Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells.
Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.
Key Laboratory for Biomechanics and Mechanobiology of the Ministry of Education, School of Biological Science and Biomedical Engineering, Beihang University, Beijing, China.
Cuihong
C
Zhou
Wu
W
Zhong
Jun
J
Zhou
Fugeng
F
Sheng
Ziyuan
Z
Fang
Yue
Y
Wei
Yingyu
Y
Chen
Xiaoyan
X
Deng
Bin
B
Xia
Jian
J
Lin
D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002945:Cisplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*; D019311:Staurosporine / Q000494:pharmacology; D000097573:Red Fluorescent Protein
D002945:Cisplatin; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D019311:Staurosporine; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein