Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time-consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency (CBF) in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]-CBF over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ~2.0 mM in the light and ~1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and non-invasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms.

中文翻译：

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从绿藻莱茵衣藻纤毛跳动频率快速估计细胞溶质 ATP 浓度

细胞 ATP 水平的测定是代谢状态的关键指标，对于代谢的定量分析至关重要。双纤毛绿藻莱茵衣藻是研究 ATP 生产途径（包括光合作用和呼吸）的极好实验生物，特别是因为它可以光自养或异养培养。此外，它的细胞 ATP 浓度 [ATP] 反映在其纤毛的跳动中。然而，目前用于量化细胞 ATP 水平的方法既耗时又具有侵入性。在这项研究中，我们建立了一种根据莱茵衣藻纤毛搏动频率 (CBF) 估计胞质 [ATP] 的快速方法。在脱膜细胞模型中使用改进的运动再激活方法，我们获得了 [ATP]-CBF 在 ATP 浓度生理范围内的校准曲线。这些曲线允许快速估计活野生型细胞中的细胞溶质 [ATP]，在光照下为 ~2.0 mM，在黑暗中为 ~1.5 mM：与其他方法获得的值相当。此外，我们使用这种方法来定量和非侵入性地评估基因突变或光合作用或呼吸抑制剂的影响。这种无传感器的方法是快速估计细胞质 [ATP] 和研究莱茵衣藻或其他纤毛生物中 ATP 产生机制的便捷工具。此外，我们使用这种方法来定量和非侵入性地评估基因突变或光合作用或呼吸抑制剂的影响。这种无传感器的方法是快速估计细胞质 [ATP] 和研究莱茵衣藻或其他纤毛生物中 ATP 产生机制的便捷工具。此外，我们使用这种方法来定量和非侵入性地评估基因突变或光合作用或呼吸抑制剂的影响。这种无传感器的方法是快速估计细胞质 [ATP] 和研究莱茵衣藻或其他纤毛生物中 ATP 产生机制的便捷工具。