Markus Grebe - Establishment of Epidermal Cell and Tissue Polarity

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Many cells in diverse organisms need to acquire specific shapes in order to fulfil their functions within the organism. Cells commonly become polarised with one end being asymmetrically oriented in a certain direction. Moreover, the polarities of individual cells are often coordinated within the tissue layer. However, the mechanisms underlying the establishment of cell polarity and its coordination in plants are poorly understood. We have introduced the root epidermis of the model plant Arabidopsis thaliana as a system to study cell polarity and its coordination within the plane of the tissue layer (planar polarity). Here, we employ the polar localisation of Rho-of-plant (ROP) proteins to, and the polar outgrowth of root hairs from, near the basal ends of the outer epidermal membrane as polarity markers. Similarly, we use the polar localisation of the PIN2 auxin efflux carrier to apical epidermal membranes as a polarity indicator. By combining forward and reverse genetic, molecular, cell biological and physiological approaches, we have started to unravel mechanisms underlying the coordination and execution of cell polarity in root epidermal cells.
Markus_Grebe_portrait

Planar polarity in the Arabidopsis root epidermis We have established the Arabidopsis root epidermis as a system for the functional dissection of planar polarity of root-hair positioning. Our recent work shows that the combinatorial action of three genes (AUXIN RESISTANT1, AUX1, ETHYLENE INSENSITIVE2, EIN2 and GNOM) is required to coordinate root-hair positioning within the epidermal layer. These genes act to establish a concentration gradient of the plant hormone auxin in the root tip. This auxin gradient to some extent instructs the coordinated polar positioning of root hairs by acting prior to or at the level of the polar accumulation of small Rho-of-Plant (ROP) GTPases at the polar site of hair initiation (Fischer et al. 2006). However, the regulation of this gradient, the functional connection to downstream molecules, and the signalling cascade between auxin action and polar ROP or actin recruitment, remain a black box in terms of our understanding. We are currently characterising novel planar polarity mutants and interactions between known genes to elucidate the molecular genetic framework of factors regulating cell and tissue polarity in the root epidermis (Ikeda et al. 2009).

The confocal laser scanning microscope

Left panel, green fluorescence of the PIN2 protein fused to enhanced Green-Fluorescent Protein (PIN2-EGFP). Right panel, overlay with filipin-sterol fluorescence (false colour red). Upper row, arrowheads indicate polar PIN2-EGFP localisation only on the upper (apical) membranes of root epidermal cells. Middle row, epidermal cell during division. Lower row, an epidermal cell just after division. Note, both newly formed membranes show PIN2-EGFP fluorescence (Men et al. 2008)

Sterol function in epidermal cell polarity
In a second line of research, we address the role of membrane sterols in the establishment of epidermal cell polarity. The common plant sterol sitosterol is a lipid similar to cholesterol in animals. In collaboration with Ben Scheres's group, we have shown that correct membrane sterol composition is needed for cell polarity (Willemsen et al. 2003). Furthermore, I have established methods for the subcellular visualisation of plant sterols and their endocytic transport in Arabidopsis (Grebe et al. 2003). Recently, my group demonstrated that the correct sterol composition of plant membranes is essential for establishment of the specific polar localisation of the auxin transporter PIN2, just after cell division. During cell division, PIN2 is integrated into cell-plate membranes and is located at both newly formed membranes directly after cell division. In order to acquire its polar apical localisation, PIN2 needs to be removed from one end of the cell by internalisation (endocytosis) from the membrane. This can only occur when the membrane has the correct sterol composition. In a sterol biosynthesis mutant, which contains a large amount of precursor sterols that normally do not accumulate in the membrane, PIN2 is not appropriately removed from one end of the cell and apical PIN2 polarity cannot be established (Men et al. 2008). We are currently addressing the sub-cellular mechanisms underlying this phenomenon, as well as the role of sterols during cell division.

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Key publications

Ikeda Y, Men S, Fischer U, Stepanova AN, Alonso JM, Ljung K & Grebe M (2009). Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis. Nature Cell Biology 11: 731-738.

Men S, Boutté Y, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann MA, Moritz T & Grebe M (2008). Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nature Cell Biology 10: 237-244.

Fischer U, Ikeda Y, Ljung K, Serralbo O, Singh M, Heidstra R, Palme K, Scheres B & Grebe M (2006). Vectorial information for Arabidopsis planar polarity is mediated by combined AUX1, EIN2, and GNOM activity. Current Biology 16: 2143-2149.

Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze HJ, Rook MB & Scheres B (2003). Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Current Biology 13: 1378-1387.

Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K & Scheres B (2003). Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function. Plant Cell 15: 612-625.

Grebe M, Friml J, Swarup R, Ljung K, Sandberg G, Terlou M, Palme K, Bennett MJ & Scheres B (2002). Cell polarity signaling in Arabidopsis involves a BFA-sensitive auxin influx pathway. Current Biology 12: 329-334.

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May 2012

Friday, May 18, 2012
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