TY - JOUR
AB - Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.
AU - Schauer, Alexandra
AU - Pranjic-Ferscha, Kornelija
AU - Hauschild, Robert
AU - Heisenberg, Carl-Philipp J
ID - 15048
IS - 4
JF - Development
SN - 0950-1991
TI - Robust axis elongation by Nodal-dependent restriction of BMP signaling
VL - 151
ER -
TY - COMP
AU - Hauschild, Robert
ID - 14926
TI - Matlab script for analysis of clone dispersal
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 - JOUR
AB - Interstitial fluid (IF) accumulation between embryonic cells is thought to be important for embryo patterning and morphogenesis. Here, we identify a positive mechanical feedback loop between cell migration and IF relocalization and find that it promotes embryonic axis formation during zebrafish gastrulation. We show that anterior axial mesendoderm (prechordal plate [ppl]) cells, moving in between the yolk cell and deep cell tissue to extend the embryonic axis, compress the overlying deep cell layer, thereby causing IF to flow from the deep cell layer to the boundary between the yolk cell and the deep cell layer, directly ahead of the advancing ppl. This IF relocalization, in turn, facilitates ppl cell protrusion formation and migration by opening up the space into which the ppl moves and, thereby, the ability of the ppl to trigger IF relocalization by pushing against the overlying deep cell layer. Thus, embryonic axis formation relies on a hydraulic feedback loop between cell migration and IF relocalization.
AU - Huljev, Karla
AU - Shamipour, Shayan
AU - Nunes Pinheiro, Diana C
AU - Preusser, Friedrich
AU - Steccari, Irene
AU - Sommer, Christoph M
AU - Naik, Suyash
AU - Heisenberg, Carl-Philipp J
ID - 12830
IS - 7
JF - Developmental Cell
SN - 1534-5807
TI - A hydraulic feedback loop between mesendoderm cell migration and interstitial fluid relocalization promotes embryonic axis formation in zebrafish
VL - 58
ER -
TY - JOUR
AB - Current methods for assessing cell proliferation in 3D scaffolds rely on changes in metabolic activity or total DNA, however, direct quantification of cell number in 3D scaffolds remains a challenge. To address this issue, we developed an unbiased stereology approach that uses systematic-random sampling and thin focal-plane optical sectioning of the scaffolds followed by estimation of total cell number (StereoCount). This approach was validated against an indirect method for measuring the total DNA (DNA content); and the Bürker counting chamber, the current reference method for quantifying cell number. We assessed the total cell number for cell seeding density (cells per unit volume) across four values and compared the methods in terms of accuracy, ease-of-use and time demands. The accuracy of StereoCount markedly outperformed the DNA content for cases with ~ 10,000 and ~ 125,000 cells/scaffold. For cases with ~ 250,000 and ~ 375,000 cells/scaffold both StereoCount and DNA content showed lower accuracy than the Bürker but did not differ from each other. In terms of ease-of-use, there was a strong advantage for the StereoCount due to output in terms of absolute cell numbers along with the possibility for an overview of cell distribution and future use of automation for high throughput analysis. Taking together, the StereoCount method is an efficient approach for direct cell quantification in 3D collagen scaffolds. Its major benefit is that automated StereoCount could accelerate research using 3D scaffolds focused on drug discovery for a wide variety of human diseases.
AU - Zavadakova, Anna
AU - Vistejnova, Lucie
AU - Belinova, Tereza
AU - Tichanek, Filip
AU - Bilikova, Dagmar
AU - Mouton, Peter R.
