Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.

• MeSH
• Arabidopsis * metabolism   genetics   growth & development   MeSH
• Biological Transport MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   genetics   MeSH
• Plant Roots metabolism   growth & development   genetics   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Membrane Transport Proteins metabolism   genetics   MeSH
• Protein Serine-Threonine Kinases * MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Xylem metabolism   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• AT1G66150 protein, Arabidopsis MeSH Browser
• BREVIS RADIX protein, Arabidopsis MeSH Browser
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Indoleacetic Acids * MeSH
• Membrane Transport Proteins MeSH
• Protein Serine-Threonine Kinases * MeSH
• Arabidopsis Proteins * MeSH

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

• MeSH
• Cell Membrane metabolism   MeSH
• Endocytosis * MeSH
• Clathrin * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Clathrin * MeSH

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

• Keywords
• Golgi, endocytosis, exocytosis, microscopy, proteomics, vesicle,
• MeSH
• Biological Transport MeSH
• Endocytosis MeSH
• Mass Spectrometry methods   MeSH
• Proteome * analysis   metabolism   MeSH
• Proteomics * methods   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Proteome * MeSH

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.

• Keywords
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Biological Transport MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids MeSH
• Membrane Transport Proteins genetics   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Cells metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• Arabidopsis Proteins * MeSH

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.

• MeSH
• ADP-Ribosylation Factors metabolism   MeSH
• Arabidopsis drug effects   metabolism   MeSH
• Biological Transport drug effects   MeSH
• Brefeldin A pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Epistasis, Genetic drug effects   MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Mutation genetics   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Phospholipid Transfer Proteins metabolism   MeSH
• Nicotiana metabolism   MeSH
• trans-Golgi Network drug effects   metabolism   MeSH
• Protein Binding drug effects   MeSH
• Guanine Nucleotide Exchange Factors metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADP-Ribosylation Factors MeSH
• Brefeldin A MeSH
• GTP Phosphohydrolases MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Phospholipid Transfer Proteins MeSH
• Guanine Nucleotide Exchange Factors MeSH

SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.

• Keywords
• cell plate, cytokinesis, evolution, formin, interaction specificity, phylogeny,
• MeSH
• Arabidopsis MeSH
• Cytokinesis * MeSH
• Dynamins metabolism   MeSH
• Phylogeny MeSH
• Evolution, Molecular * MeSH
• Arabidopsis Proteins classification   genetics   metabolism   MeSH
• Carrier Proteins classification   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Dynamins MeSH
• Arabidopsis Proteins MeSH
• SH3P2 protein, Arabidopsis MeSH Browser
• Carrier Proteins MeSH

The plant-specific proteins named PIN-FORMED (PIN) efflux carriers facilitate the direction of auxin flow and thus play a vital role in the establishment of local auxin maxima within plant tissues that subsequently guide plant ontogenesis. They are membrane integral proteins with two hydrophobic regions consisting of alpha-helices linked with a hydrophilic loop, which is usually longer for the plasma membrane-localized PINs. The hydrophilic loop harbors molecular cues important for the subcellular localization and thus auxin efflux function of those transporters. The three-dimensional structure of PIN has not been solved yet. However, there are scattered but substantial data concerning the functional characterization of amino acid strings that constitute these carriers. These sequences include motifs vital for vesicular trafficking, residues regulating membrane diffusion, cellular polar localization, and activity of PINs. Here, we summarize those bits of information striving to provide a reference to structural motifs that have been investigated experimentally hoping to stimulate the efforts toward unraveling of PIN structure-function connections.

• Keywords
• PIN efflux carriers, auxin transport, protein domains, sequence motifs, subcellular trafficking,
• Publication type
• Journal Article MeSH
• Review MeSH

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.

• Keywords
• PIN auxin efflux carriers, actin, cell polarity, cytoskeleton, live-cell imaging, microtubules, polarity establishment,
• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Cytoskeleton metabolism   MeSH
• Intracellular Space metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• PIN2 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH

Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.

• MeSH
• Arabidopsis drug effects   metabolism   MeSH
• Biological Transport drug effects   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Endocytosis * drug effects   MeSH
• Phenotype MeSH
• Phenylacetates pharmacology   MeSH
• Gravitropism drug effects   MeSH
• Hypocotyl drug effects   growth & development   MeSH
• Plant Roots drug effects   growth & development   MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Signal Transduction MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phenylacetates MeSH
• Indoleacetic Acids MeSH
• phenylacetic acid MeSH Browser
• pinstatic acid MeSH Browser
• Arabidopsis Proteins MeSH

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.

• MeSH
• Arabidopsis cytology   genetics   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Division * MeSH
• Cytokinesis MeSH
• Endocytosis MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Plant Roots cytology   genetics   physiology   MeSH
• Cell Polarity * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Recombinant Fusion Proteins MeSH
• Genes, Reporter MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• PIN2 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Recombinant Fusion Proteins MeSH