RESUMO
Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiões de Interação com a Matriz , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Both filamentous pathogens' hyphae and pollen tube penetrate the host's outer layer and involve growth within the host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla of Arabidospis thaliana's stigma, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcomed pollen tube. We showed that Phytophtora parasitica, a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cells, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganisation of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion.
RESUMO
Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.
Assuntos
Proteínas de Transporte , Proteínas de Plantas , Proteínas de Transporte/metabolismo , Proteínas de Plantas/química , Membrana Celular/metabolismo , Plantas/metabolismo , Bicamadas Lipídicas/metabolismoRESUMO
Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.
Assuntos
Óvulo Vegetal/crescimento & desenvolvimento , Fosfolipases/metabolismo , Proteínas de Plantas/metabolismo , Pólen/enzimologia , Reprodução , Zea mays/fisiologia , Regulação da Expressão Gênica de Plantas , Fosfolipases/deficiência , Zea mays/enzimologiaRESUMO
Early events occurring at the surface of the female organ are critical for plant reproduction, especially in species with a dry stigma. After landing on the stigmatic papilla cells, the pollen hydrates and germinates a tube, which penetrates the cell wall and grows towards the ovules to convey the male gametes to the embryo sac. In self-incompatible species within the Brassicaceae, these processes are blocked when the stigma encounters an incompatible pollen. Based on the generation of self-incompatible Arabidopsis lines and by setting up a live imaging system, we showed that control of pollen hydration has a central role in pollen selectivity. The faster the pollen pumps water from the papilla during an initial period of 10 min, the faster it germinates. Furthermore, we found that the self-incompatibility barriers act to block the proper hydration of incompatible pollen and, when hydration is promoted by high humidity, an additional control prevents pollen tube penetration into the stigmatic wall. In papilla cells, actin bundles focalize at the contact site with the compatible pollen but not with the incompatible pollen, raising the possibility that stigmatic cells react to the mechanical pressure applied by the invading growing tube.
Assuntos
Arabidopsis , Percepção , Pólen , Tubo Polínico , PolinizaçãoRESUMO
In Arabidopsis thaliana, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high-affinity phosphate transporters. They are transcriptionally induced by phosphate starvation and require PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1) to exit the endoplasmic reticulum (ER), indicating intracellular traffic as an additional level of regulation of PHT1 activity. Our study revealed that PHF1 acts on PHT1, upstream of vesicle coat protein COPII formation, and that additional regulatory events occur during PHT1 trafficking and determine its ER exit and plasma membrane stability. Phosphoproteomic and mutagenesis analyses revealed modulation of PHT1;1 ER export by Ser-514 phosphorylation status. Confocal microscopy analysis of root tip cells showed that PHT1;1 is localized to the plasma membrane and is present in intracellular endocytic compartments. More precisely, PHT1;1 was localized to sorting endosomes associated with prevacuolar compartments. Kinetic analysis of PHT1;1 stability and targeting suggested a modulation of PHT1 internalization from the plasma membrane to the endosomes, followed by either subsequent recycling (in low Pi) or vacuolar degradation (in high Pi). For the latter condition, we identified a rapid mechanism that reduces the pool of PHT1 proteins present at the plasma membrane. This mechanism is regulated by the Pi concentration in the medium and appears to be independent of degradation mechanisms potentially regulated by the PHO2 ubiquitin conjugase. We propose a model for differential trafficking of PHT1 to the plasma membrane or vacuole as a function of phosphate concentration.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Fosfato/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas de Transporte de Fosfato/química , Fosfatos/deficiência , Fosfatos/metabolismo , Fosforilação , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In plants, as in animals, organ growth depends on mechanical interactions between cells and tissues, and is controlled by both biochemical and mechanical cues. Here, we investigate the control of seed size, a key agronomic trait, by mechanical interactions between two compartments: the endosperm and the testa. By combining experiments with computational modelling, we present evidence that endosperm pressure plays two antagonistic roles: directly driving seed growth, but also indirectly inhibiting it through tension it generates in the surrounding testa, which promotes wall stiffening. We show that our model can recapitulate wild type growth patterns, and is consistent with the small seed phenotype of the haiku2 mutant, and the results of osmotic treatments. Our work suggests that a developmental regulation of endosperm pressure is required to prevent a precocious reduction of seed growth rate induced by force-dependent seed coat stiffening.
