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Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis

  • Authors:
    • Eleni I. Theotoki
    • Panos Kakoulidis
    • Konstantinos-Stylianos Nikolakopoulos
    • Eleni N. Vlachou
    • Ourania E. Tsitsilonis
    • Gerassimos E. Voutsinas
    • Ema Anastasiadou
    • Dimitrios J. Stravopodis
  • View Affiliations / Copyright

    Affiliations: Section of Cell Biology and Biophysics, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens 15701, Greece, Department of Cancer Genetics, Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece, Section of Animal and Human Physiology, Department of Biology, School of Science, National and Kapodistrian University of Athens, Athens 15701, Greece, Laboratory of Molecular Carcinogenesis and Rare Disease Genetics, Institute of Biosciences and Applications, National Center for Scientific Research ‘Demokritos’, Athens 15310, Greece
    Copyright: © Theotoki et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 244
    |
    Published online on: July 7, 2025
       https://doi.org/10.3892/mmr.2025.13609
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Abstract

Argonaute RNA‑induced silencing complex catalytic component 2 (AGO2) is an evolutionary conserved protein involved in microRNA‑dependent gene expression regulation via the RNA interference (RNAi) mechanism. Nevertheless, AGO2 may also be involved in other key processes, such as histone modification, DNA methylation and alternative splicing. Its role in the proper development of organisms is key and no homologue is able to compensate for its loss. Therefore, using advanced immunofluorescence, transient transfection and molecular bioinformatics, the present study aimed to investigate novel, non‑canonical, RNAi‑dependent functions of AGO2 protein in mRNA/protein local homeostasis. The data revealed microtubule network‑dependent, localization of AGO2 in both centrosome and mitotic spindle assemblies during cell division and in the cytokinetic bridge formed during the last stage of mitosis (cytokinesis). Detection of AGO2 protein in these mitosis‑specific compartments, regardless of the presence of malignant phenotypes or multiple centrosomes/mitotic spindles in liver cells, indicates the cardinal role of AGO2 in centrosome biosynthesis, mitotic spindle formation and function, potentially controlling locality‑dependent homeostasis, in a novel non‑canonical, RNAi‑dependent manner. This novel AGO2/centrosome/mitotic spindle/cytokinetic bridge pathway may serve as a versatile molecular ‘toolbox’ for targeted therapy of human malignancy, including liver cancer.
View Figures

Figure 1

Punctuate patterning of AGO2 protein
in the cytoplasm of LX-2 liver cells during interphase. Confocal
laser scanning immunofluorescence images of LX-2 cells at the
interphase stage overexpressing the human AGO2 protein being fused
with the GFP reporter protein (eGFP-hAGO2), after immuno-staining
with an anti-AGO2 primary antibody. Overlap (yellow) demonstrated
the specificity of the anti-AGO2 primary antibody. Nuclei were
visualized with DAPI staining (blue). Scale bar, 10 µm. AGO2,
argonaute RNA-induced silencing complex catalytic component 2.

Figure 2

AGO2 distribution profiling in LX-2
liver cells undergoing mitosis. Confocal laser scanning microscopy
of LX-2 cells during interphase and mitosis demonstrating
endogenous AGO2 protein expression and distribution in the
cytoplasm of interphase cells (punctuate pattern), and in the
centrosome, mitotic spindle and cytokinetic bridge of mitotic cells
(white arrowheads). Nuclear/chromosomal DNA is indicated in blue by
DAPI. Yellow is produced by the overlap of green and red. Scale
bar, 10 µm. AGO2, argonaute RNA-induced silencing complex catalytic
component 2.

Figure 3

Localization of AGO2 protein in HepG2
liver cancer cells during mitosis. Confocal laser scanning
microscopy immunofluorescence images of HepG2 cells at the
interphase and mitotic stages, demonstrating AGO2 protein
expression and distribution in the cytoplasm at interphase, and in
the centrosome, mitotic spindle and cytokinetic bridge (white
arrowheads) during mitosis of dividing cells. Nuclear/chromosomal
DNA is indicated in blue by DAPI. Yellow, merger of green and red.
Scale bar, 10 µm. AGO2, argonaute RNA-induced silencing complex
catalytic component 2.

