Molecular Mechanisms for Polar Growth in Pollen Tubes

细胞极性产生的机制对于多细胞生物的发育和形态发生是基本的，特别是植物，因为它们的细胞是不运动的。 本研究的目的是利用顶端生长的花粉管作为模型系统，阐明植物生长发育过程中极性的产生。 先前NSF资助的工作已经证明，植物特异性Rho家族GTdR，Rop，作为控制花粉管极性生长的中心开关。 这种开关由质膜上Rop激活和募集的正反馈回路控制，该正反馈回路由未知机制局部启动并横向放大。 该反馈环被Rop GT3活化蛋白和鸟嘌呤核苷酸解离抑制剂全面抑制，以在质膜处产生活性Rop的尖端聚焦梯度。 最近的研究结果表明，积极的罗普指定顶端质膜域尖端生长，然后通过调节动态尖端F-肌动蛋白和尖端聚焦的细胞质Ca 2+梯度激活生长。 两个活跃的Rop相互作用蛋白（ARIPs）已被确定，可能连接Rop顶端肌动蛋白和Ca 2+，分别。 这些进展在很大程度上依赖于生物化学和细胞的Rop信号传导测定，如基于荧光的Rop活性测定，肌动蛋白成像，和互补的拟南芥和烟草花粉管系统，适合于多方面的功能方法，包括遗传学，蛋白质的瞬时表达，和显微注射。 这项研究将继续使用类似的方法，但也开发新的测定和方法，以解决几个重要的悬而未决的问题，在花粉管极性生长的Rop依赖机制：1）什么是Rop，Ca 2+和肌动蛋白之间的功能相互作用？ 2)Rop如何调节Ca 2+和肌动蛋白？ 3)Rop正反馈环的分子基础是什么？ 具体的实验将包括识别的Rop效应和潜在的因素，可能会激活和招募Rop在质膜上使用交互式克隆和突变体分离，改变特定的Rop信号的组成部分，使用遗传和化学方法，和Rop的招聘和激活的变化和信号转导的目标，如尖端肌动蛋白和Ca 2+的分析。 这项工作应该揭示Rop依赖的信号转导对花粉管生长的关键组成部分和机制，并可能导致对细胞如何控制发育，极性生长和形态建成的新见解。

The mechanisms by which cell polarity arises are fundamental to development and morphogenesis of multicellular organisms, particularly of plants because their cells are non-motile. The goal of this project is to use tip-growing pollen tubes as a model system to elucidate how polarity arises during plant growth and development. Work from prior NSF funding has demonstrated that a plant-specific Rho family GTPase, Rop, acts as a central switch to control polar growth in pollen tubes. This switch is controlled by a positive feedback loop of Rop activation and recruitment at the plasma membrane that is initiated locally and amplified laterally by unknown mechanisms. This feedback loop is inhibited globally by Rop GTPase activating proteins and guanine nucleotide dissociation inhibitors to generate a tip-focused gradient of active Rop at the plasma membrane. Recent findings indicate that the active Rop specifies the apical plasma membrane domain for tip growth and then activates growth via the regulation of both dynamic tip F-actin and tip-focused cytosolic Ca2+ gradients. Two active Rop interacting proteins (ARIPs) have been identified that may link Rop to tip actin and Ca2+, respectively. These advances have relied heavily on biochemical and cellular Rop signaling assays such as a fluorescence-based Rop activity assay, actin imaging, and complementary Arabidopsis and tobacco pollen tube systems that are suited for a multifaceted functional approach that includes genetics, transient expression of proteins, and microinjection. This research will continue to use a similar approach but also develop new assays and methods to address several significant outstanding questions regarding Rop-dependent mechanisms for polar growth in pollen tubes: 1) What is the functional interplay between Rop, Ca2+ and actin? 2) How does Rop regulate Ca2+ and actin? 3) What is the molecular basis underlying the Rop positive feedback loop? Specific experiments will include identification of Rop effectors and potential factors that may activate and recruit Rop at the plasma membrane using interactive cloning and mutant isolation, alteration of specific components in Rop signaling using genetic and chemical methods, and analysis of changes in Rop recruitment and activation and in signaling targets such as tip actin and Ca2+. This work should uncover key components and mechanisms in Rop-dependent signaling to pollen tube growth and may lead to new insights into how cells control development, polar growth and morphogenesis.