@phdthesis{7902, abstract = {Mosaic genetic analysis has been widely used in different model organisms such as the fruit fly to study gene-function in a cell-autonomous or tissue-specific fashion. More recently, and less easily conducted, mosaic genetic analysis in mice has also been enabled with the ambition to shed light on human gene function and disease. These genetic tools are of particular interest, but not restricted to, the study of the brain. Notably, the MADM technology offers a genetic approach in mice to visualize and concomitantly manipulate small subsets of genetically defined cells at a clonal level and single cell resolution. MADM-based analysis has already advanced the study of genetic mechanisms regulating brain development and is expected that further MADM-based analysis of genetic alterations will continue to reveal important insights on the fundamental principles of development and disease to potentially assist in the development of new therapies or treatments. In summary, this work completed and characterized the necessary genome-wide genetic tools to perform MADM-based analysis at single cell level of the vast majority of mouse genes in virtually any cell type and provided a protocol to perform lineage tracing using the novel MADM resource. Importantly, this work also explored and revealed novel aspects of biologically relevant events in an in vivo context, such as the chromosome-specific bias of chromatid sister segregation pattern, the generation of cell-type diversity in the cerebral cortex and in the cerebellum and finally, the relevance of the interplay between the cell-autonomous gene function and cell-non-autonomous (community) effects in radial glial progenitor lineage progression. This work provides a foundation and opens the door to further elucidating the molecular mechanisms underlying neuronal diversity and astrocyte generation.}, author = {Contreras, Ximena}, issn = {2663-337X}, pages = {214}, publisher = {Institute of Science and Technology Austria}, title = {{Genetic dissection of neural development in health and disease at single cell resolution}}, doi = {10.15479/AT:ISTA:7902}, year = {2020}, } @article{8002, abstract = {Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity.}, author = {Hörmayer, Lukas and Montesinos López, Juan C and Marhavá, Petra and Benková, Eva and Yoshida, Saiko and Friml, Jiří}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {26}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots}}, doi = {10.1073/pnas.2003346117}, volume = {117}, year = {2020}, } @article{8139, abstract = {Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.}, author = {Johnson, Alexander J and Gnyliukh, Nataliia and Kaufmann, Walter and Narasimhan, Madhumitha and Vert, G and Bednarek, SY and Friml, Jiří}, issn = {1477-9137}, journal = {Journal of Cell Science}, number = {15}, publisher = {The Company of Biologists}, title = {{Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis}}, doi = {10.1242/jcs.248062}, volume = {133}, year = {2020}, } @article{8142, abstract = {Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine‐tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‐organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin‐sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells.}, author = {Montesinos López, Juan C and Abuzeineh, A and Kopf, Aglaja and Juanes Garcia, Alba and Ötvös, Krisztina and Petrášek, J and Sixt, Michael K and Benková, Eva}, issn = {1460-2075}, journal = {The Embo Journal}, number = {17}, publisher = {Embo Press}, title = {{Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage}}, doi = {10.15252/embj.2019104238}, volume = {39}, year = {2020}, } @article{8162, abstract = {In mammalian genomes, a subset of genes is regulated by genomic imprinting, resulting in silencing of one parental allele. Imprinting is essential for cerebral cortex development, but prevalence and functional impact in individual cells is unclear. Here, we determined allelic expression in cortical cell types and established a quantitative platform to interrogate imprinting in single cells. We created cells with uniparental chromosome disomy (UPD) containing two copies of either the maternal or the paternal chromosome; hence, imprinted genes will be 2-fold overexpressed or not expressed. By genetic labeling of UPD, we determined cellular phenotypes and transcriptional responses to deregulated imprinted gene expression at unprecedented single-cell resolution. We discovered an unexpected degree of cell-type specificity and a novel function of imprinting in the regulation of cortical astrocyte survival. More generally, our results suggest functional relevance of imprinted gene expression in glial astrocyte lineage and thus for generating cortical cell-type diversity.