Figure 4

Co-localization of AGO2 with
centrin-2 and γ-tubulin centrosomal proteins during cell cycle.
Confocal laser scanning microscopy immunofluorescence images of
LX-2 liver cells, showing co-localization patterns (white
arrowheads) of AGO2 protein with key (A-D) centrosomal components
centrin-2 (centriolar protein) and (I-L) γ-tubulin (PCM protein).
(E-H) PCM-1 did not co-localize with AGO2. Cell nucleus
(chromosomal DNA) is presented in blue by DAPI. Yellow, merge of
green and red. Scale bar, 10 µm. AGO2, argonaute RNA-induced
silencing complex catalytic component 2; PCM-1,
peri-centriolar-material 1.

Figure 5

AGO2 protein distribution in LX-2
cells undergoing mitosis. Confocal laser scanning microscopy
immunofluorescence images of LX-2 hepatic/liver cells, presenting
AGO2 localization patterns at different stages of mitosis.
Nuclear/chromosomal DNA is presented in blue by DAPI. Yellow,
merger of green and red. Scale bar, 10 µm. AGO2, argonaute
RNA-induced silencing complex catalytic component 2.

Figure 6

Co-localization of AGO2 and DICER
proteins in LX-2 liver cells undergoing mitosis. Immunofluorescence
confocal laser scanning microscopy of centrosome- and cytokinetic
bridge-specific co-localization of the AGO2 and DICER proteins in
LX-2 mitotic cells (white arrowheads). Nuclear/chromosomal DNA is
shown in blue by DAPI. Yellow, merger of green and red. Scale bar,
10 µm. AGO2, argonaute RNA-induced silencing complex catalytic
component 2; DICER, double-stranded RNA endoribonuclease.

Figure 7

TRBP2 does not co-compartmentalize
with AGO2 in LX-2 hepatic/liver cells. Confocal laser scanning
microscopy immunofluorescence images of LX-2 dividing cells
demonstrating the absence of AGO2 and TRBP2 co-localization in the
centrosome, mitotic spindle and cytokinetic bridge apparatuses
during mitosis. There was no TRBP2 detection at the anaphase stage
in LX-2 mitotic cells (middle panels). Nuclear/chromosomal DNA is
presented in blue by DAPI. Scale bar, 10 µm. AGO2, argonaute
RNA-induced silencing complex catalytic component 2; TRBP2,
transactivation response element RNA-binding protein.

Figure 8

AGO2 and UPF1 distribution in LX-2
interphase cells. Confocal laser scanning microscopy
immunofluorescence images of LX-2 hepatic/liver cells at the
interphase stage, showing cytoplasmic localization patterns of
endogenous (A) AGO2 and UPF1 protein expression in control, (B)
UPF1 and eGFP-hAGO2 protein expression in the AGO2-overexpressing
cells and (C) AGO2 and eGFP-UPF1 protein expression in
UPF1-overexpressing cells. Nuclear/chromosomal DNA is presented in
blue by DAPI. Yellow (punctate cytoplasmic patterning), merge of
green and red. Scale bar, 10 µm. AGO2, argonaute RNA-induced
silencing complex catalytic component 2; UPF1, up-frameshift
protein 1.

Figure 9

UPF1 does not compartmentalize with
α-tubulin in mitotic apparatuses during LX-2 cell division.
Confocal laser scanning microscopy immunofluorescence images of
centrosome, mitotic spindle and cytokinetic bridge machineries
(containing α-tubulin as a major structural component) (white
arrowheads) lacking UPF1 localization in LX-2 hepatic/liver cells
undergoing mitosis. Nuclear/chromatin DNA is shown in blue by DAPI.
Scale bar, 10 µm. UPF1, up-frameshift protein 1.

Figure 10

Microtubule network disintegration
causes disruption of AGO2 mitotic patterning in LX-2 dividing
cells. Confocal laser scanning microscopy immunofluorescence images
of LX-2 hepatic/liver cells, presenting AGO2 distribution, in
control (DMSO) and microtubule polymerization inhibitor
demecolcine-treated (0.4 µg/µl; 6 h) mitotic and interphase cells.
In response to demecolcine, AGO2 protein loses its
centrosome/mitotic spindle axis localization pattern at mitosis and
its cytoplasmic compartmentalization at interphase.
Nuclear/chromatin DNA is indicated in blue by DAPI. Scale bar, 10
µm. AGO2, argonaute RNA-induced silencing complex catalytic
component 2.

