@article{6508, abstract = {Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.}, author = {Shamipour, Shayan and Kardos, Roland and Xue, Shi-lei and Hof, Björn and Hannezo, Edouard B and Heisenberg, Carl-Philipp J}, issn = {10974172}, journal = {Cell}, number = {6}, pages = {1463--1479.e18}, publisher = {Elsevier}, title = {{Bulk actin dynamics drive phase segregation in zebrafish oocytes}}, doi = {10.1016/j.cell.2019.04.030}, volume = {177}, year = {2019}, } @phdthesis{6546, abstract = {Invasive migration plays a crucial role not only during development and homeostasis but also in pathological states, such as tumor metastasis. Drosophila macrophage migration into the extended germband is an interesting system to study invasive migration. It carries similarities to immune cell transmigration and cancer cell invasion, therefore studying this process could also bring new understanding of invasion in higher organisms. In our work, we uncover a highly conserved member of the major facilitator family that plays a role in tissue invasion through regulation of glycosylation on a subgroup of proteins and/or by aiding the precise timing of DN-Cadherin downregulation. Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis. }, author = {Valosková, Katarina}, issn = {2663-337X}, pages = {141}, publisher = {Institute of Science and Technology Austria}, title = {{The role of a highly conserved major facilitator superfamily member in Drosophila embryonic macrophage migration}}, doi = {10.15479/AT:ISTA:6546}, year = {2019}, } @article{6611, abstract = {Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.}, author = {Glanc, Matous and Fendrych, Matyas and Friml, Jiří}, journal = {Biomolecules}, number = {6}, publisher = {MDPI}, title = {{PIN2 polarity establishment in arabidopsis in the absence of an intact cytoskeleton}}, doi = {10.3390/biom9060222}, volume = {9}, year = {2019}, } @phdthesis{6849, abstract = {Brain function is mediated by complex dynamical interactions between excitatory and inhibitory cell types. The Cholecystokinin-expressing inhibitory cells (CCK-interneurons) are one of the least studied types, despite being suspected to play important roles in cognitive processes. We studied the network effects of optogenetic silencing of CCK-interneurons in the CA1 hippocampal area during exploration and sleep states. The cell firing pattern in response to light pulses allowed us to classify the recorded neurons in 5 classes, including disinhibited and non-responsive pyramidal cell and interneurons, and the inhibited interneurons corresponding to the CCK group. The light application, which inhibited the activity of CCK interneurons triggered wider changes in the firing dynamics of cells. We observed rate changes (i.e. remapping) of pyramidal cells during the exploration session in which the light was applied relative to the previous control session that was not restricted neither in time nor space to the light delivery. Also, the disinhibited pyramidal cells had higher increase in bursting than in single spike firing rate as a result of CCK silencing. In addition, the firing activity patterns during exploratory periods were more weakly reactivated in sleep for those periods in which CCK-interneuron were silenced than in the unaffected periods. Furthermore, light pulses during sleep disrupted the reactivation of recent waking patterns. Hence, silencing CCK neurons during exploration suppressed the reactivation of waking firing patterns in sleep and CCK interneuron activity was also required during sleep for the normal reactivation of waking patterns. These findings demonstrate the involvement of CCK cells in reactivation-related memory consolidation. An important part of our analysis was to test the relationship of the identified CCKinterneurons to brain oscillations. Our findings showed that these cells exhibited different oscillatory behaviour during anaesthesia and natural waking and sleep conditions. We showed that: 1) Contrary to the past studies performed under anaesthesia, the identified CCKinterneurons fired on the descending portion of the theta phase in waking exploration. 2) CCKinterneuron preferred phases around the trough of gamma oscillations. 3) Contrary to anaesthesia conditions, the average firing rate of the CCK-interneurons increased around the peak activity of the sharp-wave ripple (SWR) events in natural sleep, which is congruent with new reports about their functional connectivity. We also found that light driven CCK-interneuron silencing altered the dynamics on the CA1 network oscillatory activity: 1) Pyramidal cells negatively shifted their preferred theta phases when the light was applied, while interneurons responses were less consistent. 