@article{8988, abstract = {The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1’s functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1’s enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.}, author = {Düllberg, Christian F and Auer, Albert and Canigova, Nikola and Loibl, Katrin and Loose, Martin}, issn = {10916490}, journal = {PNAS}, number = {1}, publisher = {National Academy of Sciences}, title = {{In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1}}, doi = {10.1073/pnas.2010054118}, volume = {118}, year = {2021}, } @article{9259, abstract = {Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.}, author = {Vaahtomeri, Kari and Moussion, Christine and Hauschild, Robert and Sixt, Michael K}, issn = {1664-3224}, journal = {Frontiers in Immunology}, publisher = {Frontiers}, title = {{Shape and function of interstitial chemokine CCL21 gradients are independent of heparan sulfates produced by lymphatic endothelium}}, doi = {10.3389/fimmu.2021.630002}, volume = {12}, year = {2021}, } @article{9294, abstract = {In this issue of Developmental Cell, Doyle and colleagues identify periodic anterior contraction as a characteristic feature of fibroblasts and mesenchymal cancer cells embedded in 3D collagen gels. This contractile mechanism generates a matrix prestrain required for crawling in fibrous 3D environments.}, author = {Gärtner, Florian R and Sixt, Michael K}, issn = {18781551}, journal = {Developmental Cell}, number = {6}, pages = {723--725}, publisher = {Elsevier}, title = {{Engaging the front wheels to drive through fibrous terrain}}, doi = {10.1016/j.devcel.2021.03.002}, volume = {56}, year = {2021}, } @article{9822, abstract = {Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science.}, author = {Zisis, Themistoklis and Schwarz, Jan and Balles, Miriam and Kretschmer, Maibritt and Nemethova, Maria and Chait, Remy P and Hauschild, Robert and Lange, Janina and Guet, Calin C and Sixt, Michael K and Zahler, Stefan}, issn = {19448252}, journal = {ACS Applied Materials and Interfaces}, number = {30}, pages = {35545–35560}, publisher = {American Chemical Society}, title = {{Sequential and switchable patterning for studying cellular processes under spatiotemporal control}}, doi = {10.1021/acsami.1c09850}, volume = {13}, year = {2021}, } @article{10834, abstract = {Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.}, author = {Stahnke, Stephanie and Döring, Hermann and Kusch, Charly and de Gorter, David J.J. and Dütting, Sebastian and Guledani, Aleks and Pleines, Irina and Schnoor, Michael and Sixt, Michael K and Geffers, Robert and Rohde, Manfred and Müsken, Mathias and Kage, Frieda and Steffen, Anika and Faix, Jan and Nieswandt, Bernhard and Rottner, Klemens and Stradal, Theresia E.B.}, issn = {0960-9822}, journal = {Current Biology}, keywords = {General Agricultural and Biological Sciences, General Biochemistry, Genetics and Molecular Biology}, number = {10}, pages = {2051--2064.e8}, publisher = {Elsevier}, title = {{Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion}}, doi = {10.1016/j.cub.2021.02.043}, volume = {31}, year = {2021}, } @article{9094, abstract = {Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell–cell contact, the immunological synapse (IS). While it is established that F-actin dynamics regulates signaling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. Here, we show that the DC actin cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. DC actin at the IS appears in transient foci that are dynamized by the WAVE regulatory complex (WRC). The absence of the WRC in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin–mediated cell–cell adhesion. This results in lower numbers of activated and proliferating T cells, demonstrating an important role for DC actin in the regulation of immune synapse functionality.}, author = {Leithner, Alexander F and Altenburger, LM and Hauschild, R and Assen, Frank P and Rottner, K and TEB, Stradal and Diz-Muñoz, A and Stein, JV and Sixt, Michael K}, issn = {1540-8140}, journal = {Journal of Cell Biology}, number = {4}, publisher = {Rockefeller University Press}, title = {{Dendritic cell actin dynamics control contact duration and priming efficiency at the immunological synapse}}, doi = {10.1083/jcb.202006081}, volume = {220}, year = {2021}, } @article{9429, abstract = {De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.