@article{11995, abstract = {G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands.}, author = {Schulz, Rouven and Korkut, Medina and Venturino, Alessandro and Colombo, Gloria and Siegert, Sandra}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses}}, doi = {10.1038/s41467-022-32390-1}, volume = {13}, year = {2022}, } @article{12082, abstract = {Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.}, author = {Artan, Murat and Hartl, Markus and Chen, Weiqiang and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {9}, publisher = {Elsevier}, title = {{Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans}}, doi = {10.1016/j.jbc.2022.102343}, volume = {298}, year = {2022}, } @article{12117, abstract = {To understand how potential gene manipulations affect in vitro microglia, we provide a set of short protocols to evaluate microglia identity and function. We detail steps for immunostaining to determine microglia identity. We describe three functional assays for microglia: phagocytosis, calcium response following ATP stimulation, and cytokine expression upon inflammatory stimuli. We apply these protocols to human induced-pluripotent-stem-cell (hiPSC)-derived microglia, but they can be also applied to other in vitro microglial models including primary mouse microglia. For complete details on the use and execution of this protocol, please refer to Bartalska et al. (2022).1}, author = {Hübschmann, Verena and Korkut, Medina and Siegert, Sandra}, issn = {2666-1667}, journal = {STAR Protocols}, keywords = {General Immunology and Microbiology, General Biochemistry, Genetics and Molecular Biology, General Neuroscience}, number = {4}, publisher = {Elsevier}, title = {{Assessing human iPSC-derived microglia identity and function by immunostaining, phagocytosis, calcium activity, and inflammation assay}}, doi = {10.1016/j.xpro.2022.101866}, volume = {3}, year = {2022}, } @article{12144, abstract = {The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.}, author = {Qi, Linlin and Kwiatkowski, Mateusz and Chen, Huihuang and Hörmayer, Lukas and Sinclair, Scott A and Zou, Minxia and del Genio, Charo I. and Kubeš, Martin F. and Napier, Richard and Jaworski, Krzysztof and Friml, Jiří}, issn = {1476-4687}, journal = {Nature}, number = {7934}, pages = {133--138}, publisher = {Springer Nature}, title = {{Adenylate cyclase activity of TIR1/AFB auxin receptors in plants}}, doi = {10.1038/s41586-022-05369-7}, volume = {611}, year = {2022}, } @article{12209, abstract = {Embryo development requires biochemical signalling to generate patterns of cell fates and active mechanical forces to drive tissue shape changes. However, how these processes are coordinated, and how tissue patterning is preserved despite the cellular flows occurring during morphogenesis, remains poorly understood. Gastrulation is a crucial embryonic stage that involves both patterning and internalization of the mesendoderm germ layer tissue. Here we show that, in zebrafish embryos, a gradient in Nodal signalling orchestrates pattern-preserving internalization movements by triggering a motility-driven unjamming transition. In addition to its role as a morphogen determining embryo patterning, graded Nodal signalling mechanically subdivides the mesendoderm into a small fraction of highly protrusive leader cells, able to autonomously internalize via local unjamming, and less protrusive followers, which need to be pulled inwards by the leaders. The Nodal gradient further enforces a code of preferential adhesion coupling leaders to their immediate followers, resulting in a collective and ordered mode of internalization that preserves mesendoderm patterning. Integrating this dual mechanical role of Nodal signalling into minimal active particle simulations quantitatively predicts both physiological and experimentally perturbed internalization movements. This provides a quantitative framework for how a morphogen-encoded unjamming transition can bidirectionally couple tissue mechanics with patterning during complex three-dimensional morphogenesis.}, author = {Nunes Pinheiro, Diana C and Kardos, Roland and Hannezo, Edouard B and Heisenberg, Carl-Philipp J}, issn = {1745-2481}, journal = {Nature Physics}, keywords = {General Physics and Astronomy}, number = {12}, pages = {1482--1493}, publisher = {Springer Nature}, title = {{Morphogen gradient orchestrates pattern-preserving tissue morphogenesis via motility-driven unjamming}}, doi = {10.1038/s41567-022-01787-6}, volume = {18}, year = {2022}, } @article{12239, abstract = {Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.}, author = {Johnson, Alexander J and Kaufmann, Walter and Sommer, Christoph M and Costanzo, Tommaso and Dahhan, Dana A. and Bednarek, Sebastian Y. and Friml, Jiří}, issn = {1674-2052}, journal = {Molecular Plant}, keywords = {Plant Science, Molecular Biology}, number = {10}, pages = {1533--1542}, publisher = {Elsevier}, title = {{Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution}}, doi = {10.1016/j.molp.2022.09.003}, volume = {15}, 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}, } @article{12288, abstract = {To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of ‘starter’ AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.