@article{7473, abstract = {How structural and functional properties of synapses relate to each other is a fundamental question in neuroscience. Electrophysiology has elucidated mechanisms of synaptic transmission, and electron microscopy (EM) has provided insight into morphological properties of synapses. Here we describe an enhanced method for functional EM (“flash and freeze”), combining optogenetic stimulation with high-pressure freezing. We demonstrate that the improved method can be applied to intact networks in acute brain slices and organotypic slice cultures from mice. As a proof of concept, we probed vesicle pool changes during synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapse. Our findings show overlap of the docked vesicle pool and the functionally defined readily releasable pool and provide evidence of fast endocytosis at this synapse. Functional EM with acute slices and slice cultures has the potential to reveal the structural and functional mechanisms of transmission in intact, genetically perturbed, and disease-affected synapses.}, author = {Borges Merjane, Carolina and Kim, Olena and Jonas, Peter M}, issn = {0896-6273}, journal = {Neuron}, pages = {992--1006}, publisher = {Elsevier}, title = {{Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices}}, doi = {10.1016/j.neuron.2019.12.022}, volume = {105}, 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{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 of the United States of America}, number = {26}, publisher = {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{9160, abstract = {Auxin is a key hormonal regulator, that governs plant growth and development in concert with other hormonal pathways. The unique feature of auxin is its polar, cell-to-cell transport that leads to the formation of local auxin maxima and gradients, which coordinate initiation and patterning of plant organs. The molecular machinery mediating polar auxin transport is one of the important points of interaction with other hormones. Multiple hormonal pathways converge at the regulation of auxin transport and form a regulatory network that integrates various developmental and environmental inputs to steer plant development. In this review, we discuss recent advances in understanding the mechanisms that underlie regulation of polar auxin transport by multiple hormonal pathways. Specifically, we focus on the post-translational mechanisms that contribute to fine-tuning of the abundance and polarity of auxin transporters at the plasma membrane and thereby enable rapid modification of the auxin flow to coordinate plant growth and development.}, author = {Semeradova, Hana and Montesinos López, Juan C and Benková, Eva}, issn = {2590-3462}, journal = {Plant Communications}, number = {3}, publisher = {Elsevier}, title = {{All roads lead to auxin: Post-translational regulation of auxin transport by multiple hormonal pathways}}, doi = {10.1016/j.xplc.2020.100048}, volume = {1}, year = {2020}, } @unpublished{8831, abstract = {Holes in planar Ge have high mobilities, strong spin-orbit interaction and electrically tunable g-factors, and are therefore emerging as a promising candidate for hybrid superconductorsemiconductor devices. This is further motivated by the observation of supercurrent transport in planar Ge Josephson Field effect transistors (JoFETs). A key challenge towards hybrid germanium quantum technology is the design of high quality interfaces and superconducting contacts that are robust against magnetic fields. By combining the assets of Al, which has a long superconducting coherence, and Nb, which has a significant superconducting gap, we form low-disordered JoFETs with large ICRN products that are capable of withstanding high magnetic fields. We furthermore demonstrate the ability of phase-biasing individual JoFETs opening up an avenue to explore topological superconductivity in planar Ge. The persistence of superconductivity in the reported hybrid devices beyond 1.8 T paves the way towards integrating spin qubits and proximity-induced superconductivity on the same chip.}, author = {Aggarwal, Kushagra and Hofmann, Andrea C and Jirovec, Daniel and Prieto Gonzalez, Ivan and Sammak, Amir and Botifoll, Marc and Marti-Sanchez, Sara and Veldhorst, Menno and Arbiol, Jordi and Scappucci, Giordano and Katsaros, Georgios}, booktitle = {arXiv}, title = {{Enhancement of proximity induced superconductivity in planar Germanium}}, doi = {10.48550/arXiv.2012.00322}, year = {2020}, } @article{8532, abstract = {The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.