ID - 13033
IS - 1
JF - Scientific Reports
KW - Multidisciplinary
SN - 2045-2322
TI - Novel stereological method for estimation of cell counts in 3D collagen scaffolds
VL - 13
ER -
TY - JOUR
AB - Motile cells moving in multicellular organisms encounter microenvironments of locally heterogeneous mechanochemical composition. Individual compositional parameters like chemotactic signals, adhesiveness, and pore sizes are well known to be sensed by motile cells, providing individual guidance cues for cellular pathfinding. However, motile cells encounter diverse mechanochemical signals at the same time, raising the question of how cells respond to locally diverse and potentially competing signals on their migration routes. Here, we reveal that motile amoeboid cells require nuclear repositioning, termed nucleokinesis, for adaptive pathfinding in heterogeneous mechanochemical microenvironments. Using mammalian immune cells and the amoebaDictyostelium discoideum, we discover that frequent, rapid and long-distance nucleokinesis is a basic component of amoeboid pathfinding, enabling cells to reorientate quickly between locally competing cues. Amoeboid nucleokinesis comprises a two-step cell polarity switch and is driven by myosin II-forces, sliding the nucleus from a ‘losing’ to the ‘winning’ leading edge to re-adjust the nuclear to the cellular path. Impaired nucleokinesis distorts fast path adaptions and causes cellular arrest in the microenvironment. Our findings establish that nucleokinesis is required for amoeboid cell navigation. Given that motile single-cell amoebae, many immune cells, and some cancer cells utilize an amoeboid migration strategy, these results suggest that amoeboid nucleokinesis underlies cellular navigation during unicellular biology, immunity, and disease.
AU - Kroll, Janina
AU - Hauschild, Robert
AU - Kuznetcov, Arthur
AU - Stefanowski, Kasia
AU - Hermann, Monika D.
AU - Merrin, Jack
AU - Shafeek, Lubuna B
AU - Müller-Taubenberger, Annette
AU - Renkawitz, Jörg
ID - 13342
JF - EMBO Journal
SN - 0261-4189
TI - Adaptive pathfinding by nucleokinesis during amoeboid migration
ER -
TY - JOUR
AB - Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
AU - Cikes, Domagoj
AU - Elsayad, Kareem
AU - Sezgin, Erdinc
AU - Koitai, Erika
AU - Ferenc, Torma
AU - Orthofer, Michael
AU - Yarwood, Rebecca
AU - Heinz, Leonhard X.
AU - Sedlyarov, Vitaly
AU - Darwish-Miranda, Nasser
AU - Taylor, Adrian
AU - Grapentine, Sophie
AU - al-Murshedi, Fathiya
AU - Abot, Anne
AU - Weidinger, Adelheid
AU - Kutchukian, Candice
AU - Sanchez, Colline
AU - Cronin, Shane J. F.
AU - Novatchkova, Maria
AU - Kavirayani, Anoop
AU - Schuetz, Thomas
AU - Haubner, Bernhard
AU - Haas, Lisa
AU - Hagelkruys, Astrid
AU - Jackowski, Suzanne
AU - Kozlov, Andrey
AU - Jacquemond, Vincent
AU - Knauf, Claude
AU - Superti-Furga, Giulio
AU - Rullman, Eric
AU - Gustafsson, Thomas
AU - McDermot, John
AU - Lowe, Martin
AU - Radak, Zsolt
AU - Chamberlain, Jeffrey S.
AU - Bakovic, Marica
AU - Banka, Siddharth
AU - Penninger, Josef M.
ID - 12747
JF - Nature Metabolism
KW - Cell Biology
KW - Physiology (medical)
KW - Endocrinology
KW - Diabetes and Metabolism
KW - Internal Medicine
SN - 2522-5812
TI - PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing
VL - 5
ER -
TY - JOUR
AB - Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.
AU - Méhes, Elod
AU - Mones, Enys
AU - Varga, Máté
AU - Zsigmond, Áron
AU - Biri-Kovács, Beáta
AU - Nyitray, László
AU - Barone, Vanessa
AU - Krens, Gabriel
AU - Heisenberg, Carl-Philipp J
AU - Vicsek, Tamás
ID - 14041
JF - Communications Biology
TI - 3D cell segregation geometry and dynamics are governed by tissue surface tension regulation
VL - 6
ER -
TY - JOUR
AB - Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.
AU - Velicky, Philipp
AU - Miguel Villalba, Eder
AU - Michalska, Julia M
AU - Lyudchik, Julia
AU - Wei, Donglai
AU - Lin, Zudi
AU - Watson, Jake
AU - Troidl, Jakob
AU - Beyer, Johanna
AU - Ben Simon, Yoav
AU - Sommer, Christoph M
AU - Jahr, Wiebke
AU - Cenameri, Alban
AU - Broichhagen, Johannes
AU - Grant, Seth G.N.