Assuntos
Endosperma , Sementes , Endosperma/genética , Regulação da Expressão Gênica de PlantasRESUMO
Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfatidilserinas/metabolismo , Transdução de Sinais/fisiologia , Arabidopsis/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Membrana Celular/metabolismo , Plantas/metabolismoRESUMO
Extracellular matrices contain fibril-like polymers often organized in parallel arrays. Although their role in morphogenesis has been long recognized, it remains unclear how the subcellular control of fibril synthesis translates into organ shape. We address this question using the Arabidopsis sepal as a model organ. In plants, cell growth is restrained by the cell wall (extracellular matrix). Cellulose microfibrils are the main load-bearing wall component, thought to channel growth perpendicularly to their main orientation. Given the key function of CELLULOSE SYNTHASE INTERACTIVE1 (CSI1) in guidance of cellulose synthesis, we investigate the role of CSI1 in sepal morphogenesis. We observe that sepals from csi1 mutants are shorter, although their newest cellulose microfibrils are more aligned compared to wild-type. Surprisingly, cell growth anisotropy is similar in csi1 and wild-type plants. We resolve this apparent paradox by showing that CSI1 is required for spatial consistency of growth direction across the sepal.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte , Microtúbulos/metabolismo , Celulose/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , MorfogêneseRESUMO
Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.
Assuntos
Arabidopsis , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Phosphate is a crucial and often limiting nutrient for plant growth. To obtain inorganic phosphate (P(i) ), which is very insoluble, and is heterogeneously distributed in the soil, plants have evolved a complex network of morphological and biochemical processes. These processes are controlled by a regulatory system triggered by P(i) concentration, not only present in the medium (external P(i) ), but also inside plant cells (internal P(i) ). A 'split-root' assay was performed to mimic a heterogeneous environment, after which a transcriptomic analysis identified groups of genes either locally or systemically regulated by P(i) starvation at the transcriptional level. These groups revealed coordinated regulations for various functions associated with P(i) starvation (including P(i) uptake, P(i) recovery, lipid metabolism, and metal uptake), and distinct roles for members in gene families. Genetic tools and physiological analyses revealed that genes that are locally regulated appear to be modulated mostly by root development independently of the internal P(i) content. By contrast, internal P(i) was essential to promote the activation of systemic regulation. Reducing the flow of P(i) had no effect on the systemic response, suggesting that a secondary signal, independent of P(i) , could be involved in the response. Furthermore, our results display a direct role for the transcription factor PHR1, as genes systemically controlled by low P(i) have promoters enriched with P1BS motif (PHR1-binding sequences). These data detail various regulatory systems regarding P(i) starvation responses (systemic versus local, and internal versus external P(i) ), and provide tools to analyze and classify the effects of P(i) starvation on plant physiology.
Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica , Fosfatos/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA de Plantas/genética , Transdução de Sinais , Transcrição GênicaRESUMO
Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3-4 h of imaging followed by 3-4 d of image processing and data analysis.
Assuntos
Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Arabidopsis/metabolismo , Difusão , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Membrana/isolamento & purificação , Imagem Óptica/métodos , Células Vegetais/química , Plantas/química , Plantas/metabolismo , Espectrometria de Fluorescência/métodosRESUMO
Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1-3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1-4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7-10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.
Assuntos
Proteínas de Arabidopsis/metabolismo , Gravitropismo/fisiologia , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Mutação com Ganho de Função , Gravitação , Mutação com Perda de Função , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/fisiologiaRESUMO
Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity.
Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.
Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Organogênese Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Técnicas Biossensoriais , Regulação da Expressão Gênica de Plantas , Genes Reporter , Microscopia Confocal , Organogênese Vegetal/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Fatores de Tempo , Transcrição GênicaRESUMO
In the course of their growth and development, plants have to constantly perceive and react to their environment. This is achieved in cells by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependent nanodomains within the plasma membrane (PM). These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS) that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, osmotic signal triggers interaction between ROP6 and two NADPH oxidases that subsequently generate ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensures signal specificity downstream of independent stimuli.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Pressão Osmótica/fisiologia , Adaptação Fisiológica , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , NADPH Oxidases/metabolismo , Osmose/fisiologia , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfatidilserinas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/metabolismo , Endocitose/genética , Regulação da Expressão Gênica de Plantas , Gravitropismo/genética , Ácidos Indolacéticos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfatidilserinas/farmacologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Imagem Individual de MoléculaRESUMO
Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Eletricidade Estática , Arabidopsis/crescimento & desenvolvimento , Organelas , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transdução de SinaisRESUMO
In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species.
RESUMO
Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amendable to automatically track non-plant structures with an apical growth.
RESUMO
Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.