Slowest possible replicative life at frigid temperatures for yeast. Slowest possible replicative life at frigid temperatures for yeast

Microbes and animals often inhabit frigid environments. Little is known about the design principles that govern a cell’s slow progression in life – how it proliferates, ages and dies – and potential constraints on slowly progressing life. Challenges lie in monitoring slow, intracellular processes and quantifying their combined effects on cells. Here we use budding yeast to quantitatively establish principles that govern cell proliferation and survival at near-freezing temperatures (0C – 5C). By monitoring individual cells for months at near-freezing temperatures, we found small numbers of cells dividing with weeks-long doubling times, while most others died. We found that cells die and cannot divide primarily from having high levels of Reactive Oxygen Species (ROS). Reducing ROS levels or enabling G1 exit with abundant ROS largely prevented deaths and enabled cell divisions with shortened doubling times. These perturbations yielded a comprehensive view of cell's life at near-freezing temperatures that integrated our measurements of single cells’ lifespans, days-to-months-long cell-cycle durations, protein-synthesis rates, and genome-wide transcription rates. With a mathematical model, this systems-level view revealed that protein-synthesis dynamics becomes more rate-limiting for cell duplication while mRNA-synthesis dynamics becomes less rate-limiting as temperature approaches 0C. Consequently, the protein-synthesis rate and ROS together establish low-speed and high-speed limits for life at near-freezing temperatures: shortest and longest possible doubling times. Progressing through cell cycle more slowly than the low-speed limit ensures death. This work establishes a quantitative, systems-level foundation for engineering organisms to live in frigid environments and elucidating fundamental limits to slowing down life. Overall design: Data represent time-series to determine genome-wide transcription rates for various conditions. Wild-type yeast was incubated at 1.0C, 5.0C, or 30.0C with or without 250uM GSH added (3 biological replicates for each condition). The synthetic nucleotide 4-thiouracil ("4tU") was added to medium after two weeks of incubation. The abundance of 4tU-labelled mRNA was then quantified over time using next-generation sequencing (“4tU-seq”). The series has 6 timepoints at 1C, 8 time points at 5C, and 5 time points at 30C. Time point 0 is the reference for each condition. A steady-state sample was also included for each condition. All samples contained the same amount of 4tU-labelled spike-in RNA from Schizosaccharomyces pombe to normalize samples over time and across conditions.

微生物与动物常栖息于寒冷环境中。目前学界对调控细胞生命慢进程（包括增殖、衰老与死亡）的设计原则，以及慢进程生命所受的潜在限制，仍知之甚少。监测缓慢的胞内过程并量化其对细胞的综合影响，是当前面临的核心挑战。本研究以出芽酵母（budding yeast）为模型，定量阐明了近冰点温度（0℃~5℃）下调控细胞增殖与存活的核心原则。通过在近冰点环境下对单个细胞进行长达数月的监测，我们发现少量细胞的倍增周期长达数周，而其余绝大多数细胞最终死亡。我们明确，细胞死亡且无法分裂的主要诱因是活性氧（Reactive Oxygen Species, ROS）水平过高。降低ROS水平，或在ROS水平过高时促进细胞退出G1期，可大幅减少细胞死亡，并使细胞分裂的倍增周期显著缩短。这些干预手段让我们得以全面解析近冰点温度下的细胞生命活动，整合了单细胞寿命、数天至数月的细胞周期时长、蛋白质合成速率以及全基因组转录速率的多维度测量数据。借助数学模型，这一系统层面的分析揭示：随着温度趋近0℃，蛋白质合成动态对细胞复制的限速作用逐渐增强，而mRNA合成动态的限速作用则逐渐减弱。因此，蛋白质合成速率与ROS共同划定了近冰点环境下生命活动的快慢极限：即最短与最长的可能倍增周期。若细胞周期进程慢于该低速极限，则必然导致细胞死亡。本研究为改造生物体以适应寒冷环境，以及阐明生命进程放缓的基本极限，奠定了定量的系统级研究基础。

实验整体设计：本数据集为时间序列数据，用于测定不同条件下的全基因组转录速率。将野生型酵母（wild-type yeast）分别置于1.0℃、5.0℃或30.0℃环境中培养，同时添加或不添加250 μM 谷胱甘肽（GSH），每种条件设置3个生物学重复。在培养两周后，向培养基中加入合成核苷酸4-硫尿嘧啶（4-thiouracil, 4tU）。随后借助下一代测序技术（next-generation sequencing），通过"4tU-seq"方法对4tU标记的mRNA丰度进行时间维度的定量检测。该时间序列在1℃条件下设置6个时间点，5℃条件下设置8个时间点，30℃条件下设置5个时间点；每个条件下的时间点0均作为参考样本。每种条件还设置了稳态样本。所有样本均加入等量来自粟酒裂殖酵母（Schizosaccharomyces pombe）的4tU标记的外源掺入RNA，用于跨时间与跨条件的样本标准化校正。