Pankaj Dhonukshe

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.

Clathrin-Mediated Constitutive Endocytosis of PIN Auxin Efflux Carriers in Arabidopsis

Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.

ABP1 Mediates Auxin Inhibition of Clathrin-Dependent Endocytosis in Arabidopsis

Spatial distribution of the plant hormone auxin regulates multiple aspects of plant development. These self-regulating auxin gradients are established by the action of PIN auxin transporters, whose activity is regulated by their constitutive cycling between the plasma membrane and endosomes. Here, we show that auxin signaling by the auxin receptor AUXIN-BINDING PROTEIN 1 (ABP1) inhibits the clathrin-mediated internalization of PIN proteins. ABP1 acts as a positive factor in clathrin recruitment to the plasma membrane, thereby promoting endocytosis. Auxin binding to ABP1 interferes with this action and leads to the inhibition of clathrin-mediated endocytosis. Our study demonstrates that ABP1 mediates a nontranscriptional auxin signaling that regulates the evolutionarily conserved process of clathrin-mediated endocytosis and suggests that this signaling may be essential for the developmentally important feedback of auxin on its own transport.

Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development.

Abstract.The plant hormone auxin plays crucial roles in regulating plant growth development, including embryo and root patterning, organ formation, vascular tissue differentiation and growth responses to environmental stimuli. Asymmetric auxin distribution patterns have been observed within tissues, and these so-called auxin gradients change dynamically during different developmental processes. Most auxin is synthesized in the shoot and distributed directionally throughout the plant. This polar auxin transport is mediated by auxin influx and efflux facilitators, whose subcellular polar localizations guide the direction of auxin flow. The polar localization of PIN auxin efflux carriers changes in response to developmental and external cues in order to channel auxin flow in a regulated manner for organized growth. Auxin itself modulates the expression and subcellular localization of PIN proteins, contributing to a complex pattern of feedback regulation. Here we review the available information mainly from studies of a model plant, Arabidopsis thaliana, on the generation of auxin gradients, the regulation of polar auxin transport and further downstream cellular events.

Subcellular Trafficking of the Arabidopsis Auxin Influx Carrier AUX1 Uses a Novel Pathway Distinct from PIN1

The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.

ARF GEF-Dependent Transcytosis and Polar Delivery of PIN Auxin Carriers in Arabidopsis

Cell polarity manifested by the polar cargo delivery to different plasma-membrane domains is a fundamental feature of multicellular organisms. Pathways for polar delivery have been identified in animals; prominent among them is transcytosis, which involves cargo movement between different sides of the cell [1]. PIN transporters are prominent polar cargoes in plants, whose polar subcellular localization determines the directional flow of the signaling molecule auxin [2, 3]. In this study, we address the cellular mechanisms of PIN polar targeting and dynamic polarity changes. We show that apical and basal PIN targeting pathways are interconnected but molecularly distinct by means of ARF GEF vesicle-trafficking regulators. Pharmacological or genetic interference with the Arabidopsis ARF GEF GNOM leads specifically to apicalization of basal cargoes such as PIN1. We visualize the translocation of PIN proteins between the opposite sides of polarized cells in vivo and show that this PIN transcytosis occurs by endocytic recycling and alternative recruitment of the same cargo molecules by apical and basal targeting machineries. Our data suggest that an ARF GEF-dependent transcytosis-like mechanism is operational in plants and provides a plausible mechanism to trigger changes in PIN polarity and hence auxin fluxes during embryogenesis and organogenesis.

Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role.

Microtubule plus-ends reveal essential links between intracellular polarization and localized modulation of endocytosis during division-plane establishment in plant cells.

BackgroundA key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane.ResultsEarly mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes.ConclusionOur observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event.

Cellular and Molecular Requirements for Polar PIN Targeting and Transcytosis in Plants

The polar, sub-cellular localization of PIN auxin efflux carriers determines the direction of intercellular auxin flow, thus defining the spatial aspect of auxin signalling. Dynamic, transcytosis-like relocalizations of PIN proteins occur in response to external and internal signals, integrating these signals into changes in auxin distribution. Here, we examine the cellular and molecular mechanisms of polar PIN delivery and transcytosis. The mechanisms of the ARF-GEF-dependent polar targeting and transcytosis are well conserved and show little variations among diverse Arabidopsis ecotypes consistent with their fundamental importance in regulating plant development. At the cellular level, we refine previous findings on the role of the actin cytoskeleton in apical and basal PIN targeting, and identify a previously unknown role for microtubules, specifically in basal targeting. PIN protein delivery to different sides of the cell is mediated by ARF-dependent trafficking with a previously unknown complex level of distinct ARF-GEF vesicle trafficking regulators. Our data suggest that alternative recruitment of PIN proteins by these distinct pathways can account for cell type- and cargo-specific aspects of polar targeting, as well as for polarity changes in response to different signals. The resulting dynamic PIN positioning to different sides of cells defines a three-dimensional pattern of auxin fluxes within plant tissues.

Cell Plate Restricted Association of DRP1A and PIN Proteins Is Required for Cell Polarity Establishment in Arabidopsis

The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [2, 3], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4,xa05]. The PIN polar localization results from clathrin-mediated endocytosis (CME) from the plasma membrane and subsequent polar recycling [6]. The Arabidopsis genome encodes two groups of dynamin-related proteins (DRPs) that show homology to mammalian dynamin-a protein required for fission of endocytic vesicles during CME [7, 8]. Here we show by coimmunoprecipitation (coIP), bimolecular fluorescence complementation (BiFC), and Förster resonance energy transfer (FRET) that members of the DRP1 group closely associate with PIN proteins at the cell plate. Localization and phenotypic analysis of novel drp1 mutants revealed a requirement for DRP1 function in correct PIN distribution and in auxin-mediated development. We propose that rapid and specific internalization of PIN proteins mediated by the DRP1 proteins and the associated CME machinery from the cell plate membranes during cytokinesis is an important mechanism for proper polar PIN positioning in interphase cells.