@article{9259, abstract = {Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.}, author = {Vaahtomeri, Kari and Moussion, Christine and Hauschild, Robert and Sixt, Michael K}, issn = {1664-3224}, journal = {Frontiers in Immunology}, publisher = {Frontiers}, title = {{Shape and function of interstitial chemokine CCL21 gradients are independent of heparan sulfates produced by lymphatic endothelium}}, doi = {10.3389/fimmu.2021.630002}, volume = {12}, year = {2021}, } @article{9361, abstract = {The multimeric matrix (M) protein of clinically relevant paramyxoviruses orchestrates assembly and budding activity of viral particles at the plasma membrane (PM). We identified within the canine distemper virus (CDV) M protein two microdomains, potentially assuming α-helix structures, which are essential for membrane budding activity. Remarkably, while two rationally designed microdomain M mutants (E89R, microdomain 1 and L239D, microdomain 2) preserved proper folding, dimerization, interaction with the nucleocapsid protein, localization at and deformation of the PM, the virus-like particle formation, as well as production of infectious virions (as monitored using a membrane budding-complementation system), were, in sharp contrast, strongly impaired. Of major importance, raster image correlation spectroscopy (RICS) revealed that both microdomains contributed to finely tune M protein mobility specifically at the PM. Collectively, our data highlighted the cornerstone membrane budding-priming activity of two spatially discrete M microdomains, potentially by coordinating the assembly of productive higher oligomers at the PM.}, author = {Gast, Matthieu and Kadzioch, Nicole P. and Milius, Doreen and Origgi, Francesco and Plattet, Philippe}, issn = {23795042}, journal = {mSphere}, number = {2}, publisher = {American Society for Microbiology}, title = {{Oligomerization and cell egress controlled by two microdomains of canine distemper virus matrix protein}}, doi = {10.1128/mSphere.01024-20}, volume = {6}, year = {2021}, } @article{9429, abstract = {De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.}, author = {Morandell, Jasmin and Schwarz, Lena A and Basilico, Bernadette and Tasciyan, Saren and Dimchev, Georgi A and Nicolas, Armel and Sommer, Christoph M and Kreuzinger, Caroline and Dotter, Christoph and Knaus, Lisa and Dobler, Zoe and Cacci, Emanuele and Schur, Florian KM and Danzl, Johann G and Novarino, Gaia}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {1}, publisher = {Springer Nature}, title = {{Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development}}, doi = {10.1038/s41467-021-23123-x}, volume = {12}, year = {2021}, } @article{10179, abstract = {Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling.}, author = {Bajaj, Sunanjay and Bagley, Joshua A. and Sommer, Christoph M and Vertesy, Abel and Nagumo Wong, Sakurako and Krenn, Veronica and Lévi-Strauss, Julie and Knoblich, Juergen A.}, issn = {1460-2075}, journal = {EMBO Journal}, number = {23}, publisher = {Embo Press}, title = {{Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration}}, doi = {10.15252/embj.2021108714}, volume = {40}, year = {2021}, } @article{9822, abstract = {Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science.}, author = {Zisis, Themistoklis and Schwarz, Jan and Balles, Miriam and Kretschmer, Maibritt and Nemethova, Maria and Chait, Remy P and Hauschild, Robert and Lange, Janina and Guet, Calin C and Sixt, Michael K and Zahler, Stefan}, issn = {19448252}, journal = {ACS Applied Materials and Interfaces}, number = {30}, pages = {35545–35560}, publisher = {American Chemical Society}, title = {{Sequential and switchable patterning for studying cellular processes under spatiotemporal control}}, doi = {10.1021/acsami.1c09850}, volume = {13}, year = {2021}, } @article{9911, abstract = {A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.