线粒体生物能量过程是发育的基础,应激反应,和健康。秀丽隐杆线虫被广泛用于研究发育生物学,线粒体疾病,和线粒体毒性。氧化磷酸化通常在许多物种的发育过程中增加,遗传和环境因素可能会改变这种正常的轨迹。发育过程中线粒体功能的改变会导致两种剧烈的,短期反应,包括被逮捕的发展和死亡,以及可能持续一生和后代的微妙后果。目前,有关该物种发育过程中线粒体生物能参数如何变化的不完整和矛盾的报道限制了对秀丽隐杆线虫正常和改变的发育线粒体生物学的理解。我们使用海马XFe24细胞外通量(XF)分析仪对秀丽隐杆线虫幼虫发育过程中的线粒体和非线粒体耗氧率(OCR)进行了全面分析。我们优化并描述了基础OCR分析的条件,基底线粒体OCR,ATP连接的OCR,备用和最大呼吸能力,质子泄漏,和非线粒体OCR。一个关键的考虑因素是正常化,我们按照每个蠕虫的归一化结果来呈现和讨论,蛋白质含量,蠕虫体积,线粒体DNA(mtDNA)计数,核DNA(ncDNA)计数,和mtDNA:ncDNA的比例。哪个规范化过程最好取决于被问到的问题,标准化的差异解释了以前报道的秀丽隐杆线虫OCR发育变化的一些差异。广义上,当归一化为蠕虫数时,我们的结果与以前的报告一致,即在整个开发过程中OCR急剧增加。然而,当归一化为总蛋白质时,蠕虫体积,或ncDNA或mtDNA计数,在从L1到L2阶段显著增加2-3倍之后,我们发现从L2到L4阶段的大多数OCR参数变化很小或没有变化,除了L3时备用和最大呼吸容量的边际增加。总的来说,我们的结果表明,细胞向氧化代谢的转变比以前大多数文献中提出的更早。
Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.