CAREER: The Regulation of Cytokinesis by Calcium

本奖项全部或部分由《2021年美国救援计划法案》（公法117- 2）资助。胚胎要发育成完全发育的人，需要进行数百万轮细胞分裂。细胞如何在精确的时间和空间线索下如此可靠地分裂是一个基本的生物学问题。关于细胞分裂的一个长期未解之谜仍然是钙的参与。我们早就知道，在细胞分裂的最后一步——细胞质分裂过程中，由钙离子组成的两条快速电流穿过胚胎细胞。这些瞬时电流可能潜在地充当触发信号，就像电灯开关一样，用于分离两个复制的子细胞。然而，由于技术上的挑战，它们的起源和功能仍然知之甚少。这个项目将利用最近的两个突破来解决这个问题。首先是利用简单裂变酵母研究细胞分裂的概念上的进步。这种酵母菌在基因上很容易操作，但它的细胞质与动物细胞相似。二是定量显微镜技术的进步，可以高精度地测量活细胞中的钙电流。该项目将利用这些进展来揭示钙在细胞分裂过程中的作用。更广泛的影响活动包括研究的内在价值，因为所有真核细胞在分裂过程中都可能利用钙信号。其他活动包括对高中生、本科生和研究生进行研究培训。他将在学年期间为高中学生提供远程遥控显微镜的体验，并为经济困难的学生准备和吸引他们上大学而开展活动。美国救援计划的资金为研究人员在其职业生涯的关键阶段提供支持。在这个项目中，研究人员将在早期进展的基础上确定钙峰值的机制。研究人员将确定在分裂分裂酵母细胞时钙峰是如何产生的。我们将测试一种叫做机械敏感通道的特殊离子通道是否会引发钙离子峰值。然后，他们将测试两个钙泵Pmr1和Pmc1是否清除了过量的钙，从而使峰值是短暂的。2)研究人员将确定钙突如何调节细胞动力学的力发生器-收缩环。他们将测试钙突是否会促进钙敏感分子Ppb1的活性，Ppb1随后会激活环中的运动蛋白II型肌球蛋白。他们将测量Ppb1如何在环中被招募和激活，并确定缺乏Ppb1的突变细胞中肌球蛋白的活性和数量。接下来的实验将确定肌球蛋白调节轻链是否是Ppb1的底物。研究人员将确定钙峰如何调节细胞质分裂的最后一步——细胞分离。他们将确定刺突是否有助于酵母细胞壁的构建，并通过钙敏感分子Cam1的活性来维持细胞膜的完整性。他们将在项目中使用酵母遗传学、钙成像、原子力显微镜和定量显微镜。研究人员希望它最终能解决长期存在的问题，即这些细胞动力学钙电流从何而来以及它们的功能是什么。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。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117- 2).Millions of rounds of cell division are required for an embryo to become a fully developed human. How a cell can divide so reliably with precise timing and spatial cues is a fundamental biological question. One enduring mystery about cell division remains the involvement of calcium. We have long known that two quick currents consisting of calcium ions run through an embryonic cell during the last step of cell division called cytokinesis. These transient currents could potentially serve as the triggering signal, like a light switch, for the separation of two replicated daughter cells. However, their origin and function remain poorly understood due to technological challenges. This project will tackle this question by taking advantage of two fairly recent breakthroughs. The first is the conceptual advance of employing the simple fission yeast to study cytokinesis. This yeast is easy to manipulate genetically, but its cytokinesis is similar to that of the animal cells. The second is the technical advance of quantitative microscopy that can measure calcium current in live cells with high precision. The project will take advantage of these advances to uncover how calcium works during cytokinesis. Broader Impact activities include the intrinsic merit of the research as all eukaryotic cells likely utilize calcium signals during division. Additional activities involve the training of high school, undergraduate, and graduate students in research. The PI will extend the experience for high school students with remote controlled microscopes during the school year and he will also engage in activities to prepare and attract economically disadvantaged students for college. American Rescue Plan funding provides support for the investigator at a critical stage in his career.In this project, investigators will build on early advances to determine the mechanism of the calcium spikes. 1) The investigators will determine how the calcium spike arises in dividing fission yeast cells. We will test whether a special type of ion channels called mechanosensitive channels initiate the calcium spike. Then they will test whether two calcium pumps Pmr1 and Pmc1 clear out the excessive calcium so that the spikes are transient. 2) The investigators will determine how the calcium spike regulates the force-generator of cytokinesis the contractile ring. They will test whether the calcium spike promotes the activity of calcium-sensitive molecule Ppb1 which then proceeds to activate the motor protein type II myosins in the ring. They will measure how Ppb1 is recruited and activated in the ring and determine the activity and number of myosins in the mutant cells without Ppb1. This will be followed by the experiment to determine whether the myosin regulatory light chain is a substrate of Ppb1. 3) The investigators will determine how the calcium spike regulates the last step of cytokinesis the cell separation. They will determine whether the spike helps build the cell wall for yeast cells and maintain the integrity of the membrane through the activity of the calcium-sensitive molecule Cam1. They will employ yeast genetics, calcium imaging, atomic force microscopy and quantitative microscopy in the project. The investigators expect it will finally address the long-standing questions, where these cytokinetic calcium currents come from and what are their functions.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.