Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.     

Auxin transport inhibitors block PIN1 cycling and vesicle trafficking

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.     

Auxin inhibits endocytosis and promotes its own efflux from cells

One of the mechanisms by which signalling molecules regulate cellular behaviour is modulating subcellular protein translocation. This mode of regulation is often based on specialized vesicle trafficking, termed constitutive cycling, which consists of repeated internalization and recycling of proteins to and from the plasma membrane. No such mechanism of hormone action has been shown in plants although several proteins, including the PIN auxin efflux facilitators, exhibit constitutive cycling. Here we show that a major regulator of plant development, auxin, inhibits endocytosis. This effect is specific to biologically active auxins and requires activity of the Calossin-like protein BIG. By inhibiting the internalization step of PIN constitutive cycling, auxin increases levels of PINs at the plasma membrane. Concomitantly, auxin promotes its own efflux from cells by a vesicle-trafficking-dependent mechanism. Furthermore, asymmetric auxin translocation during gravitropism is correlated with decreased PIN internalization. Our data imply a previously undescribed mode of plant hormone action: by modulating PIN protein trafficking, auxin regulates PIN abundance and activity at the cell surface, providing a mechanism for the feedback regulation of auxin transport.     

Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis

Long-standing models propose that plant growth responses to light or gravity are mediated by asymmetric distribution of the phytohormone auxin. Physiological studies implicated a specific transport system that relocates auxin laterally, thereby effecting differential growth; however, neither the molecular components of this system nor the cellular mechanism of auxin redistribution on light or gravity perception have been identified. Here, we show that auxin accumulates asymmetrically during differential growth in an efflux-dependent manner. Mutations in the Arabidopsis gene PIN3, a regulator of auxin efflux, alter differential growth. PIN3 is expressed in gravity-sensing tissues, with PIN3 protein accumulating predominantly at the lateral cell surface. PIN3 localizes to the plasma membrane and to vesicles that cycle in an actin-dependent manner. In the root columella, PIN3 is positioned symmetrically at the plasma membrane but rapidly relocalizes laterally on gravity stimulation. Our data indicate that PIN3 is a component of the lateral auxin transport system regulating tropic growth. In addition, actin-dependent relocalization of PIN3 in response to gravity provides a mechanism for redirecting auxin flux to trigger asymmetric growth.     

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.     

生长素调控极性运输载体PIN2质膜丰度与降解

主要观察了外源生长素长时间(8 h)处理对拟南芥生长素极性输出载体PIN2-GFP质膜丰度的影响,低温、KCN处理和生长素受体突变对生长素调控质膜PIN2-GFP丰度的影响,以及液泡H+-ATPase抑制剂ConA对野生型和生长素受体四突变体tir1afb1,2,3液泡中PIN2-GFP积累的影响.结果表明:外源生长素通过其受体TIR1/AFB介导的信号途径下调质膜PIN2-GFP的丰度,促进PIN2-GFP的内吞、胞内运输和液泡降解.暗示生长素通过TIR1/AFB介导的信号途径反馈调控质膜PIN2-GFP的水平,从而阻止了胞内生长素的过多输出.     

Regulated portals of entry into the cell

The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.     

AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis

Polarized cellular distribution of the phytohormone auxin and its carriers is essential for normal plant growth and development. Polar auxin transport is maintained by a network of auxin influx (AUX) and efflux (PIN) carriers. Both auxin transport and PIN protein cycling between the plasma membrane and endosomes require the activity of the endosomal GNOM; however, intracellular routes taken by these carriers remain largely unknown. Here we show that Arabidopsis thaliana SORTING NEXIN 1 (AtSNX1) is involved in the auxin pathway and that PIN2, but not PIN1 or AUX1, is transported through AtSNX1-containing endosomes. We demonstrate that the snx1-null mutant exhibits multiple auxin-related defects and that loss of function of AtSNX1 severely enhances the phenotype of a weak gnom mutant. In root cells, we further show that AtSNX1 localizes to an endosomal compartment distinct from GNOM-containing endosomes, and that PIN2 accumulates in this compartment after treatment with the phosphatidylinositol-3-OH kinase inhibitor wortmannin or after a gravity stimulus. Our data reveal the existence of a novel endosomal compartment involved in PIN2 endocytic sorting and plant development.     

Dishevelled controls cell polarity during Xenopus gastrulation

Although cell movements are vital for establishing the normal architecture of embryos, it is unclear how these movements are regulated during development in vertebrates. Inhibition of Xenopus Dishevelled (Xdsh) function disrupts convergent extension movements of cells during gastrulation, but the mechanism of this effect is unclear, as cell fates are not affected. In Drosophila, Dishevelled controls both cell fate and cell polarity, but whether Dishevelled is involved in controlling cell polarity in vertebrate embryos has not been investigated. Here we show, using time-lapse confocal microscopy, that the failure of cells lacking Xdsh function to undergo convergent extension results from defects in cell polarity. Furthermore, Xdsh mutations that inhibit convergent extension correspond to mutations in Drosophila Dishevelled that selectively perturb planar cell polarity. Finally, the localization of Xdsh at the membrane of normal dorsal mesodermal cells is consistent with Xdsh controlling cell polarity. Our results show that polarized cell behaviour is essential for convergent extension and is controlled by vertebrate Dishevelled. Thus, a vertebrate equivalent of the Drosophila planar cell polarity signalling cascade may be required for normal gastrulation.     

