TY - JOUR AB - 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. AU - Leithner, Alexander F AU - Altenburger, LM AU - Hauschild, R AU - Assen, Frank P AU - Rottner, K AU - TEB, Stradal AU - Diz-Muñoz, A AU - Stein, JV AU - Sixt, Michael K ID - 9094 IS - 4 JF - Journal of Cell Biology SN - 0021-9525 TI - Dendritic cell actin dynamics control contact duration and priming efficiency at the immunological synapse VL - 220 ER - TY - JOUR AB - 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. AU - Morandell, Jasmin AU - Schwarz, Lena A AU - Basilico, Bernadette AU - Tasciyan, Saren AU - Dimchev, Georgi A AU - Nicolas, Armel AU - Sommer, Christoph M AU - Kreuzinger, Caroline AU - Dotter, Christoph AU - Knaus, Lisa AU - Dobler, Zoe AU - Cacci, Emanuele AU - Schur, Florian KM AU - Danzl, Johann G AU - Novarino, Gaia ID - 9429 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology TI - Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development VL - 12 ER - TY - THES AB - 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. AU - Tomasek, Kathrin ID - 10307 SN - 2663-337X TI - Pathogenic Escherichia coli hijack the host immune response ER - TY - GEN AB - 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. AU - Tomasek, Kathrin AU - Leithner, Alexander F AU - Glatzová, Ivana AU - Lukesch, Michael S. AU - Guet, Calin C AU - Sixt, Michael K ID - 10316 T2 - bioRxiv TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 ER - TY - JOUR AB - 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. AU - Obeidy, Peyman AU - Ju, Lining A. AU - Oehlers, Stefan H. AU - Zulkhernain, Nursafwana S. AU - Lee, Quintin AU - Galeano Niño, Jorge L. AU - Kwan, Rain Y.Q. AU - Tikoo, Shweta AU - Cavanagh, Lois L. AU - Mrass, Paulus AU - Cook, Adam J.L. AU - Jackson, Shaun P. AU - Biro, Maté AU - Roediger, Ben AU - Sixt, Michael K AU - Weninger, Wolfgang ID - 7234 IS - 2 JF - Immunology and Cell Biology SN - 08189641 TI - Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes VL - 98 ER - TY - JOUR AB - 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. AU - Jankowiak, Gaspard AU - Peurichard, Diane AU - Reversat, Anne AU - Schmeiser, Christian AU - Sixt, Michael K ID - 7623 IS - 3 JF - Mathematical Models and Methods in Applied Sciences SN - 02182025 TI - Modeling adhesion-independent cell migration VL - 30 ER - TY - JOUR AB - 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. AU - Kopf, Aglaja AU - Renkawitz, Jörg AU - Hauschild, Robert AU - Girkontaite, Irute AU - Tedford, Kerry AU - Merrin, Jack AU - Thorn-Seshold, Oliver AU - Trauner, Dirk AU - Häcker, Hans AU - Fischer, Klaus Dieter AU - Kiermaier, Eva AU - Sixt, Michael K ID - 7875 IS - 6 JF - The Journal of Cell Biology TI - Microtubules control cellular shape and coherence in amoeboid migrating cells VL - 219 ER - TY - JOUR AB - 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. AU - Sixt, Michael K AU - Lämmermann, Tim ID - 7876 IS - 5 JF - Immunity SN - 10747613 TI - T cells: Bridge-and-channel commute to the white pulp VL - 52 ER - TY - JOUR AB - 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. AU - Damiano-Guercio, Julia AU - Kurzawa, Laëtitia AU - Müller, Jan AU - Dimchev, Georgi A AU - Schaks, Matthias AU - Nemethova, Maria AU - Pokrant, Thomas AU - Brühmann, Stefan AU - Linkner, Joern AU - Blanchoin, Laurent AU - Sixt, Michael K AU - Rottner, Klemens AU - Faix, Jan ID - 7909 JF - eLife TI - Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion VL - 9 ER - TY - JOUR AB - 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. AU - Salzer, Elisabeth AU - Zoghi, Samaneh AU - Kiss, Máté G. AU - Kage, Frieda AU - Rashkova, Christina AU - Stahnke, Stephanie AU - Haimel, Matthias AU - Platzer, René AU - Caldera, Michael AU - Ardy, Rico Chandra AU - Hoeger, Birgit AU - Block, Jana AU - Medgyesi, David AU - Sin, Celine AU - Shahkarami, Sepideh AU - Kain, Renate AU - Ziaee, Vahid AU - Hammerl, Peter