Polymerized actin localization in arpc2/distorted2 mutant Arabidopsis thaliana leaf trichomes during the transition to branch elongation. The "distorted group" of trichome morphology mutants were originally identified as a class of mutants that failed to maintain polarized growth following branch initiation. Subsequent analyses indicated that distorted mutants are specifically affected in actin-dependent morphogenesis and fail to organize a polarized cytoskeleton in developing branches. Genetic experiments later proved that DISTORTED genes encode subunits of the evolutionarily conserved WAVE and ARP2/3 complexes. These heteromeric complexes translate Rho family small GTPase signals into actin filament nucleation and a coordinated growth response. Detailed methods: Intact Arabidopsis shoots were fixed in 100 mM PIPES/KOH pH 6.9, 5 mM EGTA, 4 mM MgCl2 (PEM) containing 2 % formaldehyde. Actin filaments were detected with overnight incubation with 0.2 µM Alexa Fluor 488® phalloidin. Confocal images were obtained using a Biorad Radiance 2100 confocal head mounted on a Nikon E800 stand, excitation wavelength was the 488 line of a mixed gas Argon ion laser. The objective used was a 60X 1.2 NA water immersion lens, with optical zoom. Acquisition Parameters: Z-series (0.3 µm step size, 29 planes) of confocal images of the actin cytoskeleton in the ARP2/3 complex subunit mutant arpc2/distorted2. This raw image stack accompanies the maximum projection, composite image in this image group. Trichomes are unicellular structures that arise from single protodermal cells and usually form three highly elongated branches. Additional information can be found in these background references: Hulskamp, M., Misra, S., Jurgens, G. Genetic dissection of trichome cell development in Arabidopsis, Cell 1994, 76: 555-566. Szymanski, D. Breaking the WAVE complex: the point of Arabidopsis trichomes. Curr Opin Plant Biol 2005, 8: 103-112.
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