TY - JOUR AB - The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage. AU - Kuhn, Andre AU - Roosjen, Mark AU - Mutte, Sumanth AU - Dubey, Shiv Mani AU - Carrillo Carrasco, Vanessa Polet AU - Boeren, Sjef AU - Monzer, Aline AU - Koehorst, Jasper AU - Kohchi, Takayuki AU - Nishihama, Ryuichi AU - Fendrych, Matyas AU - Sprakel, Joris AU - Friml, Jiří AU - Weijers, Dolf ID - 14826 IS - 1 JF - Cell KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0092-8674 TI - RAF-like protein kinases mediate a deeply conserved, rapid auxin response VL - 187 ER - TY - JOUR AB - 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. AU - Glanc, Matous AU - Van Gelderen, K AU - Hörmayer, Lukas AU - Tan, Shutang AU - Naramoto, S AU - Zhang, Xixi AU - Domjan, David AU - Vcelarova, L AU - Hauschild, Robert AU - Johnson, Alexander J AU - de Koning, E AU - van Dop, M AU - Rademacher, E AU - Janson, S AU - Wei, X AU - Molnar, Gergely AU - Fendrych, Matyas AU - De Rybel, B AU - Offringa, R AU - Friml, Jiří ID - 9290 IS - 9 JF - Current Biology SN - 0960-9822 TI - AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells VL - 31 ER - TY - JOUR AB - 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. AU - Gelová, Zuzana AU - Gallei, Michelle C AU - Pernisová, Markéta AU - Brunoud, Géraldine AU - Zhang, Xixi AU - Glanc, Matous AU - Li, Lanxin AU - Michalko, Jaroslav AU - Pavlovicova, Zlata AU - Verstraeten, Inge AU - Han, Huibin AU - Hajny, Jakub AU - Hauschild, Robert AU - Čovanová, Milada AU - Zwiewka, Marta AU - Hörmayer, Lukas AU - Fendrych, Matyas AU - Xu, Tongda AU - Vernoux, Teva AU - Friml, Jiří ID - 8931 JF - Plant Science KW - Agronomy and Crop Science KW - Plant Science KW - Genetics KW - General Medicine SN - 0168-9452 TI - Developmental roles of auxin binding protein 1 in Arabidopsis thaliana VL - 303 ER - TY - JOUR AB - Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking. AU - Glanc, Matous AU - Fendrych, Matyas AU - Friml, Jiří ID - 6611 IS - 6 JF - Biomolecules TI - PIN2 polarity establishment in arabidopsis in the absence of an intact cytoskeleton VL - 9 ER - TY - JOUR AB - The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants(1). It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription(2). Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition(3) by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB-Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway. AU - Fendrych, Matyas AU - Akhmanova, Maria AU - Merrin, Jack AU - Glanc, Matous AU - Hagihara, Shinya AU - Takahashi, Koji AU - Uchida, Naoyuki AU - Torii, Keiko U AU - Friml, Jirí ID - 192 IS - 7 JF - Nature Plants TI - Rapid and reversible root growth inhibition by TIR1 auxin signalling VL - 4 ER - TY - JOUR AB - Cell polarity, manifested by the localization of proteins to distinct polar plasma membrane domains, is a key prerequisite of multicellular life. In plants, PIN auxin transporters are prominent polarity markers crucial for a plethora of developmental processes. Cell polarity mechanisms in plants are distinct from other eukaryotes and still largely elusive. In particular, how the cell polarities are propagated and maintained following cell division remains unknown. Plant cytokinesis is orchestrated by the cell plate—a transient centrifugally growing endomembrane compartment ultimately forming the cross wall1. Trafficking of polar membrane proteins is typically redirected to the cell plate, and these will consequently have opposite polarity in at least one of the daughter cells2–5. Here, we provide mechanistic insights into post-cytokinetic re-establishment of cell polarity as manifested by the apical, polar localization of PIN2. We show that the apical domain is defined in a cell-intrinsic manner and that re-establishment of PIN2 localization to this domain requires de novo protein secretion and endocytosis, but not basal-to-apical transcytosis. Furthermore, we identify a PINOID-related kinase WAG1, which phosphorylates PIN2 in vitro6 and is transcriptionally upregulated specifically in dividing cells, as a crucial regulator of post-cytokinetic PIN2 polarity re-establishment. AU - Glanc, Matous AU - Fendrych, Matyas AU - Friml, Jirí ID - 5673 IS - 12 JF - Nature Plants SN - 2055-0278 TI - Mechanistic framework for cell-intrinsic re-establishment of PIN2 polarity after cell division VL - 4 ER - TY - JOUR AB - The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH. AU - Li, Lanxin AU - Krens, Gabriel AU - Fendrych, Matyas AU - Friml, Jirí ID - 442 IS - 1 JF - Bio-protocol TI - Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls VL - 8 ER - TY - JOUR AB - The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes. AU - Synek, Lukáš AU - Vukašinović, Nemanja AU - Kulich, Ivan AU - Hála, Michal AU - Aldorfová, Klára AU - Fendrych, Matyas AU - Žárský, Viktor ID - 669 IS - 1 JF - Plant Physiology SN - 00320889 TI - EXO70C2 is a key regulatory factor for optimal tip growth of pollen VL - 174 ER - TY - JOUR AB - Roots navigate through soil integrating environmental signals to orient their growth. The Arabidopsis root is a widely used model for developmental, physiological and cell biological studies. Live imaging greatly