AU - Bock, Christoph AU - Menche, Jörg AU - Dupré, Loïc AU - Huppa, Johannes B. AU - Sixt, Michael K AU - Lomakin, Alexis AU - Rottner, Klemens AU - Binder, Christoph J. AU - Stradal, Theresia E.B. AU - Rezaei, Nima AU - Boztug, Kaan ID - 8132 IS - 49 JF - Science Immunology TI - The cytoskeletal regulator HEM1 governs B cell development and prevents autoimmunity VL - 5 ER - TY - JOUR AB - 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. AU - Nicolai, Leo AU - Schiefelbein, Karin AU - Lipsky, Silvia AU - Leunig, Alexander AU - Hoffknecht, Marie AU - Pekayvaz, Kami AU - Raude, Ben AU - Marx, Charlotte AU - Ehrlich, Andreas AU - Pircher, Joachim AU - Zhang, Zhe AU - Saleh, Inas AU - Marel, Anna-Kristina AU - Löf, Achim AU - Petzold, Tobias AU - Lorenz, Michael AU - Stark, Konstantin AU - Pick, Robert AU - Rosenberger, Gerhild AU - Weckbach, Ludwig AU - Uhl, Bernd AU - Xia, Sheng AU - Reichel, Christoph Andreas AU - Walzog, Barbara AU - Schulz, Christian AU - Zheden, Vanessa AU - Bender, Markus AU - Li, Rong AU - Massberg, Steffen AU - Gärtner, Florian R ID - 8787 JF - Nature Communications TI - Vascular surveillance by haptotactic blood platelets in inflammation and infection VL - 11 ER - TY - JOUR AB - 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. AU - Montesinos López, Juan C AU - Abuzeineh, A AU - Kopf, Aglaja AU - Juanes Garcia, Alba AU - Ötvös, Krisztina AU - Petrášek, J AU - Sixt, Michael K AU - Benková, Eva ID - 8142 IS - 17 JF - The Embo Journal SN - 0261-4189 TI - Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage VL - 39 ER - TY - JOUR AB - 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. AU - Reversat, Anne AU - Gärtner, Florian R AU - Merrin, Jack AU - Stopp, Julian A AU - Tasciyan, Saren AU - Aguilera Servin, Juan L AU - De Vries, Ingrid AU - Hauschild, Robert AU - Hons, Miroslav AU - Piel, Matthieu AU - Callan-Jones, Andrew AU - Voituriez, Raphael AU - Sixt, Michael K ID - 7885 JF - Nature SN - 00280836 TI - Cellular locomotion using environmental topography VL - 582 ER - TY - JOUR AU - Sixt, Michael K AU - Huttenlocher, Anna ID - 8190 IS - 8 JF - The Journal of Cell Biology TI - Zena Werb (1945-2020): Cell biology in context VL - 219 ER - TY - JOUR AB - 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. AU - Gärtner, Florian R AU - Massberg, Steffen ID - 6824 IS - 12 JF - Nature Reviews Immunology SN - 1474-1733 TI - Patrolling the vascular borders: Platelets in immunity to infection and cancer VL - 19 ER - TY - JOUR AB - 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. AU - Yamada, KM AU - Sixt, Michael K ID - 7009 IS - 12 JF - Nature Reviews Molecular Cell Biology SN - 1471-0072 TI - Mechanisms of 3D cell migration VL - 20 ER - TY - JOUR AB - 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. AU - Nicolai, Leo AU - Gärtner, Florian R AU - Massberg, Steffen ID - 6988 IS - 10 JF - Trends in Immunology SN - 1471-4906 TI - Platelets in host defense: Experimental and clinical insights VL - 40 ER - TY - JOUR AU - Kopf, Aglaja AU - Sixt, Michael K ID - 6979 IS - 20 JF - Current Biology SN - 0960-9822 TI - Gut homeostasis: Active migration of intestinal epithelial cells in tissue renewal VL - 29 ER - TY - JOUR AB - 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. AU - Yolland, Lawrence AU - Burki, Mubarik AU - Marcotti, Stefania AU - Luchici, Andrei AU - Kenny, Fiona N. AU - Davis, John Robert AU - Serna-Morales, Eduardo AU - Müller, Jan AU - Sixt, Michael K AU - Davidson, Andrew AU - Wood, Will AU - Schumacher, Linus J. AU - Endres, Robert G. AU - Miodownik, Mark AU - Stramer, Brian M. ID - 7105 IS - 11 JF - Nature Cell Biology SN - 1465-7392 TI - Persistent and polarized global actin flow is essential for directionality during cell migration VL - 21 ER - TY - JOUR AB - 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. AU - Sahgal, Pranshu AU - Alanko, Jonna H AU - Icha, Jaroslav AU - Paatero, Ilkka