AU - Jonas, Peter M
AU - Novarino, Gaia
AU - Pfister, Hanspeter
AU - Bickel, Bernd
AU - Danzl, Johann G
ID - 13267
JF - Nature Methods
SN - 1548-7091
TI - Dense 4D nanoscale reconstruction of living brain tissue
VL - 20
ER -
TY - JOUR
AB - Germ granules, condensates of phase-separated RNA and protein, are organelles that are essential for germline development in different organisms. The patterning of the granules and their relevance for germ cell fate are not fully understood. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the localization of RNA molecules to the periphery of the granules, where ribosomes are localized, depends on translational activity at this location. In addition, we find that the vertebrate-specific Dead end (Dnd1) protein is essential for nanos3 RNA localization at the condensates’ periphery. Accordingly, in the absence of Dnd1, or when translation is inhibited, nanos3 RNA translocates into the granule interior, away from the ribosomes, a process that is correlated with the loss of germ cell fate. These findings highlight the relevance of sub-granule compartmentalization for post-transcriptional control and its importance for preserving germ cell totipotency.
AU - Westerich, Kim Joana
AU - Tarbashevich, Katsiaryna
AU - Schick, Jan
AU - Gupta, Antra
AU - Zhu, Mingzhao
AU - Hull, Kenneth
AU - Romo, Daniel
AU - Zeuschner, Dagmar
AU - Goudarzi, Mohammad
AU - Gross-Thebing, Theresa
AU - Raz, Erez
ID - 14781
IS - 17
JF - Developmental Cell
KW - Developmental Biology
KW - Cell Biology
KW - General Biochemistry
KW - Genetics and Molecular Biology
KW - Molecular Biology
SN - 1534-5807
TI - Spatial organization and function of RNA molecules within phase-separated condensates in zebrafish are controlled by Dnd1
VL - 58
ER -
TY - DATA
AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here, we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.
AU - Danzl, Johann G
ID - 13126
TI - Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales"
ER -
TY - JOUR
AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.
AU - Michalska, Julia M
AU - Lyudchik, Julia
AU - Velicky, Philipp
AU - Korinkova, Hana
AU - Watson, Jake
AU - Cenameri, Alban
AU - Sommer, Christoph M
AU - Amberg, Nicole
AU - Venturino, Alessandro
AU - Roessler, Karl
AU - Czech, Thomas
AU - Höftberger, Romana
AU - Siegert, Sandra
AU - Novarino, Gaia
AU - Jonas, Peter M
AU - Danzl, Johann G
ID - 14257
JF - Nature Biotechnology
SN - 1087-0156
TI - Imaging brain tissue architecture across millimeter to nanometer scales
ER -
TY - JOUR
AB - Singlet oxygen (1O2) formation is now recognised as a key aspect of non-aqueous oxygen redox chemistry. For identifying 1O2, chemical trapping via 9,10-dimethylanthracene (DMA) to form the endoperoxide (DMA-O2) has become the mainstay method due to its sensitivity, selectivity, and ease of use. While DMA has been shown to be selective for 1O2, rather than forming DMA-O2 with a wide variety of potentially reactive O-containing species, false positives might hypothetically be obtained in the presence of previously overlooked species. Here, we first give unequivocal direct spectroscopic proof by the 1O2-specific near infrared (NIR) emission at 1270 nm for the previously proposed 1O2 formation pathways, which centre around superoxide disproportionation. We then show that peroxocarbonates, common intermediates in metal-O2 and metal carbonate electrochemistry, do not produce false-positive DMA-O2. Moreover, we identify a previously unreported 1O2-forming pathway through the reaction of CO2 with superoxide. Overall, we give unequivocal proof for 1O2 formation in non-aqueous oxygen redox and show that chemical trapping with DMA is a reliable method to assess 1O2 formation.
