@article{10223, abstract = {Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.}, author = {Li, Lanxin and Verstraeten, Inge and Roosjen, Mark and Takahashi, Koji and Rodriguez Solovey, Lesia and Merrin, Jack and Chen, Jian and Shabala, Lana and Smet, Wouter and Ren, Hong and Vanneste, Steffen and Shabala, Sergey and De Rybel, Bert and Weijers, Dolf and Kinoshita, Toshinori and Gray, William M. and Friml, Jiří}, issn = {14764687}, journal = {Nature}, keywords = {Multidisciplinary}, number = {7884}, pages = {273--277}, publisher = {Springer Nature}, title = {{Cell surface and intracellular auxin signalling for H+ fluxes in root growth}}, doi = {10.1038/s41586-021-04037-6}, volume = {599}, year = {2021}, } @inbook{10268, abstract = {The analysis of dynamic cellular processes such as plant cytokinesis stands and falls with live-cell time-lapse confocal imaging. Conventional approaches to time-lapse imaging of cell division in Arabidopsis root tips are tedious and have low throughput. Here, we describe a protocol for long-term time-lapse simultaneous imaging of multiple root tips on a vertical-stage confocal microscope with automated root tracking. We also provide modifications of the basic protocol to implement this imaging method in the analysis of genetic, pharmacological or laser ablation wounding-mediated experimental manipulations. Our method dramatically improves the efficiency of cell division time-lapse imaging by increasing the throughput, while reducing the person-hour requirements of such experiments.}, author = {Hörmayer, Lukas and Friml, Jiří and Glanc, Matous}, booktitle = {Plant Cell Division}, isbn = {978-1-0716-1743-4}, issn = {1940-6029}, pages = {105--114}, publisher = {Humana Press}, title = {{Automated time-lapse imaging and manipulation of cell divisions in Arabidopsis roots by vertical-stage confocal microscopy}}, doi = {10.1007/978-1-0716-1744-1_6}, volume = {2382}, year = {2021}, } @article{10290, abstract = {A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.}, author = {Dimchev, Georgi A and Amiri, Behnam and Fäßler, Florian and Falcke, Martin and Schur, Florian KM}, issn = {1047-8477}, journal = {Journal of Structural Biology}, keywords = {Structural Biology}, number = {4}, publisher = {Elsevier }, title = {{Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data}}, doi = {10.1016/j.jsb.2021.107808}, volume = {213}, year = {2021}, } @phdthesis{10303, abstract = {Nitrogen is an essential macronutrient determining plant growth, development and affecting agricultural productivity. Root, as a hub that perceives and integrates local and systemic signals on the plant’s external and endogenous nitrogen resources, communicates with other plant organs to consolidate their physiology and development in accordance with actual nitrogen balance. Over the last years, numerous studies demonstrated that these comprehensive developmental adaptations rely on the interaction between pathways controlling nitrogen homeostasis and hormonal networks acting globally in the plant body. However, molecular insights into how the information about the nitrogen status is translated through hormonal pathways into specific developmental output are lacking. In my work, I addressed so far poorly understood mechanisms underlying root-to-shoot communication that lead to a rapid re-adjustment of shoot growth and development after nitrate provision. Applying a combination of molecular, cell, and developmental biology approaches, genetics and grafting experiments as well as hormonal analytics, I identified and characterized an unknown molecular framework orchestrating shoot development with a root nitrate sensory system. }, author = {Abualia, Rashed}, issn = {2663-337X}, pages = {139}, publisher = {Institute of Science and Technology Austria}, title = {{Role of hormones in nitrate regulated growth}}, doi = {10.15479/at:ista:10303}, year = {2021}, } @phdthesis{10307, abstract = {Bacteria-host interactions represent a continuous trade-off between benefit and risk. Thus, the host immune response is faced with a non-trivial problem – accommodate beneficial commensals and remove harmful pathogens. This is especially difficult as molecular patterns, such as lipopolysaccharide or specific surface organelles such as pili, are conserved in both, commensal and pathogenic bacteria. Type 1 pili, tightly regulated by phase variation, are considered an important virulence factor of pathogenic bacteria as they facilitate invasion into host cells. While invasion represents a de facto passive mechanism for pathogens to escape the host immune response, we demonstrate a fundamental role of type 1 pili as active modulators of the innate and adaptive immune response.