@article{11341, abstract = {Intragenic regions that are removed during maturation of the RNA transcript—introns—are universally present in the nuclear genomes of eukaryotes1. The budding yeast, an otherwise intron-poor species, preserves two sets of ribosomal protein genes that differ primarily in their introns2,3. Although studies have shed light on the role of ribosomal protein introns under stress and starvation4,5,6, understanding the contribution of introns to ribosome regulation remains challenging. Here, by combining isogrowth profiling7 with single-cell protein measurements8, we show that introns can mediate inducible phenotypic heterogeneity that confers a clear fitness advantage. Osmotic stress leads to bimodal expression of the small ribosomal subunit protein Rps22B, which is mediated by an intron in the 5′ untranslated region of its transcript. The two resulting yeast subpopulations differ in their ability to cope with starvation. Low levels of Rps22B protein result in prolonged survival under sustained starvation, whereas high levels of Rps22B enable cells to grow faster after transient starvation. Furthermore, yeasts growing at high concentrations of sugar, similar to those in ripe grapes, exhibit bimodal expression of Rps22B when approaching the stationary phase. Differential intron-mediated regulation of ribosomal protein genes thus provides a way to diversify the population when starvation threatens in natural environments. Our findings reveal a role for introns in inducing phenotypic heterogeneity in changing environments, and suggest that duplicated ribosomal protein genes in yeast contribute to resolving the evolutionary conflict between precise expression control and environmental responsiveness9.}, author = {Lukacisin, Martin and Espinosa-Cantú, Adriana and Bollenbach, Mark Tobias}, issn = {1476-4687}, journal = {Nature}, pages = {113--118}, publisher = {Springer Nature}, title = {{Intron-mediated induction of phenotypic heterogeneity}}, doi = {10.1038/s41586-022-04633-0}, volume = {605}, year = {2022}, } @article{9794, abstract = {Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion.}, author = {Assen, Frank P and Abe, Jun and Hons, Miroslav and Hauschild, Robert and Shamipour, Shayan and Kaufmann, Walter and Costanzo, Tommaso and Krens, Gabriel and Brown, Markus and Ludewig, Burkhard and Hippenmeyer, Simon and Heisenberg, Carl-Philipp J and Weninger, Wolfgang and Hannezo, Edouard B and Luther, Sanjiv A. and Stein, Jens V. and Sixt, Michael K}, issn = {1529-2916}, journal = {Nature Immunology}, pages = {1246--1255}, publisher = {Springer Nature}, title = {{Multitier mechanics control stromal adaptations in swelling lymph nodes}}, doi = {10.1038/s41590-022-01257-4}, volume = {23}, year = {2022}, } @article{10766, 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 grow [J.-L. Maître et al., Science 338, 253–256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell–cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell–cell contact size is limited by tension-stabilizing E-cadherin–actin complexes at the contact.}, author = {Slovakova, Jana and Sikora, Mateusz K and Arslan, Feyza N and Caballero Mancebo, Silvia and Krens, Gabriel and Kaufmann, Walter and Merrin, Jack and Heisenberg, Carl-Philipp J}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {8}, publisher = {National Academy of Sciences}, title = {{Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells}}, doi = {10.1073/pnas.2122030119}, volume = {119}, year = {2022}, } @article{10826, abstract = {Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. We probe such mechanisms by performing a forward genetic screen for mutants with enhanced O2 perception in Caenorhabditis elegans. Multiple mutants exhibiting increased O2 responsiveness concomitantly show defects in other sensory responses. One mutant, qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+ responses in the ADL pheromone-sensing neurons. At the same time, ADL responsiveness to pre-synaptic input from O2-sensing neurons is heightened in qui-1, and other sensory defective mutants, resulting in enhanced neurosecretion although not increased Ca2+ responses. Expressing qui-1 selectively in ADL rescues both the qui-1 ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling ADL neurons in qui-1 mutants highlights extensive changes in gene expression, notably of many neuropeptide receptors. We show that elevated ADL expression of the conserved neuropeptide receptor NPR-22 is necessary for enhanced ADL neurosecretion in qui-1 mutants, and is sufficient to confer increased ADL neurosecretion in control animals. Sensory loss can thus confer cross-modal plasticity by changing the peptidergic connectome.