Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

Abstract

These authors contributed equally to this work.Keywords: autophagy, autophagosome, BECN1, LC3, PtdIns3KAbbreviations: ATG, autophagy related; BafA1, bafilomycin A 1 ; BCL2L1/Bcl -xL, BCL2-like 1; BECN1, Beclin 1, autophagy related; BECN1P1/BECN2, Beclin 1, autophagy related, pseudogene 1; EM, electron microscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; KO, knockout; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MAP1LC3-I/LC3-I, soluble, proteolytically processed microtubule-associated protein 1 light chain 3; MAP1LC3-II/LC3-II, proteolytically processed and lipid-modified microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; PtdIns3K, phosphatidylinositol 3-kinase; PIK3C3/VPS34, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4/VPS15, phosphoinositide-3-kinase, regulatory subunit 4; SQSTM1/p62, sequestosome 1; TUBB, tubulin, b class I; TALEN, transcription activator-like effector nuclease; UVRAG, UV radiation resistance associated; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1 ¡/¡ cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.

Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

“…Our data thus suggest that functional autophagy remain intact when beclin 1 was deleted while DNA DSB repair is dependent on Beclin 1. These results are in contrast to the results of a recent study that reported that deletion of beclin 1 using TALEN disrupted autophagic flux by disrupting the formation of autophagosomes but not by disrupting LC3b lipidation in human Hela cells33. However, the present data appear to be consistent with our recent finding that somatic cells, but not stem cells, can acquire an alternative form of autophagy when their canonical autophagy pathway is disrupted3435.…”

Bcl-xL Affects Group A Streptococcus-Induced Autophagy Directly, by Inhibiting Fusion between Autophagosomes and Lysosomes, and Indirectly, by Inhibiting Bacterial Internalization via Interaction with Beclin 1-UVRAG

“…We confirmed the attenuation of starvation-induced autophagic activity by showing the reduction in LC3 puncta formation and LC3-II levels (S3D, S3E and S3F Fig) in Beclin 1 KO cells. These results were not inconsistent with that of a previous study [30]. …”

Effect of PI3K-mediated autophagy in human osteosarcoma MG63 cells on sensitivity to chemotherapy with cisplatin

“…LC3 appears as LC3-I and LC3-II in the cytoplasm, but LC3-I is converted to LC3-II when autophagy occurs. LC3-II is the significant protein of autophagosome which is considered as the molecular marker of autophagosome, so the ratio of LC3-II/ LC3-I was employed to reflect the degree of cell autophagy [15]. Our findings showed that the ratio of LC3-II/LC3-I in MG63 cell was remarkably lower than that in osteoblast, revealing the lower autophagy activity of MG63 cells compared with normal cells.…”