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Ultrastructural analysis and quantification of autophagic vacuoles in wild-type and atg5 knockout mouse embryonic fibroblast cells

정상 및 atg5 유전자 제거 섬유아세포에서 자가포식체의 미세구조 및 이들의 정량적 분석

  • Received : 2018.09.11
  • Accepted : 2018.10.10
  • Published : 2018.10.25

Abstract

Autophagy is a cellular process whereby cytosolic materials or organelles are taken up in a double-membrane vesicle structure known as an autophagosome and transported into a lysosome for degradation. Although autophagy has been studied at the genetic, cellular, or biochemical level, systematic ultrastructural quantitative analysis of autophagosomes during the autophagy process by using transmission electron microscopy (TEM) has not yet been reported. In this study, we performed ultrastructural analysis of autophagosomes in wild-type (WT) mouse embryonic fibroblasts (MEFs) and autophagy essential gene (atg5) knockout (KO) MEFs. First, we performed ultrastructural analysis of autophagosomes in WT MEFs compared to atg5 KO MEFs in basal autophagy or starvation-induced autophagy. Although we observed phagopore, early, late autophagosomes, or autolysosomes in WT MEFs, atg5 KO MEFs had immature autophagosomes that showed incomplete closure. Upon starvation, late autophagosomes accumulated in WT MEFs while the number of immature autophagosomes significantly increased in atg5 KO MEF indicating that atg5 plays an important role in the maturation of autophagosomes. Next, we examined autophagosomes in the cell model expressing polyQ-expanded N-terminal fragment of huntingtin. Our TEM analysis indicates that the number of late autophagosomes was significantly increased in the cells expressing the mutant huntingtin, indicating that improving the fusion of autophagosome with lysosome may be effective to enhance autophagy for the treatment of Huntington's disease. Taken together, the results of our study indicate that ultrastructural and quantitative analysis of autophagosomes using TEM can be applied to various human cellular disease models, and that they will provide an important insight for cellular pathogenesis of human diseases associated with autophagy.

Keywords

autophagy;autophagosome;ultrastructure;huntington's disease;transmission electron microscopy (TEM)

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Acknowledgement

Supported by : 한국기초과학지원연구원, 한국연구재단