NSF/MCB-BSF:Elucidating the role of ERM proteins in cytoskeletal orientation in a contractile tissue

动物体内充满了生物管道：血管、肺气道、肠、生殖系统以及各种导管和腺体。这些管道由细胞挤压和放松组成，以正确的方向，以正确的速度和协调的方式移动内容物通过管道。这种挤压是由一种叫做肌动蛋白细胞骨架的分子机器驱动的，它由微小的马达组成，它拉动一个束状的纤维网来收缩细胞。“腰带”必须在细胞之间正确排列和协调，以使管道正常工作。然而，关于这种“腰带”如何知道如何在正确的时间挤压正确的量，或者它的纤维排列如何随着机械条件的变化而变化，人们知之甚少。为了更好地理解这个过程是如何工作的，克拉姆和扎伊德尔-巴尔实验室研究了一种叫做秀丽隐杆线虫的小线虫的生殖系统。这种蠕虫每天可以生产与体重相当的婴儿，所以许多卵必须以一种协调而强健的方式挤出生殖系统。之所以选择这个系统，是因为蠕虫是透明的，这样很容易在活体动物身上实时看到“束带”的反应，而且该系统使用的细胞成分与其他动物相同。这个项目将从分子上确定蠕虫的肌凝蛋白“腰带”是如何对进入和退出系统的卵的拉伸做出反应的，以及它是如何恰到好处地收缩以将卵推向正确的方向而不会损坏它们的。由于相似的基因在许多动物中调控肌球蛋白收缩，因此该结果应该广泛适用于包括灵长类动物在内的动物。这个项目更广泛的影响包括开发帮助本科生学习如何成为科学家的材料，让高中教师参与研究经验，帮助他们设计可以带回课堂的项目。收缩细胞中的肌动蛋白网络，如血管的平滑肌和内皮细胞，对细胞的收缩性、运动性和组织功能至关重要。但是，细胞是如何组织它们的肌动蛋白细胞骨架来响应不断变化的机械条件的呢？细胞骨架排列如何在细胞间协调以产生内聚的组织水平反应？为了解决这些问题，Cram和Zaidel-Bar实验室开发了一种新的体内模型系统：秀丽隐杆线虫的精囊，线虫生殖系统中的一种拉伸反应和收缩组织。克拉姆实验室发现，在第一次排卵期间，肌凝蛋白被激活，并将松散的肌动蛋白纤维网络拉成整齐定向的应力纤维，就像肌动-肌凝蛋白束一样。在线虫所有肌动蛋白结合蛋白的筛选中，鉴定出Ezrin -radixin-moesin （ERM）蛋白Merlin （NFM-1）和Ezrin （ERM-1）是肌动蛋白纤维定向的关键组织水平调节因子。ERM蛋白可以结合质膜、肌动蛋白和跨膜蛋白，理想地调节细胞对拉伸的反应。利用实时成像、生化、遗传学和光遗传学方法，该合作团队将阐明ERM蛋白在精囊细胞内和细胞之间的肌动蛋白组织中的作用。目的1将确定精索拉伸和收缩过程中ERM蛋白的动态，并发现这些蛋白调节顶端和底部肌动蛋白网络以及肌动蛋白细胞骨架的组织水平组织的机制。目的2将确定ERM蛋白如何调节和受小GTPase Rho的调节，包括分析一种新的调节因子C45G9.7/Tip1，以促进细胞骨架组织和定向。该项目将揭示Merlin和Ezrin，肌动蛋白细胞骨架的重要调节因子，不仅在单个细胞内起作用，而且在肌动蛋白细胞骨架排列的组织水平协调中起作用。ERM蛋白在细胞对生理水平应变的反应中的新作用将被揭示，包括细胞骨架如何适应其方向和排列以获得最佳的组织收缩性。该项目的更广泛影响包括：1)为本科生研究开发一种结构化的指导方法；2)让科学教师参与独立研究，目标是加强K-12 STEM教育。这个美国/以色列合作项目由美国国家科学基金会和以色列两国科学基金会支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Animal bodies are full of biological tubing: blood vessels, lung airways, intestines, the reproductive system, and ducts and glands of various sorts. These tubes are composed of cells that squeeze and relax to move contents through the tubes in the correct direction, at the correct rate, and in a coordinated manner. The squeezing is driven by a molecular machine called the acto-myosin cytoskeleton, which is comprised of tiny motors that pull on a girdle-like mesh of fibers to contract the cells. The "girdle" must be lined up properly and coordinated between cells for tubes to work properly. However, little is known about how this "girdle" knows how to squeeze the right amount at the right time or how its fiber alignment changes in response to changing mechanical conditions. To better understand how this process works, the Cram and Zaidel-Bar labs study the reproductive system of a small nematode worm called C. elegans. This worm can produce its body weight in babies every day, so many eggs have to be squeezed through the reproductive system in a coordinated and robust manner. This system was chosen because the worm is transparent, which makes it easy to see the response of the "girdle" in real time in a living animal, and the system uses the same cellular components as do other animals. This project will determine, molecularly, how the worm's acto-myosin "girdle" responds to the stretch of eggs entering and exiting the system, and how it contracts just the right amount to push the eggs through in the right direction and without mangling them. Because similar genes regulate acto-myosin contraction in many animals, the results should be broadly applicable up to and including primates. Broader impacts of this project include developing materials to help undergraduates learn how to be scientists, and involving high school teachers in research experiences and helping them design projects that they can take back to their classrooms.Actin networks in contractile cells, such as the smooth muscle and endothelial cells of the vasculature, are critical for cell contractility, motility, and tissue function. But, how do cells organize their actin cytoskeletons in response to changing mechanical conditions? And how is cytoskeletal alignment coordinated between cells to produce a cohesive tissue-level response? To address these questions, the Cram and Zaidel-Bar labs have developed a new in vivo model system: the C. elegans spermatheca, a stretch-responsive and contractile tissue in the nematode reproductive system. The Cram lab has discovered that during the first ovulation, myosin becomes activated and pulls a network of loose actin fibers into aligned and oriented stress-fiber like acto-myosin bundles. In a screen through all actin binding proteins in C. elegans, the ezrin-radixin-moesin (ERM) proteins Merlin (NFM-1) and Ezrin (ERM-1) were identified as key tissue-level regulators of actin fiber orientation. ERM proteins can bind plasma membrane, actin, and transmembrane proteins, placing them ideally to regulate cell responses to stretch. Using live imaging, biochemical, genetic, and optogenetic approaches, this collaborative team will elucidate the role ERM proteins play in actin organization within and between cells of the spermatheca. Aim 1 will determine the dynamics of the ERM proteins during spermathecal stretch and contraction and discover the mechanism by which these proteins regulate the apical and basal actin networks and tissue-level organization of the actin cytoskeleton. Aim 2 will determine how ERM proteins both regulate and are regulated by the small GTPase Rho, including analysis of a novel regulator C45G9.7/Tip1, to promote cytoskeletal organization and orientation. This project will reveal that Merlin and Ezrin, important regulators of the actin cytoskeleton, act not only within individual cells, but in tissue-level coordination of actin cytoskeletal alignment. Novel roles for ERM proteins in cell response to physiological levels of strain will be revealed, including, how the cytoskeleton adapts its orientation and alignment for optimal tissue contractility. The Broader Impacts of this project include 1) developing a structured mentoring approach for undergraduate research and 2) involving science teachers in independent research with the goal of enhancing K-12 STEM education.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.