自噬
自噬体
细胞生物学
绿色荧光蛋白
融合蛋白
细胞器
溶酶体
细胞质
化学
生物
生物化学
酶
重组DNA
细胞凋亡
基因
作者
Pavan P. Adiseshaiah,Sarah L. Skoczen,Jamie Rodriguez,Timothy Potter,Krishna P. Kota,Stęphan T. Stern
出处
期刊:Methods in molecular biology
日期:2017-10-17
卷期号:: 211-219
被引量:6
标识
DOI:10.1007/978-1-4939-7352-1_18
摘要
Autophagy is a catabolic process involved in the degradation and recycling of long-lived proteins and damaged organelles for maintenance of cellular homeostasis, and it has also been proposed as a type II cell death pathway. The cytoplasmic components targeted for catabolism are enclosed in a double-membrane autophagosome that merges with lysosomes, to form autophagosomes, and are finally degraded by lysosomal enzymes. There is substantial evidence that several nanomaterials can cause autophagy and lysosomal dysfunction, either by prevention of autophagolysosome formation, biopersistence or inhibition of lysosomal enzymes. Such effects have emerged as a potential mechanism of cellular toxicity, which is also associated with various pathological conditions. In this chapter, we describe a method to monitor autophagy by fusion of the modifier protein MAP LC3 with green fluorescent protein (GFP; GFP-LC3). This method enables imaging of autophagosome formation in real time by fluorescence microscopy without perturbing the MAP LC3 protein function and the process of autophagy. With the GFP-LC3 protein fusion construct, a longitudinal study of autophagy can be performed in cells after treatment with nanomaterials.
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