}, author = {Sumser, Anton L and Jösch, Maximilian A and Jonas, Peter M and Ben Simon, Yoav}, issn = {2050-084X}, journal = {eLife}, keywords = {General Immunology and Microbiology, General Biochemistry, Genetics and Molecular Biology, General Medicine, General Neuroscience}, publisher = {eLife Sciences Publications}, title = {{Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling}}, doi = {10.7554/elife.79848}, volume = {11}, year = {2022}, } @article{12291, abstract = {The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1,2,3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.}, author = {Friml, Jiří and Gallei, Michelle C and Gelová, Zuzana and Johnson, Alexander J and Mazur, Ewa and Monzer, Aline and Rodriguez Solovey, Lesia and Roosjen, Mark and Verstraeten, Inge and Živanović, Branka D. and Zou, Minxia and Fiedler, Lukas and Giannini, Caterina and Grones, Peter and Hrtyan, Mónika and Kaufmann, Walter and Kuhn, Andre and Narasimhan, Madhumitha and Randuch, Marek and Rýdza, Nikola and Takahashi, Koji and Tan, Shutang and Teplova, Anastasiia and Kinoshita, Toshinori and Weijers, Dolf and Rakusová, Hana}, issn = {1476-4687}, journal = {Nature}, number = {7927}, pages = {575--581}, publisher = {Springer Nature}, title = {{ABP1–TMK auxin perception for global phosphorylation and auxin canalization}}, doi = {10.1038/s41586-022-05187-x}, volume = {609}, year = {2022}, } @phdthesis{12368, abstract = {Metazoan development relies on the formation and remodeling of cell-cell contacts. The binding of adhesion receptors and remodeling of the actomyosin cell cortex at cell-cell interaction sites have been implicated in cell-cell contact formation. Yet, how these two processes functionally interact to drive cell-cell contact expansion and strengthening remains unclear. Here, we study how primary germ layer progenitor cells from zebrafish bind to supported lipid bilayers (SLB) functionalized with E-cadherin ectodomains as an assay system for monitoring cell-cell contact formation at high spatiotemporal resolution. We show that cell-cell contact formation represents a two-tiered process: E-cadherinmediated downregulation of the small GTPase RhoA at the forming contact leads to both depletion of Myosin-2 and decrease of F-actin. This is followed by centrifugal actin network flows at the contact triggered by a sharp gradient of Myosin-2 at the rim of the contact zone, with Myosin-2 displaying higher cortical localization outside than inside of the contact. These centrifugal cortical actin flows, in turn, not only further dilute the actin network at the contact disc, but also lead to an accumulation of both F-actin and Ecadherin at the contact rim. Eventually, this combination of actomyosin downregulation and flows at the contact contribute to the characteristic molecular organization implicated in contact formation and maintenance: depletion of cortical actomyosin at the contact disc, driving contact expansion by lowering interfacial tension at the contact, and accumulation of both E-cadherin and F-actin at the contact rim, mechanically linking the contractile cortices of the adhering cells. Thus, using a biomimetic assay, we exemplify how adhesion signaling and cell mechanics function together to modulate the spatial organization of cell-cell contacts.}, author = {Arslan, Feyza N}, isbn = { 978-3-99078-025-1 }, issn = {2663-337X}, pages = {113}, publisher = {Institute of Science and Technology Austria}, title = {{Remodeling of E-cadherin-mediated contacts via cortical flows}}, doi = {10.15479/at:ista:12153}, 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{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{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 = {14698137}, 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{8931, abstract = {Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy.}, author = {Gelová, Zuzana and Gallei, Michelle C and Pernisová, Markéta and Brunoud, Géraldine and Zhang, Xixi and Glanc, Matous and Li, Lanxin and Michalko, Jaroslav and Pavlovicova, Zlata and Verstraeten, Inge and Han, Huibin and Hajny, Jakub and Hauschild, Robert and Čovanová, Milada and Zwiewka, Marta and Hörmayer, Lukas and Fendrych, Matyas and Xu, Tongda and Vernoux, Teva and Friml, Jiří}, issn = {0168-9452}, journal = {Plant Science}, keywords = {Agronomy and Crop Science, Plant Science, Genetics, General Medicine}, publisher = {Elsevier}, title = {{Developmental roles of auxin binding protein 1 in Arabidopsis thaliana}}, doi = {10.1016/j.plantsci.2020.110750}, volume = {303}, 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 = {10916490}, journal = {PNAS}, 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}, } @article{9010, abstract = {Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.