1083/jcb.201612051}, volume = {217}, year = {2018}, } @article{5858, abstract = {Spatial patterns are ubiquitous on the subcellular, cellular and tissue level, and can be studied using imaging techniques such as light and fluorescence microscopy. Imaging data provide quantitative information about biological systems; however, mechanisms causing spatial patterning often remain elusive. In recent years, spatio-temporal mathematical modelling has helped to overcome this problem. Yet, outliers and structured noise limit modelling of whole imaging data, and models often consider spatial summary statistics. Here, we introduce an integrated data-driven modelling approach that can cope with measurement artefacts and whole imaging data. Our approach combines mechanistic models of the biological processes with robust statistical models of the measurement process. The parameters of the integrated model are calibrated using a maximum-likelihood approach. We used this integrated modelling approach to study in vivo gradients of the chemokine (C-C motif) ligand 21 (CCL21). CCL21 gradients guide dendritic cells and are important in the adaptive immune response. Using artificial data, we verified that the integrated modelling approach provides reliable parameter estimates in the presence of measurement noise and that bias and variance of these estimates are reduced compared to conventional approaches. The application to experimental data allowed the parametrization and subsequent refinement of the model using additional mechanisms. Among other results, model-based hypothesis testing predicted lymphatic vessel-dependent concentration of heparan sulfate, the binding partner of CCL21. The selected model provided an accurate description of the experimental data and was partially validated using published data. Our findings demonstrate that integrated statistical modelling of whole imaging data is computationally feasible and can provide novel biological insights.}, author = {Hross, Sabrina and Theis, Fabian J. and Sixt, Michael K and Hasenauer, Jan}, issn = {17425689}, journal = {Journal of the Royal Society Interface}, number = {149}, publisher = {Royal Society Publishing}, title = {{Mechanistic description of spatial processes using integrative modelling of noise-corrupted imaging data}}, doi = {10.1098/rsif.2018.0600}, volume = {15}, year = {2018}, } @inbook{153, abstract = {Cells migrating in multicellular organisms steadily traverse complex three-dimensional (3D) environments. To decipher the underlying cell biology, current experimental setups either use simplified 2D, tissue-mimetic 3D (e.g., collagen matrices) or in vivo environments. While only in vivo experiments are truly physiological, they do not allow for precise manipulation of environmental parameters. 2D in vitro experiments do allow mechanical and chemical manipulations, but increasing evidence demonstrates substantial differences of migratory mechanisms in 2D and 3D. Here, we describe simple, robust, and versatile “pillar forests” to investigate cell migration in complex but fully controllable 3D environments. Pillar forests are polydimethylsiloxane-based setups, in which two closely adjacent surfaces are interconnected by arrays of micrometer-sized pillars. Changing the pillar shape, size, height and the inter-pillar distance precisely manipulates microenvironmental parameters (e.g., pore sizes, micro-geometry, micro-topology), while being easily combined with chemotactic cues, surface coatings, diverse cell types and advanced imaging techniques. Thus, pillar forests combine the advantages of 2D cell migration assays with the precise definition of 3D environmental parameters.}, author = {Renkawitz, Jörg and Reversat, Anne and Leithner, Alexander F and Merrin, Jack and Sixt, Michael K}, booktitle = {Methods in Cell Biology}, issn = {0091679X}, pages = {79 -- 91}, publisher = {Academic Press}, title = {{Micro-engineered “pillar forests” to study cell migration in complex but controlled 3D environments}}, doi = {10.1016/bs.mcb.2018.07.004}, volume = {147}, year = {2018}, } @article{276, abstract = {Directed migration of cells relies on their ability to sense directional guidance cues and to interact with pericellular structures in order to transduce contractile cytoskeletal- into mechanical forces. These biomechanical processes depend highly on microenvironmental factors such as exposure to 2D surfaces or 3D matrices. In vivo, the majority of cells are exposed to 3D environments. Data on 3D cell migration are mostly derived from intravital microscopy or collagen-based in vitro assays. Both approaches offer only limited controlla-bility of experimental conditions. Here, we developed an automated microfluidic system that allows positioning of cells in 3D microenvironments containing highly controlled diffusion-based chemokine gradients. Tracking migration in such gradients was feasible in real time at the single cell level. Moreover, the setup allowed on-chip immunocytochemistry and thus linking of functional with phenotypical properties in individual cells. Spatially defined retrieval of cells from the device allows down-stream off-chip analysis. Using dendritic cells as a model, our setup specifically allowed us for the first time to quantitate key migration characteristics of cells exposed to identical gradients of the chemokine CCL19 yet placed on 2D vs in 3D environments. Migration properties between 2D and 3D migration were distinct. Morphological features of cells migrating in an in vitro 3D environment were similar to those of cells migrating in animal tissues, but different from cells migrating on a surface. Our system thus offers a highly controllable in vitro-mimic of a 3D environment that cells traffic in vivo.}, author = {Frick, Corina and Dettinger, Philip and Renkawitz, Jörg and Jauch, Annaïse and Berger, Christoph and Recher, Mike and Schroeder, Timm and Mehling, Matthias}, journal = {PLoS One}, number = {6}, publisher = {Public Library of Science}, title = {{Nano-scale microfluidics to study 3D chemotaxis at the single cell level}}, doi = {10.1371/journal.pone.0198330}, volume = {13}, year = {2018}, } @article{5861, abstract = {In zebrafish larvae, it is the cell type that determines how the cell responds to a chemokine signal.}, author = {Alanko, Jonna H and Sixt, Michael K}, issn = {2050084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{The cell sets the tone}}, doi = {10.7554/eLife.37888}, volume = {7}, year = {2018}, } @article{5984, abstract = {G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs.}, author = {Morri, Maurizio and Sanchez-Romero, Inmaculada and Tichy, Alexandra-Madelaine and Kainrath, Stephanie and Gerrard, Elliot J. and Hirschfeld, Priscila and Schwarz, Jan and Janovjak, Harald L}, issn = {2041-1723}, journal = {Nature Communications}, number = {1}, publisher = {Springer Nature}, title = {{Optical functionalization of human class A orphan G-protein-coupled receptors}}, doi = {10.1038/s41467-018-04342-1}, volume = {9}, year = {2018}, } @article{5992, abstract = {Lamellipodia are flat membrane protrusions formed during mesenchymal motion. Polymerization at the leading edge assembles the actin filament network and generates protrusion force. How this force is supported by the network and how the assembly rate is shared between protrusion and network retrograde flow determines the protrusion rate. We use mathematical modeling to understand experiments changing the F-actin density in lamellipodia of B16-F1 melanoma cells by modulation of Arp2/3 complex activity or knockout of the formins FMNL2 and FMNL3. Cells respond to a reduction of density with a decrease of protrusion velocity, an increase in the ratio of force to filament number, but constant network assembly rate. The relation between protrusion force and tension gradient in the F-actin network and the density dependency of friction, elasticity, and viscosity of the network explain the experimental observations. The formins act as filament nucleators and elongators with differential rates. Modulation of their activity suggests an effect on network assembly rate. Contrary to these expectations, the effect of changes in elongator composition is much weaker than the consequences of the density change. We conclude that the force acting on the leading edge membrane is the force required to drive F-actin network retrograde flow.}, author = {Dolati, Setareh and Kage, Frieda and Mueller, Jan and Müsken, Mathias and Kirchner, Marieluise and Dittmar, Gunnar and Sixt, Michael K and Rottner, Klemens and Falcke, Martin}, issn = {1939-4586}, journal = {Molecular Biology of the Cell}, number = {22}, pages = {2674--2686}, publisher = {American Society for Cell Biology }, title = {{On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility}}, doi = {10.1091/mbc.e18-02-0082}, volume = {29}, year = {2018}, } @article{6497, abstract = {T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b−/− CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b−/− CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b−/− CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue–resident T cell populations.}, author = {Moalli, Federica and Ficht, Xenia and Germann, Philipp and Vladymyrov, Mykhailo and Stolp, Bettina and de Vries, Ingrid and Lyck, Ruth and Balmer, Jasmin and Fiocchi, Amleto and Kreutzfeldt, Mario and Merkler, Doron and Iannacone, Matteo and Ariga, Akitaka and Stoffel, Michael H. and Sharpe, James and Bähler, Martin and Sixt, Michael K and Diz-Muñoz, Alba and Stein, Jens V.}, issn = {1540-9538}, journal = {The Journal of Experimental Medicine}, number = {7}, pages = {1869–1890}, publisher = {Rockefeller University Press}, title = {{The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+T cells}}, doi = {10.1084/jem.20170896}, volume = {2015}, year = {2018}, } @article{402, abstract = {During metastasis, malignant cells escape the primary tumor, intravasate lymphatic vessels, and reach draining sentinel lymph nodes before they colonize distant organs via the blood circulation. Although lymph node metastasis in cancer patients correlates with poor prognosis, evidence is lacking as to whether and how tumor cells enter the bloodstream via lymph nodes. To investigate this question, we delivered carcinoma cells into the lymph nodes of mice by microinfusing the cells into afferent lymphatic vessels. We found that tumor cells rapidly infiltrated the lymph node parenchyma, invaded blood vessels, and seeded lung metastases without involvement of the thoracic duct. These results suggest that the lymph node blood vessels can serve as an exit route for systemic dissemination of cancer cells in experimental mouse models. Whether this form of tumor cell spreading occurs in cancer patients remains to be determined.}, author = {Brown, Markus and Assen, Frank P and Leithner, Alexander F and Abe, Jun and Schachner, Helga and Asfour, Gabriele and Bagó Horváth, Zsuzsanna and Stein, Jens and Uhrin, Pavel and Sixt, Michael K and Kerjaschki, Dontscho}, journal = {Science}, number = {6382}, pages = {1408 -- 1411}, publisher = {American Association for the Advancement of Science}, title = {{Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice}}, doi = {10.1126/science.aal3662}, volume = {359}, year = {2018}, } @phdthesis{323, abstract = {In the here presented thesis, we explore the role of branched actin networks in cell migration and antigen presentation, the two most relevant processes in dendritic cell biology. Branched actin networks construct lamellipodial protrusions at the leading edge of migrating cells. These are typically seen as adhesive structures, which mediate force transduction to the extracellular matrix that leads to forward locomotion. We ablated Arp2/3 nucleation promoting factor WAVE in DCs and found that the resulting cells lack lamellipodial protrusions. Instead, depending on the maturation state, one or multiple filopodia were formed. By challenging these cells in a variety of migration assays we found that lamellipodial protrusions are dispensable for the locomotion of leukocytes and actually dampen the speed of migration. However, lamellipodia are critically required to negotiate complex environments that DCs experience while they travel to the next draining lymph node. Taken together our results suggest that leukocyte lamellipodia have rather a sensory- than a force transducing function. Furthermore, we show for the first time structure and dynamics of dendritic cell F-actin at the immunological synapse with naïve T cells. Dendritic cell F-actin appears as dynamic foci that are nucleated by the Arp2/3 complex. WAVE ablated dendritic cells show increased membrane tension, leading to an altered ultrastructure of the immunological synapse and severe T cell priming defects. These results point towards a previously unappreciated role of the cellular mechanics of dendritic cells in T cell activation. Additionally, we present a novel cell culture based system for the differentiation of dendritic cells from conditionally immortalized hematopoietic precursors. These precursor cells are genetically tractable via the CRISPR/Cas9 system while they retain their ability to differentiate into highly migratory dendritic cells and other immune cells. This will foster the study of all aspects of dendritic cell biology and beyond. }, author = {Leithner, Alexander F}, issn = {2663-337X}, pages = {99}, publisher = {Institute of Science and Technology Austria}, title = {{Branched actin networks in dendritic cell biology}}, doi = {10.15479/AT:ISTA:th_998}, year = {2018}, } @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}, }