TY - JOUR AB - Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components. AU - Pimenta-Marques, Ana AU - Perestrelo, Tania AU - Dos Reis Rodrigues, Patricia AU - Duarte, Paulo AU - Ferreira-Silva, Ana AU - Lince-Faria, Mariana AU - Bettencourt-Dias, Mónica ID - 14933 IS - 1 JF - EMBO reports TI - Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase and the PCM VL - 25 ER - TY - JOUR AB - Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole—a protuberance of the zygote’s vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces. AU - Caballero Mancebo, Silvia AU - Shinde, Rushikesh AU - Bolger-Munro, Madison AU - Peruzzo, Matilda AU - Szep, Gregory AU - Steccari, Irene AU - Labrousse Arias, David AU - Zheden, Vanessa AU - Merrin, Jack AU - Callan-Jones, Andrew AU - Voituriez, Raphaël AU - Heisenberg, Carl-Philipp J ID - 14846 JF - Nature Physics SN - 1745-2473 TI - Friction forces determine cytoplasmic reorganization and shape changes of ascidian oocytes upon fertilization ER - TY - JOUR AB - The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly. AU - Zens, Bettina AU - Fäßler, Florian AU - Hansen, Jesse AU - Hauschild, Robert AU - Datler, Julia AU - Hodirnau, Victor-Valentin AU - Zheden, Vanessa AU - Alanko, Jonna H AU - Sixt, Michael K AU - Schur, Florian KM ID - 15146 IS - 6 JF - Journal of Cell Biology SN - 0021-9525 TI - Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix VL - 223 ER - TY - CHAP AB - Imaging of the immunological synapse (IS) between dendritic cells (DCs) and T cells in suspension is hampered by suboptimal alignment of cell-cell contacts along the vertical imaging plane. This requires optical sectioning that often results in unsatisfactory resolution in time and space. Here, we present a workflow where DCs and T cells are confined between a layer of glass and polydimethylsiloxane (PDMS) that orients the cells along one, horizontal imaging plane, allowing for fast en-face-imaging of the DC-T cell IS. AU - Leithner, Alexander F AU - Merrin, Jack AU - Sixt, Michael K ED - Baldari, Cosima ED - Dustin, Michael ID - 13052 SN - 1064-3745 T2 - The Immune Synapse TI - En-Face Imaging of T Cell-Dendritic Cell Immunological Synapses VL - 2654 ER - TY - JOUR AB - The intricate regulatory processes behind actin polymerization play a crucial role in cellular biology, including essential mechanisms such as cell migration or cell division. However, the self-organizing principles governing actin polymerization are still poorly understood. In this perspective article, we compare the Belousov-Zhabotinsky (BZ) reaction, a classic and well understood chemical oscillator known for its self-organizing spatiotemporal dynamics, with the excitable dynamics of polymerizing actin. While the BZ reaction originates from the domain of inorganic chemistry, it shares remarkable similarities with actin polymerization, including the characteristic propagating waves, which are influenced by geometry and external fields, and the emergent collective behavior. Starting with a general description of emerging patterns, we elaborate on single droplets or cell-level dynamics, the influence of geometric confinements and conclude with collective interactions. Comparing these two systems sheds light on the universal nature of self-organization principles in both living and inanimate systems. AU - Riedl, Michael AU - Sixt, Michael K ID - 14555 JF - Frontiers in Cell and Developmental Biology TI - The excitable nature of polymerizing actin and the Belousov-Zhabotinsky reaction VL - 11 ER - TY - THES AB - Most motions of many-body systems at any scale in nature with sufficient degrees of freedom tend to be chaotic; reaching from the orbital motion of planets, the air currents in our atmosphere, down to the water flowing through our pipelines or the movement of a population of bacteria. To the observer it is therefore intriguing when a moving collective exhibits order. Collective motion of flocks of birds, schools of fish or swarms of self-propelled particles or robots have been studied extensively over the past decades but the