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Cited by 183Autophagy in Disease and TherapyUbiquitin and proteasome pathwaysCellular transport and secretion

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Autophagosome proteins LC3A, LC3B and LC3C have distinct subcellular distribution kinetics and expression in cancer cell lines.

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Koukourakis, Michael I., Kalamida, Dimitra, Giatromanolaki, Alexandra, Zois, Christos E., Sivridis, Efthimios, Pouliliou, Stamatia, Mitrakas, Achilleas, Gatter, Kevin C., Harris, Adrian L.·2015·PLoS ONE
DOI 10.1371/journal.pone.0137675PMC PMC4574774
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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.

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Fig 1 (A) Specificity of various anti-LC3 antibodies tested against recombinant LC3A and LC3B proteins. (B) The LC3A and LC3B processing after 24h of Bafilomycin (100nM) treatment in glioma and breast cancer cell lines, using the LC3A specific ab62720 and of the LC3B specific 5F10 antibodies (C) Double immunofluorecence in the A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the 5F10 antibody that reacts exclusively with LC3B. Noted a clearly distinct expression of LC3A in green vacuoles with perinuclear/nuclear localization and of LC3B red vacuoles that have a diffuse, throughout the cytoplasm, localization. There are no autophagosomes composed of both LC3A and LC3B proteins (c1,c2), whilst LC3A and LAMP2a show extensive colocalization (c3,c4). The distinct identity of LC3A and LC3B autophagosomes was also confirmed under acidic conditions (c5) and after exposure to bafilomycin (c6). (D) Double immunofluorecence in A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the NB600-1384 that reacts with both LC3A and LC3B proteins. Noted that the majority of the perinuclear/nuclear LC3A (green) vacuoles show double immunofluorescence (yellow), a result of double LC3A/LC3B reactivity that produces the NB600-1384 antibody. (E) LC3A and LC3B siRNAs specifically block the expression of the LC3A and LC3B proteins, respectively, in A549 cell line (E1,2,3). In E4 the reactivity of LC3A (ab62720 Ab) and of the LC3B (5F10 antibody) is shown, following silencing of the LC3A or of the LC3B genes, in the A549 cell line.
Fig 1 (A) Specificity of various anti-LC3 antibodies tested against recombinant LC3A and LC3B proteins. (B) The LC3A and LC3B processing after 24h of Bafilomycin (100nM) treatment in glioma and breast cancer cell lines, using the LC3A specific ab62720 and of the LC3B specific 5F10 antibodies (C) Double immunofluorecence in the A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the 5F10 antibody that reacts exclusively with LC3B. Noted a clearly distinct expression of LC3A in green vacuoles with perinuclear/nuclear localization and of LC3B red vacuoles that have a diffuse, throughout the cytoplasm, localization. There are no autophagosomes composed of both LC3A and LC3B proteins (c1,c2), whilst LC3A and LAMP2a show extensive colocalization (c3,c4). The distinct identity of LC3A and LC3B autophagosomes was also confirmed under acidic conditions (c5) and after exposure to bafilomycin (c6). (D) Double immunofluorecence in A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the NB600-1384 that reacts with both LC3A and LC3B proteins. Noted that the majority of the perinuclear/nuclear LC3A (green) vacuoles show double immunofluorescence (yellow), a result of double LC3A/LC3B reactivity that produces the NB600-1384 antibody. (E) LC3A and LC3B siRNAs specifically block the expression of the LC3A and LC3B proteins, respectively, in A549 cell line (E1,2,3). In E4 the reactivity of LC3A (ab62720 Ab) and of the LC3B (5F10 antibody) is shown, following silencing of the LC3A or of the LC3B genes, in the A549 cell line.CC BY · PubMed Central
Fig 2 (A) Confocal double immunofluorescence for LC3A (green) and LC3B (red) in various cell lines. Noted the LC3A accumulation in the perinuclear area, while LC3B is distributed throughout the cytoplasm (A1-7). LC3 aggregates, suggestive of small LC3A+ autophagosomes included in LC3B+ autophagosomes, are occasionally present (boxes in A1,2). Western blots confirm the nuclear presence of LC3A in the nuclei, mainly with the LC3A-II form (A8). (B) Nucleoli are stained with the MIB1/green (B1), the LC3B/red (B2), but not the LC3A/violet antibody (B3); LC3A specific ap1805a and of the LC3B specific 5F10 antibodies were used. This is confirmed in double immunostaining for LC3A/MIB1 (B4), LC3B/MIB1 (B5) and LC3A/LC3B (B6). Noted that LC3B stains the internal nucleolar areas, while MIB1 the peripheral. (C) Actinomycin D damages nucleoli and disrupts the nucleaolar LC3B and MIB1 localization (C1). Acidic conditions (pH = 6.5; C2) or hyperthermia (40°C; C3) do not abrogate the distribution of LC3B in the nucleoli nor of LC3A in the nuclei. Lamin is also used as a control to confirm the presence of nuclei only in the nuclear fraction.
Fig 2 (A) Confocal double immunofluorescence for LC3A (green) and LC3B (red) in various cell lines. Noted the LC3A accumulation in the perinuclear area, while LC3B is distributed throughout the cytoplasm (A1-7). LC3 aggregates, suggestive of small LC3A+ autophagosomes included in LC3B+ autophagosomes, are occasionally present (boxes in A1,2). Western blots confirm the nuclear presence of LC3A in the nuclei, mainly with the LC3A-II form (A8). (B) Nucleoli are stained with the MIB1/green (B1), the LC3B/red (B2), but not the LC3A/violet antibody (B3); LC3A specific ap1805a and of the LC3B specific 5F10 antibodies were used. This is confirmed in double immunostaining for LC3A/MIB1 (B4), LC3B/MIB1 (B5) and LC3A/LC3B (B6). Noted that LC3B stains the internal nucleolar areas, while MIB1 the peripheral. (C) Actinomycin D damages nucleoli and disrupts the nucleaolar LC3B and MIB1 localization (C1). Acidic conditions (pH = 6.5; C2) or hyperthermia (40°C; C3) do not abrogate the distribution of LC3B in the nucleoli nor of LC3A in the nuclei. Lamin is also used as a control to confirm the presence of nuclei only in the nuclear fraction.CC BY · PubMed Central
Fig 3 (A,B,C) 24h incubation with Leptomycin B at 10 ng/ml, results in nuclear accumulation of LC3A, LC3C and Beclin-1 in lung A549,H1299 and glioblastom U87,T98 cell lines. (D) 24h incubation with Ivermectin at 100μg/ml, reduced the nuclear expression of LC3A/Beclin-1 in A549 and H1299 cell lines and increased the perinuclear accumulation of LC3A in the H1299 cell line. (E) Western blots of the nuclear cell fraction confirm accumulation of LC3s and Beclin-1 in lung and glioblastoma cell lines after incubation with Leptomycin B. The reduction of proteins after incubation with Ivermectin was not consistent in all cell lines.
Fig 3 (A,B,C) 24h incubation with Leptomycin B at 10 ng/ml, results in nuclear accumulation of LC3A, LC3C and Beclin-1 in lung A549,H1299 and glioblastom U87,T98 cell lines. (D) 24h incubation with Ivermectin at 100μg/ml, reduced the nuclear expression of LC3A/Beclin-1 in A549 and H1299 cell lines and increased the perinuclear accumulation of LC3A in the H1299 cell line. (E) Western blots of the nuclear cell fraction confirm accumulation of LC3s and Beclin-1 in lung and glioblastoma cell lines after incubation with Leptomycin B. The reduction of proteins after incubation with Ivermectin was not consistent in all cell lines.CC BY · PubMed Central
Fig 4 (A) Cytoplasmic and nuclear staining of LC3C (red) in U87 cell line. (B) Double LC3C (red) and LC3B (green) immunostaining in U87 cells showing lack of co-localization between the two proteins, both in the cytoplasm and the nuclear/nucleolar regions. (C,D) Double LC3C (red) and LC3A (green) immunostaining showing co-localization between the two proteins, mainly in the nuclear area (U87 and T98 cell lines). (E) Western blot for LC3C in the nuclear (N), Pellet (P) and Soluble (S) fraction of U87 and T98 cells. ‘M’ is the marker and Lamin is used as a control to confirm the presence of nuclei only in the nuclear fraction.
Fig 4 (A) Cytoplasmic and nuclear staining of LC3C (red) in U87 cell line. (B) Double LC3C (red) and LC3B (green) immunostaining in U87 cells showing lack of co-localization between the two proteins, both in the cytoplasm and the nuclear/nucleolar regions. (C,D) Double LC3C (red) and LC3A (green) immunostaining showing co-localization between the two proteins, mainly in the nuclear area (U87 and T98 cell lines). (E) Western blot for LC3C in the nuclear (N), Pellet (P) and Soluble (S) fraction of U87 and T98 cells. ‘M’ is the marker and Lamin is used as a control to confirm the presence of nuclei only in the nuclear fraction.CC BY · PubMed Central

