CAREER: Elucidating the spatiotemporal dynamics of the cyanobacterial circadian clock

我们生活在一个绕轴旋转的星球上，每天都在我们的环境中产生非常可预测的变化;包括光照强度，温度和湿度的波动。为了科普这些变化，许多生物体已经进化出昼夜节律，这使得它们能够在一天中协调它们的生物活动。由24小时生物钟驱动的昼夜节律是生物活动的振荡，每天大约达到一次峰值，并且在自然界中无处不在。该项目将阐明生物钟如何在蓝藻中发挥作用，蓝藻是最简单的生物体，也是唯一已知拥有强大生物钟的细菌。该项目的目的是获得对蓝藻生物钟的近乎全面的了解，并帮助为利用这些细菌进行广泛的应用奠定基础，包括生物修复，生物技术和生态/环境问题。此外，该项目将通过提供本科生和研究生研究助学金为学生提供研究培训机会，但也将通过在入门和高级本科课程的实验室模块中引入基于探究的研究元素来吸引更多的学生。这些经验将为学生提供实验生物学的实践经验，并且概念的重复和渐进发展将使学生对材料有更深入的理解。此外，学生将向当地中小学学生展示他们学期项目的成果，以促进当地社区K-12教育中的STEM。该项目旨在研究蓝细菌Synechococcus elongatus PCC 7942的核心振荡器如何整合到三维细胞中，以及振荡器的时空动力学变化如何有助于生物钟的同步和鲁棒性以及与其他细胞过程的整合。虽然已知在空间和时间上变化的时钟蛋白质的亚细胞定位和共定位对生物钟机制贡献了一定程度的复杂性，但很少了解所观察到的以24小时周期发生的时空动态的生物学意义。该研究旨在通过调查亚细胞定位的变化如何实现以及扩展时钟网络中的时空变化如何有助于时钟功能来填补这些空白，通过使用分子遗传学，生物化学和细胞生物学，包括荧光显微镜，以确定扩展的时钟网络和时间的成员的亚细胞定位，lapse imaging来研究细胞分裂期间时钟蛋白的遗传如何有助于生物钟的鲁棒性和细胞分裂期间时间戳的遗传。一个简短的候选人名单，确定了免疫沉淀质谱分析，已被确定为可能的贡献，以促进核心振荡蛋白的亚细胞定位的变化和初步删除或耗尽这些基因的结果在各种昼夜节律的缺陷。这些候选人之前都没有涉及昼夜节律机制，因此代表了了解生物钟在体内如何运作以及生物钟如何与各种细胞过程相结合的新途径。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

We live on a planet that rotates on its axis, creating daily and very predictable changes in our environment; including fluctuations in light intensity, temperature and humidity. In order to cope with these changes many organisms have evolved circadian rhythms, which allow them to coordinate their biological activity over the course of the day. Circadian rhythms, driven by 24-hour biological clocks, are oscillations in biological activity that peak approximately once per day, and are found ubiquitously throughout nature. This project will elucidate how the circadian clock functions in cyanobacteria, which are the simplest organisms and the only bacteria known to possess a robust circadian clock. The aim of this project is to gain a near comprehensive understanding of the cyanobacterial circadian clock, and help set the foundation for leveraging these bacteria for broad ranging applications including bioremediation, biotechnology, and ecological/environmental issues. Additionally the project will provide research training opportunities for students both by providing undergraduate and graduate research assistantships, but will also reach a greater number of students by introducing an element of inquiry-based research into laboratory modules for both introductory and advanced undergraduate courses. These experiences will provide students with hands-on experience in experimental biology, and also the repetitive and progressive development of concepts will allow students to gain a deeper understanding of the material. Moreover, students will present the results of their semester long projects to local elementary and middle school students to promote STEM in K-12 education in the local community. The project aims to investigate how the core oscillator of the cyanobacterium, Synechococcus elongatus PCC 7942, is integrated into a three-dimensional cell and how changes in the spatiotemporal dynamics of the oscillator contribute to the synchronization and robustness of the circadian clock as well as its integration with other cellular processes. While it is known that the subcellular localization and co-localizations of clock proteins that vary in space and time contribute a level of complexity to the circadian clock mechanism, very little is understood about the biological significance of the observed spatiotemporal dynamics occurring with 24-hour periodicity. The research aims to fill in these gaps by investigating how the changes in subcellular localization are achieved and how spatiotemporal changes in the extended clock network contribute to clock function, by using a combination of molecular genetics, biochemistry and cell biology including fluorescence microscopy to determine the subcellular localization of members of the extended clock network and time-lapse imaging to investigate how the inheritance of clock proteins during cell division contributes to the robustness of the circadian clock and the inheritance of timestamps during cell division. A short list of candidates, identified by immunoprecipitation mass-spectrometry analysis, has been identified for possible contributions to promoting changes in the subcellular localization of the core oscillator proteins and preliminary deletion or depletion of these genes results in various circadian defects. None of these candidates have been previously implicated in the circadian mechanism and thus represent novel avenues for understanding how the clock functions in vivo and how the clock is integrated with various cellular processes.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.