}, author = {Kleindienst, David and Montanaro-Punzengruber, Jacqueline-Claire and Bhandari, Pradeep and Case, Matthew J and Fukazawa, Yugo and Shigemoto, Ryuichi}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {18}, publisher = {MDPI}, title = {{Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses}}, doi = {10.3390/ijms21186737}, volume = {21}, year = {2020}, } @phdthesis{8657, abstract = {Synthesis of proteins – translation – is a fundamental process of life. Quantitative studies anchor translation into the context of bacterial physiology and reveal several mathematical relationships, called “growth laws,” which capture physiological feedbacks between protein synthesis and cell growth. Growth laws describe the dependency of the ribosome abundance as a function of growth rate, which can change depending on the growth conditions. Perturbations of translation reveal that bacteria employ a compensatory strategy in which the reduced translation capability results in increased expression of the translation machinery. Perturbations of translation are achieved in various ways; clinically interesting is the application of translation-targeting antibiotics – translation inhibitors. The antibiotic effects on bacterial physiology are often poorly understood. Bacterial responses to two or more simultaneously applied antibiotics are even more puzzling. The combined antibiotic effect determines the type of drug interaction, which ranges from synergy (the effect is stronger than expected) to antagonism (the effect is weaker) and suppression (one of the drugs loses its potency). In the first part of this work, we systematically measure the pairwise interaction network for translation inhibitors that interfere with different steps in translation. We find that the interactions are surprisingly diverse and tend to be more antagonistic. To explore the underlying mechanisms, we begin with a minimal biophysical model of combined antibiotic action. We base this model on the kinetics of antibiotic uptake and binding together with the physiological response described by the growth laws. The biophysical model explains some drug interactions, but not all; it specifically fails to predict suppression. In the second part of this work, we hypothesize that elusive suppressive drug interactions result from the interplay between ribosomes halted in different stages of translation. To elucidate this putative mechanism of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using in- ducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks partially causes these interactions. We extend this approach by varying two translation bottlenecks simultaneously. This approach reveals the suppression of translocation inhibition by inhibited translation. We rationalize this effect by modeling dense traffic of ribosomes that move on transcripts in a translation factor-mediated manner. This model predicts a dissolution of traffic jams caused by inhibited translocation when the density of ribosome traffic is reduced by lowered initiation. We base this model on the growth laws and quantitative relationships between different translation and growth parameters. In the final part of this work, we describe a set of tools aimed at quantification of physiological and translation parameters. We further develop a simple model that directly connects the abundance of a translation factor with the growth rate, which allows us to extract physiological parameters describing initiation. We demonstrate the development of tools for measuring translation rate. This thesis showcases how a combination of high-throughput growth rate mea- surements, genetics, and modeling can reveal mechanisms of drug interactions. Furthermore, by a gradual transition from combinations of antibiotics to precise genetic interventions, we demonstrated the equivalency between genetic and chemi- cal perturbations of translation. These findings tile the path for quantitative studies of antibiotic combinations and illustrate future approaches towards the quantitative description of translation.}, author = {Kavcic, Bor}, isbn = {978-3-99078-011-4}, issn = {2663-337X}, pages = {271}, publisher = {Institute of Science and Technology Austria}, title = {{Perturbations of protein synthesis: from antibiotics to genetics and physiology}}, doi = {10.15479/AT:ISTA:8657}, year = {2020}, } @article{8569, abstract = {Concerted radial migration of newly born cortical projection neurons, from their birthplace to their final target lamina, is a key step in the assembly of the cerebral cortex. The cellular and molecular mechanisms regulating the specific sequential steps of radial neuronal migration in vivo are however still unclear, let alone the effects and interactions with the extracellular environment. In any in vivo context, cells will always be exposed to a complex extracellular environment