}, author = {Tomasek, Kathrin}, issn = {2663-337X}, pages = {73}, publisher = {Institute of Science and Technology Austria}, title = {{Pathogenic Escherichia coli hijack the host immune response}}, doi = {10.15479/at:ista:10307}, year = {2021}, } @unpublished{10316, abstract = {A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease.}, author = {Tomasek, Kathrin and Leithner, Alexander F and Glatzová, Ivana and Lukesch, Michael S. and Guet, Calin C and Sixt, Michael K}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14}}, doi = {10.1101/2021.10.18.464770}, year = {2021}, } @article{10321, abstract = {Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice. MADM enables concomitant fluorescent cell labeling and introduction of a mutation of a gene of interest with single-cell resolution. This protocol highlights major steps for the generation of genetic mosaic tissue and the isolation and processing of respective tissues for downstream histological analysis. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).}, author = {Amberg, Nicole and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {4}, publisher = {Cell Press}, title = {{Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers}}, doi = {10.1016/j.xpro.2021.100939}, volume = {2}, year = {2021}, } @article{10565, abstract = {Enzymatic digestion of the extracellular matrix with chondroitinase-ABC reinstates juvenile-like plasticity in the adult cortex as it also disassembles the perineuronal nets (PNNs). The disadvantage of the enzyme is that it must be applied intracerebrally and it degrades the ECM for several weeks. Here, we provide two minimally invasive and transient protocols for microglia-enabled PNN disassembly in mouse cortex: repeated treatment with ketamine-xylazine-acepromazine (KXA) anesthesia and 60-Hz light entrainment. We also discuss how to analyze PNNs within microglial endosomes-lysosomes. For complete details on the use and execution of this protocol, please refer to Venturino et al. (2021).}, author = {Venturino, Alessandro and Siegert, Sandra}, issn = {2666-1667}, journal = {STAR Protocols}, number = {4}, publisher = {Elsevier ; Cell Press}, title = {{Minimally invasive protocols and quantification for microglia-mediated perineuronal net disassembly in mouse brain}}, doi = {10.1016/j.xpro.2021.101012}, volume = {2}, year = {2021}, } @article{10606, abstract = {Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).}, author = {Godard, Benoit G and Dumollard, Remi and Heisenberg, Carl-Philipp J and Mcdougall, Alex}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Combined effect of cell geometry and polarity domains determines the orientation of unequal division}}, doi = {10.7554/eLife.75639}, volume = {10}, year = {2021}, } @article{10655, abstract = {Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency. }, author = {Maes, Margaret E and Wögenstein, Gabriele M. and Colombo, Gloria and Casado Polanco, Raquel and Siegert, Sandra}, issn = {2329-0501}, journal = {Molecular Therapy - Methods and Clinical Development}, pages = {210--224}, publisher = {Elsevier}, title = {{Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment}}, doi = {10.1016/j.omtm.2021.09.006}, volume = {23}, year = {2021}, } @article{9603, abstract = {Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.}, author = {Contreras, Ximena and Amberg, Nicole and Davaatseren, Amarbayasgalan and Hansen, Andi H and Sonntag, Johanna and Andersen, Lill and Bernthaler, Tina and Streicher, Carmen and Heger, Anna-Magdalena and Johnson, Randy L. and Schwarz, Lindsay A. and Luo, Liqun and Rülicke, Thomas and Hippenmeyer, Simon}, issn = {22111247}, journal = {Cell Reports}, number = {12}, publisher = {Cell Press}, title = {{A genome-wide library of MADM mice for single-cell genetic mosaic analysis}}, doi = {10.1016/j.celrep.2021.109274}, volume = {35}, year = {2021}, } @phdthesis{9623, abstract = {Cytoplasmic reorganizations are essential for morphogenesis. In large cells like oocytes, these reorganizations become crucial in patterning the oocyte for later stages of embryonic development. Ascidians oocytes reorganize their cytoplasm (ooplasm) in a spectacular manner. Ooplasmic reorganization is initiated