Collaborative Research: Regulation of Cell Expansion and Microtubule Function by SPR1

科学价值：微管在植物生长和发育的许多方面发挥着核心作用，包括细胞扩张、分裂和信号传递。在植物中，微管被组织成四个不同的阵列，具有不同的功能。最近的研究表明，皮质阵列中的微管通过跑步机运动机制重新定位，该机制由微管加末端生长、停顿和缩短(称为动态不稳定性)和间歇性负端解聚的间歇性周期驱动。阐明微管末端行为对于理解微管组织和功能的重要方面是至关重要的。在动物和真菌中，动态不稳定性由一组称为+TIPS的蛋白质(加上末端跟踪蛋白)调控，这些蛋白质优先定位于微管+末端，在那里它们也参与捕获和移动细胞组件。人们对植物中存在哪些+TIPS知之甚少，更不用说它们如何调节动态不稳定性和影响微管功能了。首席研究人员最近在正向遗传学筛查中发现了一个新的植物特异性+TIP，命名为SPIRAL1(SPR1)，以寻找影响根细胞扩张的拟南芥突变体。SPR1突变体的器官既表现出触摸诱导的细胞定向扩张缺陷，又表现出结构性的定向细胞扩张缺陷，这种缺陷可被较低的温度增强，并被抗微管药物抑制。序列分析表明，12kD的SPR1蛋白在拟南芥中有5个同源蛋白，这些蛋白在其氨基和羧基末端具有高度保守的直接重复序列，被一个低复杂性的区域隔开，该区域被预测形成棒状结构。鉴于稳定表达的SPR1：GFP融合蛋白的行为与其他GFP标记的+TIPS相似，因为它优先定位于生长中的微管+末端，并在微管缩短时分散，首席研究人员预测，与其他+TIPS一样，SPR1调节微管动态不稳定性，将蛋白质招募到微管+末端，和/或将微管连接到其他细胞结构。为了验证这些假设，将使用时间推移共聚焦显微镜和荧光微管标记来分析SPR1-6零等位基因和SPR1高表达植物中动态微管行为的重要参数。这将通过进行体外微管下拉试验来确定SPR1是否直接与微管结合。酵母双杂交系统将用于筛选SPR1相互作用蛋白的文库，并测试SPR1相互作用的候选图谱。鉴于动物EB1蛋白有许多结合伙伴，而拟南芥AtEB1蛋白表现出与SPR1相似的定位动力学，因此将特别关注鉴定SPR1和拟南芥同源蛋白与功能良好的动物EB1类蛋白之间的关系。总之，这些实验将使首席研究人员能够确定SPR1和其他植物+尖端如何参与微管+末端功能。这个项目还应该提供对微管和植物中定向细胞扩张之间鲜为人知的联系的见解。在植物中获得的关于+TIPS的知识可能为理解+TIPS在动物和真菌中的功能提供了一个视角。更广泛的影响：私人投资机构将积极从科学界代表性不足的群体中招募个人参与这一项目并接受基础研究方面的培训。项目参与者将包括塞德布鲁克博士实验室的一名女研究生和一名女本科生，以及埃哈特博士实验室从当地社区大学招募的学生。这些实验的一个子集将被整合到塞德布鲁克博士开设的生物技术实验室课程中，该课程旨在向本科生和研究生传授基本的分子技术，以培养他们的批判性思维和故障排除技能。伊利诺伊州立大学生物科学系最近用来自NSF设备赠款的资金购买了徕卡共焦显微镜系统，因此该项目将使研究人员能够充分利用这一重要仪器进行研究和教学。这些研究产生的活植物细胞内动态微管的图像和电影将被用于加强伊利诺伊州立大学PI教授的各种课程，并将被整合到由Ehrhardt博士创建的植物细胞成像网站。这个网站展示了活生生的植物细胞的图片和电影，揭示了它们迷人的内在生命。这个网站已经在《科学》杂志上发表了简介，并被用作全国几所中学和高等教育机构教授细胞生物学的资源。

Scientific merit: Microtubules play a central role in many aspects of plant growth and development including cell expansion, division and signaling. In plants, microtubules become organized into four distinct arrays with varying functions. It has recently been shown that microtubules in the cortical array become repositioned by a treadmilling motility mechanism that is driven by frequent cycles of microtubule plus end growth, pausing, and shortening (termed dynamic instability), and intermittent episodes of minus end depolymerization. An elucidation of microtubule end behavior is essential for understanding important aspects of microtubule organization and function. In animals and fungi, dynamic instability is regulated by a collection of proteins called +TIPS (plus end tracking proteins) that are preferentially localized to microtubule plus ends, where they also participate in capturing and moving cellular components. Little is known about what +TIPS exist in plants, let alone how they regulate dynamic instability and affect microtubule function. The Principal Investigators have recently identified a novel plant specific +TIP named SPIRAL1 (SPR1) in a forward genetics screen looking for Arabidopsis mutants affected in root cell expansion. The organs of spr1 mutants exhibit both touch induced and constitutive directional cell expansion defects that are enhanced by lower temperatures and suppressed by anti-microtubule drugs. Sequence analyses show that the 12 kD SPR1 protein has five homologues in Arabidopsis, with these proteins having highly conserved direct repeat sequences at their amino and carboxy termini, separated by a region of low complexity