Regulation of the polarity of protein trafficking by phosphorylation

The asymmetry of environmental stimuli and the execution of developmental programs at the organism level require a corresponding polarity at the cellular level, in both unicellular and multicellular organisms. In plants, cell polarity is important in major developmental processes such as cell division, cell enlargement, cell morphogenesis, embryogenesis, axis formation, organ development, and defense. One of the most important factors controlling cell polarity is the asymmetric distribution of polarity determinants. In particular, phosphorylation is implicated in the polar distribution of the determinant protein factors, a mechanism conserved in both prokaryotes and eukaryotes. In plants, formation of local gradients of auxin, the morphogenic hormone, is critical for plant developmental processes exhibiting polarity. The auxin efflux carriers PIN-FORMEDs (PINs) localize asymmetrically in the plasma membrane and cause the formation of local auxin gradients throughout the plant. The asymmetry of PIN distribution in the plasma membrane is determined by phosphorylationmediated polar trafficking of PIN proteins. This review discusses recent studies on the role of phosphorylation in polar PIN trafficking.     

Cell polarity: Regulators and mechanisms in plants

Cell polarity plays an important role in a wide range of biological processes in plant growth and development.Cell polarity is manifested as the asymmetric distribution of molecules,for example,proteins and lipids,at the plasma membrane and inside of a cell.Here,we summarize a few polarized proteins that have been characterized in plants and we review recent advances towards understanding the molecular mechanism for them to polarize at the plasma membrane.Multiple mechanisms,including membrane trafficking,cytoskeletal activities,and protein phosphorylation,and so forth define the polarized plasma membrane domains.Recent discoveries suggest that the polar positioning of the proteo-lipid membrane domain may instruct the formation of polarity complexes in plants.In this review,we highlight the factors and regulators for their functions in establishing the membrane asymmetries in plant development.Furthermore,we discuss a few outstanding questions to be addressed to better understand the mechanisms by which cell polarity is regulated in plants.     

Recycling,clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.     

Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity

The polarization of yeast and animal cells relies on membrane sterols for polar targeting of proteins to the plasma membrane, their polar endocytic recycling and restricted lateral diffusion. However, little is known about sterol function in plant-cell polarity. Directional root growth along the gravity vector requires polar transport of the plant hormone auxin. In Arabidopsis, asymmetric plasma membrane localization of the PIN-FORMED2 (PIN2) auxin transporter directs root gravitropism. Although the composition of membrane sterols influences gravitropism and localization of two other PIN proteins, it remains unknown how sterols contribute mechanistically to PIN polarity. Here, we show that correct membrane sterol composition is essential for the acquisition of PIN2 polarity. Polar PIN2 localization is defective in the sterol-biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) which displays altered sterol composition, PIN2 endocytosis, and root gravitropism. At the end of cytokinesis, PIN2 localizes initially to both newly formed membranes but subsequently disappears from one. By contrast, PIN2 frequently remains at both daughter membranes in endocytosis-defective cpi1-1 cells. Hence, sterol composition affects post-cytokinetic acquisition of PIN2 polarity by endocytosis, suggesting a mechanism for sterol action on establishment of asymmetric protein localization.     

PIN polarity maintenance by the cell wall in Arabidopsis

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.     

Polar-localized NPH3-like proteins regulate polarity and endocytosis of PIN-FORMED auxin efflux carriers

PIN-FORMED (PIN)-dependent auxin transport is essential for plant development and its modulation in response to the environment or endogenous signals. A NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)-like protein, MACCHI-BOU 4 (MAB4), has been shown to control PIN1 localization during organ formation, but its contribution is limited. The Arabidopsis genome contains four genes, MAB4/ENP/NPY1-LIKE1 (MEL1), MEL2, MEL3 and MEL4, highly homologous to MAB4. Genetic analysis disclosed functional redundancy between MAB4 and MEL genes in regulation of not only organ formation but also of root gravitropism, revealing that NPH3 family proteins have a wider range of functions than previously suspected. Multiple mutants showed severe reduction in PIN abundance and PIN polar localization, leading to defective expression of an auxin responsive marker DR5rev::GFP. Pharmacological analyses and fluorescence recovery after photo-bleaching experiments showed that mel mutations increase PIN2 internalization from the plasma membrane, but affect neither intracellular PIN2 trafficking nor PIN2 lateral diffusion at the plasma membrane. Notably, all MAB4 subfamily proteins show polar localization at the cell periphery in plants. The MAB4 polarity was almost identical to PIN polarity. Our results suggest that the MAB4 subfamily proteins specifically retain PIN proteins in a polarized manner at the plasma membrane, thus controlling directional auxin transport and plant development.     

Vesicular cycling mechanisms that control auxin transport polarity

The polar transport of auxin controls many important plant growth and developmental processes. The polarity of auxin movement has long been suggested to be mediated by asymmetric distribution of auxin transport proteins, yet, until recently, little was known about the mechanisms that establish protein asymmetry in auxin-transporting cells. Now, a recent paper provides significant insight into the mechanism by which the GNOM protein controls the cycling of an auxin efflux carrier protein, PIN1, between the endosome and the plasma membrane. The dynamic movement of auxin transport proteins between internal compartments and the plasma membrane suggests mechanisms for alterations in auxin transport polarity in response to changing developmental or environmental regulation.     

PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte

The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.     

Overexpression of partner of numb induces asymmetric distribution of the PI4P 5-Kinase Skittles in mitotic sensory organ precursor cells in Drosophila

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::Pon(LD)) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::Pon(LD) is often used as a polarity marker in dividing progenitors.     

Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.     

Lipid rafts in lymphocyte activation and migration

Functional polarization of leukocytes is a requisite to accomplish immune function. Immune synapse formation or chemotaxis requires asymmetric redistribution of membrane receptors, signaling molecules and the actin cytoskeleton. There is increasing evidence that compartmentalization of the plasma membrane into distinct lipid microdomains is pivotal in establishing and maintaining leukocyte polarity. Specific rafts assemble into large-scale domains to create plasma membrane asymmetries at specific cell locations, thus coordinating temporally and spatially cell signaling in these processes. In this review we discuss the roles of lipid rafts as organizers of T lymphocyte polarity during cell activation and migration.     

Drosophila Ric-8 is essential for plasma-membrane localization of heterotrimeric G proteins

Heterotrimeric G proteins act during signal transduction in response to extracellular ligands. They are also required for spindle orientation and cell polarity during asymmetric cell division. We show here that, in Drosophila, both functions require the Galpha interaction partner Ric-8. Drosophila Ric-8 is a cytoplasmic protein that binds both the GDP- and GTP-bound form of the G-protein alpha-subunit Galphai. In ric-8 mutants, neither Galphai nor its associated beta-subunit Gbeta13F are localized at the plasma membrane, which leads to their degradation in the cytosol. During asymmetric cell division, this leads to various defects: apico-basal polarity is not maintained, mitotic spindles are misoriented and the size of the two daughter cells becomes nearly equal. ric-8 mutants also have defects in gastrulation that resemble mutants in the Galpha protein concertina or the extracellular ligand foldedgastrulation. Our results indicate a model in which both receptor-dependent and receptor-independent G-protein functions are executed at the plasma membrane and require the Ric-8 protein.     

An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.     

Mechanisms of auxin-dependent cell and tissue polarity

The establishment of cellular asymmetries and their coordination within the tissue layer are fundamental to the development of multicellular organisms. In plants, the induction and coordination of cell polarity have classically been attributed to involve the hormone auxin and its flow. However, the underlying mechanisms have only recently been addressed at the molecular level. We review progress on the characterisation of the auxin influx and efflux carrier properties of specific plasma membrane proteins, mechanisms underlying their delivery to and internalisation from the plasma membrane, their endocytic transport and degradation. We discuss mechanisms of auxin gradient, transport and response action during the coordination of polarity, along with the downstream involvement of Rho-of-plant small GTPases during the execution of cell polarity.     

Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport--phlorizin, which inhibits brush border (apical) Na+/glucose symporters, and ouabain, which inhibits Na+/K+-ATPase, thereby modifying the transmembrane Na+ gradient--were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain (L.M. Wiley, 1984, Dev. Biol. 105, 330-342) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10(-5) M; phlorizin, 10(-6) M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10(-4) M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.     

Polar delivery in plants; commonalities and differences to animal epithelial cells

Although plant and animal cells use a similar core mechanism to deliver proteins to the plasma membrane, their different lifestyle, body organization and specific cell structures resulted in the acquisition of regulatory mechanisms that vary in the two kingdoms. In particular, cell polarity regulators do not seem to be conserved, because genes encoding key components are absent in plant genomes. In plants, the broad knowledge on polarity derives from the study of auxin transporters, the PIN-FORMED proteins, in the model plant Arabidopsis thaliana. In animals, much information is provided from the study of polarity in epithelial cells that exhibit basolateral and luminal apical polarities, separated by tight junctions. In this review, we summarize the similarities and differences of the polarization mechanisms between plants and animals and survey the main genetic approaches that have been used to characterize new genes involved in polarity establishment in plants, including the frequently used forward and reverse genetics screens as well as a novel chemical genetics approach that is expected to overcome the limitation of classical genetics methods.     

Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.     

Ups and downs of tissue and planar polarity in plants

The polar orientation of cells within a tissue is an intensively studied research area in animal cells. The term planar polarity refers to the common polar arrangement of cells within the plane of an epithelium. In plants, the subcellular analysis of tissue polarity has been limited by the lack of appropriate markers. Recently, research on plant tissue polarity has come of age. Advances are based on studies of Arabidopsis patterning, cell polarity and auxin transport mutants employing the coordinated, polar localization of auxin transporters and the planar polarity of root epidermal hairs as markers. These approaches have revealed auxin transport and response, vesicular trafficking, membrane sterol and cytoskeletal requirements of tissue polarity. This review summarizes recent progress in research on vascular tissue and planar epidermal polarity in the Arabidopsis root and compares it to findings on planar polarity in animals and cell polarity in yeast.