at fertilization with the contraction of the actomyosin cortex along the animal-vegetal axis of the oocyte, driving the accumulation of cortical endoplasmic reticulum (cER), maternal mRNAs associated to it and a mitochondria-rich subcortical layer – the myoplasm – in a region of the vegetal pole termed contraction pole (CP). Here we have used the species Phallusia mammillata to investigate the changes in cell shape that accompany these reorganizations and the mechanochemical mechanisms underlining CP formation. We report that the length of the animal-vegetal (AV) axis oscillates upon fertilization: it first undergoes a cycle of fast elongation-lengthening followed by a slow expansion of mainly the vegetal pole (VP) of the cell. We show that the fast oscillation corresponds to a dynamic polarization of the actin cortex as a result of a fertilization-induced increase in cortical tension in the oocyte that triggers a rupture of the cortex at the animal pole and the establishment of vegetal-directed cortical flows. These flows are responsible for the vegetal accumulation of actin causing the VP to flatten. We find that the slow expansion of the VP, leading to CP formation, correlates with a relaxation of the vegetal cortex and that the myoplasm plays a role in the expansion. We show that the myoplasm is a solid-like layer that buckles under compression forces arising from the contracting actin cortex at the VP. Straightening of the myoplasm when actin flows stops, facilitates the expansion of the VP and the CP. Altogether, our results present a previously unrecognized role for the myoplasm in ascidian ooplasmic segregation. }, author = {Caballero Mancebo, Silvia}, isbn = {978-3-99078-012-1}, issn = {2663-337X}, pages = {111}, publisher = {Institute of Science and Technology Austria}, title = {{Fertilization-induced deformations are controlled by the actin cortex and a mitochondria-rich subcortical layer in ascidian oocytes}}, doi = {10.15479/at:ista:9623}, year = {2021}, } @article{9642, abstract = {Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.}, author = {Venturino, Alessandro and Schulz, Rouven and De Jesús-Cortés, Héctor and Maes, Margaret E and Nagy, Balint and Reilly-Andújar, Francis and Colombo, Gloria and Cubero, Ryan J and Schoot Uiterkamp, Florianne E and Bear, Mark F. and Siegert, Sandra}, issn = {22111247}, journal = {Cell Reports}, number = {1}, publisher = {Elsevier}, title = {{Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain}}, doi = {10.1016/j.celrep.2021.109313}, volume = {36}, year = {2021}, } @article{9887, abstract = {Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.}, author = {Johnson, Alexander J and Dahhan, Dana A and Gnyliukh, Nataliia and Kaufmann, Walter and Zheden, Vanessa and Costanzo, Tommaso and Mahou, Pierre and Hrtyan, Mónika and Wang, Jie and Aguilera Servin, Juan L and van Damme, Daniël and Beaurepaire, Emmanuel and Loose, Martin and Bednarek, Sebastian Y and Friml, Jiří}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {51}, publisher = {National Academy of Sciences}, title = {{The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis}}, doi = {10.1073/pnas.2113046118}, volume = {118}, year = {2021}, } @article{9907, abstract = {DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane. }, author = {Labajová, Naďa and Baranova, Natalia S. and Jurásek, Miroslav and Vácha, Robert and Loose, Martin and Barák, Imrich}, issn = {14220067}, journal = {International Journal of Molecular Sciences}, number = {15}, publisher = {MDPI}, title = {{Cardiolipin-containing lipid membranes attract the bacterial cell division protein diviva}}, doi = {10.3390/ijms22158350}, volume = {22}, year = {2021}, } @phdthesis{9992, abstract = {Blood – this is what animals use to heal wounds fast and efficient. Plants do not have blood circulation and their cells cannot move. However, plants have evolved remarkable capacities to regenerate tissues and organs preventing further damage. In my PhD research, I studied the wound healing in the Arabidopsis root. I used a UV laser to ablate single cells in the root tip and observed the consequent wound healing. Interestingly, the inner adjacent cells induced a division plane switch and subsequently adopted the cell type of the killed cell to replace it. We termed this form of wound healing “restorative divisions”. This initial observation triggered the questions of my PhD studies: How and why do cells orient their division