Palmisano Nicholas J, Meléndez Alicia
Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot086504. doi: 10.1101/pdb.prot086504.
Autophagy plays an active role during the early stages of embryogenesis in the nematode Caenorhabditis elegans. Although their exact function is unknown, P granules are ribonucleoprotein particles that play a role in germ cell specification. The localization of P granules is restricted to the germline precursor cells in wild-type embryos, as a result of their degradation in the somatic cell lineage. Autophagy is known to be required for the degradation of P granules, as mutations in various autophagy genes, including those encoding the adaptor SEPA-1 and the p62-like adaptor SQST-1, result in the accumulation of the P granule components PGL-1 and PGL-3 (termed PGL granules) in the somatic cells of C. elegans embryos. In this protocol, we present a methodology for using fusion reporters of SEPA-1, SQST-1, and PGL-1 that have aided in the identification of new genes for normal autophagy activity by screening for mutant animals that lack the degradation of these autophagy substrates.
自噬在秀丽隐杆线虫胚胎发生的早期阶段发挥着积极作用。尽管其确切功能尚不清楚，但P颗粒是核糖核蛋白颗粒，在生殖细胞特化中发挥作用。由于P颗粒在体细胞谱系中降解，其定位在野生型胚胎中仅限于生殖系前体细胞。已知自噬是P颗粒降解所必需的，因为各种自噬基因的突变，包括编码衔接蛋白SEPA-1和p62样衔接蛋白SQST-1的基因，会导致秀丽隐杆线虫胚胎体细胞中P颗粒成分PGL-1和PGL-3（称为PGL颗粒）的积累。在本方案中，我们介绍了一种使用SEPA-1、SQST-1和PGL-1融合报告基因的方法，该方法通过筛选缺乏这些自噬底物降解的突变动物，有助于鉴定正常自噬活性的新基因。
