Subcellular Cycling of PIN Proteins + BFA - BFA Geldner et al., 2001 UV-activated PIN2-EosFP Protoplasts + BFA - BFA Dhonukshe et al., 2007 Cellular Polarity of PIN Localisation and Directionality of Intercellular Auxin Flow Molecular Components of PIN Polar Targeting Ser/Thr protein kinase PINOID (PID) Christensen et al., 2000; Benjamins et al., 2001; Friml et al., 2004 pinoidCol-0 PP2A Phosphatase and PIN Apical-Basal Targeting Michniewicz et al., 2007 PID Phosphorylates PINs Phosphorylation assays in protoplast in vitro phosphorylation Michniewicz et al., 2007 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 Friml et al., 2004 Polar delivery of proteins Animal Plant “Plant Epithelium”: root-soil interface PINs IBA Cd BPEN3 Langowski et al., 2010 Mechanistic Insight into Polar Targeting in Plants • Sterol-dependent reduced lateral diffusion • Super polar exocytosis PIN2-GFP Lateral Diffusion PIN2-GFP 10 min 10 s/frameunpublished Super Polar Delivery PIN2-GFP unpublished Internalisation Hot Spots unpublished Pick-up Service at Internalisation Hot Spots unpublished Mechanistic insights into cell polarity in plants unpublished Genetic approaches: Forward Genetic Screens Tanaka et al., 2009, Feraru et al., 2010; Feraru et al., 2011, unpublished Endocytosis ben ...5 Exocytosis bex ...8 Vacuolar Function deg ...3 Apical/Basal Targeting dpt ...4 Outer Polar Targeting dol ...2 Auxin – Endocytosis eon ...6 Chemical Genetic Screens Endocytosis Polar Targeting Reverse Genetics Marker:FP mutant lines novel genes Deep sequencing EMS mutagenesis. Epifluorescence Screening So far mapped in the lab: 11 mutants Beauty of forward genetics gravitropic mutant - epid cells agravitropicgravitropic WT - epid cells P2::P1:HA in pin2 - epid cells Polarity screen - design PIN1 PIN1 PIN2 PIN2 PIN1 PIN1 EMS Feraru et al., 2011 Elena P2P1HAWT-COL A good hit!! Polarity screen Elena Feraru et al., 2011 P2::P1:HA repp1 repp2 repp3 repp4 repp5 regulator of PIN polarity (repp) Feraru et al., 2011 repp3 rescues gravitropism and PIN polarity Gravistimulated Wild type epidermis repp3 (50-60%) PIN2::PIN1 PIN2::PIN1 x dpt3 Feraru et al., 2011 REPP3 encodes cellulose synthase 3 (CESA3/CEV1/IXR1/ELI1) je5(P2P1HAeir1-1)x repp3 F1 repp3 nga139 PHYC LFY3 Chromosome 5 BAC K2A11 BAC K2A11 1.52Mb 1.53Mb 1.54Mb 1.55Mb At5g05170.1 repp3je5 Mapping Allelic test PIN2::PIN1-HA repp3 je5(PIN2::PIN1HAeir1-1)xdpt3 F1 (43%) PIN2::PIN1-HA dpt3 repp3 Feraru et al., 2011 Degradation of cell wall results in loss of polarity 10’ protoplasting 15’ protoplasting PIN2::PIN2-GFP Feraru et al., 2011 Before After 30 min After 1.5 hours PIN2::PIN2-GFP After 1.5 hours PIN proteins are attached to cell wall Partial degradation of cell wall Feraru et al., 2011 Partial degradation of cell wall Polar cargos are attached to cell wall LTI6b Before After 2 hoursAfter 2 hours PIN 1 PIP2 a Feraru et al., 2011 PIN Proteins Display Reduced Mobility FRAP analysis Mean StDev Repetitio n T test two tail PIN2 untreated 14.1 5.3 18 PIN2 IX treated 20.1 5.8 11 0.00545 Feraru et al., 2011 Patterning in Plant Development Jiří Friml, ZMBP Tübingen Plants and Animals Live Different Lives Arabidopsis Embryogenesis Mutant screen at seedling level Auxin in Early Embryogenesis DR5::GFP IAA localisation DR5::GFP IAA localisation Auxin in Embryogenesis GUS mRNA Protein PIN7 in Embryogenesis PIN1 Analysis of DR5 activity in pin7 Col-0 pin7 NPA DR5 Embryo Phenotype of pin7 Mutants Col-0 pin7 PIN1 in Early Embryogenesis GUS mRNA Protein pin1 30,4% Enk 6,1% Phenotypes of pin Multiple Mutants 4x7 1x3x4 1x3x4x7 1x3x4x7 gnom gnom 1x3x4x7 1x3x4x7 1x3x4x7 1x3x4x7 Auxin and Embryogenesis GlobularTwo-Cell GNOM PIN1 Apical pole specification Root pole specification MP BDL PIN1 MP BDL PIN4 PIN7 PIN7 Organogenesis Auxin in Cotyledon Formation PIN1 MP BDL GNOM DR5 BFA pins gnom IAA pin1 PIN1 Polarity in Cotyledon Formation Outer layer Globular Heart HeartHeart Inner layers BFA treatmen Auxin in Flower and Leave Formation PIN1 localisation pin1 DR5rev::GFP + NPA + NPA Lateral Root Development Lateral Root Development in Time DR5 in Lateral Root Formation DR5rev::GUS IAA PIN1:GFP PIN1+ NPA Relocation > Gradients > Primordia PIN1 primordiaDR5 CycBmargins + IAA + IAA + NPA Wt pin1,3 pin1,3,4 + IAA + IAA Common module for organ formation Aerial organogenesis Underground organogenesis Cotyledons, leaves, flowers, ral organs, ovules, integuments Lateral roots PIN-dependent Auxin Gradients in Plant Development TROPISMS Tropisms: „Movements“ in Plants Phototropism Gravitropism Col-0 Atpin3 PIN3 – Lateral Auxin Transport pin3 mutant PIN3 protein Auxin response Friml et al. 2002 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 PIN3 Polarity Switch in Gravitropic Response Nucleus StatolitsAtPIN3 Root turned on its side Friml et al. 02 FRAP of PIN3-GFP Gravity-induced PIN3 transcytosis Kleine-Vehn et al., 2010 PIN3 in Phototropic Response Ding et al., 2011 Light-dependent PIN3 relocation 0‘ 2 hours 6 hours Auxin response Col-0 Shoot gravitropic response Bending PIN3 polarizationDR5 response unpublished DNA RNA PID ARF GN BIG Cell-biological Determinants Auxin Gradients Signal s Gravity Light Tissue context Auxin Friml et al. 2002 unpublished Develop. context Benková et al. 2003 Friml et al. 2003 Reinhardt et al. 2003 Wisniewska et al., 2006 Sauer et al., 2006Sauer et al., 2006 Paciorek et al., 2005 unpublished unpublished