planes, how do they feel the wound and why does this happen only in inner adjacent cells. For answering these questions, I used a quite simple experimental setup: 5 day - old seedlings were stained with propidium iodide to visualize cell walls and dead cells; ablation was carried out using a special laser cutter and a confocal microscope. Adaptation of the novel vertical microscope system made it possible to observe wounds in real time. This revealed that restorative divisions occur at increased frequency compared to normal divisions. Additionally, the major plant hormone auxin accumulates in wound adjacent cells and drives the expression of the wound-stress responsive transcription factor ERF115. Using this as a marker gene for wound responses, we found that an important part of wound signalling is the sensing of the collapse of the ablated cell. The collapse causes a radical pressure drop, which results in strong tissue deformations. These deformations manifest in an invasion of the now free spot specifically by the inner adjacent cells within seconds, probably because of higher pressure of the inner tissues. Long-term imaging revealed that those deformed cells continuously expand towards the wound hole and that this is crucial for the restorative division. These wound-expanding cells exhibit an abnormal, biphasic polarity of microtubule arrays before the division. Experiments inhibiting cell expansion suggest that it is the biphasic stretching that induces those MT arrays. Adapting the micromanipulator aspiration system from animal scientists at our institute confirmed the hypothesis that stretching influences microtubule stability. In conclusion, this shows that microtubules react to tissue deformation and this facilitates the observed division plane switch. This puts mechanical cues and tensions at the most prominent position for explaining the growth and wound healing properties of plants. Hence, it shines light onto the importance of understanding mechanical signal transduction. }, author = {Hörmayer, Lukas}, issn = {2663-337X}, pages = {168}, publisher = {Institute of Science and Technology Austria}, title = {{Wound healing in the Arabidopsis root meristem}}, doi = {10.15479/at:ista:9992}, year = {2021}, } @article{8336, abstract = {Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.}, author = {Kubiasova, Karolina and Montesinos López, Juan C and Šamajová, Olga and Nisler, Jaroslav and Mik, Václav and Semeradova, Hana and Plíhalová, Lucie and Novák, Ondřej and Marhavý, Peter and Cavallari, Nicola and Zalabák, David and Berka, Karel and Doležal, Karel and Galuszka, Petr and Šamaj, Jozef and Strnad, Miroslav and Benková, Eva and Plíhal, Ondřej and Spíchal, Lukáš}, issn = {20411723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Cytokinin fluoroprobe reveals multiple sites of cytokinin perception at plasma membrane and endoplasmic reticulum}}, doi = {10.1038/s41467-020-17949-0}, volume = {11}, year = {2020}, } @article{8337, abstract = {Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.}, author = {Antoniadi, Ioanna and Novák, Ondřej and Gelová, Zuzana and Johnson, Alexander J and Plíhal, Ondřej and Simerský, Radim and Mik, Václav and Vain, Thomas and Mateo-Bonmatí, Eduardo and Karady, Michal and Pernisová, Markéta and Plačková, Lenka and Opassathian, Korawit and Hejátko, Jan and Robert, Stéphanie and Friml, Jiří and Doležal, Karel and Ljung, Karin and Turnbull, Colin}, issn = {20411723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Cell-surface receptors enable perception of extracellular cytokinins}}, doi = {10.1038/s41467-020-17700-9}, volume = {11}, year = {2020}, } @phdthesis{8341, abstract = {One of the most striking hallmarks of the eukaryotic cell is the presence of intracellular vesicles and organelles. Each of these membrane-enclosed compartments has a distinct composition of lipids and proteins, which is essential for accurate membrane traffic and homeostasis. Interestingly, their biochemical identities are achieved with the help of small GTPases of the Rab family, which cycle between GDP- and GTP-bound forms on the selected membrane surface. While this activity switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate decisive signal propagation in space and time is unclear. In my PhD thesis, I present in vitro reconstitution experiments with theoretical modeling to systematically study a minimal Rab5 activation network from bottom-up. We find that positive