2) As a population, pyramidal cells negatively shifted their preferred activity during gamma oscillations, albeit we did not find gamma modulation differences related to the light application when pyramidal cells were subdivided into the disinhibited and unaffected groups. 3) During the peak of SWR events, all but the CCK-interneurons had a reduction in their relative firing rate change during the light application as compared to the change observed at SWR initiation. Finally, regarding to the place field activity of the recorded pyramidal neurons, we showed that the disinhibited pyramidal cells had reduced place field similarity, coherence and spatial information, but only during the light application. The mechanisms behind such observed behaviours might involve eCB signalling and plastic changes in CCK-interneuron synapses. In conclusion, the observed changes related to the light-mediated silencing of CCKinterneurons have unravelled characteristics of this interneuron subpopulation that might change the understanding not only of their particular network interactions, but also of the current theories about the emergence of certain cognitive processes such as place coding needed for navigation or hippocampus-dependent memory consolidation. }, author = {Rangel Guerrero, Dámaris K}, isbn = {9783990780039}, issn = {2663-337X}, pages = {97}, publisher = {Institute of Science and Technology Austria}, title = {{The role of CCK-interneurons in regulating hippocampal network dynamics}}, doi = {10.15479/AT:ISTA:6849}, year = {2019}, } @article{6897, abstract = {The apical hook is a transiently formed structure that plays a protective role when the germinating seedling penetrates through the soil towards the surface. Crucial for proper bending is the local auxin maxima, which defines the concave (inner) side of the hook curvature. As no sign of asymmetric auxin distribution has been reported in embryonic hypocotyls prior to hook formation, the question of how auxin asymmetry is established in the early phases of seedling germination remains largely unanswered. Here, we analyzed the auxin distribution and expression of PIN auxin efflux carriers from early phases of germination, and show that bending of the root in response to gravity is the crucial initial cue that governs the hypocotyl bending required for apical hook formation. Importantly, polar auxin transport machinery is established gradually after germination starts as a result of tight root-hypocotyl interaction and a proper balance between abscisic acid and gibberellins.}, author = {Zhu, Qiang and Gallemi, Marçal and Pospíšil, Jiří and Žádníková, Petra and Strnad, Miroslav and Benková, Eva}, issn = {14779129}, journal = {Development}, number = {17}, publisher = {The Company of Biologists}, title = {{Root gravity response module guides differential growth determining both root bending and apical hook formation in Arabidopsis}}, doi = {10.1242/dev.175919}, volume = {146}, year = {2019}, } @phdthesis{323, abstract = {In the here presented thesis, we explore the role of branched actin networks in cell migration and antigen presentation, the two most relevant processes in dendritic cell biology. Branched actin networks construct lamellipodial protrusions at the leading edge of migrating cells. These are typically seen as adhesive structures, which mediate force transduction to the extracellular matrix that leads to forward locomotion. We ablated Arp2/3 nucleation promoting factor WAVE in DCs and found that the resulting cells lack lamellipodial protrusions. Instead, depending on the maturation state, one or multiple filopodia were formed. By challenging these cells in a variety of migration assays we found that lamellipodial protrusions are dispensable for the locomotion of leukocytes and actually dampen the speed of migration. However, lamellipodia are critically required to negotiate complex environments that DCs experience while they travel to the next draining lymph node. Taken together our results suggest that leukocyte lamellipodia have rather a sensory- than a force transducing function. Furthermore, we show for the first time structure and dynamics of dendritic cell F-actin at the immunological synapse with naïve T cells. Dendritic cell F-actin appears as dynamic foci that are nucleated by the Arp2/3 complex. WAVE ablated dendritic cells show increased membrane tension, leading to an altered ultrastructure of the immunological synapse and severe T cell