AU - Mondal, Soumyadip
AU - Jethwa, Rajesh B
AU - Pant, Bhargavi
AU - Hauschild, Robert
AU - Freunberger, Stefan Alexander
ID - 13044
JF - Faraday Discussions
KW - Physical and Theoretical Chemistry
SN - 1359-6640
TI - Singlet oxygen in non-aqueous oxygen redox: Direct spectroscopic evidence for formation pathways and reliability of chemical probes
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 - 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 grow [J.-L. Maître et al., Science 338, 253–256 (2012)]. 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. After 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.
AU - Slovakova, Jana
AU - Sikora, Mateusz K
AU - Arslan, Feyza N
AU - Caballero Mancebo, Silvia
AU - Krens, Gabriel
AU - Kaufmann, Walter
AU - Merrin, Jack
AU - Heisenberg, Carl-Philipp J
ID - 10766
IS - 8
JF - Proceedings of the National Academy of Sciences of the United States of America
TI - Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells
VL - 119
ER -
TY - JOUR
AB - Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.
AU - Johnson, Alexander J
AU - Kaufmann, Walter
AU - Sommer, Christoph M
AU - Costanzo, Tommaso
AU - Dahhan, Dana A.
AU - Bednarek, Sebastian Y.
AU - Friml, Jiří
ID - 12239
IS - 10
JF - Molecular Plant
KW - Plant Science
KW - Molecular Biology
SN - 1674-2052
TI - Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution
VL - 15
ER -
TY - JOUR
AB - Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis.
AU - Weier, Ann-Kathrin
AU - Homrich, Mirka
AU - Ebbinghaus, Stephanie
AU - Juda, Pavel
AU - Miková, Eliška
AU - Hauschild, Robert
AU - Zhang, Lili
AU - Quast, Thomas
AU - Mass, Elvira
AU - Schlitzer, Andreas
AU - Kolanus, Waldemar
AU - Burgdorf, Sven
AU - Gruß, Oliver J.
AU - Hons, Miroslav
AU - Wieser, Stefan
AU - Kiermaier, Eva
ID - 12122
IS - 12
JF - Journal of Cell Biology
KW - Cell Biology
SN - 0021-9525
TI - Multiple centrosomes enhance migration and immune cell effector functions of mature dendritic cells
VL - 221
ER -
TY - JOUR
AB - Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear.
Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains.
Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane.
This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.
AU - Li, Hongjiang
AU - von Wangenheim, Daniel
AU - Zhang, Xixi
AU - Tan, Shutang
AU - Darwish-Miranda, Nasser
AU - Naramoto, Satoshi
AU - Wabnik, Krzysztof T
AU - de Rycke, Riet
AU - Kaufmann, Walter
AU - Gütl, Daniel J
AU - Tejos, Ricardo
AU - Grones, Peter
AU - Ke, Meiyu
AU - Chen, Xu
AU - Dettmer, Jan
AU - Friml, Jiří
ID - 8582
IS - 1
JF - New Phytologist
SN - 0028646X
TI - Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana
VL - 229
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 - 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.
AU - Gast, Matthieu
AU - Kadzioch, Nicole P.
AU - Milius, Doreen
AU - Origgi, Francesco
AU - Plattet, Philippe
ID - 9361
IS - 2
JF - mSphere
TI - Oligomerization and cell egress controlled by two microdomains of canine distemper virus matrix protein
VL - 6
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 - 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.
AU - Nelson, Glyn
AU - Boehm, Ulrike
AU - Bagley, Steve
AU - Bajcsy, Peter
AU - Bischof, Johanna
AU - Brown, Claire M.
AU - Dauphin, Aurélien
AU - Dobbie, Ian M.
AU - Eriksson, John E.
AU - Faklaris, Orestis
AU - Fernandez-Rodriguez, Julia
AU - Ferrand, Alexia
AU - Gelman, Laurent
AU - Gheisari, Ali
AU - Hartmann, Hella
AU - Kukat, Christian
AU - Laude, Alex
AU - Mitkovski, Miso
AU - Munck, Sebastian
AU - North, Alison J.
AU - Rasse, Tobias M.