that is predicted to form a rod-like structure. Given that a stably expressed SPR1:GFP fusion protein behaves like other GFP labeled +TIPS in that it preferentially localizes to growing microtubule plus ends and disperses upon microtubule shortening, the Principal Investigators predict that like those other +TIPS, SPR1 regulates microtubule dynamic instability, recruits proteins to microtubule plus ends, and/or links microtubules to other cellular structures. To test these hypotheses, important parameters of dynamic microtubule behavior will be analyzed in the spr1-6 null allele as well as in SPR1 over-expressing plants, using time-lapse confocal microscopy and fluorescent microtubule markers. It will be determined if SPR1 binds directly to microtubules by performing in vitro microtubule pull-down assays. A yeast two-hybrid system will be used to screen libraries for SPR1 interacting proteins and to test candidate MAPs for SPR1 interactions. Particular focus will be placed on identifying the relationship between SPR1 and Arabidopsis homologues to the well-characterized animal EB1-like proteins, given that animal EB1 proteins have many binding partners and the Arabidopsis AtEB1 proteins exhibit localization dynamics similar to SPR1. Together, these experiments will allow the Principal Investigators to determine how SPR1 and other plant +TIPS participate in microtubule plus end function. This project should also provide insights into the poorly understood connection between microtubules and directional cell expansion in plants. The knowledge gained on +TIPS in plants may provide a perspective that aids in the understanding of +TIPS function in animals and fungi. Broader impacts: The PIs will actively recruit individuals from underrepresented groups in science to participate in this project and receive training in basic research. Project participants will include a female graduate student and a female undergraduate in Dr. Sedbrook's laboratory, and students recruited from local community colleges in Dr. Ehrhardt's laboratory. A subset of the experiments will be integrated into a biotechnology lab course run by Dr. Sedbrook that is designed to teach undergraduates and graduate students basic molecular techniques in a way that develops their critical thinking and troubleshooting skills. The Biological Sciences department at Illinois State University has recently purchased a Leica confocal microscope system with funds derived from an NSF equipment grant, so this project will allow the investigators to take full advantage of this important instrument for both research and teaching. Images and movies of dynamic microtubules in living plant cells generated from these studies will be used to enhance a variety of courses taught by the PI at Illinois State University and will be integrated into the Plant Cell Imaging web site created by the Dr. Ehrhardt. This web site features images and movies of living plant cells that reveal their fascinating inner life. This site has been profiled in Science magazine, and is used as a resource for teaching cell biology at several secondary and higher educational institutions around the country.