consisting of (1) secreted factors acting as potential signaling cues, (2) the extracellular matrix, and (3) other cells providing cell–cell interaction through receptors and/or direct physical stimuli. Most studies so far have described and focused mainly on intrinsic cell-autonomous gene functions in neuronal migration but there is accumulating evidence that non-cell-autonomous-, local-, systemic-, and/or whole tissue-wide effects substantially contribute to the regulation of radial neuronal migration. These non-cell-autonomous effects may differentially affect cortical neuron migration in distinct cellular environments. However, the cellular and molecular natures of such non-cell-autonomous mechanisms are mostly unknown. Furthermore, physical forces due to collective migration and/or community effects (i.e., interactions with surrounding cells) may play important roles in neocortical projection neuron migration. In this concise review, we first outline distinct models of non-cell-autonomous interactions of cortical projection neurons along their radial migration trajectory during development. We then summarize experimental assays and platforms that can be utilized to visualize and potentially probe non-cell-autonomous mechanisms. Lastly, we define key questions to address in the future.}, author = {Hansen, Andi H and Hippenmeyer, Simon}, issn = {2296-634X}, journal = {Frontiers in Cell and Developmental Biology}, number = {9}, publisher = {Frontiers}, title = {{Non-cell-autonomous mechanisms in radial projection neuron migration in the developing cerebral cortex}}, doi = {10.3389/fcell.2020.574382}, volume = {8}, year = {2020}, } @article{8250, abstract = {Antibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we explain these interactions by “translation bottlenecks”: points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further show that growth laws, combined with drug uptake and binding kinetics, enable the direct prediction of a large fraction of observed interactions, yet fail to predict suppression. However, varying two translation bottlenecks simultaneously supports that dense traffic of ribosomes and competition for translation factors account for the previously unexplained suppression. These results highlight the importance of “continuous epistasis” in bacterial physiology.}, author = {Kavcic, Bor and Tkačik, Gašper and Bollenbach, Tobias}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Mechanisms of drug interactions between translation-inhibiting antibiotics}}, doi = {10.1038/s41467-020-17734-z}, volume = {11}, 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}, } @unpublished{7673, abstract = {Combining drugs can improve the efficacy of treatments. However, predicting the effect of drug combinations is still challenging. The combined potency of drugs determines the drug interaction, which is classified as synergistic, additive, antagonistic, or suppressive. While probabilistic, non-mechanistic models exist, there is currently no biophysical model that can predict antibiotic interactions. Here, we present a physiologically relevant model of the combined action of antibiotics that inhibit protein synthesis by targeting the ribosome. This model captures the kinetics of antibiotic binding and transport, and uses bacterial growth laws to predict growth in the presence of antibiotic combinations. We find that this biophysical model can produce all drug interaction types except suppression. We show analytically that antibiotics which cannot bind to the ribosome simultaneously generally act as substitutes for one another, leading to additive drug interactions. Previously proposed null expectations for higher-order drug interactions follow as a limiting case of our model. We further extend the model to include the effects of direct physical or allosteric interactions between individual drugs on the ribosome. Notably, such direct interactions profoundly change the combined drug effect, depending on the kinetic parameters of the drugs used. The model makes additional predictions for the effects of resistance genes on drug interactions and for interactions between ribosome-targeting antibiotics and antibiotics with other targets. These findings enhance our understanding of the interplay between drug action and cell physiology and are a key step toward a general framework for predicting drug interactions.