}, author = {Nelson, Glyn and Boehm, Ulrike and Bagley, Steve and Bajcsy, Peter and Bischof, Johanna and Brown, Claire M. and Dauphin, Aurélien and Dobbie, Ian M. and Eriksson, John E. and Faklaris, Orestis and Fernandez-Rodriguez, Julia and Ferrand, Alexia and Gelman, Laurent and Gheisari, Ali and Hartmann, Hella and Kukat, Christian and Laude, Alex and Mitkovski, Miso and Munck, Sebastian and North, Alison J. and Rasse, Tobias M. and Resch-Genger, Ute and Schuetz, Lucas C. and Seitz, Arne and Strambio-De-Castillia, Caterina and Swedlow, Jason R. and Alexopoulos, Ioannis and Aumayr, Karin and Avilov, Sergiy and Bakker, Gert Jan and Bammann, Rodrigo R. and Bassi, Andrea and Beckert, Hannes and Beer, Sebastian and Belyaev, Yury and Bierwagen, Jakob and Birngruber, Konstantin A. and Bosch, Manel and Breitlow, Juergen and Cameron, Lisa A. and Chalfoun, Joe and Chambers, James J. and Chen, Chieh Li and Conde-Sousa, Eduardo and Corbett, Alexander D. and Cordelieres, Fabrice P. and Nery, Elaine Del and Dietzel, Ralf and Eismann, Frank and Fazeli, Elnaz and Felscher, Andreas and Fried, Hans and Gaudreault, Nathalie and Goh, Wah Ing and Guilbert, Thomas and Hadleigh, Roland and Hemmerich, Peter and Holst, Gerhard A. and Itano, Michelle S. and Jaffe, Claudia B. and Jambor, Helena K. and Jarvis, Stuart C. and Keppler, Antje and Kirchenbuechler, David and Kirchner, Marcel and Kobayashi, Norio and Krens, Gabriel and Kunis, Susanne and Lacoste, Judith and Marcello, Marco and Martins, Gabriel G. and Metcalf, Daniel J. and Mitchell, Claire A. and Moore, Joshua and Mueller, Tobias and Nelson, Michael S. and Ogg, Stephen and Onami, Shuichi and Palmer, Alexandra L. and Paul-Gilloteaux, Perrine and Pimentel, Jaime A. and Plantard, Laure and Podder, Santosh and Rexhepaj, Elton and Royon, Arnaud and Saari, Markku A. and Schapman, Damien and Schoonderwoert, Vincent and Schroth-Diez, Britta and Schwartz, Stanley and Shaw, Michael and Spitaler, Martin and Stoeckl, Martin T. and Sudar, Damir and Teillon, Jeremie and Terjung, Stefan and Thuenauer, Roland and Wilms, Christian D. and Wright, Graham D. and Nitschke, Roland}, issn = {1365-2818}, journal = {Journal of Microscopy}, number = {1}, pages = {56--73}, publisher = {Wiley}, title = {{QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy}}, doi = {10.1111/jmi.13041}, volume = {284}, year = {2021}, } @article{7864, abstract = {Purpose of review: Cancer is one of the leading causes of death and the incidence rates are constantly rising. The heterogeneity of tumors poses a big challenge for the treatment of the disease and natural antibodies additionally affect disease progression. The introduction of engineered mAbs for anticancer immunotherapies has substantially improved progression-free and overall survival of cancer patients, but little efforts have been made to exploit other antibody isotypes than IgG. Recent findings: In order to improve these therapies, ‘next-generation antibodies’ were engineered to enhance a specific feature of classical antibodies and form a group of highly effective and precise therapy compounds. Advanced antibody approaches include among others antibody-drug conjugates, glyco-engineered and Fc-engineered antibodies, antibody fragments, radioimmunotherapy compounds, bispecific antibodies and alternative (non-IgG) immunoglobulin classes, especially IgE. Summary: The current review describes solutions for the needs of next-generation antibody therapies through different approaches. Careful selection of the best-suited engineering methodology is a key factor in developing personalized, more specific and more efficient mAbs against cancer to improve the outcomes of cancer patients. We highlight here the large evidence of IgE exploiting a highly cytotoxic effector arm as potential next-generation anticancer immunotherapy.