}, author = {Morandell, Jasmin and Schwarz, Lena A and Basilico, Bernadette and Tasciyan, Saren and Dimchev, Georgi A and Nicolas, Armel and Sommer, Christoph M and Kreuzinger, Caroline and Dotter, Christoph and Knaus, Lisa and Dobler, Zoe and Cacci, Emanuele and Schur, Florian KM and Danzl, Johann G and Novarino, Gaia}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {1}, publisher = {Springer Nature}, title = {{Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development}}, doi = {10.1038/s41467-021-23123-x}, volume = {12}, year = {2021}, } @phdthesis{10307, abstract = {Bacteria-host interactions represent a continuous trade-off between benefit and risk. Thus, the host immune response is faced with a non-trivial problem – accommodate beneficial commensals and remove harmful pathogens. This is especially difficult as molecular patterns, such as lipopolysaccharide or specific surface organelles such as pili, are conserved in both, commensal and pathogenic bacteria. Type 1 pili, tightly regulated by phase variation, are considered an important virulence factor of pathogenic bacteria as they facilitate invasion into host cells. While invasion represents a de facto passive mechanism for pathogens to escape the host immune response, we demonstrate a fundamental role of type 1 pili as active modulators of the innate and adaptive immune response.}, author = {Tomasek, Kathrin}, issn = {2663-337X}, pages = {73}, publisher = {Institute of Science and Technology Austria}, title = {{Pathogenic Escherichia coli hijack the host immune response}}, doi = {10.15479/at:ista:10307}, year = {2021}, } @unpublished{10316, abstract = {A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease.}, author = {Tomasek, Kathrin and Leithner, Alexander F and Glatzová, Ivana and Lukesch, Michael S. and Guet, Calin C and Sixt, Michael K}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14}}, doi = {10.1101/2021.10.18.464770}, year = {2021}, } @article{7234, abstract = {T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.}, author = {Obeidy, Peyman and Ju, Lining A. and Oehlers, Stefan H. and Zulkhernain, Nursafwana S. and Lee, Quintin and Galeano Niño, Jorge L. and Kwan, Rain Y.Q. and Tikoo, Shweta and Cavanagh, Lois L. and Mrass, Paulus and Cook, Adam J.L. and Jackson, Shaun P. and Biro, Maté and Roediger, Ben and Sixt, Michael K and Weninger, Wolfgang}, issn = {14401711}, journal = {Immunology and Cell Biology}, number = {2}, pages = {93--113}, publisher = {Wiley}, title = {{Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes}}, doi = {10.1111/imcb.12304}, volume = {98}, year = {2020}, } @article{7623, abstract = {A two-dimensional mathematical model for cells migrating without adhesion capabilities is presented and analyzed. Cells are represented by their cortex, which is modeled as an elastic curve, subject to an internal pressure force. Net polymerization or depolymerization in the cortex is modeled via local addition or removal of material, driving a cortical flow. The model takes the form of a fully nonlinear degenerate parabolic system. An existence analysis is carried out by adapting ideas from the theory of gradient flows. Numerical simulations show that these simple rules can account for the behavior observed in experiments, suggesting a possible mechanical mechanism for adhesion-independent motility.}, author = {Jankowiak, Gaspard and Peurichard, Diane and Reversat, Anne and Schmeiser, Christian and Sixt, Michael K}, issn = {02182025}, journal = {Mathematical Models and Methods in Applied Sciences}, number = {3}, pages = {513--537}, publisher = {World Scientific}, title = {{Modeling adhesion-independent cell migration}}, doi = {10.1142/S021820252050013X}, volume = {30}, year = {2020}, } @article{7875, abstract = {Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.}, author = {Kopf, Aglaja and Renkawitz, Jörg and Hauschild, Robert and Girkontaite, Irute and Tedford, Kerry and Merrin, Jack and Thorn-Seshold, Oliver and Trauner, Dirk and Häcker, Hans and Fischer, Klaus Dieter and Kiermaier, Eva and Sixt, Michael K}, issn = {1540-8140}, journal = {The Journal of Cell Biology}, number = {6}, publisher = {Rockefeller University Press}, title = {{Microtubules control cellular shape and coherence in amoeboid migrating cells}}, doi = {10.1083/jcb.201907154}, volume = {219}, year = {2020}, } @article{7876, abstract = {In contrast to lymph nodes, the lymphoid regions of the spleen—the white pulp—are located deep within the organ, yielding the trafficking paths of T cells in the white pulp largely invisible. In an intravital microscopy tour de force reported in this issue of Immunity, Chauveau et al. show that T cells perform unidirectional, perivascular migration through the enigmatic marginal zone bridging channels. }, author = {Sixt, Michael K and Lämmermann, Tim}, issn = {10974180}, journal = {Immunity}, number = {5}, pages = {721--723}, publisher = {Elsevier}, title = {{T cells: Bridge-and-channel commute to the white pulp}}, doi = {10.1016/j.immuni.2020.04.020}, volume = {52}, year = {2020}, } @article{7909, abstract = {Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.