}, author = {Laukoter, Susanne and Pauler, Florian and Beattie, Robert J and Amberg, Nicole and Hansen, Andi H and Streicher, Carmen and Penz, Thomas and Bock, Christoph and Hippenmeyer, Simon}, issn = {0896-6273}, journal = {Neuron}, number = {6}, pages = {1160--1179.e9}, publisher = {Elsevier}, title = {{Cell-type specificity of genomic imprinting in cerebral cortex}}, doi = {10.1016/j.neuron.2020.06.031}, volume = {107}, year = {2020}, } @unpublished{9750, abstract = {Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow1,2. Here we show in zebrafish primary germ layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase, and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. Once tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension stabilizing E-cadherin-actin complexes at the contact.}, author = {Slovakova, Jana and Sikora, Mateusz K and Caballero Mancebo, Silvia and Krens, Gabriel and Kaufmann, Walter and Huljev, Karla and Heisenberg, Carl-Philipp J}, booktitle = {bioRxiv}, pages = {41}, publisher = {Cold Spring Harbor Laboratory}, title = {{Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion}}, doi = {10.1101/2020.11.20.391284}, year = {2020}, } @phdthesis{6947, abstract = {Lymph nodes are es s ential organs of the immune s ys tem where adaptive immune responses originate, and consist of various leukocyte populations and a stromal backbone. Fibroblastic reticular cells (FRCs) are the main stromal cells and form a sponge-like extracellular matrix network, called conduits , which they thems elves enwrap and contract. Lymph, containing s oluble antigens , arrive in lymph nodes via afferent lymphatic vessels that connect to the s ubcaps ular s inus and conduit network. According to the current paradigm, the conduit network dis tributes afferent lymph through lymph nodes and thus provides acces s for immune cells to lymph-borne antigens. An elas tic caps ule s urrounds the organ and confines the immune cells and FRC network. Lymph nodes are completely packed with lymphocytes and lymphocyte numbers directly dictates the size of the organ. Although lymphocytes cons tantly enter and leave the lymph node, its s ize remains remarkedly s table under homeostatic conditions. It is only partly known how the cellularity and s ize of the lymph node is regulated and how the lymph node is able to swell in inflammation. The role of the FRC network in lymph node s welling and trans fer of fluids are inves tigated in this thes is. Furthermore, we s tudied what trafficking routes are us ed by cancer cells in lymph nodes to form distal metastases.We examined the role of a mechanical feedback in regulation of lymph node swelling. Using parallel plate compression and UV-las er cutting experiments we dis s ected the mechanical force dynamics of the whole lymph node, and individually for FRCs and the caps ule. Physical forces generated by packed lymphocytes directly affect the tens ion on the FRC network and capsule, which increases its resistance to swelling. This implies a feedback mechanism between tis s ue pres s ure and ability of lymphocytes to enter the organ. Following inflammation, the lymph node swells ∼10 fold in two weeks . Yet, what is the role for tens ion on the FRC network and caps ule, and how are lymphocytes able to enter in conditions that resist swelling remain open ques tions . We s how that tens ion on the FRC network is important to limit the swelling rate of the organ so that the FRC network can grow in a coordinated fashion. This is illustrated by interfering with FRC contractility, which leads to faster swelling rates and a dis organized FRC network in the inflamed lymph node. Growth of the FRC network in turn is expected to releas e tens ion on thes e s tructures and lowers the res is tance to swelling, thereby allowing more lymphocytes to enter the organ and drive more swelling. Halt of swelling coincides with a thickening of the caps ule, which forms a thick res is tant band around the organ and lowers tens ion on the FRC network to form a new force equilibrium.The FRC and conduit network are further believed to be a privileged s ite of s oluble information within the lymph node, although many details remain uns olved. We s how by 3D ultra-recons truction that FRCs and antigen pres enting cells cover the s urface of conduit s ys tem for more than 99% and we dis cus s the implications for s oluble information exchangeat the conduit level.Finally, there is an ongoing debate in the cancer field whether and how cancer cells in lymph nodes s eed dis tal metas tas es . We s how that cancer cells infus ed into the lymph node can utilize trafficking routes of immune cells and rapidly migrate to blood vessels. Once in the blood circulation, these cells are able to form metastases in distal tissues.