Yu Feiqiao Brian, Willis Lisa, Chau Rosanna Man Wah, Zambon Alessandro, Horowitz Mark, Bhaya Devaki, Huang Kerwyn Casey, Quake Stephen R
Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA.
Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.
BMC Biol. 2017 Feb 14;15(1):11. doi: 10.1186/s12915-016-0344-4.
Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.
To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control.
Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.
蓝藻是全球碳和氮循环中的重要参与者，在生物技术应用方面具有巨大潜力。诸如聚球藻属（Synechocystis sp.）和集胞藻属（Synechococcus sp.）等模式生物增进了我们对光合能力和昼夜节律行为的理解，这主要是通过群体水平的测量实现的，在此过程中无法监测个体行为。聚球藻属细胞体积小且分裂缓慢，需要长期实验来追踪单个细胞。因此，长时间的漂移累积效应会给监测和量化细胞生长及分裂动态带来困难。
为克服这一挑战，我们改进了一种微流控细胞培养装置，并开发了用于稳健谱系重建的图像分析流程。这使得能够在多代过程中同时追踪多个细胞，并揭示细胞在整个细胞周期中呈指数级扩张。姐妹细胞的世代时间高度相关，但母细胞和子细胞之间则不然。出生大小、分裂大小和世代时间之间的关系表明，细胞大小控制不符合“大小控制”规则（即分裂时间基于细胞大小）或“时间控制”规则（即分裂在固定时间间隔后发生）。相反，单细胞生长统计数据最符合“加法器”规则，即细胞体积在恒定增加后发生分裂。暴露于明暗循环的细胞仅在光照期生长和分裂；黑暗阶段暂停但不扰乱细胞周期控制。
我们的分析表明，“加法器”模型可以解释单个聚球藻细胞的生长相关统计数据以及姐妹细胞世代时间之间的相关性。我们还在单细胞水平观察到对明暗转换的快速表型反应，突出了光在蓝藻细胞周期控制中的关键作用。我们的研究结果表明，通过监测单个细胞的生长动力学，我们可以建立可测试的蓝藻细胞周期昼夜控制模型。