mechanisms involved in the transition from chaos to order remain unclear. Here, the interactions, that in most systems give rise to chaos, sustain order. In this thesis we investigate mechanisms that preserve, destabilize or lead to the ordered state. We show that endothelial cells migrating in circular confinements transition to a collective rotating state and concomitantly synchronize the frequencies of nucleating actin waves within individual cells. Consequently, the frequency dependent cell migration speed uniformizes across the population. Complementary to the WAVE dependent nucleation of traveling actin waves, we show that in leukocytes the actin polymerization depending on WASp generates pushing forces locally at stationary patches. Next, in pipe flows, we study methods to disrupt the self--sustaining cycle of turbulence and therefore relaminarize the flow. While we find in pulsating flow conditions that turbulence emerges through a helical instability during the decelerating phase. Finally, we show quantitatively in brain slices of mice that wild-type control neurons can compensate the migratory deficits of a genetically modified neuronal sub--population in the developing cortex. AU - Riedl, Michael ID - 14530 KW - Synchronization KW - Collective Movement KW - Active Matter KW - Cell Migration KW - Active Colloids SN - 2663 - 337X TI - Synchronization in collectively moving active matter ER - TY - JOUR AB - Whether one considers swarming insects, flocking birds, or bacterial colonies, collective motion arises from the coordination of individuals and entails the adjustment of their respective velocities. In particular, in close confinements, such as those encountered by dense cell populations during development or regeneration, collective migration can only arise coordinately. Yet, how individuals unify their velocities is often not understood. Focusing on a finite number of cells in circular confinements, we identify waves of polymerizing actin that function as a pacemaker governing the speed of individual cells. We show that the onset of collective motion coincides with the synchronization of the wave nucleation frequencies across the population. Employing a simpler and more readily accessible mechanical model system of active spheres, we identify the synchronization of the individuals’ internal oscillators as one of the essential requirements to reach the corresponding collective state. The mechanical ‘toy’ experiment illustrates that the global synchronous state is achieved by nearest neighbor coupling. We suggest by analogy that local coupling and the synchronization of actin waves are essential for the emergent, self-organized motion of cell collectives. AU - Riedl, Michael AU - Mayer, Isabelle D AU - Merrin, Jack AU - Sixt, Michael K AU - Hof, Björn ID - 14361 JF - Nature Communications TI - Synchronization in collectively moving inanimate and living active matter VL - 14 ER - TY - JOUR AB - To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells’ capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment. AU - Sitarska, Ewa AU - Almeida, Silvia Dias AU - Beckwith, Marianne Sandvold AU - Stopp, Julian A AU - Czuchnowski, Jakub AU - Siggel, Marc AU - Roessner, Rita AU - Tschanz, Aline AU - Ejsing, Christer AU - Schwab, Yannick AU - Kosinski, Jan AU - Sixt, Michael K AU - Kreshuk, Anna AU - Erzberger, Anna AU - Diz-Muñoz, Alba ID - 14360 JF - Nature Communications TI - Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles VL - 14 ER - TY - JOUR AB - Immune responses rely on the rapid and coordinated migration of leukocytes. Whereas it is well established that single-cell migration is often guided by gradients of chemokines and other chemoattractants, it remains poorly understood how these gradients are generated, maintained, and modulated. By combining experimental data with theory on leukocyte chemotaxis guided by the G protein–coupled receptor (GPCR) CCR7, we demonstrate that in addition to its role as the sensory receptor that steers migration, CCR7 also acts as a generator and a modulator of chemotactic