}, author = {Singer, Judit and Singer, Josef and Jensen-Jarolim, Erika}, issn = {14736322}, journal = {Current opinion in allergy and clinical immunology}, number = {3}, pages = {282--289}, publisher = {Wolters Kluwer}, title = {{Precision medicine in clinical oncology: the journey from IgG antibody to IgE}}, doi = {10.1097/ACI.0000000000000637}, volume = {20}, year = {2020}, } @article{7875, abstract = {Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.}, author = {Kopf, Aglaja and Renkawitz, Jörg and Hauschild, Robert and Girkontaite, Irute and Tedford, Kerry and Merrin, Jack and Thorn-Seshold, Oliver and Trauner, Dirk and Häcker, Hans and Fischer, Klaus Dieter and Kiermaier, Eva and Sixt, Michael K}, issn = {1540-8140}, journal = {The Journal of Cell Biology}, number = {6}, publisher = {Rockefeller University Press}, title = {{Microtubules control cellular shape and coherence in amoeboid migrating cells}}, doi = {10.1083/jcb.201907154}, volume = {219}, year = {2020}, } @article{7885, abstract = {Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.}, author = {Reversat, Anne and Gärtner, Florian R and Merrin, Jack and Stopp, Julian A and Tasciyan, Saren and Aguilera Servin, Juan L and De Vries, Ingrid and Hauschild, Robert and Hons, Miroslav and Piel, Matthieu and Callan-Jones, Andrew and Voituriez, Raphael and Sixt, Michael K}, issn = {14764687}, journal = {Nature}, pages = {582–585}, publisher = {Springer Nature}, title = {{Cellular locomotion using environmental topography}}, doi = {10.1038/s41586-020-2283-z}, volume = {582}, year = {2020}, } @article{7888, abstract = {Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order.}, author = {Schauer, Alexandra and Nunes Pinheiro, Diana C and Hauschild, Robert and Heisenberg, Carl-Philipp J}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Zebrafish embryonic explants undergo genetically encoded self-assembly}}, doi = {10.7554/elife.55190}, volume = {9}, year = {2020}, } @misc{8181, author = {Hauschild, Robert}, publisher = {IST Austria}, title = {{Amplified centrosomes in dendritic cells promote immune cell effector functions}}, doi = {10.15479/AT:ISTA:8181}, year = {2020}, } @misc{8294, abstract = {Automated root growth analysis and tracking of root tips. }, author = {Hauschild, Robert}, publisher = {IST Austria}, title = {{RGtracker}}, doi = {10.15479/AT:ISTA:8294}, year = {2020}, } @unpublished{9750, abstract = {Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow1,2. Here we show in zebrafish primary germ layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase, and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. Once tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension stabilizing E-cadherin-actin complexes at the contact.}, author = {Slovakova, Jana and Sikora, Mateusz K and Caballero Mancebo, Silvia and Krens, Gabriel and Kaufmann, Walter and Huljev, Karla and Heisenberg, Carl-Philipp J}, booktitle = {bioRxiv}, pages = {41}, publisher = {Cold Spring Harbor Laboratory}, title = {{Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion}}, doi = {10.1101/2020.11.20.391284}, year = {2020}, } @article{6867, abstract = {A novel magnetic scratch method achieves repeatability, reproducibility and geometric control greater than pipette scratch assays and closely approximating the precision of cell exclusion assays while inducing the cell injury inherently necessary for wound healing assays. The magnetic scratch is affordable, easily implemented and standardisable and thus may contribute toward better comparability of data generated in different studies and laboratories.}, author = {Fenu, M. and Bettermann, T. and Vogl, C. and Darwish-Miranda, Nasser and Schramel, J. and Jenner, F. and Ribitsch, I.}, issn = {20452322}, journal = {Scientific Reports}, number = {1}, publisher = {Springer Nature}, title = {{A novel magnet-based scratch method for standardisation of wound-healing assays}}, doi = {10.1038/s41598-019-48930-7}, volume = {9}, year = {2019}, } @article{7406, abstract = {Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.