Mello Danielle F, Perez Luiza, Bergemann Christina M, Morton Katherine S, Ryde Ian T, Meyer Joel N
Nicholas School of the Environment, Duke University, Box 90328, Durham, NC, 27708-0328 United States of America.
bioRxiv. 2024 Jun 30:2024.06.26.600841. doi: 10.1101/2024.06.26.600841.
Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. 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 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 . 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 . 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.
线粒体生物能量学过程对于发育、应激反应和健康至关重要。它被广泛用于研究发育生物学、线粒体疾病和线粒体毒性。在许多物种的发育过程中，氧化磷酸化通常会增加，遗传和环境因素可能会改变这一正常轨迹。发育过程中线粒体功能的改变可能导致剧烈的短期反应，包括发育停滞和死亡，以及可能贯穿一生并延续至后代的细微后果。目前，关于该物种发育过程中线粒体生物能量学参数如何变化的报道并不完整且相互矛盾，这限制了我们对正常和异常发育的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，对该物种幼虫发育过程中的线粒体和非线粒体氧消耗率（OCR）进行了全面分析。我们优化并描述了分析基础OCR、基础线粒体OCR、ATP相关OCR、备用和最大呼吸能力、质子泄漏以及非线粒体OCR的条件。一个关键的考虑因素是标准化，我们将结果呈现并讨论为按单个蠕虫、蛋白质含量、蠕虫体积、线粒体DNA（mtDNA）计数、核DNA（ncDNA）计数以及mtDNA:ncDNA比率进行标准化。哪种标准化过程最佳取决于所提出的问题，标准化的差异解释了先前报道的该物种OCR发育变化中的一些差异。总体而言，当按蠕虫数量进行标准化时，我们的结果与先前的报道一致，表明整个发育过程中OCR显著增加。然而，当按总蛋白质、蠕虫体积或ncDNA或mtDNA计数进行标准化时，从L1到L2阶段显著增加2 - 3倍后，我们发现从L2到L4阶段，大多数OCR参数变化很小或没有变化，除了L3阶段备用和最大呼吸能力略有增加。总体而言，我们的结果表明细胞向氧化代谢的转变比大多数先前文献所暗示的更早。