gradients. Upon exposure to the CCR7 ligand CCL19, dendritic cells (DCs) effectively internalize the receptor and ligand as part of the canonical GPCR desensitization response. We show that CCR7 internalization also acts as an effective sink for the chemoattractant, dynamically shaping the spatiotemporal distribution of the chemokine. This mechanism drives complex collective migration patterns, enabling DCs to create or sharpen chemotactic gradients. We further show that these self-generated gradients can sustain the long-range guidance of DCs, adapt collective migration patterns to the size and geometry of the environment, and provide a guidance cue for other comigrating cells. Such a dual role of CCR7 as a GPCR that both senses and consumes its ligand can thus provide a novel mode of cellular self-organization. AU - Alanko, Jonna H AU - Ucar, Mehmet C AU - Canigova, Nikola AU - Stopp, Julian A AU - Schwarz, Jan AU - Merrin, Jack AU - Hannezo, Edouard B AU - Sixt, Michael K ID - 14274 IS - 87 JF - Science Immunology KW - General Medicine KW - Immunology SN - 2470-9468 TI - CCR7 acts as both a sensor and a sink for CCL19 to coordinate collective leukocyte migration VL - 8 ER - TY - THES AU - Stopp, Julian A ID - 14697 SN - 2663 - 337X TI - Neutrophils on the hunt: Migratory strategies employed by neutrophils to fulfill their effector function ER - TY - CHAP AB - Regulating protein states is considered the core function of chaperones. However, despite their importance to all major cellular processes, the conformational changes that chaperones impart on polypeptide chains are difficult to study directly due to their heterogeneous, dynamic, and multi-step nature. Here, we review recent advances towards this aim using single-molecule manipulation methods, which are rapidly revealing new mechanisms of conformational control and helping to define a different perspective on the chaperone function. AU - Wruck, F. AU - Avellaneda Sarrió, Mario AU - Naqvi, M. M. AU - Koers, E. J. AU - Till, K. AU - Gross, L. AU - Moayed, F. AU - Roland, A. AU - Heling, L. W. H. J. AU - Mashaghi, A. AU - Tans, S. J. ED - Hiller, Sebastian ED - Liu, Maili ED - He, Lichun ID - 14848 SN - 9781839162824 T2 - Biophysics of Molecular Chaperones TI - Probing Single Chaperone Substrates VL - 29 ER - TY - JOUR AB - Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion. AU - Assen, Frank P AU - Abe, Jun AU - Hons, Miroslav AU - Hauschild, Robert AU - Shamipour, Shayan AU - Kaufmann, Walter AU - Costanzo, Tommaso AU - Krens, Gabriel AU - Brown, Markus AU - Ludewig, Burkhard AU - Hippenmeyer, Simon AU - Heisenberg, Carl-Philipp J AU - Weninger, Wolfgang AU - Hannezo, Edouard B AU - Luther, Sanjiv A. AU - Stein, Jens V. AU - Sixt, Michael K ID - 9794 JF - Nature Immunology SN - 1529-2908 TI - Multitier mechanics control stromal adaptations in swelling lymph nodes VL - 23 ER - TY - JOUR AB - Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level. AU - Nicolai, Leo AU - Kaiser, Rainer AU - Escaig, Raphael AU - Hoffknecht, Marie Louise AU - Anjum, Afra AU - Leunig, Alexander AU - Pircher, Joachim AU - Ehrlich, Andreas AU - Lorenz, Michael AU - Ishikawa-Ankerhold, Hellen AU - Aird, William C. AU - Massberg, Steffen AU - Gärtner, Florian R ID - 11588 IS - 7 JF - Haematologica SN - 0390-6078 TI - Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo VL - 107 ER - TY - JOUR 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 mouse dendritic cells (DCs) as a 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 the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. 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 - 11843 JF - eLife TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 VL - 11 ER - TY - JOUR AB - Molecular catch bonds are ubiquitous in biology and essential for processes like leucocyte extravasion1 and cellular mechanosensing2. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this feature provides ‘strength on demand3’, thus enabling cells to increase rigidity under stress1,4,5,6. However, catch bonds are often weaker than slip bonds because they have cryptic binding sites that are usually buried7,8. Here we show that catch bonds render reconstituted cytoskeletal actin networks stronger than slip bonds, even though the individual bonds are weaker. Simulations show that slip bonds remain trapped in stress-free areas, whereas weak binding allows catch bonds to mitigate crack initiation by moving to high-tension areas. This ‘dissociation on demand’ explains how cells combine mechanical strength with the adaptability required for shape change, and is relevant to diseases where catch bonding is compromised7,9, including focal segmental glomerulosclerosis10 caused by the α-actinin-4 mutant studied here. We surmise that catch bonds are the key to create life-like materials. AU - Mulla, Yuval AU - Avellaneda Sarrió, Mario AU - Roland, Antoine AU - Baldauf, Lucia AU - Jung, Wonyeong AU - Kim, Taeyoon AU - Tans, Sander J. AU - Koenderink, Gijsje H. ID - 12085 IS - 9 JF - Nature Materials SN - 1476-1122 TI - Weak catch bonds make strong networks VL - 21 ER - TY - JOUR AB - Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils “plucked” intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events. AU - Petzold, Tobias AU - Zhang, Zhe AU - Ballesteros, Iván AU - Saleh, Inas AU - Polzin, Amin AU - Thienel, Manuela AU - Liu, Lulu AU - Ul Ain, Qurrat AU - Ehreiser, Vincent AU - Weber, Christian AU - Kilani, Badr AU - Mertsch, Pontus AU - Götschke, Jeremias AU - Cremer, Sophie AU - Fu, Wenwen AU - Lorenz, Michael AU - Ishikawa-Ankerhold, Hellen AU - Raatz, Elisabeth AU - El-Nemr, Shaza AU - Görlach, Agnes AU - Marhuenda, Esther AU - Stark, Konstantin AU - Pircher, Joachim AU - Stegner, David AU - Gieger, Christian AU - Schmidt-Supprian, Marc AU - Gärtner, Florian R AU - Almendros, Isaac AU - Kelm, Malte AU - Schulz, Christian AU - Hidalgo, Andrés AU - Massberg, Steffen ID - 12119 IS - 12 JF - Immunity KW - Infectious Diseases KW - Immunology KW - Immunology and Allergy SN - 1074-7613 TI - Neutrophil “plucking” on megakaryocytes drives platelet production and boosts cardiovascular disease VL - 55 ER - TY - JOUR AB - Social distancing is an effective way to prevent the spread of disease in societies, whereas infection elimination is a key element of organismal immunity. Here, we discuss how the study of social insects such as ants — which form a superorganism of unconditionally cooperative individuals and thus represent a level of organization that is intermediate between a classical society of individuals and an organism of cells — can help to determine common principles of disease defence across levels of organization. AU - Cremer, Sylvia AU - Sixt, Michael K ID - 12133 IS - 12 JF - Nature Reviews Immunology KW - Energy Engineering and Power Technology KW - Fuel Technology SN - 1474-1733 TI - Principles of disease defence in organisms, superorganisms and societies VL - 22 ER - TY - JOUR AB - Reading, interpreting and crawling along gradients of chemotactic cues is one of the most complex questions in cell biology. In this issue, Georgantzoglou et al. (2022. J. Cell. Biol.https://doi.org/10.1083/jcb.202103207) use in vivo models to map the temporal sequence of how neutrophils respond to an acutely arising gradient of chemoattractant. AU - Stopp, Julian A AU - Sixt, Michael K ID - 12272 IS - 8 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Plan your trip before you leave: The neutrophils’ search-and-run journey VL - 221 ER - TY - JOUR AB - When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes. AU - Gaertner, Florian AU - Reis-Rodrigues, Patricia AU - De Vries, Ingrid AU - Hons, Miroslav AU - Aguilera, Juan AU - Riedl, Michael AU - Leithner, Alexander F AU - Tasciyan, Saren AU - Kopf, Aglaja AU - Merrin, Jack AU - Zheden, Vanessa AU - Kaufmann, Walter AU - Hauschild, Robert AU - Sixt, Michael K ID - 10703 IS - 1 JF - Developmental Cell SN - 1534-5807 TI - WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues VL - 57 ER - TY - THES AB - Detachment of the cancer cells from the bulk of the tumor is the first step of metastasis, which is the primary cause of cancer related deaths. It is unclear, which factors contribute to this step. Recent studies indicate a crucial role of the tumor microenvironment in malignant transformation and metastasis. Studying cancer cell invasion and detachments quantitatively in the context of its physiological microenvironment is technically challenging. Especially, precise control of microenvironmental properties in vivo is currently not possible. Here, I studied the role of microenvironment geometry in the invasion and detachment of cancer cells from the bulk with a simplistic and reductionist approach. In this approach, I engineered microfluidic devices to mimic a pseudo 3D extracellular matrix environment, where I was able to quantitatively tune the geometrical configuration of the microenvironment and follow tumor cells with fluorescence live imaging. To aid quantitative analysis I developed a widely applicable software application to automatically analyze and visualize particle tracking data. Quantitative analysis of tumor cell invasion in isotropic and anisotropic microenvironments showed that heterogeneity in the microenvironment promotes faster invasion and more frequent detachment of cells. These observations correlated with overall higher speed of cells at the edge of the bulk of the cells. In heterogeneous microenvironments cells preferentially passed through larger pores, thus invading areas of least resistance and generating finger-like invasive structures. The detachments occurred mostly at the tips of these structures. To investigate the potential mechanism, we established a two dimensional model to simulate active Brownian particles representing the cell nuclei dynamics. These simulations backed our in vitro observations without the need of precise fitting the simulation parameters. Our model suggests the importance of the pore heterogeneity in the direction perpendicular to the orientation of bias field (lateral heterogeneity), which causes the interface roughening. AU - Tasciyan, Saren ID - 12401 SN - 2663-337X TI - Role of microenvironment heterogeneity in cancer cell invasion ER - TY - JOUR AB - 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. AU - Düllberg, Christian F AU - Auer, Albert AU - Canigova, Nikola AU - Loibl, Katrin AU - Loose, Martin ID - 8988 IS - 1 JF - PNAS SN - 00278424 TI - In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1 VL - 118 ER - TY - JOUR AB - 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. AU - Vaahtomeri, Kari AU - Moussion, Christine AU - Hauschild, Robert AU - Sixt, Michael K ID - 9259 JF - Frontiers in Immunology TI - Shape and function of interstitial chemokine CCL21 gradients are independent of heparan sulfates produced by lymphatic endothelium VL - 12 ER - TY - JOUR AB - 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. AU - Gärtner, Florian R AU - Sixt, Michael K ID - 9294 IS - 6 JF - Developmental Cell SN - 15345807 TI - Engaging the front wheels to drive through fibrous terrain VL - 56 ER - TY - JOUR AB - 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. AU - Zisis, Themistoklis AU - Schwarz, Jan AU - Balles, Miriam AU - Kretschmer, Maibritt AU - Nemethova, Maria AU - Chait, Remy P AU - Hauschild, Robert AU - Lange, Janina AU - Guet, Calin C AU - Sixt, Michael K AU - Zahler, Stefan ID - 9822 IS - 30 JF - ACS Applied Materials and Interfaces SN - 19448244 TI - Sequential and switchable patterning for studying cellular processes under spatiotemporal control VL - 13 ER - TY - JOUR AB - 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. AU - Stahnke, Stephanie AU - Döring, Hermann AU - Kusch, Charly AU - de Gorter, David J.J. AU - Dütting, Sebastian AU - Guledani, Aleks AU - Pleines, Irina AU - Schnoor, Michael AU - Sixt, Michael K AU - Geffers, Robert AU - Rohde, Manfred AU - Müsken, Mathias AU - Kage, Frieda AU - Steffen, Anika AU - Faix, Jan AU - Nieswandt, Bernhard AU - Rottner, Klemens AU - Stradal, Theresia E.B. ID - 10834 IS - 10 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion VL - 31 ER - 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 -