Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

<div><p>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 <i>BECN1</i> 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 <i>BECN1</i>^−/− cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.</p></div

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

<div><p>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 <i>BECN1</i> 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 <i>BECN1</i>^−/− cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.</p></div

Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.

<p>(<b>A</b>, <b>D</b> and <b>G</b>) Partial sequences of <i>HBEGF</i>, <i>ANTXR1</i>, and <i>LRP1</i> genes in genome containing TALENs binding regions (overlined for TALEN^L and underlined for TALEN^R). Restriction enzyme cutting sites are highlighted in yellow. (<b>B</b>, <b>E</b> and <b>H</b>) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting <i>ANTXR2</i> gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). (<b>C</b>, <b>F</b> and <b>I</b>) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.</p