}, author = {Valperga, Giulio and De Bono, Mario}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans}}, doi = {10.7554/eLife.68040}, volume = {11}, year = {2022}, } @misc{10934, abstract = {FtsA is crucial for assembly of the E. coli divisome, as it dynamically links cytoplasmic FtsZ filaments with transmembrane cell division proteins. FtsA allegedly initiates cell division by switching from an inactive polymeric to an active monomeric confirmation, which recruits downstream proteins and stabilizes FtsZ filaments. Here, we use biochemical reconstitution experiments combined with quantitative fluorescence microscopy to study divisome activation in vitro. We compare wildtype-FtsA with FtsA-R286W, a constantly active gain-of-function mutant and find that R286W outperforms the wildtype protein in replicating FtsZ treadmilling dynamics, stabilizing FtsZ filaments and recruiting FtsN. We attribute these differences to a faster membrane exchange of FtsA-R286W and its higher packing density below FtsZ filaments. Using FRET microscopy, we find that FtsN binding does not compete with, but promotes FtsA self-interaction. Our findings suggest a model where FtsA always forms dynamic polymers on the membrane, which re-organize during assembly and activation of the divisome. }, author = {Radler, Philipp}, keywords = {Bacterial cell division, in vitro reconstitution, FtsZ, FtsN, FtsA}, publisher = {Institute of Science and Technology Austria}, title = {{In vitro reconstitution of Escherichia coli divisome activation}}, doi = {10.15479/AT:ISTA:10934}, year = {2022}, } @article{11373, abstract = {The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.}, author = {Radler, Philipp and Baranova, Natalia S. and Dos Santos Caldas, Paulo R and Sommer, Christoph M and Lopez Pelegrin, Maria D and Michalik, David and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry}, publisher = {Springer Nature}, title = {{In vitro reconstitution of Escherichia coli divisome activation}}, doi = {10.1038/s41467-022-30301-y}, volume = {13}, year = {2022}, } @phdthesis{11879, abstract = {As the overall global mean surface temperature is increasing due to climate change, plant adaptation to those stressful conditions is of utmost importance for their survival. Plants are sessile organisms, thus to compensate for their lack of mobility, they evolved a variety of mechanisms enabling them to flexibly adjust their physiological, growth and developmental processes to fluctuating temperatures and to survive in harsh environments. While these unique adaptation abilities provide an important evolutionary advantage, overall modulation of plant growth and developmental program due to non-optimal temperature negatively affects biomass production, crop productivity or sensitivity to pathogens. Thus, understanding molecular processes underlying plant adaptation to increased temperature can provide important resources for breeding strategies to ensure sufficient agricultural food production. An increase in ambient temperature by a few degrees leads to profound changes in organ growth including enhanced hypocotyl elongation, expansion of petioles, hyponastic growth of leaves and cotyledons, collectively named thermomorphogenesis (Casal & Balasubramanian, 2019). Auxin, one of the best-studied growth hormones, plays an essential role in this process by direct activation of transcriptional and non-transcriptional processes resulting in elongation growth (Majda & Robert, 2018).To modulate hypocotyl growth in response to high ambient temperature (hAT), auxin needs to be redistributed accordingly. PINs, auxin efflux transporters, are key components of the polar auxin transport (PAT) machinery, which controls the amount and direction of auxin translocated in the plant tissues and organs(Adamowski & Friml, 2015). Hence, PIN-mediated transport is tightly linked with thermo-morphogenesis, and interference with PAT through either chemical or genetic means dramatically affecting the adaptive responses to hAT. Intriguingly, despite the key role of PIN mediated transport in growth response to hAT, whether and how PINs at the level of expression adapt to fluctuation in temperature is scarcely understood. With genetic, molecular and advanced bio-imaging approaches, we demonstrate the role of PIN auxin transporters in the regulation of hypocotyl growth in response to hAT. We show that via adjustment of PIN3, PIN4 and PIN7 expression in cotyledons and hypocotyls, auxin distribution is modulated thereby determining elongation pattern of epidermal cells at hAT. Furthermore, we identified three Zinc-Finger (ZF) transcription factors as novel molecular components of the thermo-regulatory network, which through negative regulation of PIN transcription adjust the transport of auxin at hAT. Our results suggest that the ZF-PIN module might be a part of the negative feedback loop attenuating the activity of the thermo-sensing pathway to restrain exaggerated growth and developmental responses to hAT.}, author = {Artner, Christina}, isbn = {978-3-99078-022-0}, issn = {2663-337X}, keywords = {high ambient temperature, auxin, PINs, Zinc-Finger proteins, thermomorphogenesis, stress}, pages = {128}, publisher = {Institute of Science and Technology Austria}, title = {{Modulation of auxin transport via ZF proteins adjust plant response to high ambient temperature}}, doi = {10.15479/at:ista:11879}, year = {2022}, } @article{11160, abstract = {Mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are a frequent cause of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly, implicating cortical abnormalities, how CHD8 haploinsufficiency affects neurodevelopmental is unclear. Here, employing human cerebral organoids, we find that CHD8 haploinsufficiency disrupted neurodevelopmental trajectories with an accelerated and delayed generation of, respectively, inhibitory and excitatory neurons that yields, at days 60 and 120, symmetrically opposite expansions in their proportions. This imbalance is consistent with an enlargement of cerebral organoids as an in vitro correlate of patients’ macrocephaly. Through an isogenic design of patient-specific mutations and mosaic organoids, we define genotype-phenotype relationships and uncover their cell-autonomous nature. Our results define cell-type-specific CHD8-dependent molecular defects related to an abnormal program of proliferation and alternative splicing. By identifying cell-type-specific effects of CHD8 mutations, our study uncovers reproducible developmental alterations that may be employed for neurodevelopmental disease modeling.}, author = {Villa, Carlo Emanuele and Cheroni, Cristina and Dotter, Christoph and López-Tóbon, Alejandro and Oliveira, Bárbara and Sacco, Roberto and Yahya, Aysan Çerağ and Morandell, Jasmin and Gabriele, Michele and Tavakoli, Mojtaba and Lyudchik, Julia and Sommer, Christoph M and Gabitto, Mariano and Danzl, Johann G and Testa, Giuseppe and Novarino, Gaia}, issn = {2211-1247}, journal = {Cell Reports}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {1}, publisher = {Elsevier}, title = {{CHD8 haploinsufficiency links autism to transient alterations in excitatory and inhibitory trajectories}}, doi = {10.1016/j.celrep.2022.110615}, volume = {39}, year = {2022}, } @article{12244, abstract = {Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes and overcomes feature-selection biases and biological variability. We extract spatially heterogeneous and sexually dimorphic morphological phenotypes for seven adult mouse brain regions. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration mouse models, with females showing a faster morphological shift in affected brain regions. Remarkably, microglia morphologies reflect an adaptation upon repeated exposure to ketamine anesthesia and do not recover to control morphologies. Finally, we demonstrate that both long primary processes and short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs opens a new perspective to characterize microglial morphology.