}, author = {Assen, Frank P}, issn = {2663-337X}, pages = {142}, publisher = {Institute of Science and Technology Austria}, title = {{Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking}}, doi = {10.15479/AT:ISTA:6947}, year = {2019}, } @article{7001, author = {Schwayer, Cornelia and Shamipour, Shayan and Pranjic-Ferscha, Kornelija and Schauer, Alexandra and Balda, M and Tada, M and Matter, K and Heisenberg, Carl-Philipp J}, issn = {1097-4172}, journal = {Cell}, number = {4}, pages = {937--952.e18}, publisher = {Cell Press}, title = {{Mechanosensation of tight junctions depends on ZO-1 phase separation and flow}}, doi = {10.1016/j.cell.2019.10.006}, volume = {179}, year = {2019}, } @phdthesis{7172, abstract = {The development and growth of Arabidopsis thaliana is regulated by a combination of genetic programing and also by the environmental influences. An important role in these processes play the phytohormones and among them, auxin is crucial as it controls many important functions. It is transported through the whole plant body by creating local and temporal concentration maxima and minima, which have an impact on the cell status, tissue and organ identity. Auxin has the property to undergo a directional and finely regulated cell-to-cell transport, which is enabled by the transport proteins, localized on the plasma membrane. An important role in this process have the PIN auxin efflux proteins, which have an asymmetric/polar subcellular localization and determine the directionality of the auxin transport. During the last years, there were significant advances in understanding how the trafficking molecular machineries function, including studies on molecular interactions, function, subcellular localization and intracellular distribution. However, there is still a lack of detailed characterization on the steps of endocytosis, exocytosis, endocytic recycling and degradation. Due to this fact, I focused on the identification of novel trafficking factors and better characterization of the intracellular trafficking pathways. My PhD thesis consists of an introductory chapter, three experimental chapters, a chapter containing general discussion, conclusions and perspectives and also an appendix chapter with published collaborative papers. The first chapter is separated in two different parts: I start by a general introduction to auxin biology and then I introduce the trafficking pathways in the model plant Arabidopsis thaliana. Then, I explain also the phosphorylation-signals for polar targeting and also the roles of the phytohormone strigolactone. The second chapter includes the characterization of bar1/sacsin mutant, which was identified in a forward genetic screen for novel trafficking components in Arabidopsis thaliana, where by the implementation of an EMS-treated pPIN1::PIN1-GFP marker line and by using the established inhibitor of ARF-GEFs, Brefeldin A (BFA) as a tool to study trafficking processes, we identified a novel factor, which is mediating the adaptation of the plant cell to ARF-GEF inhibition. The mutation is in a previously uncharacterized gene, encoding a very big protein that we, based on its homologies, called SACSIN with domains suggesting roles as a molecular chaperon or as a component of the ubiquitin-proteasome system. Our physiology and imaging studies revealed that SACSIN is a crucial plant cell component of the adaptation to the ARF-GEF inhibition. The third chapter includes six subchapters, where I focus on the role of the phytohormone strigolactone, which interferes with auxin feedback on PIN internalization. Strigolactone moderates the polar auxin transport by increasing the internalization of the PIN auxin efflux carriers, which reduces the canalization related growth responses. In addition, I also studied the role of phosphorylation in the strigolactone regulation of auxin feedback on PIN internalization. In this chapter I also present my results on the MAX2-dependence of strigolactone-mediated root growth inhibition and I also share my results on the auxin metabolomics profiling after application of GR24. In the fourth chapter I studied the effect of two small molecules ES-9 and ES9-17, which were identified from a collection of small molecules with the property to impair the clathrin-mediated endocytosis. In the fifth chapter, I discuss all my observations and experimental findings and suggest alternative hypothesis to interpret my results. In the appendix there are three collaborative published projects. In the first, I participated in the characterization of the role of ES9 as a small molecule, which is inhibitor of clathrin- mediated endocytosis in different model organisms. In the second paper, I contributed to the characterization of another small molecule ES9-17, which is a non-protonophoric analog of ES9 and also impairs the clathrin-mediated endocytosis not only in plant cells, but also in mammalian HeLa cells. Last but not least, I also attach another paper, where I tried to establish the grafting method as a technique in our lab to study canalization related processes.