AU - Resch-Genger, Ute
AU - Schuetz, Lucas C.
AU - Seitz, Arne
AU - Strambio-De-Castillia, Caterina
AU - Swedlow, Jason R.
AU - Alexopoulos, Ioannis
AU - Aumayr, Karin
AU - Avilov, Sergiy
AU - Bakker, Gert Jan
AU - Bammann, Rodrigo R.
AU - Bassi, Andrea
AU - Beckert, Hannes
AU - Beer, Sebastian
AU - Belyaev, Yury
AU - Bierwagen, Jakob
AU - Birngruber, Konstantin A.
AU - Bosch, Manel
AU - Breitlow, Juergen
AU - Cameron, Lisa A.
AU - Chalfoun, Joe
AU - Chambers, James J.
AU - Chen, Chieh Li
AU - Conde-Sousa, Eduardo
AU - Corbett, Alexander D.
AU - Cordelieres, Fabrice P.
AU - Nery, Elaine Del
AU - Dietzel, Ralf
AU - Eismann, Frank
AU - Fazeli, Elnaz
AU - Felscher, Andreas
AU - Fried, Hans
AU - Gaudreault, Nathalie
AU - Goh, Wah Ing
AU - Guilbert, Thomas
AU - Hadleigh, Roland
AU - Hemmerich, Peter
AU - Holst, Gerhard A.
AU - Itano, Michelle S.
AU - Jaffe, Claudia B.
AU - Jambor, Helena K.
AU - Jarvis, Stuart C.
AU - Keppler, Antje
AU - Kirchenbuechler, David
AU - Kirchner, Marcel
AU - Kobayashi, Norio
AU - Krens, Gabriel
AU - Kunis, Susanne
AU - Lacoste, Judith
AU - Marcello, Marco
AU - Martins, Gabriel G.
AU - Metcalf, Daniel J.
AU - Mitchell, Claire A.
AU - Moore, Joshua
AU - Mueller, Tobias
AU - Nelson, Michael S.
AU - Ogg, Stephen
AU - Onami, Shuichi
AU - Palmer, Alexandra L.
AU - Paul-Gilloteaux, Perrine
AU - Pimentel, Jaime A.
AU - Plantard, Laure
AU - Podder, Santosh
AU - Rexhepaj, Elton
AU - Royon, Arnaud
AU - Saari, Markku A.
AU - Schapman, Damien
AU - Schoonderwoert, Vincent
AU - Schroth-Diez, Britta
AU - Schwartz, Stanley
AU - Shaw, Michael
AU - Spitaler, Martin
AU - Stoeckl, Martin T.
AU - Sudar, Damir
AU - Teillon, Jeremie
AU - Terjung, Stefan
AU - Thuenauer, Roland
AU - Wilms, Christian D.
AU - Wright, Graham D.
AU - Nitschke, Roland
ID - 9911
IS - 1
JF - Journal of Microscopy
SN - 0022-2720
TI - QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy
VL - 284
ER -
TY - JOUR
AB - 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.
AU - Bajaj, Sunanjay
AU - Bagley, Joshua A.
AU - Sommer, Christoph M
AU - Vertesy, Abel
AU - Nagumo Wong, Sakurako
AU - Krenn, Veronica
AU - Lévi-Strauss, Julie
AU - Knoblich, Juergen A.
ID - 10179
IS - 23
JF - EMBO Journal
SN - 0261-4189
TI - Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration
VL - 40
ER -
TY - JOUR
AU - Pranger, Christina L.
AU - Fazekas-Singer, Judit
AU - Köhler, Verena K.
AU - Pali‐Schöll, Isabella
AU - Fiocchi, Alessandro
AU - Karagiannis, Sophia N.
