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.     

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.     

Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis

Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.     

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.     

The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells

Development of plants and their adaptive capacity towards ever‐changing environmental conditions largely depend on the spatial distribution of the plant hormone auxin. At the cellular level, various internal and external signals are translated into specific changes in the polar, subcellular localization of auxin transporters from the PIN family thereby directing and redirecting the intercellular fluxes of auxin. The current model of polar targeting of PIN proteins towards different plasma membrane domains encompasses apolar secretion of newly synthesized PINs followed by endocytosis and recycling back to the plasma membrane in a polarized manner. In this review, we follow the subcellular march of the PINs and highlight the cellular and molecular mechanisms behind polar foraging and subcellular trafficking pathways. Also, the entry points for different signals and regulations including by auxin itself will be discussed within the context of morphological and developmental consequences of polar targeting and subcellular trafficking.     

SNARE proteins VAMP721 and VAMP722 mediate the post-Golgi trafficking required for auxin-mediated development in Arabidopsis

The plant hormone auxin controls many aspects of plant development. Membrane trafficking processes, such as secretion, endocytosis and recycling, regulate the polar localization of auxin transporters in order to establish an auxin concentration gradient. Here, we investigate the function of the Arabidopsis thaliana R-SNAREs VESICLE-ASSOCIATED MEMBRANE PROTEIN 721 (VAMP721) and VAMP722 in the post-Golgi trafficking required for proper auxin distribution and seedling growth. We show that multiple growth phenotypes, such as cotyledon development, vein patterning and lateral root growth, were defective in the double homozygous vamp721 vamp722 mutant. Abnormal auxin distribution and root patterning were also observed in the mutant seedlings. Fluorescence imaging revealed that three auxin transporters, PIN-FORMED 1 (PIN1), PIN2 and AUXIN RESISTANT 1 (AUX1), aberrantly accumulate within the cytoplasm of the double mutant, impairing the polar localization at the plasma membrane (PM). Analysis of intracellular trafficking demonstrated the involvement of VAMP721 and VAMP722 in the endocytosis of FM4-64 and the secretion and recycling of the PIN2 transporter protein to the PM, but not its trafficking to the vacuole. Furthermore, vamp721 vamp722 mutant roots display enlarged trans-Golgi network (TGN) structures, as indicated by the subcellular localization of a variety of marker proteins and the ultrastructure observed using transmission electron microscopy. Thus, our results suggest that the R-SNAREs VAMP721 and VAMP722 mediate the post-Golgi trafficking of auxin transporters to the PM from the TGN subdomains, substantially contributing to plant growth.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

PIN Auxin Efflux Carrier Polarity Is Regulated by PINOID Kinase-Mediated Recruitment into GNOM-Independent Trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development.     

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.     

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,?5]. 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 F?rster 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.     

Diversification of sterol methyltransferase enzymes in plants and a role for β‐sitosterol in oriented cell plate formation and polarized growth

Phytosterols are classified into C24‐ethylsterols and C24‐methylsterols according to the different C24‐alkylation levels conferred by two types of sterol methyltransferases (SMTs). The first type of SMT (SMT1) is widely conserved, whereas the second type (SMT2) has diverged in charophytes and land plants. The Arabidopsis smt2 smt3 mutant is defective in the SMT2 step, leading to deficiency in C24‐ethylsterols while the C24‐methylsterol pathway is unchanged. smt2 smt3 plants exhibit severe dwarfism and abnormal development throughout their life cycle, with irregular cell division followed by collapsed cell files. Preprophase bands are occasionally formed in perpendicular directions in adjacent cells, and abnormal phragmoplasts with mislocalized KNOLLE syntaxin and tubulin are observed. Defects in auxin‐dependent processes are exemplified by mislocalizations of the PIN2 auxin efflux carrier due to disrupted cell division and failure to distribute PIN2 asymmetrically after cytokinesis. Although endocytosis of PIN2–GFP from the plasma membrane (PM) is apparently unaffected in smt2 smt3, strong inhibition of the endocytic recycling is associated with a remarkable reduction in the level of PIN2–GFP on the PM. Aberrant localization of the cytoplasmic linker associated protein (CLASP) and microtubules is implicated in the disrupted endocytic recycling in smt2 smt3. Exogenous C24‐ethylsterols partially recover lateral root development and auxin distribution in smt2 smt3 roots. These results indicate that C24‐ethylsterols play a crucial role in division plane determination, directional auxin transport, and polar growth. It is proposed that the divergence of SMT2 genes together with the ability to produce C24‐ethylsterols were critical events to achieve polarized growth in the plant lineage.     

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.     

Plant cell polarity: sterols enter into action after cytokinesis

In yeast and animal cells, the sterol composition of membranes is a key factor that controls the polarity of membrane proteins by regulating their intracellular trafficking or lateral diffusion. A recent study in Nature Cell Biology demonstrates that plant sterols play a major role in the acquisition of cell polarity by modulating endocytosis after cell division.     

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.     

Quantitative Analysis of Membrane Trafficking in Regulation of Cdc42 Polarity

Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post‐Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP‐tag placed on the N‐terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post‐Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.      

Subcellular trafficking of PIN auxin efflux carriers in auxin transport

The directional transport of the plant hormone auxin is a unique process mediating a wide variety of developmental processes. Auxin movement between cells depends on AUX1/LAX, PGP and PIN protein families that mediate auxin transport across the plasma membrane. The directionality of auxin flow within tissues is largely determined by polar, subcellular localization of PIN auxin efflux carriers. PIN proteins undergo rapid subcellular dynamics that is important for the process of auxin transport and its directionality. Furthermore, various environmental and endogenous signals can modulate trafficking and polarity of PIN proteins and by this mechanism change auxin distribution. Thus, the subcellular dynamics of auxin transport proteins represents an important interface between cellular processes and development of the whole plant. This review summarizes our recent contributions to the field of PIN trafficking and auxin transport regulation.     

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.