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，每天都会给我们的环境带来非常可预测的变化；包括光照强度、温度和湿度的波动。为了应对这些变化，许多生物体进化出了昼夜节律，使它们能够在一天中协调自己的生物活动。昼夜节律由24小时生物钟驱动，是生物活动的振荡，大约每天一次达到峰值，在自然界中无处不在。这个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，也是唯一已知拥有强大生物钟的细菌。该项目的目的是对蓝藻生物钟有一个近乎全面的了解，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理奖学金，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单元中引入探究性研究的元素，接触到更多的学生。这些经验将为学生提供实验生物学的实践经验，概念的重复和渐进发展将使学生对材料有更深的理解。此外，学生还将向当地中小学生展示他们为期一学期的项目的成果，以促进当地社区在K-12教育中推广STEM。该项目旨在研究蓝藻的核心振荡器--细长聚球藻PCC7942是如何整合到三维细胞中的，以及振荡器时空动力学的变化如何有助于生物钟的同步性和稳健性以及它与其他细胞过程的整合。虽然已知时钟蛋白的亚细胞定位和共定位在空间和时间上的变化对生物钟机制造成了一定程度的复杂性，但人们对观察到的具有24小时周期性的时空动力学的生物学意义知之甚少。本研究旨在通过结合分子遗传学、生物化学和细胞生物学(包括荧光显微镜)来确定扩展时钟网络成员的亚细胞定位，并通过时间推移成像来研究细胞分裂过程中时钟蛋白的遗传如何有助于昼夜节律时钟的稳健性和细胞分裂过程中时间戳的遗传，从而通过研究扩展时钟网络中的亚细胞定位变化以及扩展时钟网络中的时空变化对时钟功能的影响来填补这些空白。通过免疫沉淀质谱分析确定了一系列候选基因，这些候选基因可能对促进核心振荡器蛋白亚细胞定位的变化做出了贡献，这些基因的初步缺失或耗尽会导致各种昼夜节律缺陷。这些候选人以前都没有被牵连到生理机制中，因此代表着理解时钟如何在体内发挥作用以及时钟如何与各种细胞过程整合的新途径。这一奖项反映了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。

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.