}, author = {Vasileva, Mina K}, issn = {2663-337X}, pages = {192}, publisher = {Institute of Science and Technology Austria}, title = {{Molecular mechanisms of endomembrane trafficking in Arabidopsis thaliana}}, doi = {10.15479/AT:ISTA:7172}, year = {2019}, } @phdthesis{7186, abstract = {Tissue morphogenesis in developmental or physiological processes is regulated by molecular and mechanical signals. While the molecular signaling cascades are increasingly well described, the mechanical signals affecting tissue shape changes have only recently been studied in greater detail. To gain more insight into the mechanochemical and biophysical basis of an epithelial spreading process (epiboly) in early zebrafish development, we studied cell-cell junction formation and actomyosin network dynamics at the boundary between surface layer epithelial cells (EVL) and the yolk syncytial layer (YSL). During zebrafish epiboly, the cell mass sitting on top of the yolk cell spreads to engulf the yolk cell by the end of gastrulation. It has been previously shown that an actomyosin ring residing within the YSL pulls on the EVL tissue through a cable-constriction and a flow-friction motor, thereby dragging the tissue vegetal wards. Pulling forces are likely transmitted from the YSL actomyosin ring to EVL cells; however, the nature and formation of the junctional structure mediating this process has not been well described so far. Therefore, our main aim was to determine the nature, dynamics and potential function of the EVL-YSL junction during this epithelial tissue spreading. Specifically, we show that the EVL-YSL junction is a mechanosensitive structure, predominantly made of tight junction (TJ) proteins. The process of TJ mechanosensation depends on the retrograde flow of non-junctional, phase-separated Zonula Occludens-1 (ZO-1) protein clusters towards the EVL-YSL boundary. Interestingly, we could demonstrate that ZO-1 is present in a non-junctional pool on the surface of the yolk cell, and ZO-1 undergoes a phase separation process that likely renders the protein responsive to flows. These flows are directed towards the junction and mediate proper tension-dependent recruitment of ZO-1. Upon reaching the EVL-YSL junction ZO-1 gets incorporated into the junctional pool mediated through its direct actin-binding domain. When the non-junctional pool and/or ZO-1 direct actin binding is absent, TJs fail in their proper mechanosensitive responses resulting in slower tissue spreading. We could further demonstrate that depletion of ZO proteins within the YSL results in diminished actomyosin ring formation. This suggests that a mechanochemical feedback loop is at work during zebrafish epiboly: ZO proteins help in proper actomyosin ring formation and actomyosin contractility and flows positively influence ZO-1 junctional recruitment. Finally, such a mesoscale polarization process mediated through the flow of phase-separated protein clusters might have implications for other processes such as immunological synapse formation, C. elegans zygote polarization and wound healing.}, author = {Schwayer, Cornelia}, issn = {2663-337X}, pages = {107}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanosensation of tight junctions depends on ZO-1 phase separation and flow}}, doi = {10.15479/AT:ISTA:7186}, year = {2019}, } @article{7197, abstract = {During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.}, author = {Dos Santos Caldas, Paulo R and Lopez Pelegrin, Maria D and Pearce, Daniel J. G. and Budanur, Nazmi B and Brugués, Jan and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA}}, doi = {10.1038/s41467-019-13702-4}, volume = {10}, year = {2019}, } @article{7406, abstract = {Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.