Alireza Rahnama, Manibarathi Vaithiyanathan, Luis Briceno-Mena, Travis M. Dugas, Kelly L. Yates, Jose A. Romagnoli and Adam T. Melvin
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In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, <em>Chlamydomonas reinhardtii</em>, when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1–41 mM), <em>C. reinhardtii</em> cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of <em>C. reinhardtii</em> cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses.</p>","PeriodicalId":63,"journal":{"name":"Analyst","volume":null,"pages":null},"PeriodicalIF":3.6000,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/an/d4an00619d?page=search","citationCount":"0","resultStr":"{\"title\":\"A microfluidic approach to study variations in Chlamydomonas reinhardtii alkaline phosphatase activity in response to phosphate availability†\",\"authors\":\"Alireza Rahnama, Manibarathi Vaithiyanathan, Luis Briceno-Mena, Travis M. Dugas, Kelly L. Yates, Jose A. Romagnoli and Adam T. 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Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. 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引用次数: 0
摘要
藻类的生长在很大程度上依赖于作为关键营养元素的磷(P),这就强调了监测磷含量的重要性。藻类物种对磷供应量的波动表现出敏感的反应,特别是在面临磷缺乏的挑战时,藻类会表达碱性磷酸酶(AP)。因此,碱性磷酸酶活性(APA)是衡量钾可用性的重要指标,可帮助了解藻类如何利用和固定可用的钾资源。然而,目前的 APA 定量方法缺乏单细胞分辨率,而且耗费时间和试剂。微流控技术为解决这些局限性提供了一种经济有效的解决方案,为精确的单细胞分析提供了一个平台。在这项研究中,一种基于捕集器的微流控装置与一种市售的AP活染色剂相结合,研究了模式藻株--莱茵衣藻在暴露于不同外源P水平时的单细胞APA反应。在两种不同的基础 P 水平(1 毫摩尔和 21 毫摩尔)下培养的细胞中,采用了三步培养-饥饿-穗状过程来诱导 APA。当细胞受到不同加标 P 水平(0.1 - 41 mM)的挑战时,C. reinhardtii 细胞表现出高度异质性的 APA 反应。双向方差分析证实,这种反应既受加标 P 水平的影响,也受基础 P 水平的影响。利用无监督机器学习方法(HDBSCAN),确定了在单细胞水平上表现出不同水平 APA 的不同 C. reinhardtii 细胞亚群。这些亚群包括大量具有明显高或低 APA 的单个细胞群,它们对群组的整体行为做出了贡献。在平均行为相似的队列中,观察到 APA 在队列内存在相当大的差异。例如,一些队列集中分布在总体平均 APA 水平附近,而另一些队列的亚群则分散在更大的 APA 水平范围内。这凸显了批量分析少量细胞可能带来的偏差,因为批量分析可能会过度代表极端行为亚群,从而导致结果偏差。这项研究的结果突出表明,有必要采用能考虑到 APA 中单细胞异质性的分析方法,并证明微流控技术是进行此类研究的理想手段。
A microfluidic approach to study variations in Chlamydomonas reinhardtii alkaline phosphatase activity in response to phosphate availability†
Algal growth depends strongly on phosphorus (P) as a key nutrient, underscoring the significance of monitoring P levels. Algal species display a sensitive response to fluctuations in P availability, notably through the expression of alkaline phosphatase (AP) when challenged with P-depletion. As such, alkaline phosphatase activity (APA) serves as a valuable metric for P availability, offering insights into how algae utilize and fix available P resources. However, current APA quantification methods lack single cell resolution, while also being time- and reagent consuming. Microfluidics offers a promising cost-effective solution to these limitations, providing a platform for precise single-cell analysis. In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, Chlamydomonas reinhardtii, when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1–41 mM), C. reinhardtii cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of C. reinhardtii cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses.