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First published online 31 January 2007
doi: 10.1242/dev.02792

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The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis

Joachim F. Uhrig^1,*, Moola Mutondo^1,*, Ilona Zimmermann^1,*, Michael J. Deeks^2,*, Laura M. Machesky³, Philipp Thomas¹, Silke Uhrig¹, Claudia Rambke¹, Patrick J. Hussey² and Martin Hülskamp^1,

¹ University of Köln, Botanical Institute III, Gyrhofstr. 15, 50931 Köln, Germany.
² The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK.
³ School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Birmingham B15 2TT, UK.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Molecular characterisation of the AtSCAR genes. (A) Gene structures of AtSCAR genes as confirmed by sequencing, and the location of mutations in their respective genes. Black boxes represent exons and black lines represent introns. The locations of the T-DNA insertions are indicated by arrows. To the right RT-PCRs using primers 3' of the insertion site are shown to confirm that no RNA transcript is produced by the relevant alleles. The expression of elongation factor1 was used as a control. (B) Expression analysis of AtSCAR1, AtSCAR2, AtSCAR3, AtSCAR4 and SCAR-LIKE by RT-PCR in various plant organs. The expression of actin was used as a positive control.

View larger version (89K): [in this window] [in a new window]   Fig. 2. Phenotypic characterisation of wild-type and atscar mutant plants. (A) Leaf overview of the mutant SALK-039449. (B) Leaf overview of the mutant SALK-124023. (C) Wild-type leaf. (D,E) Trichomes on leaves of the mutant SALK-039449 and SALK-124023, respectively. The mutants have slightly distorted trichomes and the branches are not regularly arranged as can be seen by the larger distance between the two branching points. (F) Wild-type trichome. (G,H) Dark-grown hypocotyl of the mutants SALK-039449 and SALK-124023, respectively. Some cells are disconnected from their neighbouring cells and bend outwards. (I) Dark grown wild-type hypocotyl. (J,K) 8-day-old dark-grown seedlings of the mutant SALK-039449 and SALK-124023, respectively. The seedlings already show developed petioles, cotyledons, and the first pair of true leaves. Note the long petioles. (L) 8-day-old dark-grown wild-type seedlings. (M) Overview of rosette leaves of the atscar2; atscar4 double mutant. (N) Overview of rosette leaves of the atscar4 single mutant. (O) Trichome of the atscar2; atscar4 double mutant displaying the distorted phenotype. (P) Trichome of the atscar4 single mutant. (Q) Measurements of the distance between the lower and upper branch point of trichomes. Only atscar2 mutants show differences to wild type. Scale bars: 1 mm in L, M and N; 0.4 mm in O; 0.2 mm in P.

View larger version (5K): [in this window] [in a new window]   Fig. 3. In vitro activation of bovine ARP2-ARP3. Graph showing pyrene fluorescence (arbitrary units) plotted against time (seconds) of actin polymerisation assays (10% pyrenelated actin) in the presence of 500 nM bovine ARP2-ARP3 with 160 nM GST (triangles), 160 nM AtSCAR2 GST-VCA only (squares), and ARP2-ARP3 with AtSCAR2 GST-VCA (diamonds).

View larger version (24K): [in this window] [in a new window]   Fig. 4. Localisation of AtSCAR2-GFP. In wild-type cells, SCAR2 protein fused to GFP (A), localises to the cytoplasm, which is labelled with DsRed (B). (C) is a merged image of (A) and (B). Scale bar: 20 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Protein interactions in planta detected by BiFC. Plasmids encoding fusion constructs with the N- or C-terminal part of YFP (YFP-N or -C), respectively, were co-bombarded into onion cells (A,B,D,F,G) or were transiently expressed in Arabidopsis protoplasts (C,E,H). The nuclear blue fluorescence of co-expressed GL3:CFP served as a transformation control (A,B,D,F). YFP fluorescence indicates direct interaction in planta between AtSCAR2 and ROP7 (A,C), AtSCAR2 and the constant active ROP7CA (B and E). The signal is localised to the plasma membrane. The dominant negative form ROP7DN does not interact with AtSCAR2 (D). Interactions between SPIKE1 and AtSCAR2 (F) and homodimerisation of HSPC300/BRICK1 (G,H) take place in the cytoplasm.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Protein interaction network of ARP2-ARP3 complex and regulatory components. The subunits of the ARP2-ARP3 complex and upstream regulatory components have been tested for direct interactions using the yeast two-hybrid system (black lines) and Bimolecular Fluorescence Complementation (BiFC, yellow lines). In the yeast two-hybrid system, interactions were detected by growth of yeast cells on triple dropout media containing 3-20 mM 3-AT.

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