AU - Zenarruzabeitia, Olatz
AU - Borrego, Francisco
AU - Jensen‐Jarolim, Erika
ID - 10836
IS - 5
JF - Allergy
KW - Immunology
KW - Immunology and Allergy
SN - 0105-4538
TI - PIPE‐cloned human IgE and IgG4 antibodies: New tools for investigating cow's milk allergy and tolerance
VL - 76
ER -
TY - JOUR
AB - A semiconducting nanowire fully wrapped by a superconducting shell has been proposed as a platform for obtaining Majorana modes at small magnetic fields. In this study, we demonstrate that the appearance of subgap states in such structures is actually governed by the junction region in tunneling spectroscopy measurements and not the full-shell nanowire itself. Short tunneling regions never show subgap states, whereas longer junctions always do. This can be understood in terms of quantum dots forming in the junction and hosting Andreev levels in the Yu-Shiba-Rusinov regime. The intricate magnetic field dependence of the Andreev levels, through both the Zeeman and Little-Parks effects, may result in robust zero-bias peaks—features that could be easily misinterpreted as originating from Majorana zero modes but are unrelated to topological superconductivity.
AU - Valentini, Marco
AU - Peñaranda, Fernando
AU - Hofmann, Andrea C
AU - Brauns, Matthias
AU - Hauschild, Robert
AU - Krogstrup, Peter
AU - San-Jose, Pablo
AU - Prada, Elsa
AU - Aguado, Ramón
AU - Katsaros, Georgios
ID - 8910
IS - 6550
JF - Science
SN - 00368075
TI - Nontopological zero-bias peaks in full-shell nanowires induced by flux-tunable Andreev states
VL - 373
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 - JOUR
AB - Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear.
Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation.
The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.
AU - Gelová, Zuzana
AU - Gallei, Michelle C
AU - Pernisová, Markéta
AU - Brunoud, Géraldine
AU - Zhang, Xixi
AU - Glanc, Matous
AU - Li, Lanxin
AU - Michalko, Jaroslav
AU - Pavlovicova, Zlata
AU - Verstraeten, Inge
AU - Han, Huibin
AU - Hajny, Jakub
AU - Hauschild, Robert
AU - Čovanová, Milada
AU - Zwiewka, Marta
AU - Hörmayer, Lukas
AU - Fendrych, Matyas
AU - Xu, Tongda
AU - Vernoux, Teva
AU - Friml, Jiří
ID - 8931
JF - Plant Science
KW - Agronomy and Crop Science
KW - Plant Science
KW - Genetics
KW - General Medicine
SN - 0168-9452
TI - Developmental roles of auxin binding protein 1 in Arabidopsis thaliana
VL - 303
ER -
TY - COMP
AU - Hauschild, Robert
ID - 8181
TI - Amplified centrosomes in dendritic cells promote immune cell effector functions
ER -
TY - COMP
AB - Automated root growth analysis and tracking of root tips.
AU - Hauschild, Robert
ID - 8294
TI - RGtracker
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 - 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.
AU - Schauer, Alexandra
AU - Nunes Pinheiro, Diana C
AU - Hauschild, Robert
AU - Heisenberg, Carl-Philipp J
ID - 7888
JF - eLife
SN - 2050-084X
TI - Zebrafish embryonic explants undergo genetically encoded self-assembly
VL - 9
ER -
TY - JOUR
AB - 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.
AU - Singer, Judit
AU - Singer, Josef
AU - Jensen-Jarolim, Erika
ID - 7864
IS - 3
JF - Current opinion in allergy and clinical immunology
TI - Precision medicine in clinical oncology: the journey from IgG antibody to IgE
VL - 20
ER -
TY - GEN
AB - 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.
AU - Slovakova, Jana
AU - Sikora, Mateusz K
AU - Caballero Mancebo, Silvia
AU - Krens, Gabriel
AU - Kaufmann, Walter
AU - Huljev, Karla
AU - Heisenberg, Carl-Philipp J
ID - 9750
T2 - bioRxiv
TI - Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion
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
AB - 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.
AU - Truckenbrodt, Sven M
AU - Sommer, Christoph M
AU - Rizzoli, Silvio O
AU - Danzl, Johann G
ID - 6052
IS - 3
JF - Nature Protocols
TI - A practical guide to optimization in X10 expansion microscopy
VL - 14
ER -
TY - JOUR
AB - 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.
AU - Fenu, M.
AU - Bettermann, T.
AU - Vogl, C.
