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、肺部、肠道、生殖系统以及各种导管和腺体。这些管子由挤压和松弛的细胞组成，以正确的方向、正确的速度和协调的方式通过管子移动内容物。挤压是由一种名为acto-myosin细胞骨架的分子机器驱动的，它由微小的马达组成，拉动一层带状的纤维网来收缩细胞。“腰带”必须正确排列，并在细胞之间协调，以便管子正常工作。然而，关于这种“腰带”如何知道如何在正确的时间挤压适量的纤维，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。为了更好地了解这一过程是如何运作的，克拉姆和扎伊德尔-巴尔实验室研究了一种名为线虫的小型线虫的生殖系统。这种蠕虫每天都能产生与婴儿体重相当的重量，因此许多卵子必须以协调和强大的方式通过生殖系统挤压。之所以选择这一系统，是因为蠕虫是透明的，这使得它很容易实时看到活着的动物体内“腰带”的反应，而且该系统使用的细胞组件与其他动物相同。这个项目将从分子上确定蠕虫的肌球蛋白“腰带”如何对进入和离开系统的卵子做出反应，以及它如何收缩适量的卵子以正确的方向推动卵子通过，而不破坏它们。由于相似的基因调节许多动物的肌球蛋白收缩，结果应该广泛适用于包括灵长类动物在内的所有动物。这个项目的更广泛的影响包括开发材料来帮助本科生学习如何成为科学家，让高中教师参与研究经验，并帮助他们设计可以带回课堂的项目。收缩细胞中的活动网络，如血管系统的平滑肌肉和内皮细胞，对细胞的收缩、运动和组织功能至关重要。但是，细胞如何组织它们的肌动蛋白细胞骨架以应对机械条件的变化呢？细胞之间的细胞骨架排列是如何协调的，以产生具有凝聚力的组织水平的反应？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：线虫受精膜，线虫生殖系统中一种拉伸反应和收缩的组织。克拉姆实验室发现，在第一次排卵时，肌球蛋白被激活，并将松散的肌动蛋白纤维网络拉成排列定向的压力纤维，就像acto-myosin束一样。在对线虫所有肌动蛋白结合蛋白的筛选中，Ezrin-Radisin-moesin(ERM)蛋白Merlin(NFM-1)和Ezrin(ERM-1)被鉴定为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，使它们成为调节细胞对拉伸的反应的理想场所。利用实时成像、生化、遗传学和光遗传学方法，这个合作小组将阐明ERM蛋白在受精囊细胞内和细胞间肌动蛋白组织中所起的作用。目的1测定精子膜拉伸和收缩过程中ERM蛋白的动态变化，揭示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组织水平的机制。目的2将确定ERM蛋白如何调节和被小GTP酶Rho调节，包括分析一个新的调节因子C45G9.7/Tip1，以促进细胞骨架的组织和定位。这个项目将揭示，肌动蛋白细胞骨架的重要调节者Merlin和Ezrin不仅在单个细胞内发挥作用，而且在组织水平上协调肌动蛋白细胞骨架的排列。ERM蛋白在细胞对生理水平的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定向和排列以获得最佳的组织收缩能力。该项目的更广泛影响包括1)为本科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。这一美国/以色列合作项目得到了美国国家科学基金会和以色列双国科学基金会的支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.