Ectopic expression of an activated RAC in Arabidopsis disrupts membrane cycling
Ectopic expression of an activated RAC in Arabidopsis disrupts membrane cycling
复制标题
DOI：
10.1091/mbc.e04-07-0562
复制
发表时间：
2005-04-01
影响因子：
3.3
通讯作者：
Yalovsky, S
中科院分区：
文献类型：
--
作者：
Bloch, D;Lavy, M;Yalovsky, S
Rho GTP酶调节肌动蛋白细胞骨架、胞吐作用、胞吞作用和其它信号级联。Rhos又分为Rho、Racs、Cdc 42四个亚家族和一个植物特异性的RAC/Rops。这项研究表明，组成型活性拟南芥RAC AtRAC 10的异位表达会破坏肌动蛋白细胞骨架的组织和膜循环。我们创建了表达野生型或组成型活性AtRAC 10融合到绿色荧光蛋白的转基因植物。激活的AtRAC 10诱导根毛和叶表皮细胞的变形，主要定位在Triton X-100不溶性组分的质膜。肌动蛋白细胞骨架重组揭示了通过创建双转基因植物表达激活AtRAC 10和肌动蛋白标记YFP-塔林。在不同的处理下，包括蛋白质运输抑制剂布雷菲德菌素A或肌动蛋白解聚剂latrunculin-B（Lat-B）和细胞松弛素-D（CD），通过用N-[3-三乙基铵丙基]-4-[p-二乙基氨基-苯基己三烯基]吡啶鎓二溴化物（FM 4 -64）进行膜染色来进一步分析植物。药物处理后，激活的AtRAC 10没有积累在布雷菲德菌素A的车厢，而是减少其数量和共定位与FM 4 -64标记的膜在大的细胞内囊泡。此外，内吞受损的激活AtRAC 10转基因植物的根毛。FM 4 -64被内吞在用肌动蛋白稳定药物jasplakinaline处理的非转基因根毛中。这些发现表明植物RAC对膜循环的复杂调节。
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-diethylamino-phenylhexatrienyl] 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.