AU - Darwish-Miranda, Nasser
AU - Schramel, J.
AU - Jenner, F.
AU - Ribitsch, I.
ID - 6867
IS - 1
JF - Scientific Reports
TI - A novel magnet-based scratch method for standardisation of wound-healing assays
VL - 9
ER -
TY - JOUR
AB - 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.
AU - Mckenzie, Catherine
AU - Spanova, Miroslava
AU - Johnson, Alexander J
AU - Kainrath, Stephanie
AU - Zheden, Vanessa
AU - Sitte, Harald H.
AU - Janovjak, Harald L
ID - 7406
JF - Journal of Neuroscience Methods
SN - 0165-0270
TI - Isolation of synaptic vesicles from genetically engineered cultured neurons
VL - 312
ER -
TY - JOUR
AB - 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.
AU - Goudarzi, Mohammad
AU - Boquet-Pujadas, Aleix
AU - Olivo-Marin, Jean Christophe
AU - Raz, Erez
ID - 6093
IS - 2
JF - PLOS ONE
TI - Fluid dynamics during bleb formation in migrating cells in vivo
VL - 14
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
AB - Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.
AU - Ratheesh, Aparna
AU - Biebl, Julia
AU - Smutny, Michael
AU - Veselá, Jana
AU - Papusheva, Ekaterina
AU - Krens, Gabriel
AU - Kaufmann, Walter
AU - György, Attila
AU - Casano, Alessandra M
AU - Siekhaus, Daria E
ID - 308
IS - 3
JF - Developmental Cell
TI - Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration
VL - 45
ER -
TY - JOUR
AB - Dendritic cells (DCs) are sentinels of the adaptive immune system that reside in peripheral organs of mammals. Upon pathogen encounter, they undergo maturation and up-regulate the chemokine receptor CCR7 that guides them along gradients of its chemokine ligands CCL19 and 21 to the next draining lymph node. There, DCs present peripherally acquired antigen to naïve T cells, thereby triggering adaptive immunity.
AU - Leithner, Alexander F
AU - Renkawitz, Jörg
AU - De Vries, Ingrid
AU - Hauschild, Robert
AU - Haecker, Hans
AU - Sixt, Michael K
ID - 437
IS - 6
JF - European Journal of Immunology
TI - Fast and efficient genetic engineering of hematopoietic precursor cells for the study of dendritic cell migration
VL - 48
ER -
TY - JOUR
AB - Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified > 1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.
AU - Brown, Markus
AU - Johnson, Louise
AU - Leone, Dario
AU - Májek, Peter
AU - Vaahtomeri, Kari
AU - Senfter, Daniel
AU - Bukosza, Nora
AU - Schachner, Helga
AU - Asfour, Gabriele
AU - Langer, Brigitte
AU - Hauschild, Robert
AU - Parapatics, Katja
AU - Hong, Young
AU - Bennett, Keiryn
AU - Kain, Renate
AU - Detmar, Michael
AU - Sixt, Michael K
AU - Jackson, David
AU - Kerjaschki, Dontscho
ID - 275
IS - 6
JF - Journal of Cell Biology
TI - Lymphatic exosomes promote dendritic cell migration along guidance cues
VL - 217
ER -
TY - DATA
AB - Script to perform a simple exponential lifetime fit of a ROI on time stacks acquired with a FLIM X16 TCSPC detector (+example data)
AU - Hauschild, Robert
ID - 5588
KW - FLIM
KW - FRET
KW - fluorescence lifetime imaging
TI - Fluorescence lifetime analysis of FLIM X16 TCSPC data
ER -
TY - JOUR
AB - Although much is known about the physiological framework of T cell motility, and numerous rate-limiting molecules have been identified through loss-of-function approaches, an integrated functional concept of T cell motility is lacking. Here, we used in vivo precision morphometry together with analysis of cytoskeletal dynamics in vitro to deconstruct the basic mechanisms of T cell migration within lymphatic organs. We show that the contributions of the integrin LFA-1 and the chemokine receptor CCR7 are complementary rather than positioned in a linear pathway, as they are during leukocyte extravasation from the blood vasculature. Our data demonstrate that CCR7 controls cortical actin flows, whereas integrins mediate substrate friction that is sufficient to drive locomotion in the absence of considerable surface adhesions and plasma membrane flux.
