Cell Biology of Auxin Transport gnom Phenotype is Mimicked by Alteration of Auxin Homeostasis split collar cotyledon collar cotyledon cucumber ball/egg data from Hadfi et al., 1998 gnom embryos display variable defects in the apical-basal axis of polarity split collar cotyledon Wild-type and gnom Arabidopsis seedling cucumber ball/egg collar cotyledon GNOM VectorGNOM Vector 25°C 32°C GNOM rescues gea2, gea1-19 temperature sensitive yeast strains GNOM Encodes a Homolog of Yeast ARF G-protein Exchange Factors GNOM Has BFA Sensitive GDP/GTP Exchange Activity on ARF 0 0,5 1 1,5 2 2,5 3 relativeGDP/GTPexchangerate ARF+GNOM ARF+GNOM 100MBFA ARF+GNOM 300MBFA ARFalone Small GTPases ­ Universal Switchers Secretory pathway Establishment of coordinated PIN1 polarity is disrupted in gnom embryos gn wt wt gn PIN Proteins Cycling BFA reveals subcellular movement of PIN1 PIN1 cycles between endosome and plasma membrane PIN1 cycling is actin but not tubulin dependent Auxin transport inhibitors interfere with protein trafficking Role for cycling ­ root gravitropism + BFA - BFA PIN1 Subcellular Movement untreated Intracellular Cycling of AtPIN1 is Independent of Newly Synthesised Protein Cycloheximide Cycloheximide + BFA BFA + Cycloheximide PIN1 Cycling is Actin Dependent Cytochelasin D Cytochelasin D + BFA BFA + Cytochelasin D GNOM regulates intracellular transport of auxin efflux carriers, such as PIN1. How can we show this? Hypothesis: BFA resistant? BFA resistant? VLS FA II M LNTSSH VLS YS II L LNTSSH GNOM * YS to FA GNOM * M to L At5g19610 At5g39500 GNOM BFA sensitive? BFA resistant? BFA sensitive VLS YS II M LNTSSH VLA YS II L LNTDQH ILC YS LI M LNTDQH VLS FA VI M LNTSLH VLS FA II M LNTSLH SLA YA VI M LNTDQH ARNO CYTOHESIN GBF1 BFA resistant BFA resistant BFA resistant S. cerevisiae Gea1p BFA sensitiveVLS YS II M LNTSSH Gea1p * YS to FA Gea1p * M to L VLS FA II M LNTSSH VLS YS II L LNTSSH BFA resistant BFA resistant Engineering BFA-resistant GNOM H. sapiens Arabidopsis Amino Acid Residues of ARF-GEFs Conferring BFA Resistance The engineered GNOM construct GNOM localises to the cytosol and some endomembrane compartment control BFA, 60 min GNwt-myc line BFA, 60 min GNM696L-myc line GNOM partially co-localises with FM4-64, an endocytic tracer GNOM and PIN1 co-localise after BFA treatment BFA 50 M, 45 min Col background BFA 50 M, 45 min GNOMBFA res. transgenic line PIN1 Localisation in BFA-resistant GNOM sensitive (control) resistant GNOM BFA-resistant lines display BFA insensitive auxin flux GNwt-myc line GNM696L-myc line 0 0,05 0,1 0,15 0,2 0,25 0 5 10 15 20 0 0,05 0,1 0,15 0,2 0,25 0 5 10 15 20 %auxinin2mmstemsegments Distance from source [mm] %auxinin2mmstemsegments Distance from source [mm] The root gravitropic response becomes BFA-resistant in GNOM BFA-resistant lines Side root formation and primary root elongation also become BFA-resistant in GNOM BFA-resistant lines Sensitive line Resistant line Sensitive line Resistant line GNOM BFA-resistant lines lead to strong phenotypic BFA-resistance GNwt-myc line GNOMM696L-myc line BFA 5 M exocytosis endocytosis BFA / TIBA TIBA Dynamic Movement of PIN1 UV-activated PIN2-EosFP Reveals Constitutive PIN Endocytosis Protoplasts + BFA - BFA Dhonukshe et al., 2007 Incidence of Clathrin and Clathrin-coated Pits at Plasma Membrane of Arabidopsis Cells anti-Clathrin Different Stages of Forming Clathrin-coated Vesicles at Plasma Membrane Dhonukshe et al., 2007 Genetic Interference with Clathrin Function Inhibits PIN Endocytosis Dhonukshe et al., 2007 Molecular Components of PIN Polarity Control Role of PINOID protein kinase Regulation of PIN Polarity AUX1/PIN1 PIN1/pin1 What are determinants of polarity of PIN localisation? Does PIN polarity determines the direction of auxin flow? Molecular mechanism of the polar targeting pathway in plants? PIN2pr::PIN2:HA PIN2pr::PIN1:HA PIN2pr::PIN3:HA PIN2pr::PIN1:GFP PIN-specific Signals for Polarity apical basal localization basal localization apical basal basal localization apical basal localization PIN Polarity Determines Direction of Auxin Flow eir1 PIN2::PIN2:HA PIN2::PIN1:HA PIN2::PIN1:GFP-3 DR5rev::GFP gravitropism PIN2::PIN1:GFP-2 Role of PINOID Kinase in PIN Polar Targeting PIN4 PIN1 Col-0 35S::PID inducible PIDCol-0 Col-0 pinoid PINOID kinase loss-of-function > basal PIN targeting Role of PID in Controlling PIN Polarity > Auxin Flow > Patterning Col-0 RPS5::PID Col-0 RPS5::PID RPS5::PID seedlings PIN1 PIN1 DR5 DR5 Link between constitutive recycling and cell polarity Two Step Mechanism of PIN Polarity Establishment unpublished BFA washout: Non-polar recovery of PIN2 FRAP: Non-polar targeting of PIN1 Attenuation of Rab5 Function Inhibits Endocytosis and Leads to PIN Polarity Defects unpublished FM4-64 uptake PIN1 polarity in embryogenesis Inhibition of Endocytosis Leads to Transformation of Leaves to Roots unpublished Rab5 dominant negatime Vps9a loss-of-function PIN2-GFP QC25 Inhibition of Endocytosis Causes Transformation of Embryonic Leaves into Root Fates unpublished in situ mRNA of root markers DR5 auxin response TROPISMS Phototropism Gravitropism - NPA - NPA+ NPA + NPA Asymmetric Auxin Distribution Underlies Tropisms Atpin3-1 Atpin3-2 AtPIN3 Gene and Atpin3 Knock-Out Mutants Col-0 Col-0Atpin3 Atpin3 Hypocotyl phototropism Hypocotyl gravitropism 0 -9090 180 Atpin3 0 -9090 180 Col-0 Root gravitr. Atpin3 Tropisms Col-0 Atpin3 Col-0 (NPA) light dark DR5 Atpin3 Hypocotyl and Apical Hook AtPIN3::GUS Transgenics The AtPIN3 protein in situ RNA hybridisation AtPIN3 in Hypocotyl The AtPIN3 protein in situ RNA hybridisation AtPIN3 in Inflorescence Axis Col-0 Atpin3 PIN3 ­ Lateral Auxin Transport pin3 phototropism PIN3 protein DR5 - phototropism pin2Col-0 PIN2 ­ Root Gravitropism PIN2 protein PIN3 Actin Dependent Cycling + BFA - BFA + Cyt D - BFA + BFA PIN3 Immunogold Electron Microscopy gravity stimulated gravity + NPA DR5rev::GFP Root Gravitropism PIN2 localizationStatoliths - gravity perception Relocation of PIN3 during Gravitropism 0 min PIN3 in vertical root PIN3 in root on its side 2 min Nucleus Statolits Vascular tissue Root cap Vertical root Root turned on its side AtPIN3 Model for Root Gravitropism DNA RNA PID ARF GN BIG Cell-biological Determinants Auxin Gradients Plant Development