Molecular Mechanisms for Polar Growth in Pollen Tubes

细胞极性产生的机制是多细胞生物体，特别是植物的发育和形态发生的基础，因为它们的细胞是非运动性的。这个项目的目标是使用顶端生长的花粉管作为模型系统来阐明植物生长和发育过程中极性是如何产生的。先前美国国家科学基金会资助的研究表明，植物特有的Rho家族GTPase，Rop，起着控制花粉管中极性生长的中央开关的作用。这种开关是由质膜上Rop激活和募集的正反馈环控制的，该环在局部启动，并由未知机制横向放大。这个反馈环被Rop GTPase激活蛋白和鸟嘌呤核苷酸解离抑制剂全局抑制，以在质膜上产生活性Rop的末端聚焦梯度。最近的发现表明，活性Rop为TIP的生长指定顶端的质膜结构域，然后通过调节动态的TIP F-肌动蛋白和TIP聚焦的胞浆钙梯度来激活生长。已鉴定出两个活性Rop相互作用蛋白(ARIP)，它们可能分别与TIP肌动蛋白和Ca~(2+)相连。这些进展在很大程度上依赖于生化和细胞Rop信号分析，如基于荧光的Rop活性分析、肌动蛋白成像，以及适合于包括遗传学、蛋白质瞬时表达和显微注射在内的多方面功能方法的拟南芥和烟草花粉管互补系统。本研究将继续使用类似的方法，但也将开发新的分析和方法来解决有关Rop依赖的花粉管极性生长机制的几个重要悬而未决的问题：1)Rop、钙和肌动蛋白之间的功能相互作用是什么？2)Rop如何调节钙和肌动蛋白？3)Rop正反馈环背后的分子基础是什么？具体的实验将包括使用交互克隆和突变分离来鉴定Rop效应分子和可能在质膜上激活和招募Rop的潜在因素，使用遗传和化学方法改变Rop信号中的特定成分，以及分析Rop招募和激活以及TIP肌动蛋白和钙离子等信号靶标的变化。这项工作将揭示Rop依赖的信号转导花粉管生长的关键成分和机制，并可能导致对细胞如何控制发育、极地生长和形态发生的新见解。

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.