An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish.
An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish.
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DOI：
10.7554/elife.64744
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发表时间：
2021-06-11
期刊：
影响因子：
7.7
通讯作者：
White RM
中科院分区：
文献类型：
--
作者：
Lumaquin D;Johns E;Montal E;Weiss JM;Ola D;Abuhashem A;White RM
脂滴是在从脂肪细胞到癌细胞的几乎所有细胞类型中发现的脂质储存细胞器。虽然越来越多地涉及疾病，目前的方法来研究脊椎动物模型中的脂滴依赖于静态成像或使用荧光染料，限制了他们的快速在体内动力学的调查。为了解决这个问题，我们通过将tdTOMATO融合到脂滴结构蛋白Perilipin-2（PLIN 2），在整个动物和细胞培养物中创建了脂滴转基因报告基因。这种转基因在透明的卡斯珀斑马鱼中的表达，使体内成像的脂肪库响应营养剥夺和高脂肪饮食。同时，我们进行了大规模的1280化合物的体外化学筛选，并确定了几个新的脂肪细胞脂解调节剂。使用我们的Tg（-3.5ubb：plin 2-tdTomato）斑马鱼系，我们验证了这些新型调节剂中的几种，并揭示了一氧化氮在调节脂肪细胞脂滴中的意想不到的作用。类似地，我们在黑色素瘤细胞中表达PLIN 2-tdTOMATO转基因，发现一氧化氮途径也调节癌症中的脂滴。该模型提供了一个易于处理的成像平台，可以使用化学，饮食或遗传扰动来研究细胞类型和疾病背景中的脂滴。生物体需要脂肪分子作为能量来源和建筑材料，但如果这些“脂质”大量存在，也会损害细胞。细胞通过安全地将脂质隔离在参与一系列生物过程的专门液滴中来防止这种毒性。例如，这些结构可以根据细胞的能量需求快速改变大小以储存或释放脂质。对脂滴成像是可能的-例如使用优先染色脂肪的染料-但这些方法通常只能产生快照：随着时间的推移跟踪脂滴动态仍然很困难。因此，Lumaquin，Johns等人开始开发一种新方法，可以标记脂滴并监测它们在小而透明的斑马鱼幼虫细胞中的行为。首先，对这些鱼进行了基因操作，使脂滴中的一种关键蛋白质携带荧光标签：这使得这些结构具有强烈的荧光，易于随着时间的推移进行跟踪。事实上，Lumaquin，Johns等人可以根据鱼的饮食监测液滴的变化，当动物获得丰富的食物时，结构会变大，当资源稀缺时，结构会缩小。最后，进行实验以筛选可能导致脂质在脂肪细胞中释放的化合物。然后，新的成像技术被用来证实这些分子在活细胞中的作用，揭示了一种称为一氧化氮的信号分子的意想不到的作用，它也被证明是调节癌细胞中的脂滴。进一步的研究表明，影响一氧化氮的药物可以调节正常细胞和肿瘤细胞中脂滴的大小。这项工作验证了一种新的方法来研究活细胞中脂滴的实时行为及其对不同刺激的反应。在未来，Lumaquin，Johns等人希望这项技术将有助于揭示脂质如何参与健康和异常的生物过程。
Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes, limiting investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Simultaneously, we performed a large-scale in vitro chemical screen of 1280 compounds and identified several novel regulators of lipolysis in adipocytes. Using our Tg(-3.5ubb:plin2-tdTomato) zebrafish line, we validated several of these novel regulators and revealed an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts using chemical, dietary, or genetic perturbations. Organisms need fat molecules as a source of energy and as building blocks, but these ‘lipids’ can also damage cells if they are present in large amounts. Cells guard against such toxicity by safely sequestering lipids in specialized droplets that participate in a range of biological processes. For instance, these structures can quickly change size to store or release lipids depending on the energy demands of a cell. It is possible to image lipid droplets – using, for example, dyes that preferentially stain fat – but often these methods can only yield a snapshot: tracking lipid droplet dynamics over time remains difficult. Lumaquin, Johns et al. therefore set out to develop a new method that could label lipid droplets and monitor their behaviour ‘live’ in the cells of small, transparent zebrafish larvae. First, the fish were genetically manipulated so that a key protein found in lipid droplets would carry a fluorescent tag: this made the structures strongly fluorescent and easy to track over time. And indeed, Lumaquin, Johns et al. could monitor changes in the droplets depending on the fish diet, with the structures getting bigger when the animal received rich food, and shrinking when resources were scarce. Finally, experiments were conducted to screen for compounds that could lead to lipids being released in fat cells. The new imaging technique was then used to confirm the effect of these molecules in live cells, revealing an unexpected role for a signalling molecule known as nitric oxide, which also turned out to be regulating lipid droplets in cancerous cells. Further work then showed that drugs affecting nitric oxide could modulate lipid droplet size in both normal and tumor cells. This work has validated a new method to study the real-time behavior of lipid droplets and their responses to different stimuli in living cells. In the future, Lumaquin, Johns et al. hope that the technique will help to shed new light on how lipids are involved in both healthy and abnormal biological processes.