aids these efforts, but the horizontal sample position and continuous root tip displacement present significant difficulties. Here, we develop a confocal microscope setup for vertical sample mounting and integrated directional illumination. We present TipTracker – a custom software for automatic tracking of diverse moving objects usable on various microscope setups. Combined, this enables observation of root tips growing along the natural gravity vector over prolonged periods of time, as well as the ability to induce rapid gravity or light stimulation. We also track migrating cells in the developing zebrafish embryo, demonstrating the utility of this system in the acquisition of high-resolution data sets of dynamic samples. We provide detailed descriptions of the tools enabling the easy implementation on other microscopes. AU - Von Wangenheim, Daniel AU - Hauschild, Robert AU - Fendrych, Matyas AU - Barone, Vanessa AU - Benková, Eva AU - Friml, Jirí ID - 946 JF - eLife TI - Live tracking of moving samples in confocal microscopy for vertically grown roots VL - 6 ER - TY - JOUR AB - Despite being composed of immobile cells, plants reorient along directional stimuli. The hormone auxin is redistributed in stimulated organs leading to differential growth and bending. Auxin application triggers rapid cell wall acidification and elongation of aerial organs of plants, but the molecular players mediating these effects are still controversial. Here we use genetically-encoded pH and auxin signaling sensors, pharmacological and genetic manipulations available for Arabidopsis etiolated hypocotyls to clarify how auxin is perceived and the downstream growth executed. We show that auxin-induced acidification occurs by local activation of H+-ATPases, which in the context of gravity response is restricted to the lower organ side. This auxin-stimulated acidification and growth require TIR1/AFB-Aux/IAA nuclear auxin perception. In addition, auxin-induced gene transcription and specifically SAUR proteins are crucial downstream mediators of this growth. Our study provides strong experimental support for the acid growth theory and clarified the contribution of the upstream auxin perception mechanisms. AU - Fendrych, Matyas AU - Leung, Jeffrey AU - Friml, Jirí ID - 1344 JF - eLife TI - TIR1 AFB Aux IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls VL - 5 ER - TY - JOUR AB - The electrostatic charge at the inner surface of the plasma membrane is strongly negative in higher organisms. A new study shows that phosphatidylinositol-4-phosphate plays a critical role in establishing plasma membrane surface charge in Arabidopsis, which regulates the correct localization of signalling components. AU - Molnar, Gergely AU - Fendrych, Matyas AU - Friml, Jirí ID - 1345 JF - Nature Plants TI - Plasma membrane: Negative attraction VL - 2 ER - TY - JOUR AB - ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. AU - Dejonghe, Wim AU - Kuenen, Sabine AU - Mylle, Evelien AU - Vasileva, Mina K AU - Keech, Olivier AU - Viotti, Corrado AU - Swerts, Jef AU - Fendrych, Matyas AU - Ortiz Morea, Fausto AU - Mishev, Kiril AU - Delang, Simon AU - Scholl, Stefan AU - Zarza, Xavier AU - Heilmann, Mareike AU - Kourelis, Jiorgos AU - Kasprowicz, Jaroslaw AU - Nguyen, Le AU - Drozdzecki, Andrzej AU - Van Houtte, Isabelle AU - Szatmári, Anna AU - Majda, Mateusz AU - Baisa, Gary AU - Bednarek, Sebastian AU - Robert, Stéphanie AU - Audenaert, Dominique AU - Testerink, Christa AU - Munnik, Teun AU - Van Damme, Daniël AU - Heilmann, Ingo AU - Schumacher, Karin AU - Winne, Johan AU - Friml, Jirí AU - Verstreken, Patrik AU - Russinova, Eugenia ID - 1346 JF - Nature Communications TI - Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification VL - 7 ER - TY - JOUR AB - A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta. AU - Olvera Carrillo, Yadira AU - Van Bel, Michiel AU - Van Hautegem, Tom AU - Fendrych, Matyas AU - Huysmans, Marlies AU - Šimášková, Mária AU - Van Durme, Matthias AU - Buscaill, Pierre AU - Rivas, Susana AU - Coll, Núria AU - Coppens, Frederik AU - Maere, Steven AU - Nowack, Moritz ID - 1543 IS - 4 JF - Plant Physiology TI - A conserved core of programmed cell death indicator genes discriminates developmentally and environmentally induced programmed cell death in plants VL - 169 ER - TY - JOUR AB - When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions. AU - Kremer, A AU - Lippens, Stefaan AU - Bartunkova, Sonia AU - Asselbergh, Bob AU - Blanpain, Cendric AU - Fendrych, Matyas AU - Goossens, A AU - Holt, Matthew AU - Janssens, Sophie AU - Krols, Michiel AU - Larsimont, Jean AU - Mc Guire, Conor AU - Nowack, Moritz AU - Saelens, Xavier AU - Schertel, Andreas AU - Schepens, B AU - Slezak, M AU - Timmerman, Vincent AU - Theunis, Clara AU - Van Brempt, Ronald AU - Visser, Y AU - Guérin, Christophe ID - 1879 IS - 2 JF - Journal of Microscopy TI - Developing 3D SEM in a broad biological context VL - 259 ER - TY - JOUR AU - Rakusová, Hana AU - Fendrych, Matyas AU - Friml, Jirí ID - 1944 IS - 2 JF - Current Opinion in Plant Biology TI - Intracellular trafficking and PIN-mediated cell polarity during tropic responses in plants VL - 23 ER -