AU - Hamidi, Hellyeh AU - Arjonen, Antti AU - Pietilä, Mika AU - Rokka, Anne AU - Ivaska, Johanna ID - 7420 IS - 11 JF - Journal of Cell Science SN - 0021-9533 TI - GGA2 and RAB13 promote activity-dependent β1-integrin recycling VL - 132 ER - TY - JOUR AB - 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. AU - Stürner, Tomke AU - Tatarnikova, Anastasia AU - Müller, Jan AU - Schaffran, Barbara AU - Cuntz, Hermann AU - Zhang, Yun AU - Nemethova, Maria AU - Bogdan, Sven AU - Small, Vic AU - Tavosanis, Gaia ID - 7404 IS - 7 JF - Development SN - 0950-1991 TI - Transient localization of the Arp2/3 complex initiates neuronal dendrite branching in vivo VL - 146 ER - TY - THES AB - 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. AU - Assen, Frank P ID - 6947 SN - 2663-337X TI - Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking ER - TY - THES AB - 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. AU - Kopf, Aglaja ID - 6891 KW - cell biology KW - immunology KW - leukocyte KW - migration KW - microfluidics SN - 978-3-99078-002-2 TI - The implication of cytoskeletal dynamics on leukocyte migration ER - TY - JOUR AB - 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. AU - Renkawitz, Jörg AU - Kopf, Aglaja AU - Stopp, Julian A AU - de Vries, Ingrid AU - Driscoll, Meghan K. AU - Merrin, Jack AU - Hauschild, Robert AU - Welf, Erik S. AU - Danuser, Gaudenz AU - Fiolka, Reto AU - Sixt, Michael K ID - 6328 JF - Nature TI - Nuclear positioning facilitates amoeboid migration along the path of least resistance VL - 568 ER - TY - JOUR AU - Kopf, Aglaja AU - Sixt, Michael K ID - 6877 IS - 1 JF - Cell SN - 0092-8674 TI - The neural crest pitches in to remove apoptotic debris VL - 179 ER - TY - JOUR AB - 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. AU - Fan, Shuxia AU - Lorenz, Michael AU - Massberg, Steffen AU - Gärtner, Florian R ID - 6354 IS - 18 JF - Bio-Protocol KW - Platelets KW - Cell migration KW - Bacteria KW - Shear flow KW - Fibrinogen KW - E. coli SN - 2331-8325 TI - Platelet migration and bacterial trapping assay under flow VL - 8 ER - TY - JOUR AB - 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. AU - Casano, Alessandra M AU - Sixt, Michael K ID - 318 IS - 4 JF - Developmental Cell TI - A fat lot of good for wound healing VL - 44 ER - TY - JOUR AB - 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. AU - Ratheesh, Aparna AU - Biebl, Julia AU - Smutny, Michael AU - Veselá, Jana AU - Papusheva, Ekaterina AU - Krens, Gabriel AU - Kaufmann, Walter AU - György, Attila AU - Casano, Alessandra M AU - Siekhaus, Daria E ID - 308 IS - 3 JF - Developmental Cell TI - Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration VL - 45 ER - TY - JOUR AB - 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. AU - Leithner, Alexander F AU - Renkawitz, Jörg AU - De Vries, Ingrid AU - Hauschild, Robert AU - Haecker, Hans AU - Sixt, Michael K ID - 437 IS - 6 JF - European Journal of Immunology TI - Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration VL - 48 ER - TY - JOUR AB - 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. AU - Reversat, Anne AU - Sixt, Michael K ID - 5672 IS - 12 JF - Journal of Experimental Medicine SN - 00221007 TI - IgM's exit route VL - 215 ER - TY - JOUR AB - 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. AU - Brown, Markus AU - Johnson, Louise AU - Leone, Dario AU - Májek, Peter AU - Vaahtomeri, Kari AU - Senfter, Daniel AU - Bukosza, Nora AU - Schachner, Helga AU - Asfour, Gabriele AU - Langer, Brigitte AU - Hauschild, Robert AU - Parapatics, Katja AU - Hong, Young AU - Bennett, Keiryn AU - Kain, Renate AU - Detmar, Michael AU - Sixt, Michael K AU - Jackson, David AU - Kerjaschki, Dontscho ID - 275 IS - 6 JF - Journal of Cell Biology TI - Lymphatic exosomes promote dendritic cell migration along guidance cues VL - 217 ER - TY - JOUR AB - 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. AU - Hross, Sabrina AU - Theis, Fabian J. AU - Sixt, Michael K AU - Hasenauer, Jan ID - 5858 IS - 149 JF - Journal of the Royal Society Interface SN - 17425689 TI - Mechanistic description of spatial processes using integrative modelling of noise-corrupted imaging data VL - 15 ER - TY - CHAP AB - 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. AU - Renkawitz, Jörg AU - Reversat, Anne AU - Leithner, Alexander F AU - Merrin, Jack AU - Sixt, Michael K ID - 153 SN - 0091679X T2 - Methods in Cell Biology TI - Micro-engineered “pillar forests” to study cell migration in complex but controlled 3D environments VL - 147 ER - TY - JOUR AB - 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. AU - Frick, Corina AU - Dettinger, Philip AU - Renkawitz, Jörg AU - Jauch, Annaïse AU - Berger, Christoph AU - Recher, Mike AU - Schroeder, Timm AU - Mehling, Matthias ID - 276 IS - 6 JF - PLoS One TI - Nano-scale microfluidics to study 3D chemotaxis at the single cell level VL - 13 ER - TY - JOUR AB - In zebrafish larvae, it is the cell type that determines how the cell responds to a chemokine signal. AU - Alanko, Jonna H AU - Sixt, Michael K ID - 5861 JF - eLife SN - 2050084X TI - The cell sets the tone VL - 7 ER - TY - JOUR AB - 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. AU - Morri, Maurizio AU - Sanchez-Romero, Inmaculada AU - Tichy, Alexandra-Madelaine AU - Kainrath, Stephanie AU - Gerrard, Elliot J. AU - Hirschfeld, Priscila AU - Schwarz, Jan AU - Janovjak, Harald L ID - 5984 IS - 1 JF - Nature Communications SN - 2041-1723 TI - Optical functionalization of human class A orphan G-protein-coupled receptors VL - 9 ER - TY - JOUR AB - 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. AU - Dolati, Setareh AU - Kage, Frieda AU - Mueller, Jan AU - Müsken, Mathias AU - Kirchner, Marieluise AU - Dittmar, Gunnar AU - Sixt, Michael K AU - Rottner, Klemens AU - Falcke, Martin ID - 5992 IS - 22 JF - Molecular Biology of the Cell TI - On the relation between filament density, force generation, and protrusion rate in mesenchymal cell motility VL - 29 ER - TY - JOUR AB - 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. AU - Moalli, Federica AU - Ficht, Xenia AU - Germann, Philipp AU - Vladymyrov, Mykhailo AU - Stolp, Bettina AU - de Vries, Ingrid AU - Lyck, Ruth AU - Balmer, Jasmin AU - Fiocchi, Amleto AU - Kreutzfeldt, Mario AU - Merkler, Doron AU - Iannacone, Matteo AU - Ariga, Akitaka AU - Stoffel, Michael H. AU - Sharpe, James AU - Bähler, Martin AU - Sixt, Michael K AU - Diz-Muñoz, Alba AU - Stein, Jens V. ID - 6497 IS - 7 JF - The Journal of Experimental Medicine SN - 0022-1007 TI - The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+T cells VL - 2015 ER - TY - JOUR AB - 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. AU - Brown, Markus AU - Assen, Frank P AU - Leithner, Alexander F AU - Abe, Jun AU - Schachner, Helga AU - Asfour, Gabriele AU - Bagó Horváth, Zsuzsanna AU - Stein, Jens AU - Uhrin, Pavel AU - Sixt, Michael K AU - Kerjaschki, Dontscho ID - 402 IS - 6382 JF - Science TI - Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice VL - 359 ER - TY - THES AB - 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. AU - Leithner, Alexander F ID - 323 SN - 2663-337X TI - Branched actin networks in dendritic cell biology ER - TY - JOUR AB - 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. AU - Hons, Miroslav AU - Kopf, Aglaja AU - Hauschild, Robert AU - Leithner, Alexander F AU - Gärtner, Florian R AU - Abe, Jun AU - Renkawitz, Jörg AU - Stein, Jens AU - Sixt, Michael K ID - 15 IS - 6 JF - Nature Immunology TI - Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells VL - 19 ER - TY - JOUR AB - 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. AU - Spira, Felix AU - Cuylen Haering, Sara AU - Mehta, Shalin AU - Samwer, Matthias AU - Reversat, Anne AU - Verma, Amitabh AU - Oldenbourg, Rudolf AU - Sixt, Michael K AU - Gerlich, Daniel ID - 569 JF - eLife SN - 2050084X TI - Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments VL - 6 ER - TY - JOUR AB - 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. AU - Gärtner, Florian R AU - Ahmad, Zerkah AU - Rosenberger, Gerhild AU - Fan, Shuxia AU - Nicolai, Leo AU - Busch, Benjamin AU - Yavuz, Gökce AU - Luckner, Manja AU - Ishikawa Ankerhold, Hellen AU - Hennel, Roman AU - Benechet, Alexandre AU - Lorenz, Michael AU - Chandraratne, Sue AU - Schubert, Irene AU - Helmer, Sebastian AU - Striednig, Bianca AU - Stark, Konstantin AU - Janko, Marek AU - Böttcher, Ralph AU - Verschoor, Admar AU - Leon, Catherine AU - Gachet, Christian AU - Gudermann, Thomas AU - Mederos Y Schnitzler, Michael AU - Pincus, Zachary AU - Iannacone, Matteo AU - Haas, Rainer AU - Wanner, Gerhard AU - Lauber, Kirsten AU - Sixt, Michael K AU - Massberg, Steffen ID - 571 IS - 6 JF - Cell Press SN - 00928674 TI - Migrating platelets are mechano scavengers that collect and bundle bacteria VL - 171 ER - TY - JOUR AB - 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. AU - Kage, Frieda AU - Winterhoff, Moritz AU - Dimchev, Vanessa AU - Müller, Jan AU - Thalheim, Tobias AU - Freise, Anika AU - Brühmann, Stefan AU - Kollasser, Jana AU - Block, Jennifer AU - Dimchev, Georgi A AU - Geyer, Matthias AU - Schnittler, Hams AU - Brakebusch, Cord AU - Stradal, Theresia AU - Carlier, Marie AU - Sixt, Michael K AU - Käs, Josef AU - Faix, Jan AU - Rottner, Klemens ID - 659 JF - Nature Communications SN - 20411723 TI - FMNL formins boost lamellipodial force generation VL - 8 ER - TY - JOUR AB - 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. AU - Horsthemke, Markus AU - Bachg, Anne AU - Groll, Katharina AU - Moyzio, Sven AU - Müther, Barbara AU - Hemkemeyer, Sandra AU - Wedlich Söldner, Roland AU - Sixt, Michael K AU - Tacke, Sebastian AU - Bähler, Martin AU - Hanley, Peter ID - 668 IS - 17 JF - Journal of Biological Chemistry SN - 00219258 TI - Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion VL - 292 ER - TY - JOUR AB - 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. AU - Vaahtomeri, Kari AU - Brown, Markus AU - Hauschild, Robert AU - De Vries, Ingrid AU - Leithner, Alexander F AU - Mehling, Matthias AU - Kaufmann, Walter AU - Sixt, Michael K ID - 672 IS - 5 JF - Cell Reports SN - 22111247 TI - Locally triggered release of the chemokine CCL21 promotes dendritic cell transmigration across lymphatic endothelia VL - 19 ER - TY - JOUR AB - 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. AU - Schwarz, Jan AU - Bierbaum, Veronika AU - Vaahtomeri, Kari AU - Hauschild, Robert AU - Brown, Markus AU - De Vries, Ingrid AU - Leithner, Alexander F AU - Reversat, Anne AU - Merrin, Jack AU - Tarrant, Teresa AU - Bollenbach, Tobias AU - Sixt, Michael K ID - 674 IS - 9 JF - Current Biology SN - 09609822 TI - Dendritic cells interpret haptotactic chemokine gradients in a manner governed by signal to noise ratio and dependent on GRK6 VL - 27 ER - TY - JOUR AB - 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. AU - Lademann, Claudio AU - Renkawitz, Jörg AU - Pfander, Boris AU - Jentsch, Stefan ID - 677 IS - 7 JF - Cell Reports SN - 22111247 TI - The INO80 complex removes H2A.Z to promote presynaptic filament formation during homologous recombination VL - 19 ER - TY - JOUR AB - 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. AU - Veß, Astrid AU - Blache, Ulrich AU - Leitner, Laura AU - Kurz, Angela AU - Ehrenpfordt, Anja AU - Sixt, Michael K AU - Posern, Guido ID - 694 IS - 13 JF - Journal of Cell Science SN - 00219533 TI - A dual phenotype of MDA MB 468 cancer cells reveals mutual regulation of tensin3 and adhesion plasticity VL - 130 ER - TY - JOUR AB - 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. AU - Müller, Jan AU - Sixt, Michael K ID - 1161 IS - 1 JF - Current Biology SN - 09609822 TI - Cell migration: Making the waves VL - 27 ER -