}, author = {Mckenzie, Catherine and Spanova, Miroslava and Johnson, Alexander J and Kainrath, Stephanie and Zheden, Vanessa and Sitte, Harald H. and Janovjak, Harald L}, issn = {0165-0270}, journal = {Journal of Neuroscience Methods}, pages = {114--121}, publisher = {Elsevier}, title = {{Isolation of synaptic vesicles from genetically engineered cultured neurons}}, doi = {10.1016/j.jneumeth.2018.11.018}, volume = {312}, year = {2019}, } @article{5789, abstract = {Tissue morphogenesis is driven by mechanical forces that elicit changes in cell size, shape and motion. The extent by which forces deform tissues critically depends on the rheological properties of the recipient tissue. Yet, whether and how dynamic changes in tissue rheology affect tissue morphogenesis and how they are regulated within the developing organism remain unclear. Here, we show that blastoderm spreading at the onset of zebrafish morphogenesis relies on a rapid, pronounced and spatially patterned tissue fluidization. Blastoderm fluidization is temporally controlled by mitotic cell rounding-dependent cell–cell contact disassembly during the last rounds of cell cleavages. Moreover, fluidization is spatially restricted to the central blastoderm by local activation of non-canonical Wnt signalling within the blastoderm margin, increasing cell cohesion and thereby counteracting the effect of mitotic rounding on contact disassembly. Overall, our results identify a fluidity transition mediated by loss of cell cohesion as a critical regulator of embryo morphogenesis.}, author = {Petridou, Nicoletta and Grigolon, Silvia and Salbreux, Guillaume and Hannezo, Edouard B and Heisenberg, Carl-Philipp J}, issn = {14657392}, journal = {Nature Cell Biology}, pages = {169–178}, publisher = {Nature Publishing Group}, title = {{Fluidization-mediated tissue spreading by mitotic cell rounding and non-canonical Wnt signalling}}, doi = {10.1038/s41556-018-0247-4}, volume = {21}, year = {2019}, } @article{6025, abstract = {Non-canonical Wnt signaling plays a central role for coordinated cell polarization and directed migration in metazoan development. While spatiotemporally restricted activation of non-canonical Wnt-signaling drives cell polarization in epithelial tissues, it remains unclear whether such instructive activity is also critical for directed mesenchymal cell migration. Here, we developed a light-activated version of the non-canonical Wnt receptor Frizzled 7 (Fz7) to analyze how restricted activation of non-canonical Wnt signaling affects directed anterior axial mesendoderm (prechordal plate, ppl) cell migration within the zebrafish gastrula. We found that Fz7 signaling is required for ppl cell protrusion formation and migration and that spatiotemporally restricted ectopic activation is capable of redirecting their migration. Finally, we show that uniform activation of Fz7 signaling in ppl cells fully rescues defective directed cell migration in fz7 mutant embryos. Together, our findings reveal that in contrast to the situation in epithelial cells, non-canonical Wnt signaling functions permissively rather than instructively in directed mesenchymal cell migration during gastrulation.}, author = {Capek, Daniel and Smutny, Michael and Tichy, Alexandra Madelaine and Morri, Maurizio and Janovjak, Harald L and Heisenberg, Carl-Philipp J}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Light-activated Frizzled7 reveals a permissive role of non-canonical wnt signaling in mesendoderm cell migration}}, doi = {10.7554/eLife.42093}, volume = {8}, year = {2019}, } @article{6046, abstract = {Sudden stress often triggers diverse, temporally structured gene expression responses in microbes, but it is largely unknown how variable in time such responses are and if genes respond in the same temporal order in every single cell. Here, we quantified timing variability of individual promoters responding to sublethal antibiotic stress using fluorescent reporters, microfluidics, and time‐lapse microscopy. We identified lower and upper bounds that put definite constraints on timing variability, which varies strongly among promoters and conditions. Timing variability can be interpreted using results from statistical kinetics, which enable us to estimate the number of rate‐limiting molecular steps underlying different responses. We found that just a few critical steps control some responses while others rely on dozens of steps. To probe connections between different stress responses, we then tracked the temporal order and response time correlations of promoter pairs in individual cells. Our results support that, when bacteria are exposed to the antibiotic nitrofurantoin, the ensuing oxidative stress and SOS responses are part of the same causal chain of molecular events. In contrast, under trimethoprim, the acid stress response and the SOS response are part of different chains of events running in parallel. Our approach reveals fundamental constraints on gene expression timing and provides new insights into the molecular events that underlie the timing of stress responses.}, author = {Mitosch, Karin and Rieckh, Georg and Bollenbach, Mark Tobias}, journal = {Molecular systems biology}, number = {2}, publisher = {Embo Press}, title = {{Temporal order and precision of complex stress responses in individual bacteria}}, doi = {10.15252/msb.20188470}, volume = {15}, year = {2019}, } @article{6087, abstract = {Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz−/− follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.