}, author = {Kavcic, Bor and Tkačik, Gašper and Bollenbach, Tobias}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{A minimal biophysical model of combined antibiotic action}}, doi = {10.1101/2020.04.18.047886}, year = {2020}, } @phdthesis{7258, abstract = {Many flows encountered in nature and applications are characterized by a chaotic motion known as turbulence. Turbulent flows generate intense friction with pipe walls and are responsible for considerable amounts of energy losses at world scale. The nature of turbulent friction and techniques aimed at reducing it have been subject of extensive research over the last century, but no definite answer has been found yet. In this thesis we show that in pipes at moderate turbulent Reynolds numbers friction is better described by the power law first introduced by Blasius and not by the Prandtl–von Kármán formula. At higher Reynolds numbers, large scale motions gradually become more important in the flow and can be related to the change in scaling of friction. Next, we present a series of new techniques that can relaminarize turbulence by suppressing a key mechanism that regenerates it at walls, the lift–up effect. In addition, we investigate the process of turbulence decay in several experiments and discuss the drag reduction potential. Finally, we examine the behavior of friction under pulsating conditions inspired by the human heart cycle and we show that under such circumstances turbulent friction can be reduced to produce energy savings.}, author = {Scarselli, Davide}, issn = {2663-337X}, pages = {174}, publisher = {Institute of Science and Technology Austria}, title = {{New approaches to reduce friction in turbulent pipe flow}}, doi = {10.15479/AT:ISTA:7258}, year = {2020}, } @article{7427, abstract = {Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense.}, author = {Tan, Shutang and Abas, Melinda F and Verstraeten, Inge and Glanc, Matous and Molnar, Gergely and Hajny, Jakub and Lasák, Pavel and Petřík, Ivan and Russinova, Eugenia and Petrášek, Jan and Novák, Ondřej and Pospíšil, Jiří and Friml, Jiří}, issn = {09609822}, journal = {Current Biology}, number = {3}, pages = {381--395.e8}, publisher = {Cell Press}, title = {{Salicylic acid targets protein phosphatase 2A to attenuate growth in plants}}, doi = {10.1016/j.cub.2019.11.058}, volume = {30}, year = {2020}, } @article{7500, abstract = {Plant survival depends on vascular tissues, which originate in a self‐organizing manner as strands of cells co‐directionally transporting the plant hormone auxin. The latter phenomenon (also known as auxin canalization) is classically hypothesized to be regulated by auxin itself via the effect of this hormone on the polarity of its own intercellular transport. Correlative observations supported this concept, but molecular insights remain limited. In the current study, we established an experimental system based on the model Arabidopsis thaliana, which exhibits auxin transport channels and formation of vasculature strands in response to local auxin application. Our methodology permits the genetic analysis of auxin canalization under controllable experimental conditions. By utilizing this opportunity, we confirmed the dependence of auxin canalization on a PIN‐dependent auxin transport and nuclear, TIR1/AFB‐mediated auxin signaling. We also show that leaf venation and auxin‐mediated PIN repolarization in the root require TIR1/AFB signaling. Further studies based on this experimental system are likely to yield better understanding of the mechanisms underlying auxin transport polarization in other developmental contexts.}, author = {Mazur, E and Kulik, Ivan and Hajny, Jakub and Friml, Jiří}, issn = {1469-8137}, journal = {New Phytologist}, number = {5}, pages = {1375--1383}, publisher = {Wiley}, title = {{Auxin canalization and vascular tissue formation by TIR1/AFB-mediated auxin signaling in arabidopsis}}, doi = {10.1111/nph.16446}, volume = {226}, year = {2020}, } @phdthesis{8822, abstract = {Self-organization is a hallmark of plant development manifested e.g. by intricate leaf vein patterns, flexible formation of vasculature during organogenesis or its regeneration following wounding. Spontaneously arising channels transporting the phytohormone auxin, created by coordinated polar localizations of PIN-FORMED 1 (PIN1) auxin exporter, provide positional cues for these as well as other plant patterning processes. To find regulators acting downstream of auxin and the TIR1/AFB auxin signaling pathway essential for PIN1 coordinated polarization during auxin canalization, we performed microarray experiments. Besides the known components of general PIN polarity maintenance, such as PID and PIP5K kinases, we identified and characterized a new regulator of auxin canalization, the transcription factor WRKY DNA-BINDING PROTEIN 23 (WRKY23). Next, we designed a subsequent microarray experiment to further uncover other molecular players, downstream of auxin-TIR1/AFB-WRKY23 involved in the regulation of auxin-mediated PIN repolarization. We identified a novel and crucial part of the molecular machinery underlying auxin canalization. The auxin-regulated malectin-type receptor-like kinase CAMEL and the associated leucine-rich repeat receptor-like kinase CANAR target and directly phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated repolarization leading to defects in auxin transport, ultimately to leaf venation and vasculature regeneration defects. Our results describe the CAMEL-CANAR receptor complex, which is required for auxin feed-back on its own transport and thus for coordinated tissue polarization during auxin canalization.