Figure 11

Centrosome/mitotic spindle
aberrations do not alter mitotic co-localization of AGO2 and
α-tubulin proteins in LX-2 dividing cells. Confocal laser scanning
microscopy immunofluorescence images of rare LX-2 liver cell
sub-populations carrying aberrant (number, geometry, topology and
architecture) centrosome and mitotic spindle structures
(multi-polar spindles). AGO2 and α-tubulin co-localization
patterning was observed in aberrant mitotic apparatuses (white
arrowheads). Nuclear/chromatin DNA is indicated in blue by DAPI.
Yellow, merge of green and red. Scale bar, 10 µm. AGO2, argonaute
RNA-induced silencing complex catalytic component 2.

Figure 12

Molecular modelling of AGO2
protein-AGO2 protein-interactor complexes. Docking tests between
AGO2 and potential interacting proteins. (A) Predicted 3D molecular
models of AGO2 protein with interactors, such as centrin-2,
γ-tubulin, UPF1, α-tubulin and DICER* proteins. (B) Dissociation
constant (Kd) values, indicating the possibility
(complex stability) of bi-molecular binding between AGO2 and
putative interactors centrin-2, γ-tubulin, UPF1, α-tubulin and
DICER*. *Constrained docking between the AGO2 PIWI (position:
517–818th aa; 301 residues) and the DICER RNase III A domain
(position: 1,295-1,596th aa; 301 residues). AGO2, argonaute
RNA-induced silencing complex catalytic component 2; aa, amino
acid; DICER, double-stranded RNA endoribonuclease; UPF1,
up-frameshift protein 1.
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Copy and paste a formatted citation
Spandidos Publications style
Theotoki EI, Kakoulidis P, Nikolakopoulos K, Vlachou EN, Tsitsilonis OE, Voutsinas GE, Anastasiadou E and Stravopodis DJ: Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis. Mol Med Rep 32: 244, 2025.
APA
Theotoki, E.I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E.N., Tsitsilonis, O.E., Voutsinas, G.E. ... Stravopodis, D.J. (2025). Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis. Molecular Medicine Reports, 32, 244. https://doi.org/10.3892/mmr.2025.13609
MLA
Theotoki, E. I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E. N., Tsitsilonis, O. E., Voutsinas, G. E., Anastasiadou, E., Stravopodis, D. J."Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis". Molecular Medicine Reports 32.3 (2025): 244.
Chicago
Theotoki, E. I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E. N., Tsitsilonis, O. E., Voutsinas, G. E., Anastasiadou, E., Stravopodis, D. J."Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis". Molecular Medicine Reports 32, no. 3 (2025): 244. https://doi.org/10.3892/mmr.2025.13609
Copy and paste a formatted citation
x
Spandidos Publications style
Theotoki EI, Kakoulidis P, Nikolakopoulos K, Vlachou EN, Tsitsilonis OE, Voutsinas GE, Anastasiadou E and Stravopodis DJ: Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis. Mol Med Rep 32: 244, 2025.
APA
Theotoki, E.I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E.N., Tsitsilonis, O.E., Voutsinas, G.E. ... Stravopodis, D.J. (2025). Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis. Molecular Medicine Reports, 32, 244. https://doi.org/10.3892/mmr.2025.13609
MLA
Theotoki, E. I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E. N., Tsitsilonis, O. E., Voutsinas, G. E., Anastasiadou, E., Stravopodis, D. J."Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis". Molecular Medicine Reports 32.3 (2025): 244.
Chicago
Theotoki, E. I., Kakoulidis, P., Nikolakopoulos, K., Vlachou, E. N., Tsitsilonis, O. E., Voutsinas, G. E., Anastasiadou, E., Stravopodis, D. J."Centrosome‑, mitotic spindle‑ and cytokinetic bridge‑specific compartmentalization of AGO2 protein in human liver cells undergoing mitosis: Non‑canonical, RNAi‑dependent, control of local homeostasis". Molecular Medicine Reports 32, no. 3 (2025): 244. https://doi.org/10.3892/mmr.2025.13609
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