Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells
March
Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells
Editor: Rafael Aldabe 0
SPAIN 0
Aleksandra Simiczyjew 0
Antonina Joanna Mazur 0
Ewelina Dratkiewicz 0
Dorota Nowak 0
0 Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw , Joliot-Curie 14a, Wroclaw , Poland
Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by nonmuscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers±ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes.
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Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: Dorota Nowak would like to thank the
Ministry of Science and Higher Education, Poland,
for Grant No. N N303337535. Aleksandra
Simiczyjew would like to thank the National Center
for Science for funds for doctoral thesis realization
on the basis of the decision number: DEC-2014/12/
T/NZ3/00503. Publication costs were supported by
Wroclaw Center of Biotechnology program of the
Leading National Research Center (KNOW) for
Introduction
Actin is an abundant protein which is essential for proper cell functioning. It takes part in many
physiological processes including cell motility, signal transduction, maintenance of cell shape,
ring formation during cytokinesis, cell adhesion, transcription and muscle contraction [
1
].
There are six actin isoforms present in vertebrates. Among them are β- and γ-non-muscle,
αskeletal, α-cardiac, and α-and γ-smooth muscle isoactins [
2
]. The α-actins as well as γ-smooth
muscle actin are present typically within muscle tissue, whereas non-muscle β- and γ-actin
isoforms, essential for cell survival, are present in almost all cell types [
3
]. Actin isoforms are
encoded by separate genes, but the primary structure of these proteins is very similar due to
2014-2018. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
high homology among their nucleotide sequences [
4,5
]. The β- and γ-actin isoforms differ only
by four amino acids present in the N-terminus part of the polypeptide chain. Within the β-actin
structure Asp-Asp-Asp tripeptide is present at positions 2, 3, 4 and Val at position 10, whereas
γ-actin contains Glu-Glu-Glu and Ile in the same positions of the polypeptide chain [2]. The
βto γ-actin ratio is distinct depending on the cell type [
4,6
]. In most cells the β- to γ-actin level
ratio is approximately 2:1 [
7,8
]. Levels of actin isoforms often change in cells under pathological
conditions [6,9±16].
It was also shown that β- and γ-actins in the form of both protein and mRNA are located in
different cytoplasmic areas [
5,17,18
]. β-actin was observed in protrusive structures responsible
for cell migrationÐlamella, pseudopod and at the tips and edges of regions involved in active
movement. This isoform is also essential during intravasation of cancer cells through the vessel
wall [
5,13,19
]. Presence of γ-actin was more often detected in the stress fibers, protrusions
which are involved in regulation of cell shape and differentiation [
5,20,21
]. In conflicting
reports Dugina et al. observed β-actin mainly in stress fibers and at cell-cell contacts, while
γactin was observed within the leading edge. The authors postulated that β-actin is essential for
cell attachment and contraction, whereas γ-actin is involved in cell motility [
22,23
]. It is well
known that they play unique roles in many physiological processes including regulation of
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