}, author = {Hajny, Jakub}, issn = {2663-337X}, pages = {249}, publisher = {Institute of Science and Technology Austria}, title = {{Identification and characterization of the molecular machinery of auxin-dependent canalization during vasculature formation and regeneration}}, doi = {10.15479/AT:ISTA:8822}, year = {2020}, } @phdthesis{8983, abstract = {Metabolic adaptation is a critical feature of migrating cells. It tunes the metabolic programs of migrating cells to allow them to efficiently exert their crucial roles in development, inflammatory responses and tumor metastasis. Cell migration through physically challenging contexts requires energy. However, how the metabolic reprogramming that underlies in vivo cell invasion is controlled is still unanswered. In my PhD project, I identify a novel conserved metabolic shift in Drosophila melanogaster immune cells that by modulating their bioenergetic potential controls developmentally programmed tissue invasion. We show that this regulation requires a novel conserved nuclear protein, named Atossa. Atossa enhances the transcription of a set of proteins, including an RNA helicase Porthos and two metabolic enzymes, each of which increases the tissue invasion of leading Drosophila macrophages and can rescue the atossa mutant phenotype. Porthos selectively regulates the translational efficiency of a subset of mRNAs containing a 5’-UTR cis-regulatory TOP-like sequence. These 5’TOPL mRNA targets encode mitochondrial-related proteins, including subunits of mitochondrial oxidative phosphorylation (OXPHOS) components III and V and other metabolic-related proteins. Porthos powers up mitochondrial OXPHOS to engender a sufficient ATP supply, which is required for tissue invasion of leading macrophages. Atossa’s two vertebrate orthologs rescue the invasion defect. In my PhD project, I elucidate that Atossa displays a conserved developmental metabolic control to modulate metabolic capacities and the cellular energy state, through altered transcription and translation, to aid the tissue infiltration of leading cells into energy demanding barriers.}, author = {Emtenani, Shamsi}, issn = {2663-337X}, pages = {141}, publisher = {Institute of Science and Technology Austria}, title = {{Metabolic regulation of Drosophila macrophage tissue invasion}}, doi = {10.15479/AT:ISTA:8983}, year = {2020}, } @unpublished{8557, abstract = {The infiltration of immune cells into tissues underlies the establishment of tissue resident macrophages, and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio which are themselves required for invasion. Cortical F-actin levels are critical as expressing a dominant active form of Diaphanous, a actin polymerizing Formin, can rescue the Dfos Dominant Negative macrophage invasion defect. In vivo imaging shows that Dfos is required to enhance the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the mechanical properties of the macrophage nucleus from affecting tissue entry. We thus identify tuning the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.}, author = {Belyaeva, Vera and Wachner, Stephanie and Gridchyn, Igor and Linder, Markus and Emtenani, Shamsi and György, Attila and Sibilia, Maria and Siekhaus, Daria E}, booktitle = {bioRxiv}, title = {{Cortical actin properties controlled by Drosophila Fos aid macrophage infiltration against surrounding tissue resistance}}, doi = {10.1101/2020.09.18.301481}, year = {2020}, } @phdthesis{8340, abstract = {Mitochondria are sites of oxidative phosphorylation in eukaryotic cells. Oxidative phosphorylation operates by a chemiosmotic mechanism made possible by redox-driven proton pumping machines which establish a proton motive force across the inner mitochondrial membrane. This electrochemical proton gradient is used to drive ATP synthesis, which powers the majority of cellular processes such as protein synthesis, locomotion and signalling. In this thesis I investigate the structures and molecular mechanisms of two inner mitochondrial proton pumping enzymes, respiratory complex I and transhydrogenase. I present the first high-resolution structure of the full transhydrogenase from any