AU - Hons, Miroslav
AU - Kopf, Aglaja
AU - Hauschild, Robert
AU - Leithner, Alexander F
AU - Gärtner, Florian R
AU - Abe, Jun
AU - Renkawitz, Jörg
AU - Stein, Jens
AU - Sixt, Michael K
ID - 15
IS - 6
JF - Nature Immunology
TI - Chemokines and integrins independently tune actin flow and substrate friction during intranodal migration of T cells
VL - 19
ER -
TY - JOUR
AB - The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.
AU - Li, Lanxin
AU - Krens, Gabriel
AU - Fendrych, Matyas
AU - Friml, Jirí
ID - 442
IS - 1
JF - Bio-protocol
TI - Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls
VL - 8
ER -
TY - JOUR
AB - Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration.
AU - Vaahtomeri, Kari
AU - Brown, Markus
AU - Hauschild, Robert
AU - De Vries, Ingrid
AU - Leithner, Alexander F
AU - Mehling, Matthias
AU - Kaufmann, Walter
AU - Sixt, Michael K
ID - 672
IS - 5
JF - Cell Reports
SN - 22111247
TI - Locally triggered release of the chemokine CCL21 promotes dendritic cell transmigration across lymphatic endothelia
VL - 19
ER -
TY - JOUR
AB - Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable “roads” ensure optimal guidance in vivo.
AU - Schwarz, Jan
AU - Bierbaum, Veronika
AU - Vaahtomeri, Kari
AU - Hauschild, Robert
AU - Brown, Markus
AU - De Vries, Ingrid
AU - Leithner, Alexander F
AU - Reversat, Anne
AU - Merrin, Jack
AU - Tarrant, Teresa
AU - Bollenbach, Tobias
AU - Sixt, Michael K
ID - 674
IS - 9
JF - Current Biology
SN - 09609822
TI - Dendritic cells interpret haptotactic chemokine gradients in a manner governed by signal to noise ratio and dependent on GRK6
VL - 27
ER -
TY - JOUR
AB - Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load.
AU - Mueller, Jan
AU - Szep, Gregory
AU - Nemethova, Maria
AU - De Vries, Ingrid
AU - Lieber, Arnon
AU - Winkler, Christoph
AU - Kruse, Karsten
AU - Small, John
AU - Schmeiser, Christian
AU - Keren, Kinneret
AU - Hauschild, Robert
AU - Sixt, Michael K
ID - 727
IS - 1
JF - Cell
SN - 00928674
TI - Load adaptation of lamellipodial actin networks
VL - 171
ER -
TY - DATA
AB - Matlab script to calculate the forward migration indexes (/) from TrackMate spot-statistics files.
AU - Hauschild, Robert
ID - 5570
KW - Cell migration
KW - tracking
KW - forward migration index
KW - FMI
TI - Forward migration indexes
ER -
TY - DATA
AB - This repository contains the data collected for the manuscript "Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity".
The data is compressed into a single archive. Within the archive, different folders correspond to figures of the main text and the SI of the related publication.
Data is saved as plain text, with each folder containing a separate readme file describing the format. Typically, the data is from fluorescence microscopy measurements of single cells growing in a microfluidic "mother machine" device, and consists of relevant values (primarily arbitrary unit or normalized fluorescence measurements, and division times / growth rates) after raw microscopy images have been processed, segmented, and their features extracted, as described in the methods section of the related publication.
AU - Bergmiller, Tobias
AU - Andersson, Anna M
AU - Tomasek, Kathrin
AU - Balleza, Enrique
AU - Kiviet, Daniel
AU - Hauschild, Robert
AU - Tkacik, Gasper
AU - Guet, Calin C
ID - 5560
KW - single cell microscopy
KW - mother machine microfluidic device
KW - AcrAB-TolC pump
KW - multi-drug efflux
KW - Escherichia coli
TI - Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity
ER -