组蛋白去乙酰化抑制剂对拟南芥根尖干细胞微环境的影响

根系发育是植物生长的重要组成部分,该过程由多种信号转导途径共同调节。组蛋白乙酰化和去乙酰化的动态变化对基因表达具有关键的调控作用,而对其在根发育中功能的研究还不深入。本研究通过组蛋白去乙酰化酶抑制剂Trichostatin A（TSA）处理拟南芥,发现其主根生长受到抑制,分生区细胞数目变少。显微观察的结果表明TSA影响了根尖干细胞微环境。根尖微环境调控相关因子SCR和SHR的表达受TSA处理的影响并不明显,而PLT1/PLT2的表达受TSA强烈抑制。我们对生长素运输途径的分析发现在TSA处理条件下,PIN1表达只受轻微影响,而PIN2表达量明显下调。pDR5:GFP的结果表明TSA可能引起生长素在根尖的积累和分布变化。综上所述,TSA影响了拟南芥根尖干细胞微环境的维持,表明组蛋白的去乙酰化在根发育过程中发挥着重要作用。     

On the spontaneous emergence of cell polarity

Diverse cell polarity networks require positive feedback for locally amplifying distributions of signalling molecules at the plasma membrane. Additional mechanisms, such as directed transport or coupled inhibitors, have been proposed to be required for reinforcing a unique axis of polarity. Here we analyse a simple model of positive feedback, with strong analogy to the 'stepping stone' model of population genetics, in which a single species of diffusible, membrane-bound signalling molecules can self-recruit from a cytoplasmic pool. We identify an intrinsic stochastic mechanism through which positive feedback alone is sufficient to account for the spontaneous establishment of a single site of polarity. We find that the polarization frequency has an inverse dependence on the number of signalling molecules: the frequency of polarization decreases as the number of molecules becomes large. Experimental observation of polarizing Cdc42 in budding yeast is consistent with this prediction. Our work suggests that positive feedback can work alone or with additional mechanisms to create robust cell polarity.     

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.     

Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos

Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.     

Asymmetric cell divisions promote stratification and differentiation of mammalian skin

The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation programme to form the barrier. The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells, most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane. Here we show that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. We further demonstrate that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3-LGN-Inscuteable complex and NuMA-dynactin to align the spindle.     

狭叶锦鸡儿(Caragana stenophylla Pojark)的胚胎发育

本文对狭叶锦鸡儿的胚胎发育过程进行了研究,主要结论如下:胚囊发育属蓼型.在卵与次生核周围的原生质中聚集多糖.卵细胞、助细胞具明显极性,幼期的卵细胞、助细胞和中央细胞具完整的细胞壁.受精时,次生核与卵器相贴近,卵与助细胞在合点端的细胞璧消失.受精后,合子在合点端重新产生不连续的细胞壁,胚胎发育属柳叶菜型.基细胞纵横分裂产生多细胞胚柄.球胚早期,胚柄呈泡沫状团块结构,珠孔端胚囊璧以及胚柄基部,与胚囊璧毗邻处都具明显的传递型细胞壁,胚柄细胞中积累网球状颗较,从球孔端至合点端其分布密度具一定梯度,珠孔端珠被细胞中富含多糖类物质,同时,与胚囊相邻的珠被细胞质膜与壁之间具壁旁体,显示珠被组织对早期胚胎发育的营养关系,胚乳细胞化完成后,胚柄即行退化.     

A mirror-symmetric cell division that orchestrates neuroepithelial morphogenesis

The development of cell polarity is an essential prerequisite for tissue morphogenesis during embryogenesis, particularly in the development of epithelia. In addition, oriented cell division can have a powerful influence on tissue morphogenesis. Here we identify a novel mode of polarized cell division that generates pairs of neural progenitors with mirror-symmetric polarity in the developing zebrafish neural tube and has dramatic consequences for the organization of embryonic tissue. We show that during neural rod formation the polarity protein Pard3 is localized to the cleavage furrow of dividing progenitors, and then mirror-symmetrically inherited by the two daughter cells. This allows the daughter cells to integrate into opposite sides of the developing neural tube. Furthermore, these mirror-symmetric divisions have powerful morphogenetic influence: when forced to occur in ectopic locations during neurulation, they orchestrate the development of mirror-image pattern formation and the consequent generation of ectopic neural tubes.     

Fidelity in planar cell polarity signalling

The polarity of Drosophila wing hairs displays remarkable fidelity. Each of the approximately 30,000 wing epithelial cells constructs an actin-rich prehair that protrudes from its distal vertex and points distally. The distal location and orientation of the hairs is virtually error free, thus forming a nearly perfect parallel array. This process is controlled by the planar cell polarity signalling pathway. Here we show that interaction between two tiers of the planar cell polarity signalling mechanism results in the observed high fidelity. The first tier, mediated by the cadherin Fat, dictates global orientation by transducing a directional signal to individual cells. The second tier, orchestrated by the 7-pass transmembrane receptor Frizzled, aligns each cell's polarity with that of its neighbours through the action of an intercellular feedback loop, enabling polarity to propagate from cell to cell. We show that all cells need not respond correctly to the presumably subtle signal transmitted by Fat. Subsequent action of the Frizzled feedback loop is sufficient to align all the cells cooperatively. This economical system is therefore highly robust, and produces virtually error-free arrays.