species, and a significantly improved structure of complex I. Improving the resolution from 3.3 Å available previously to up to 2.3 Å in this thesis allowed us to model bound water molecules, crucial in the proton pumping mechanism. For both enzymes, up to five cryo-EM datasets with different substrates and inhibitors bound were solved to delineate the catalytic cycle and understand the proton pumping mechanism. In transhydrogenase, the proton channel is gated by reversible detachment of the NADP(H)-binding domain which opens the proton channel to the opposite sites of the membrane. In complex I, the proton channels are gated by reversible protonation of key glutamate and lysine residues and breaking of the water wire connecting the proton pumps with the quinone reduction site. The tight coupling between the redox and the proton pumping reactions in transhydrogenase is achieved by controlling the NADP(H) exchange which can only happen when the NADP(H)-binding domain interacts with the membrane domain. In complex I, coupling is achieved by cycling of the whole complex between the closed state, in which quinone can get reduced, and the open state, in which NADH can induce quinol ejection from the binding pocket. On the basis of these results I propose detailed mechanisms for catalytic cycles of transhydrogenase and complex I that are consistent with a large amount of previous work. In both enzymes, conformational and electrostatic mechanisms contribute to the overall catalytic process. Results presented here could be used for better understanding of the human pathologies arising from deficiencies of complex I or transhydrogenase and could be used to develop novel therapies.}, author = {Kampjut, Domen}, isbn = {978-3-99078-008-4}, issn = {2663-337X}, pages = {242}, publisher = {Institute of Science and Technology Austria}, title = {{Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes}}, doi = {10.15479/AT:ISTA:8340}, year = {2020}, } @article{7652, abstract = {Organisms cope with change by taking advantage of transcriptional regulators. However, when faced with rare environments, the evolution of transcriptional regulators and their promoters may be too slow. Here, we investigate whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. Using real-time monitoring of gene-copy-number mutations in Escherichia coli, we show that gene duplications and amplifications enable adaptation to fluctuating environments by rapidly generating copy-number and, therefore, expression-level polymorphisms. This amplification-mediated gene expression tuning (AMGET) occurs on timescales that are similar to canonical gene regulation and can respond to rapid environmental changes. Mathematical modelling shows that amplifications also tune gene expression in stochastic environments in which transcription-factor-based schemes are hard to evolve or maintain. The fleeting nature of gene amplifications gives rise to a generic population-level mechanism that relies on genetic heterogeneity to rapidly tune the expression of any gene, without leaving any genomic signature.}, author = {Tomanek, Isabella and Grah, Rok and Lagator, M. and Andersson, A. M. C. and Bollback, Jonathan P and Tkačik, Gašper and Guet, Calin C}, issn = {2397-334X}, journal = {Nature Ecology & Evolution}, number = {4}, pages = {612--625}, publisher = {Springer Nature}, title = {{Gene amplification as a form of population-level gene expression regulation}}, doi = {10.1038/s41559-020-1132-7}, volume = {4}, year = {2020}, } @phdthesis{8653, abstract = {Mutations are the raw material of evolution and come in many different flavors. Point mutations change a single letter in the DNA sequence, while copy number mutations like duplications or deletions add or remove many letters of the DNA sequence simultaneously. Each type of mutation exhibits specific properties like its rate of formation and reversal. Gene expression is a fundamental phenotype that can be altered by both, point and copy number mutations. The following thesis is concerned with the dynamics of gene expression evolution and how it is affected by the properties exhibited by point and copy number mutations. Specifically, we are considering i) copy number mutations during adaptation to fluctuating environments and ii) the interaction of copy number and point mutations during adaptation to constant environments.  }, author = {Tomanek, Isabella}, issn = {2663-337X}, keywords = {duplication, amplification, promoter, CNV, AMGET, experimental evolution, Escherichia coli}, pages = {117}, publisher = {Institute of Science and Technology Austria}, title = {{The evolution of gene expression by copy number and point mutations}}, doi = {10.15479/AT:ISTA:8653}, year = {2020}, }