}, author = {Ötvös, Krisztina and Marconi, Marco and Vega, Andrea and O’Brien, Jose and Johnson, Alexander J and Abualia, Rashed and Antonielli, Livio and Montesinos López, Juan C and Zhang, Yuzhou and Tan, Shutang and Cuesta, Candela and Artner, Christina and Bouguyon, Eleonore and Gojon, Alain and Friml, Jiří and Gutiérrez, Rodrigo A. and Wabnik, Krzysztof T and Benková, Eva}, issn = {14602075}, journal = {EMBO Journal}, number = {3}, publisher = {Embo Press}, title = {{Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport}}, doi = {10.15252/embj.2020106862}, volume = {40}, year = {2021}, } @inbook{9245, abstract = {Tissue morphogenesis is driven by mechanical forces triggering cell movements and shape changes. Quantitatively measuring tension within tissues is of great importance for understanding the role of mechanical signals acting on the cell and tissue level during morphogenesis. Here we introduce laser ablation as a useful tool to probe tissue tension within the granulosa layer, an epithelial monolayer of somatic cells that surround the zebrafish female gamete during folliculogenesis. We describe in detail how to isolate follicles, mount samples, perform laser surgery, and analyze the data.}, author = {Xia, Peng and Heisenberg, Carl-Philipp J}, booktitle = {Germline Development in the Zebrafish}, editor = {Dosch, Roland}, isbn = {978-1-0716-0969-9}, issn = {1940-6029}, keywords = {Tissue tension, Morphogenesis, Laser ablation, Zebrafish folliculogenesis, Granulosa cells}, pages = {117--128}, publisher = {Humana}, title = {{Quantifying tissue tension in the granulosa layer after laser surgery}}, doi = {10.1007/978-1-0716-0970-5_10}, volume = {2218}, year = {2021}, } @article{9287, abstract = {The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural (IAA) and synthetic (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network (EE/TGN), rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using Total Internal Reflection Fluorescence (TIRF) microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments. }, author = {Narasimhan, Madhumitha and Gallei, Michelle C and Tan, Shutang and Johnson, Alexander J and Verstraeten, Inge and Li, Lanxin and Rodriguez Solovey, Lesia and Han, Huibin and Himschoot, E and Wang, R and Vanneste, S and Sánchez-Simarro, J and Aniento, F and Adamowski, Maciek and Friml, Jiří}, issn = {1532-2548}, journal = {Plant Physiology}, number = {2}, pages = {1122–1142}, publisher = {Oxford University Press}, title = {{Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking}}, doi = {10.1093/plphys/kiab134}, volume = {186}, year = {2021}, } @article{9290, abstract = {Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.}, author = {Glanc, Matous and Van Gelderen, K and Hörmayer, Lukas and Tan, Shutang and Naramoto, S and Zhang, Xixi and Domjan, David and Vcelarova, L and Hauschild, Robert and Johnson, Alexander J and de Koning, E and van Dop, M and Rademacher, E and Janson, S and Wei, X and Molnar, Gergely and Fendrych, Matyas and De Rybel, B and Offringa, R and Friml, Jiří}, issn = {1879-0445}, journal = {Current Biology}, number = {9}, pages = {1918--1930}, publisher = {Elsevier}, title = {{AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells}}, doi = {10.1016/j.cub.2021.02.028}, volume = {31}, year = {2021}, } @article{9316, abstract = {Embryo morphogenesis is impacted by dynamic changes in tissue material properties, which have been proposed to occur via processes akin to phase transitions (PTs). Here, we show that rigidity percolation provides a simple and robust theoretical framework to predict material/structural PTs of embryonic tissues from local cell connectivity. By using percolation theory, combined with directly monitoring dynamic changes in tissue rheology and cell contact mechanics, we demonstrate that the zebrafish blastoderm undergoes a genuine rigidity PT, brought about by a small reduction in adhesion-dependent cell connectivity below a critical value. We quantitatively predict and experimentally verify hallmarks of PTs, including power-law exponents and associated discontinuities of macroscopic observables. Finally, we show that this uniform PT depends on blastoderm cells undergoing meta-synchronous divisions causing random and, consequently, uniform changes in cell connectivity. Collectively, our theoretical and experimental findings reveal the structural basis of material PTs in an organismal context.}, author = {Petridou, Nicoletta and Corominas-Murtra, Bernat and Heisenberg, Carl-Philipp J and Hannezo, Edouard B}, issn = {10974172}, journal = {Cell}, number = {7}, pages = {1914--1928.e19}, publisher = {Elsevier}, title = {{Rigidity percolation uncovers a structural basis for embryonic tissue phase transitions}}, doi = {10.1016/j.cell.2021.02.017}, volume = {184}, year = {2021}, }