Yu Yong, Mutlu Ayse Sena, Liu Harrison, Wang Meng C
Huffington Center on Aging, Baylor College of Medicine, Houston, TX, 77030, USA.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.
Nat Commun. 2017 Oct 11;8(1):865. doi: 10.1038/s41467-017-00944-3.
High-throughput screens at microscopic resolution can uncover molecular mechanisms of cellular dynamics, but remain technically challenging in live multicellular organisms. Here we present a genetic screening method using photo-highlighting for candidate selection on microscopes. We apply this method to stimulated Raman scattering (SRS) microscopy and systematically identify 57 Caenorhabditis elegans mutants with altered lipid distribution. Four of these mutants target the components of the Bone Morphogenetic Protein (BMP) signaling pathway, revealing that BMP signaling inactivation causes exhaustion of lipid reserves in somatic tissues. Using SRS-based isotope tracing assay to quantitatively track lipid synthesis and mobilization, we discover that the BMP signaling mutants have increased rates of lipid mobilization. Furthermore, this increase is associated with the induction of mitochondrial β-oxidation and mitochondrial fusion. Together these studies demonstrate a photo-highlighting microscopic strategy for genome-scale screens, leading to the discovery of new roles for BMP signaling in linking mitochondrial homeostasis and lipid metabolism.High-throughput genetic screens in animals could benefit from an easy way to mark positive hits. Here the authors introduce photo-highlighting using a photoconvertible fluorescent protein, and in combination with stimulated Raman scattering (SRS) microscopy, define a role for BMP signaling in lipid metabolism in C. elegans.
微观分辨率下的高通量筛选能够揭示细胞动力学的分子机制，但在活的多细胞生物体中仍面临技术挑战。在此，我们展示一种利用光高亮在显微镜上进行候选选择的遗传筛选方法。我们将此方法应用于受激拉曼散射（SRS）显微镜，并系统地鉴定出57个脂质分布改变的秀丽隐杆线虫突变体。其中四个突变体靶向骨形态发生蛋白（BMP）信号通路的组分，揭示BMP信号失活会导致体细胞组织中脂质储备的耗尽。利用基于SRS的同位素示踪测定法定量追踪脂质合成和动员，我们发现BMP信号突变体的脂质动员速率增加。此外，这种增加与线粒体β-氧化和线粒体融合的诱导有关。这些研究共同证明了一种用于基因组规模筛选的光高亮显微镜策略，从而发现了BMP信号在连接线粒体稳态和脂质代谢中的新作用。动物中的高通量遗传筛选可能受益于一种标记阳性结果的简便方法。本文作者引入了使用光可转换荧光蛋白的光高亮，并结合受激拉曼散射（SRS）显微镜，确定了BMP信号在秀丽隐杆线虫脂质代谢中的作用。