}, author = {Xia, Peng and Gütl, Daniel J and Zheden, Vanessa and Heisenberg, Carl-Philipp J}, journal = {Cell}, number = {6}, pages = {1379--1392.e14}, publisher = {Elsevier}, title = {{Lateral inhibition in cell specification mediated by mechanical signals modulating TAZ activity}}, doi = {10.1016/j.cell.2019.01.019}, volume = {176}, year = {2019}, } @phdthesis{6269, abstract = {Clathrin-Mediated Endocytosis (CME) is an aspect of cellular trafficking that is constantly regulated for mediating developmental and physiological responses. The main aim of my thesis is to decipher the basic mechanisms of CME and post-endocytic trafficking in the whole multicellular organ systems of Arabidopsis. The first chapter of my thesis describes the search for new components involved in CME. Tandem affinity purification was conducted using CLC and its interacting partners were identified. Amongst the identified proteins were the Auxilin-likes1 and 2 (Axl1/2), putative uncoating factors, for which we made a full functional analysis. Over-expression of Axl1/2 causes extreme modifications in the dynamics of the machinery proteins and inhibition of endocytosis altogether. However the loss of function of the axl1/2 did not present any cellular or physiological phenotype, meaning Auxilin-likes do not form the major uncoating machinery. The second chapter of my thesis describes the establishment/utilisation of techniques to capture the dynamicity and the complexity of CME and post-endocytic trafficking. We have studied the development of endocytic pits at the PM – specifically, the mode of membrane remodeling during pit development and the role of actin in it, given plant cells possess high turgor pressure. Utilizing the improved z-resolution of TIRF and VAEM techniques, we captured the time-lapse of the endocytic events at the plasma membrane; and using particle detection software, we quantitatively analysed all the endocytic trajectories in an unbiased way to obtain the endocytic rate of the system. This together with the direct analysis of cargo internalisation from the PM provided an estimate on the endocytic potential of the cell. We also developed a methodology for ultrastructural analysis of different populations of Clathrin-Coated Structures (CCSs) in both PM and endomembranes in unroofed protoplasts. Structural analysis, together with the intensity profile of CCSs at the PM show that the mode of CCP development at the PM follows ‘Constant curvature model’; meaning that clathrin polymerisation energy is a major contributing factor of membrane remodeling. In addition, other analyses clearly show that actin is not required for membrane remodeling during invagination or any other step of CCP development, despite the prevalent high turgor pressure. However, actin is essential in orchestrating the post-endocytic trafficking of CCVs facilitating the EE formation. We also observed that the uncoating process post-endocytosis is not immediate; an alternative mechanism of uncoating – Sequential multi-step process – functions in the cell. Finally we also looked at one of the important physiological stimuli modulating the process – hormone, auxin. auxin has been known to influence CME before. We have made a detailed study on the concentration-time based effect of auxin on the machinery proteins, CCP development, and the specificity of cargoes endocytosed. To this end, we saw no general effect of auxin on CME at earlier time points. However, very low concentration of IAA, such as 50nM, accelerates endocytosis of specifically PIN2 through CME. Such a tight regulatory control with high specificity to PIN2 could be essential in modulating its polarity. }, author = {Narasimhan, Madhumitha}, issn = {2663-337X}, pages = {138}, publisher = {Institute of Science and Technology Austria}, title = {{Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants }}, doi = {10.15479/at:ista:th1075}, year = {2019}, } @article{6351, abstract = {A process of restorative patterning in plant roots correctly replaces eliminated cells to heal local injuries despite the absence of cell migration, which underpins wound healing in animals. Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing.}, author = {Marhavá, Petra and Hörmayer, Lukas and Yoshida, Saiko and Marhavy, Peter and Benková, Eva and Friml, Jiří}, issn = {10974172}, journal = {Cell}, number = {4}, pages = {957--969.e13}, publisher = {Elsevier}, title = {{Re-activation of stem cell pathways for pattern restoration in plant wound healing}}, doi = {10.1016/j.cell.2019.04.015}, volume = {177}, year = {2019}, } @phdthesis{6392, abstract = {The regulation of gene expression is one of the most fundamental processes in living systems. In recent years, thanks to advances in sequencing technology and automation, it has become possible to study gene expression quantitatively, genome-wide and in high-throughput. This leads to the possibility of exploring changes in gene expression in the context of many external perturbations and their combinations, and thus of characterising the basic principles governing gene regulation. In this thesis, I present quantitative experimental approaches to studying transcriptional and protein level changes in response to combinatorial drug treatment, as well as a theoretical data-driven approach to analysing thermodynamic principles guiding transcription of protein coding genes. In the first part of this work, I present a novel methodological framework for quantifying gene expression changes in drug combinations, termed isogrowth profiling. External perturbations through small molecule drugs influence the growth rate of the cell, leading to wide-ranging changes in cellular physiology and gene expression. This confounds the gene expression changes specifically elicited by the particular drug. Combinatorial perturbations, owing to the increased stress they exert, influence the growth rate even more strongly and hence suffer the convolution problem to a greater extent when measuring gene expression changes. Isogrowth profiling is a way to experimentally abstract non-specific, growth rate related changes, by performing the measurement using varying ratios of two drugs at such concentrations that the overall inhibition rate is constant. Using a robotic setup for automated high-throughput re-dilution culture of Saccharomyces cerevisiae, the budding yeast, I investigate all pairwise interactions of four small molecule drugs through sequencing RNA along a growth isobole. Through principal component analysis, I demonstrate here that isogrowth profiling can uncover drug-specific as well as drug-interaction-specific gene expression changes. I show that drug-interaction-specific gene expression changes can be used for prediction of higher-order drug interactions. I propose a simplified generalised framework of isogrowth profiling, with few measurements needed for each drug pair, enabling the broad application of isogrowth profiling to high-throughput screening of inhibitors of cellular growth and beyond. Such high-throughput screenings of gene expression changes specific to pairwise drug interactions will be instrumental for predicting the higher-order interactions of the drugs. In the second part of this work, I extend isogrowth profiling to single-cell measurements of gene expression, characterising population heterogeneity in the budding yeast in response to combinatorial drug perturbation while controlling for non-specific growth rate effects. Through flow cytometry of strains with protein products fused to green fluorescent protein, I discover multiple proteins with bi-modally distributed expression levels in the population in response to drug treatment. I characterize more closely the effect of an ionic stressor, lithium chloride, and find that it inhibits the splicing of mRNA, most strongly affecting ribosomal protein transcripts and leading to a bi-stable behaviour of a small ribosomal subunit protein Rps22B. Time-lapse microscopy of a microfluidic culture system revealed that the induced Rps22B heterogeneity leads to preferential survival of Rps22B-low cells after long starvation, but to preferential proliferation of Rps22B-high cells after short starvation. Overall, this suggests that yeast cells might use splicing of ribosomal genes for bet-hedging in fluctuating environments. I give specific examples of how further exploration of cellular heterogeneity in yeast in response to external perturbation has the potential to reveal yet-undiscovered gene regulation circuitry. In the last part of this thesis, a re-analysis of a published sequencing dataset of nascent elongating transcripts is used to characterise the thermodynamic constraints for RNA polymerase II (RNAP) elongation. Population-level data on RNAP position throughout the transcribed genome with single nucleotide resolution are used to infer the sequence specific thermodynamic determinants of RNAP pausing and backtracking. This analysis reveals that the basepairing strength of the eight nucleotide-long RNA:DNA duplex relative to the basepairing strength of the same sequence when in DNA:DNA duplex, and the change in this quantity during RNA polymerase movement, is the key determinant of RNAP pausing. This is true for RNAP pausing while elongating, but also of RNAP pausing while backtracking and of the backtracking length. The quantitative dependence