Abstract
Despite substantial achievements in elucidating the metabolic pathways of lipogenesis, a mechanistic representation of lipid accumulation and degradation has not been fully attained to-date. Recent evidence suggests that lipid accumulation can occur through increases of either the cytosolic copy-number of lipid droplets (LDs), or the LDs size. However, the prevailing phenotype, or how such mechanisms pertain to lipid degradation remain poorly understood. To address this shortcoming, we employed the–recently discovered–innate bioprocessing fluctuations in Yarrowia lipolytica, and performed single-cell fluctuation analysis using optical microscopy and microfluidics that generate a quasi-time invariant microenvironment. We report that lipid accumulation at early stationary phase in rich medium is substantially more likely to occur through variations in the LDs copy-number, rather than the LDs size. Critically, these mechanistics are also preserved during lipid degradation, as well as upon exposure to a protein translation inhibitor. The latter condition additionally induced a lipid accumulation phase, accompanied by the downregulation of lipid catabolism. Our results enable an in-depth mechanistic understanding of lipid biogenesis, and expand longitudinal single-cell fluctuation analyses from gene regulation to metabolism.
Highlights
Lipid droplets (LDs) are cytoplasmic emulsions, capable of storing neutral lipids such as triacylglycerols (TAG) and steryl esters (SE) at varying ratios [1,2,3,4]
Despite substantial recent progress in identifying the different biochemical pathways of lipid accumulation [19], including the transcriptional regulatory changes under nitrogen starvation [20, 21], observations similar to those of Fig 1A still question the mechanistics of lipogenesis, namely: does lipid accumulation occurs primarily through the number of cytosolic LDs, or through their size? To answer this question, we explored neutral lipid expression at the single-cell level using microfluidics and optical microscopy
For Po1g, the positive and negative median fluxes were similar, at 2.8%/hr (Fig 2B). This indicates that both lipid accumulation and degradation in Y. lipolytica occur at rates of comparable magnitudes
Summary
Lipid droplets (LDs) are cytoplasmic emulsions, capable of storing neutral lipids such as triacylglycerols (TAG) and steryl esters (SE) at varying ratios [1,2,3,4]. These stored compounds serve several cellular needs, such as membrane and lipoprotein biogenesis, as well as provide precursors towards oxidation mediated energy production. LDs have attracted significant excitement in recent years as cost-effective biodiesel precursors in renewable energy production [6]. To this end, Yarrowia lipolytica has emerged as a model oleaginous yeast due to its genetic tractability, as well as enhanced lipid accumulation capability–most in the form of TAG [7,8,9].
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