Jimenez-Lopez Jose C, Wang Xia, Kotchoni Simeon O, Huang Shanjin, Szymanski Daniel B, Staiger Christopher J
Departments of Biological Sciences (J.C.J.-L., X.W., S.H., C.J.S.) and Agronomy (S.O.K., D.B.S.), Bindley Bioscience Center (C.J.S.), Purdue University, West Lafayette, Indiana 47907.
Departments of Biological Sciences (J.C.J.-L., X.W., S.H., C.J.S.) and Agronomy (S.O.K., D.B.S.), Bindley Bioscience Center (C.J.S.), Purdue University, West Lafayette, Indiana 47907
Plant Physiol. 2014 Nov;166(3):1312-28. doi: 10.1104/pp.114.242487. Epub 2014 Sep 8.
The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction.
肌动蛋白细胞骨架是细胞形态发生以及对生物和非生物刺激作出反应的主要调节因子。细胞骨架的组织和活动由数百种辅助蛋白精心编排。许多肌动蛋白结合蛋白被认为是刺激反应调节因子，它们与信号磷脂结合，并在脂质结合后改变其活性。这些蛋白在植物细胞中是否与信号脂质结合和/或受其调节，目前仍知之甚少。异二聚体封端蛋白（CP）是一种保守且普遍存在的肌动蛋白动力学调节因子。它以高亲和力结合到细丝的带刺末端，并在体外调节细丝的组装和拆卸反应。CP与包括磷脂酸在内的磷脂直接相互作用，会导致体外细丝末端的解封。最近，对拟南芥cp突变体的活细胞成像和反向遗传学分析为一种模型提供了有力支持，该模型认为CP活性在体内受到磷脂酸的负调控。在这里，我们使用了互补的生化、亚细胞分级分离和免疫荧光显微镜方法来阐明CP与膜的结合。我们发现CP在拟南芥组织中含量适中，并存在于微粒体膜组分中。利用蔗糖密度梯度分离和已知区室标记物的免疫印迹法证明，CP在内质网和高尔基体等膜结合细胞器上富集。这种结合可能有助于肌动蛋白细胞骨架与广泛的基本细胞功能（如细胞器运动和信号转导）之间的相互作用。