priming defects. These results point towards a previously unappreciated role of the cellular mechanics of dendritic cells in T cell activation. Additionally, we present a novel cell culture based system for the differentiation of dendritic cells from conditionally immortalized hematopoietic precursors. These precursor cells are genetically tractable via the CRISPR/Cas9 system while they retain their ability to differentiate into highly migratory dendritic cells and other immune cells. This will foster the study of all aspects of dendritic cell biology and beyond. }, author = {Leithner, Alexander F}, issn = {2663-337X}, pages = {99}, publisher = {Institute of Science and Technology Austria}, title = {{Branched actin networks in dendritic cell biology}}, doi = {10.15479/AT:ISTA:th_998}, year = {2018}, } @phdthesis{395, abstract = {Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g. autism spectrum disorder, intellectual disability, epilepsy) remains a great challenge. Recent advancements in geno mics, like whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that were discovered, the etiological variability and the heterogeneous phenotypic outcomes, the need for genotype -along with phenotype- based diagnosis of individual patients becomes a requisite. Driven by this rationale, in a previous study our group described mutations, identified via whole - exome sequencing, in the gene BCKDK – encoding for a key regulator of branched chain amin o acid (BCAA) catabolism - as a cause of ASD. Following up on the role of BCAAs, in the study described here we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized mainly at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation and severe neurolo gical abnormalities. Additionally, deletion of Slc7a5 from the neural progenitor cell population leads to microcephaly. Interestingly, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Furthermore, whole - exome sequencing of patients diagnosed with neurological dis o r ders helped us identify several patients with autistic traits, microcephaly and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. In conclusion, our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for t he BCAA s in human bra in function. Together with r ecent studies (described in chapter two) that have successfully made the transition into clinical practice, our findings on the role of B CAAs might have a crucial impact on the development of novel individualized therapeutic strategies for ASD. }, author = {Tarlungeanu, Dora-Clara}, issn = {2663-337X}, pages = {88}, publisher = {Institute of Science and Technology Austria}, title = {{The branched chain amino acids in autism spectrum disorders }}, doi = {10.15479/AT:ISTA:th_992}, year = {2018}, } @article{402, abstract = {During metastasis, malignant cells escape the primary tumor, intravasate lymphatic vessels, and reach draining sentinel lymph nodes before they colonize distant organs via the blood circulation. Although lymph node metastasis in cancer patients correlates with poor prognosis, evidence is lacking as to whether and how tumor cells enter the bloodstream via lymph nodes. To investigate this question, we delivered carcinoma cells into the lymph nodes of mice by microinfusing the cells into afferent lymphatic vessels. We found that tumor cells rapidly infiltrated the lymph node parenchyma, invaded blood vessels, and seeded lung metastases without involvement of the thoracic duct. These results suggest that the lymph node blood vessels can serve as an exit route for systemic dissemination of cancer cells in experimental mouse models. Whether this form of tumor cell spreading occurs in cancer patients remains to be determined.}, author = {Brown, Markus and Assen, Frank P and Leithner, Alexander F and Abe, Jun and Schachner, Helga and Asfour, Gabriele and Bagó Horváth, Zsuzsanna and Stein, Jens and Uhrin, Pavel and Sixt, Michael K and Kerjaschki, Dontscho}, journal = {Science}, number = {6382}, pages = {1408 -- 1411}, publisher = {American Association for the Advancement of Science}, title = {{Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice}}, doi = {10.1126/science.aal3662}, volume = {359}, year = {2018}, } @article{503, abstract = {Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg2+ and Ca2+, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry.}, author = {Tomasek, Kathrin and Bergmiller, Tobias and Guet, Calin C}, journal = {Journal of Biotechnology}, pages = {40 -- 52}, publisher = {Elsevier}, title = {{Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains}}, doi = {10.1016/j.jbiotec.2018.01.008}, volume = {268}, year = {2018}, } @article{803, abstract = {Eukaryotic cells store their chromosomes in a single nucleus. This is important to maintain genomic integrity, as chromosomes packaged into separate nuclei (micronuclei) are prone to massive DNA damage. During mitosis, higher eukaryotes disassemble their nucleus and release individualized chromosomes for segregation. How numerous chromosomes subsequently reform a single nucleus has remained unclear. Using image-based screening of human cells, we identified barrier-to-autointegration factor (BAF) as a key factor guiding membranes to form a single nucleus. Unexpectedly, nuclear assembly does not require BAF?s association with inner nuclear membrane proteins but instead relies on BAF?s ability to bridge distant DNA sites. Live-cell imaging and in vitro reconstitution showed that BAF enriches around the mitotic chromosome ensemble to induce a densely cross-bridged chromatin layer that is mechanically stiff and limits membranes to the surface. Our study reveals that BAF-mediated changes in chromosome mechanics underlie nuclear assembly with broad implications for proper genome function.}, author = {Samwer, Matthias and Schneider, Maximilian and Hoefler, Rudolf and Schmalhorst, Philipp S and Jude, Julian and Zuber, Johannes and Gerlic, Daniel}, issn = {00928674}, journal = {Cell}, number = {5}, pages = {956 -- 972}, publisher = {Cell Press}, title = {{DNA cross-bridging shapes a single nucleus from a set of mitotic chromosomes}}, doi = {10.1016/j.cell.2017.07.038}, volume = {170}, year = {2017}, } @article{1078, abstract = {One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions. }, author = {Von Wangenheim, Daniel and Hauschild, Robert and Friml, Jirí}, journal = {Journal of visualized experiments JoVE}, number = {119}, publisher = {Journal of Visualized Experiments}, title = {{Light sheet fluorescence microscopy of plant roots growing on the surface of a gel}}, doi = {10.3791/55044}, volume = {2017}, year = {2017}, } @article{944, abstract = {The concerted production of neurons and glia by neural stem cells (NSCs) is essential for neural circuit assembly. In the developing cerebral cortex, radial glia progenitors (RGPs) generate nearly all neocortical neurons and certain glia lineages. RGP proliferation behavior shows a high degree of non-stochasticity, thus a deterministic characteristic of neuron and glia production. However, the cellular and molecular mechanisms controlling RGP behavior and proliferation dynamics in neurogenesis and glia generation remain unknown. By using mosaic analysis with double markers (MADM)-based genetic paradigms enabling the sparse and global knockout with unprecedented single-cell resolution, we identified Lgl1 as a critical regulatory component. We uncover Lgl1-dependent tissue-wide community effects required for embryonic cortical neurogenesis and novel cell-autonomous Lgl1 functions controlling RGP-mediated glia genesis and postnatal NSC behavior. These results suggest that NSC-mediated neuron and glia production is tightly regulated through the concerted interplay of sequential Lgl1-dependent global and cell intrinsic mechanisms.}, author = {Beattie, Robert J and Postiglione, Maria P and Burnett, Laura and Laukoter, Susanne and Streicher, Carmen and Pauler, Florian and Xiao, Guanxi and Klezovitch, Olga and Vasioukhin, Valeri and Ghashghaei, Troy and Hippenmeyer, Simon}, issn = {08966273}, journal = {Neuron}, number = {3}, pages = {517 -- 533.e3}, publisher = {Cell Press}, title = {{Mosaic analysis with double markers reveals distinct sequential functions of Lgl1 in neural stem cells}}, doi = {10.1016/j.neuron.2017.04.012}, volume = {94}, year = {2017}, } @article{946, abstract = {Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker – a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high-resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes.}, author = {Von Wangenheim, Daniel and Hauschild, Robert and Fendrych, Matyas and Barone, Vanessa and Benková, Eva and Friml, Jirí}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Live tracking of moving samples in confocal microscopy for vertically grown roots}}, doi = {10.7554/eLife.26792}, volume = {6}, year = {2017}, } @article{1117, abstract = {GABAergic synapses in brain circuits generate inhibitory output signals with submillisecond latency and temporal precision. Whether the molecular identity of the release sensor contributes to these signaling properties remains unclear. Here, we examined the Ca^2+ sensor of exocytosis at GABAergic basket cell (BC) to Purkinje cell (PC) synapses in cerebellum. Immunolabeling suggested that BC terminals selectively expressed synaptotagmin 2 (Syt2), whereas synaptotagmin 1 (Syt1) was enriched in excitatory terminals. Genetic elimination of Syt2 reduced action potential-evoked release to ∼10%, identifying Syt2 as the major Ca^2+ sensor at BC-PC synapses. Differential adenovirus-mediated rescue revealed that Syt2 triggered release with shorter latency and higher temporal precision and mediated faster vesicle pool replenishment than Syt1. Furthermore, deletion of Syt2 severely reduced and delayed disynaptic inhibition following parallel fiber stimulation. Thus, the selective use of Syt2 as release sensor at BC-PC synapses ensures fast and efficient feedforward inhibition in cerebellar microcircuits. #bioimagingfacility-author}, author = {Chen, Chong and Arai, Itaru and Satterield, Rachel and Young, Samuel and Jonas, Peter M}, issn = {22111247}, journal = {Cell Reports}, number = {3}, pages = {723 -- 736}, publisher = {Cell Press}, title = {{Synaptotagmin 2 is the fast Ca2+ sensor at a central inhibitory synapse}}, doi = {10.1016/j.celrep.2016.12.067}, volume = {18}, year = {2017}, } @inbook{1213, abstract = {Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments in the presence of GTP. Since the discovery of the Z-ring 25 years ago, various models for the role of FtsZ have been suggested. However, important information about the architecture and dynamics of FtsZ filaments during cytokinesis is still missing. One reason for this lack of knowledge has been the small size of bacteria, which has made it difficult to resolve the orientation and dynamics of individual FtsZ filaments in the Z-ring. While superresolution microscopy experiments have helped to gain more information about the organization of the Z-ring in the dividing cell, they were not yet able to elucidate a mechanism of how FtsZ filaments reorganize during assembly and disassembly of the Z-ring. In this chapter, we explain how to use an in vitro reconstitution approach to investigate the self-organization of FtsZ filaments recruited to a biomimetic lipid bilayer by its membrane anchor FtsA. We show how to perform single-molecule experiments to study the behavior of individual FtsZ monomers during the constant reorganization of the FtsZ-FtsA filament network. We describe how to analyze the dynamics of single molecules and explain why this information can help to shed light onto possible mechanism of Z-ring constriction. We believe that similar experimental approaches will be useful to study the mechanism of membrane-based polymerization of other cytoskeletal systems, not only from prokaryotic but also eukaryotic origin.}, author = {Baranova, Natalia and Loose, Martin}, booktitle = {Cytokinesis}, editor = {Echard, Arnaud }, issn = {0091679X}, pages = {355 -- 370}, publisher = {Academic Press}, title = {{Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers}}, doi = {10.1016/bs.mcb.2016.03.036}, volume = {137}, year = {2017}, } @article{1597, abstract = {Chemokines are the main guidance cues directing leukocyte migration. Opposed to early assumptions, chemokines do not necessarily act as soluble cues but are often immobilized within tissues, e.g., dendritic cell migration toward lymphatic vessels is guided by a haptotactic gradient of the chemokine CCL21. Controlled assay systems to quantitatively study haptotaxis in vitro are still missing. In this chapter, we describe an in vitro haptotaxis assay optimized for the unique properties of dendritic cells. The chemokine CCL21 is immobilized in a bioactive state, using laser-assisted protein adsorption by photobleaching. The cells follow this immobilized CCL21 gradient in a haptotaxis chamber, which provides three dimensionally confined migration conditions.}, author = {Schwarz, Jan and Sixt, Michael K}, journal = {Methods in Enzymology}, pages = {567 -- 581}, publisher = {Elsevier}, title = {{Quantitative analysis of dendritic cell haptotaxis}}, doi = {10.1016/bs.mie.2015.11.004}, volume = {570}, year = {2016}, } @phdthesis{1129, abstract = {Directed cell migration is a hallmark feature, present in almost all multi-cellular organisms. Despite its importance, basic questions regarding force transduction or directional sensing are still heavily investigated. Directed migration of cells guided by immobilized guidance cues - haptotaxis - occurs in key-processes, such as embryonic development and immunity (Middleton et al., 1997; Nguyen et al., 2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues comprise adhesive ligands, such as collagen and fibronectin (Barczyk et al., 2009), or chemokines - the main guidance cues for migratory leukocytes (Middleton et al., 1997; Weber et al., 2013). While adhesive ligands serve as attachment sites guiding cell migration (Carter, 1965), chemokines instruct haptotactic migration by inducing adhesion to adhesive ligands and directional guidance (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis of the cellular response to immobilized guidance cues requires in vitro assays that foster cell migration, offer accurate control of the immobilized cues on a subcellular scale and in the ideal case closely reproduce in vivo conditions. The exploration of haptotactic cell migration through design and employment of such assays represents the main focus of this work. Dendritic cells (DCs) are leukocytes, which after encountering danger signals such as pathogens in peripheral organs instruct naïve T-cells and consequently the adaptive immune response in the lymph node (Mellman and Steinman, 2001). To reach the lymph node from the periphery, DCs follow haptotactic gradients of the chemokine CCL21 towards lymphatic vessels (Weber et al., 2013). Questions about how DCs interpret haptotactic CCL21 gradients have not yet been addressed. The main reason for this is the lack of an assay that offers diverse haptotactic environments, hence allowing the study of DC migration as a response to different signals of immobilized guidance cue. In this work, we developed an in vitro assay that enables us to quantitatively assess DC haptotaxis, by combining precisely controllable chemokine photo-patterning with physically confining migration conditions. With this tool at hand, we studied the influence of CCL21 gradient properties and concentration on DC haptotaxis. We found that haptotactic gradient sensing depends on the absolute CCL21 concentration in combination with the local steepness of the gradient. Our analysis suggests that the directionality of migrating DCs is governed by the signal-to-noise ratio of CCL21 binding to its receptor CCR7. Moreover, the haptotactic CCL21 gradient formed in vivo provides an optimal shape for DCs to recognize haptotactic guidance cue. By reconstitution of the CCL21 gradient in vitro we were also able to study the influence of CCR7 signal termination on DC haptotaxis. To this end, we used DCs lacking the G-protein coupled receptor kinase GRK6, which is responsible for CCL21 induced CCR7 receptor phosphorylation and desensitization (Zidar et al., 2009). We found that CCR7 desensitization by GRK6 is crucial for maintenance of haptotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. In the context of the organism, immobilized haptotactic guidance cues often coincide and compete with soluble chemotactic guidance cues. During wound healing, fibroblasts are exposed and influenced by adhesive cues and soluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly, migrating DCs are exposed to both, soluble chemokines (CCL19 and truncated CCL21) inducing chemotactic behavior as well as the immobilized CCL21. To quantitatively assess these complex coinciding immobilized and soluble guidance cues, we implemented our chemokine photo-patterning technique in a microfluidic system allowing for chemotactic gradient generation. To validate the assay, we observed DC migration in competing CCL19/CCL21 environments. Adhesiveness guided haptotaxis has been studied intensively over the last century. However, quantitative studies leading to conceptual models are largely missing, again due to the lack of a precisely controllable in vitro assay. A requirement for such an in vitro assay is that it must prevent any uncontrolled cell adhesion. This can be accomplished by stable passivation of the surface. In addition, controlled adhesion must be sustainable, quantifiable and dose dependent in order to create homogenous gradients. Therefore, we developed a novel covalent photo-patterning technique satisfying all these needs. In combination with a sustainable poly-vinyl alcohol (PVA) surface coating we were able to generate gradients of adhesive cue to direct cell migration. This approach allowed us to characterize the haptotactic migratory behavior of zebrafish keratocytes in vitro. Furthermore, defined patterns of adhesive cue allowed us to control for cell shape and growth on a subcellular scale.}, author = {Schwarz, Jan}, issn = {2663-337X}, pages = {178}, publisher = {Institute of Science and Technology Austria}, title = {{Quantitative analysis of haptotactic cell migration}}, year = {2016}, } @article{1640, abstract = {Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.}, author = {Šimášková, Mária and O'Brien, José and Khan-Djamei, Mamoona and Van Noorden, Giel and Ötvös, Krisztina and Vieten, Anne and De Clercq, Inge and Van Haperen, Johanna and Cuesta, Candela and Hoyerová, Klára and Vanneste, Steffen and Marhavy, Peter and Wabnik, Krzysztof T and Van Breusegem, Frank and Nowack, Moritz and Murphy, Angus and Friml, Jiřĺ and Weijers, Dolf and Beeckman, Tom and Benková, Eva}, journal = {Nature Communications}, publisher = {Nature Publishing Group}, title = {{Cytokinin response factors regulate PIN-FORMED auxin transporters}}, doi = {10.1038/ncomms9717}, volume = {6}, year = {2015}, } @phdthesis{1406, abstract = {Epithelial spreading is a critical part of various developmental and wound repair processes. Here we use zebrafish epiboly as a model system to study the cellular and molecular mechanisms underlying the spreading of epithelial sheets. During zebrafish epiboly the enveloping cell layer (EVL), a simple squamous epithelium, spreads over the embryo to eventually cover the entire yolk cell by the end of gastrulation. The EVL leading edge is anchored through tight junctions to the yolk syncytial layer (YSL), where directly adjacent to the EVL margin a contractile actomyosin ring is formed that is thought to drive EVL epiboly. The prevalent view in the field was that the contractile ring exerts a pulling force on the EVL margin, which pulls the EVL towards the vegetal pole. However, how this force is generated and how it affects EVL morphology still remains elusive. Moreover, the cellular mechanisms mediating the increase in EVL surface area, while maintaining tissue integrity and function are still unclear. Here we show that the YSL actomyosin ring pulls on the EVL margin by two distinct force-generating mechanisms. One mechanism is based on contraction of the ring around its circumference, as previously proposed. The second mechanism is based on actomyosin retrogade flows, generating force through resistance against the substrate. The latter can function at any epiboly stage even in situations where the contraction-based mechanism is unproductive. Additionally, we demonstrate that during epiboly the EVL is subjected to anisotropic tension, which guides the orientation of EVL cell division along the main axis (animal-vegetal) of tension. The influence of tension in cell division orientation involves cell elongation and requires myosin-2 activity for proper spindle alignment. Strikingly, we reveal that tension-oriented cell divisions release anisotropic tension within the EVL and that in the absence of such divisions, EVL cells undergo ectopic fusions. We conclude that forces applied to the EVL by the action of the YSL actomyosin ring generate a tension anisotropy in the EVL that orients cell divisions, which in turn limit tissue tension increase thereby facilitating tissue spreading.}, author = {Campinho, Pedro}, issn = {2663-337X}, pages = {123}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanics of zebrafish epiboly: Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading}}, year = {2013}, } @article{2282, abstract = {Epithelial spreading is a common and fundamental aspect of various developmental and disease-related processes such as epithelial closure and wound healing. A key challenge for epithelial tissues undergoing spreading is to increase their surface area without disrupting epithelial integrity. Here we show that orienting cell divisions by tension constitutes an efficient mechanism by which the enveloping cell layer (EVL) releases anisotropic tension while undergoing spreading during zebrafish epiboly. The control of EVL cell-division orientation by tension involves cell elongation and requires myosin II activity to align the mitotic spindle with the main tension axis. We also found that in the absence of tension-oriented cell divisions and in the presence of increased tissue tension, EVL cells undergo ectopic fusions, suggesting that the reduction of tension anisotropy by oriented cell divisions is required to prevent EVL cells from fusing. We conclude that cell-division orientation by tension constitutes a key mechanism for limiting tension anisotropy and thus promoting tissue spreading during EVL epiboly.}, author = {Campinho, Pedro and Behrndt, Martin and Ranft, Jonas and Risler, Thomas and Minc, Nicolas and Heisenberg, Carl-Philipp J}, journal = {Nature Cell Biology}, pages = {1405 -- 1414}, publisher = {Nature Publishing Group}, title = {{Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly}}, doi = {10.1038/ncb2869}, volume = {15}, year = {2013}, }