feedback based on known molecular interactions gives rise to bistable GTPase activity switching on system’s scale. Furthermore, we determine that collective transition near the critical point is intrinsically stochastic and provide evidence that the inactive Rab5 abundance on the membrane can shape the network response. Finally, we demonstrate that collective switching can spread on the lipid bilayer as a traveling activation wave, representing a possible emergent activity pattern in endosomal maturation. Together, our findings reveal new insights into the self-organization properties of signaling networks away from chemical equilibrium. Our work highlights the importance of systematic characterization of biochemical systems in well-defined physiological conditions. This way, we were able to answer long-standing open questions in the field and close the gap between regulatory processes on a molecular scale and emergent responses on system’s level.}, author = {Bezeljak, Urban}, issn = {2663-337X}, pages = {215}, publisher = {Institute of Science and Technology Austria}, title = {{In vitro reconstitution of a Rab activation switch}}, doi = {10.15479/AT:ISTA:8341}, year = {2020}, } @phdthesis{8350, abstract = {Cytoplasm is a gel-like crowded environment composed of tens of thousands of macromolecules, organelles, cytoskeletal networks and cytosol. The structure of the cytoplasm is thought to be highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules is very restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the jammed nature of the cytoplasm at the microscopic scale, large-scale reorganization of cytoplasm is essential for important cellular functions, such as nuclear positioning and cell division. How such mesoscale reorganization of the cytoplasm is achieved, especially for very large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, has only begun to be understood. In this thesis, I focus on the recent advances in elucidating the molecular, cellular and biophysical principles underlying cytoplasmic organization across different scales, structures and species. First, I outline which of these principles have been identified by reductionist approaches, such as in vitro reconstitution assays, where boundary conditions and components can be modulated at ease. I then describe how the theoretical and experimental framework established in these reduced systems have been applied to their more complex in vivo counterparts, in particular oocytes and embryonic syncytial structures, and discuss how such complex biological systems can initiate symmetry breaking and establish patterning. Specifically, I examine an example of large-scale reorganizations taking place in zebrafish embryos, where extensive cytoplasmic streaming leads to the segregation of cytoplasm from yolk granules along the animal-vegetal axis of the embryo. Using biophysical experimentation and theory, I investigate the forces underlying this process, to 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 embryo. This wave functions in segregation by both pulling cytoplasm animally and pushing yolk granules vegetally. Cytoplasm pulling is mediated by bulk actin network flows exerting friction forces on the cytoplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. This study defines a novel role of bulk actin polymerization waves in embryo polarization via cytoplasmic segregation. Lastly, I describe the cytoplasmic reorganizations taking place during zebrafish oocyte maturation, where the initial segregation of the cytoplasm and yolk granules occurs. Here, I demonstrate a previously uncharacterized wave of microtubule aster formation, traveling the oocyte along the animal-vegetal axis. Further research is required to determine the role of such microtubule structures in cytoplasmic reorganizations therein. Collectively, these studies provide further evidence for the coupling between cell cytoskeleton and cell cycle machinery, which can underlie a core self-organizing mechanism for orchestrating large-scale reorganizations in a cell-cycle-tunable manner, where the modulations of the force-generating machinery and cytoplasmic mechanics can be harbored to fulfill cellular functions.}, author = {Shamipour, Shayan}, issn = {2663-337X}, pages = {107}, publisher = {Institute of Science and Technology Austria}, title = {{Bulk actin dynamics drive phase segregation in zebrafish oocytes }}, doi = {10.15479/AT:ISTA:8350}, year = {2020}, }