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Vol. 16, Issue 4, 1913-1927, April 2005

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Ectopic Expression of an Activated RAC in Arabidopsis Disrupts Membrane Cycling

Daria Bloch ^*, Meirav Lavy ^*, Yael Efrat ^*, Idan Efroni ^*, Keren Bracha-Drori ^*, Mohamad Abu-Abied , Einat Sadot , and Shaul Yalovsky ^*

^* Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel; Department of Ornamental Horticulture, Volcani Center, Bet Dagan 50250, Israel

Submitted July 7, 2004; Revised January 28, 2005; Accepted January 30, 2005
Monitoring Editor: Anne Ridley

Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100–insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64–labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Shaul Yalovsky (shauly{at}tauex.tau.ac.il).

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