}, author = {Colombo, Gloria and Cubero, Ryan J and Kanari, Lida and Venturino, Alessandro and Schulz, Rouven and Scolamiero, Martina and Agerberg, Jens and Mathys, Hansruedi and Tsai, Li-Huei and Chachólski, Wojciech and Hess, Kathryn and Siegert, Sandra}, issn = {1546-1726}, journal = {Nature Neuroscience}, keywords = {General Neuroscience}, number = {10}, pages = {1379--1393}, publisher = {Springer Nature}, title = {{A tool for mapping microglial morphology, morphOMICs, reveals brain-region and sex-dependent phenotypes}}, doi = {10.1038/s41593-022-01167-6}, volume = {25}, year = {2022}, } @phdthesis{12378, abstract = {Environmental cues influence the highly dynamic morphology of microglia. Strategies to characterize these changes usually involve user-selected morphometric features, which preclude the identification of a spectrum of context-dependent morphological phenotypes. Here, we develop MorphOMICs, a topological data analysis approach, which enables semiautomatic mapping of microglial morphology into an atlas of cue-dependent phenotypes, overcomes feature-selection bias and minimizes biological variability. First, with MorphOMICs we derive the morphological spectrum of microglia across seven brain regions during postnatal development and in two distinct Alzheimer’s disease degeneration mouse models. We uncover region-specific and sexually dimorphic morphological trajectories, with females showing an earlier morphological shift than males in the degenerating brain. Overall, we demonstrate that both long primary- and short terminal processes provide distinct insights to morphological phenotypes. Moreover, using machine learning to map novel condition on the spectrum, we observe that microglia morphologies reflect a dose-dependent adaptation upon ketamine anesthesia and do not recover to control morphologies. Next, we took advantage of MorphOMICs to build a high-resolution and layer-specific map of microglial morphological spectrum in the retina, covering postnatal development and rd10 degeneration. Here, following photoreceptor death, microglia assume an early developmentlike morphology. Finally, we map microglial morphology following optic nerve crush on the retinal spectrum and observe a layer- and sex-dependent response. Overall, MorphOMICs opens a new perspective to analyze microglial morphology across multiple conditions, and provides a novel tool to characterize microglial morphology beyond the traditionally dichotomized view of microglia.}, author = {Colombo, Gloria}, issn = {2663-337X}, pages = {142}, publisher = {Institute of Science and Technology Austria}, title = {{MorphOMICs, a tool for mapping microglial morphology, reveals brain region- and sex-dependent phenotypes}}, doi = {10.15479/at:ista:12378}, year = {2022}, } @article{10703, abstract = {When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes.}, author = {Gaertner, Florian and Dos Reis Rodrigues, Patricia and De Vries, Ingrid and Hons, Miroslav and Aguilera, Juan and Riedl, Michael and Leithner, Alexander F and Tasciyan, Saren and Kopf, Aglaja and Merrin, Jack and Zheden, Vanessa and Kaufmann, Walter and Hauschild, Robert and Sixt, Michael K}, issn = {1878-1551}, journal = {Developmental Cell}, number = {1}, pages = {47--62.e9}, publisher = {Cell Press}, title = {{WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues}}, doi = {10.1016/j.devcel.2021.11.024}, volume = {57}, year = {2022}, } @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}, 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{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 = {1476-4687}, 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}, } @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{8582, abstract = {Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.}, author = {Li, Hongjiang and von Wangenheim, Daniel and Zhang, Xixi and Tan, Shutang and Darwish-Miranda, Nasser and Naramoto, Satoshi and Wabnik, Krzysztof T and de Rycke, Riet and Kaufmann, Walter and Gütl, Daniel J and Tejos, Ricardo and Grones, Peter and Ke, Meiyu and Chen, Xu and Dettmer, Jan and Friml, Jiří}, issn = {1469-8137}, journal = {New Phytologist}, number = {1}, pages = {351--369}, publisher = {Wiley}, title = {{Cellular requirements for PIN polar cargo clustering in Arabidopsis thaliana}}, doi = {10.1111/nph.16887}, volume = {229}, year = {2021}, } @article{8988, abstract = {The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1’s functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1’s enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.}, author = {Düllberg, Christian F and Auer, Albert and Canigova, Nikola and Loibl, Katrin and Loose, Martin}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {1}, publisher = {National Academy of Sciences}, title = {{In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1}}, doi = {10.1073/pnas.2010054118}, volume = {118}, year = {2021}, }