}, author = {Mckenzie, Catherine and Spanova, Miroslava and Johnson, Alexander J and Kainrath, Stephanie and Zheden, Vanessa and Sitte, Harald H. and Janovjak, Harald L}, issn = {0165-0270}, journal = {Journal of Neuroscience Methods}, pages = {114--121}, publisher = {Elsevier}, title = {{Isolation of synaptic vesicles from genetically engineered cultured neurons}}, doi = {10.1016/j.jneumeth.2018.11.018}, volume = {312}, year = {2019}, } @article{6052, abstract = {Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.}, author = {Truckenbrodt, Sven M and Sommer, Christoph M and Rizzoli, Silvio O and Danzl, Johann G}, journal = {Nature Protocols}, number = {3}, pages = {832–863}, publisher = {Nature Publishing Group}, title = {{A practical guide to optimization in X10 expansion microscopy}}, doi = {10.1038/s41596-018-0117-3}, volume = {14}, year = {2019}, } @article{6093, abstract = {Blebs are cellular protrusions observed in migrating cells and in cells undergoing spreading, cytokinesis, and apoptosis. Here we investigate the flow of cytoplasm during bleb formation and the concurrent changes in cell volume using zebrafish primordial germ cells (PGCs) as an in vivo model. We show that bleb inflation occurs concomitantly with cytoplasmic inflow into it and that during this process the total cell volume does not change. We thus show that bleb formation in primordial germ cells results primarily from redistribution of material within the cell rather than being driven by flow of water from an external source.}, author = {Goudarzi, Mohammad and Boquet-Pujadas, Aleix and Olivo-Marin, Jean Christophe and Raz, Erez}, journal = {PLOS ONE}, number = {2}, publisher = {Public Library of Science}, title = {{Fluid dynamics during bleb formation in migrating cells in vivo}}, doi = {10.1371/journal.pone.0212699}, volume = {14}, year = {2019}, } @article{6328, abstract = {During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1,2,3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some—but not all—cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion7, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.}, author = {Renkawitz, Jörg and Kopf, Aglaja and Stopp, Julian A and de Vries, Ingrid and Driscoll, Meghan K. and Merrin, Jack and Hauschild, Robert and Welf, Erik S. and Danuser, Gaudenz and Fiolka, Reto and Sixt, Michael K}, journal = {Nature}, pages = {546--550}, publisher = {Springer Nature}, title = {{Nuclear positioning facilitates amoeboid migration along the path of least resistance}}, doi = {10.1038/s41586-019-1087-5}, volume = {568}, year = {2019}, } @article{275, abstract = {Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified > 1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.}, author = {Brown, Markus and Johnson, Louise and Leone, Dario and Májek, Peter and Vaahtomeri, Kari and Senfter, Daniel and Bukosza, Nora and Schachner, Helga and Asfour, Gabriele and Langer, Brigitte and Hauschild, Robert and Parapatics, Katja and Hong, Young and Bennett, Keiryn and Kain, Renate and Detmar, Michael and Sixt, Michael K and Jackson, David and Kerjaschki, Dontscho}, journal = {Journal of Cell Biology}, number = {6}, pages = {2205 -- 2221}, publisher = {Rockefeller University Press}, title = {{Lymphatic exosomes promote dendritic cell migration along guidance cues}}, doi = {10.1083/jcb.201612051}, volume = {217}, year = {2018}, } @article{308, abstract = {Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.}, author = {Ratheesh, Aparna and Biebl, Julia and Smutny, Michael and Veselá, Jana and Papusheva, Ekaterina and Krens, Gabriel and Kaufmann, Walter and György, Attila and Casano, Alessandra M and Siekhaus, Daria E}, journal = {Developmental Cell}, number = {3}, pages = {331 -- 346}, publisher = {Elsevier}, title = {{Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration}}, doi = {10.1016/j.devcel.2018.04.002}, volume = {45}, year = {2018}, }