of RNAP pausing on basepairing energetics is used to infer the increase in pausing due to transcriptional mismatches, leading to a hypothesis that pervasive RNA polymerase II pausing is due to basepairing energetics, as an evolutionary cost for increased RNA polymerase II fidelity. This work advances our understanding of the general principles governing gene expression, with the goal of making computational predictions of single-cell gene expression responses to combinatorial perturbations based on the individual perturbations possible. This ability would substantially facilitate the design of drug combination treatments and, in the long term, lead to our increased ability to more generally design targeted manipulations to any biological system. }, author = {Lukacisin, Martin}, isbn = {978-3-99078-001-5}, issn = {2663-337X}, pages = {103}, publisher = {IST Austria}, title = {{Quantitative investigation of gene expression principles through combinatorial drug perturbation and theory}}, doi = {10.15479/AT:ISTA:6392}, year = {2019}, } @phdthesis{6435, abstract = {Social insect colonies tend to have numerous members which function together like a single organism in such harmony that the term ``super-organism'' is often used. In this analogy the reproductive caste is analogous to the primordial germ cells of a metazoan, while the sterile worker caste corresponds to somatic cells. The worker castes, like tissues, are in charge of all functions of a living being, besides reproduction. The establishment of new super-organismal units (i.e. new colonies) is accomplished by the co-dependent castes. The term oftentimes goes beyond a metaphor. We invoke it when we speak about the metabolic rate, thermoregulation, nutrient regulation and gas exchange of a social insect colony. Furthermore, we assert that the super-organism has an immune system, and benefits from ``social immunity''. Social immunity was first summoned by evolutionary biologists to resolve the apparent discrepancy between the expected high frequency of disease outbreak amongst numerous, closely related tightly-interacting hosts, living in stable and microbially-rich environments, against the exceptionally scarce epidemic accounts in natural populations. Social immunity comprises a multi-layer assembly of behaviours which have evolved to effectively keep the pathogenic enemies of a colony at bay. The field of social immunity has drawn interest, as it becomes increasingly urgent to stop the collapse of pollinator species and curb the growth of invasive pests. In the past decade, several mechanisms of social immune responses have been dissected, but many more questions remain open. I present my work in two experimental chapters. In the first, I use invasive garden ants (*Lasius neglectus*) to study how pathogen load and its distribution among nestmates affect the grooming response of the group. Any given group of ants will carry out the same total grooming work, but will direct their grooming effort towards individuals carrying a relatively higher spore load. Contrary to expectation, the highest risk of transmission does not stem from grooming highly contaminated ants, but instead, we suggest that the grooming response likely minimizes spore loss to the environment, reducing contamination from inadvertent pickup from the substrate. The second is a comparative developmental approach. I follow black garden ant queens (*Lasius niger*) and their colonies from mating flight, through hibernation for a year. Colonies which grow fast from the start, have a lower chance of survival through hibernation, and those which survive grow at a lower pace later. This is true for colonies of naive and challenged queens. Early pathogen exposure of the queens changes colony dynamics in an unexpected way: colonies from exposed queens are more likely to grow slowly and recover in numbers only after they survive hibernation. In addition to the two experimental chapters, this thesis includes a co-authored published review on organisational immunity, where we enlist the experimental evidence and theoretical framework on which this hypothesis is built, identify the caveats and underline how the field is ripe to overcome them. In a final chapter, I describe my part in two collaborative efforts, one to develop an image-based tracker, and the second to develop a classifier for ant behaviour.}, author = {Casillas Perez, Barbara E}, issn = {2663-337X}, keywords = {Social Immunity, Sanitary care, Social Insects, Organisational Immunity, Colony development, Multi-target tracking}, pages = {183}, publisher = {Institute of Science and Technology Austria}, title = {{Collective defenses of garden ants against a fungal pathogen}}, doi = {10.15479/AT:ISTA:6435}, year = {2019}, }