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Metadata

Authors
Koukourakis, M. I., Kalamida, D., Giatromanolaki, A., Zois, C. E., Sivridis, E., Pouliliou, S., Mitrakas, A., Gatter, K. C., & Harris, A. L.
Affiliations
  • Democritus University of Thrace (GR)
  • John Radcliffe Hospital (GB)
  • University of Oxford (GB)
Corresponding author
Koukourakis, Michael I.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-09-17
Volume
10
Issue
9
Pages
e0137675
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0137675
PMC
PMC4574774
OpenAlex
W2300762239
Cited by
183
References
21
Citations by year
2026: 13 · 2025: 12 · 2024: 27 · 2023: 16 · 2022: 16 · 2021: 17 · 2020: 19 · 2019: 24 · 2018: 13 · 2017: 14 · 2016: 11 · 2015: 1
FWCI
10.15
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Cancer Research UK (11359, 16466)
  • National Institute for Health and Care Research (NF-SI-0611-10163)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
LC3A, LC3B, LC3C, autophagosomes, autophagy, subcellular localization, nuclear localization, Beclin-1, nuclear export, nuclear import, leptomycin B, ivermectin, antibody specificity, cancer cell lines
MeSH terms
Beclin-1, Antibodies, Autophagy, Fatty Acids, Unsaturated, Gene Expression Regulation, Humans, Ivermectin, Membrane Proteins, Microtubule-Associated Proteins, Neoplasms, Macrolides, Active Transport, Cell Nucleus, RNA Interference, RNA, Small Interfering, Cell Line, Tumor, Apoptosis Regulatory Proteins, Human Umbilical Vein Endothelial Cells

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