}, author = {Damiano-Guercio, Julia and Kurzawa, Laëtitia and Müller, Jan and Dimchev, Georgi A and Schaks, Matthias and Nemethova, Maria and Pokrant, Thomas and Brühmann, Stefan and Linkner, Joern and Blanchoin, Laurent and Sixt, Michael K and Rottner, Klemens and Faix, Jan}, issn = {2050084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion}}, doi = {10.7554/eLife.55351}, volume = {9}, year = {2020}, } @article{8132, abstract = {The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1−/− mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity.}, author = {Salzer, Elisabeth and Zoghi, Samaneh and Kiss, Máté G. and Kage, Frieda and Rashkova, Christina and Stahnke, Stephanie and Haimel, Matthias and Platzer, René and Caldera, Michael and Ardy, Rico Chandra and Hoeger, Birgit and Block, Jana and Medgyesi, David and Sin, Celine and Shahkarami, Sepideh and Kain, Renate and Ziaee, Vahid and Hammerl, Peter and Bock, Christoph and Menche, Jörg and Dupré, Loïc and Huppa, Johannes B. and Sixt, Michael K and Lomakin, Alexis and Rottner, Klemens and Binder, Christoph J. and Stradal, Theresia E.B. and Rezaei, Nima and Boztug, Kaan}, issn = {24709468}, journal = {Science Immunology}, number = {49}, publisher = {AAAS}, title = {{The cytoskeletal regulator HEM1 governs B cell development and prevents autoimmunity}}, doi = {10.1126/sciimmunol.abc3979}, volume = {5}, year = {2020}, } @article{8787, abstract = {Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.}, author = {Nicolai, Leo and Schiefelbein, Karin and Lipsky, Silvia and Leunig, Alexander and Hoffknecht, Marie and Pekayvaz, Kami and Raude, Ben and Marx, Charlotte and Ehrlich, Andreas and Pircher, Joachim and Zhang, Zhe and Saleh, Inas and Marel, Anna-Kristina and Löf, Achim and Petzold, Tobias and Lorenz, Michael and Stark, Konstantin and Pick, Robert and Rosenberger, Gerhild and Weckbach, Ludwig and Uhl, Bernd and Xia, Sheng and Reichel, Christoph Andreas and Walzog, Barbara and Schulz, Christian and Zheden, Vanessa and Bender, Markus and Li, Rong and Massberg, Steffen and Gärtner, Florian R}, issn = {20411723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Vascular surveillance by haptotactic blood platelets in inflammation and infection}}, doi = {10.1038/s41467-020-19515-0}, volume = {11}, year = {2020}, } @article{8142, abstract = {Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine‐tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‐organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin‐sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells.}, author = {Montesinos López, Juan C and Abuzeineh, A and Kopf, Aglaja and Juanes Garcia, Alba and Ötvös, Krisztina and Petrášek, J and Sixt, Michael K and Benková, Eva}, issn = {1460-2075}, journal = {The Embo Journal}, number = {17}, publisher = {Embo Press}, title = {{Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage}}, doi = {10.15252/embj.2019104238}, volume = {39}, year = {2020}, } @article{7885, abstract = {Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.}, author = {Reversat, Anne and Gärtner, Florian R and Merrin, Jack and Stopp, Julian A and Tasciyan, Saren and Aguilera Servin, Juan L and De Vries, Ingrid and Hauschild, Robert and Hons, Miroslav and Piel, Matthieu and Callan-Jones, Andrew and Voituriez, Raphael and Sixt, Michael K}, issn = {14764687}, journal = {Nature}, pages = {582–585}, publisher = {Springer Nature}, title = {{Cellular locomotion using environmental topography}}, doi = {10.1038/s41586-020-2283-z}, volume = {582}, year = {2020}, } @article{8190, author = {Sixt, Michael K and Huttenlocher, Anna}, issn = {1540-8140}, journal = {The Journal of Cell Biology}, number = {8}, publisher = {Rockefeller University Press}, title = {{Zena Werb (1945-2020): Cell biology in context}}, doi = {10.1083/jcb.202007029}, volume = {219}, year = {2020}, } @article{6824, abstract = {Platelets are small anucleate cellular fragments that are released by megakaryocytes and safeguard vascular integrity through a process termed ‘haemostasis’. However, platelets have important roles beyond haemostasis as they contribute to the initiation and coordination of intravascular immune responses. They continuously monitor blood vessel integrity and tightly coordinate vascular trafficking and functions of multiple cell types. In this way platelets act as ‘patrolling officers of the vascular highway’ that help to establish effective immune responses to infections and cancer. Here we discuss the distinct biological features of platelets that allow them to shape immune responses to pathogens and tumour cells, highlighting the parallels between these responses.}, author = {Gärtner, Florian R and Massberg, Steffen}, issn = {1474-1741}, journal = {Nature Reviews Immunology}, number = {12}, pages = {747–760}, publisher = {Springer Nature}, title = {{Patrolling the vascular borders: Platelets in immunity to infection and cancer}}, doi = {10.1038/s41577-019-0202-z}, volume = {19}, year = {2019}, } @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}, }