Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

Abstract

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy

“…In addition to these functions, BECN1 has been shown to localize to the nucleus (21). The nuclear function of BECN1 is unclear although it may play a role in nuclear export (21–23) and/or transcriptional regulation of autophagy proteins (22). BECN1 has been established as a locus of coordination for the cellular pathways of autophagy and apoptosis (15, 17, 24), as the anti-apoptotic BCL2 proteins bind to BECN1 and down-regulate autophagy (25, 26).…”

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma

“…BECN1 and LC3B were chosen for gene expression analysis because they are key autophagy biomarkers [33, 34]. Moreover, these genes had intermediate intensity values (~9 A.U.…”

“…Microarray data intensity values are correlated with transcript abundance and directed our experimental efforts toward qPCR analysis of BECN1 and LC3B gene expression in monolayer and hydrogel cultures because both genes had intermediate intensity values of approximately 9 (Online Resource 3). BECN1 has been identified as a key autophagy regulator protein involved in the formation, extension and maturation of autophagosome, a unique organelle which mediates autophagy phenomenon [58–60], while LC3B is one of the structural proteins involved in the formation of the autophagosomal membrane and is a biomarker routinely used for monitoring autophagy [33]. At least two reference genes are recommended to improve reliability and accuracy of qPCR normalization [61–63].…”

Autophagy gene expression profiling identifies a defective microtubule-associated protein light chain 3A mutant in cancer

“…Recently, a distinct role for LC3A in cancer cell lines has been described [35]. The authors demonstrate that LC3A-positive autophagosomes are distinct from LC3B-positive autophagosomes and cell- or tissue-specific roles for LC3A as opposed to LC3B may exist.…”