CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION
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Date
2024-05-16
Authors
Nurmagambetova, Assel
Journal Title
Journal ISSN
Volume Title
Publisher
Nazarbayev University School of Engineering and Digital Sciences
Abstract
RATIONALE: Epithelial-to-mesenchymal transition (EMT) is a process that
occurs during normal physiological processes (embryogenesis and organ
formation) and if it is inappropriately activated it can lead to pathological
processes (formation of scars, cancer metastasis, etc.). EMT is well studied at
the morphological and transcriptome level. However, cytoskeleton changes
during this process are less well understood. The cytoskeleton consists of
microtubules, actin filaments, and intermediate filaments. In addition, there are
protein complexes named focal adhesions that provide cell attachment to the
extracellular matrix, and connect the actin cytoskeleton with the extracellular
matrix. To describe the changes in the behavior of the cytoskeleton, namely
microtubules and actin cytoskeleton during EMT is of particular interest. In
addition, describing the behavior of focal adhesions during EMT is also
important.
AIM: The objective of this study is to describe quantitatively morphological
changes that occurred in post-EMT MCF-7, A-549, and HaCaT cells, analyze
microtubule dynamics, spatial organization, and its contribution to cell motility,
identify changes in actin filament organization and study focal adhesion
turnover.
HYPOTHESIS: The dynamics of microtubules in cells undergoing EMT
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might change. Cells undergoing EMT are expected to have more dynamic
microtubules. Cells undergoing EMT are expected to more efficiently adhere to
diverse substrates and therefore better spread. Focal contacts in cells
undergoing EMT are expected to be more pronounced and dynamic than in cells
not undergoing EMT.
METHODS: To study changes in post-EMT cells, EMT was induced in three
different cell models: MCF-7, A-549, and HaCaT. To evaluate that EMT
happened, western blot and quantitative polymerase chain reaction (q-PCR)
were applied to determine the level of expression of master regulators of EMT.
Cell images were recorded using bright field microscopy, and analyzed using
the Fiji Image J program.
In analyzed cells, microtubule networks and actin filaments were visualized by
immunofluorescence. To follow, describe, and measure microtubule dynamics
transfection with EB-3-RFP protein was conducted. To visualize focal
adhesions, two approaches were used: transduction with a talin red fluorescent
protein (Talin-RFP) and transient transfection with Ptag-RFP-vinculin. Films
were recorded using time-lapse fluorescent microscopy and analyzed using the
Fiji Image J program. All statistical analysis was performed using GraphPad
Prism (Dotmatics, USA) and a nonparametric Mann-Whitney U test or
parametric t-test with Welch correction. The actin filament measurements were
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completed using Matlab scripts.
CONCLUSION: This study showed morphological changes in three post-
EMT cell cultures studied. All types of cells increased in size. MCF-7 and
HaCaT became spread out, while A-549 became elongated. All three post-EMT
cell cultures had changes in microtubule organization and dynamics. Post-EMT
MCF-7 and HaCaT cells showed microtubules at a low density at cell borders,
while post-EMT A-549 cells had less covered nuclei by microtubules. In all
three studied models, the microtubule growth rate increased and the length of
the microtubule plus end tracks became longer. The average angle of
microtubule growth trajectories to cell radius decreased. Actin fibers rearranged
into stress fibers in post-EMT cells. The area of focal adhesions decreased in all
post-EMT cell cultures studied and focal adhesions appeared localized
throughout the inner areas of spread cells. These results indicate that
cytoskeletal changes make a significant contribution to the EMT process.
Description
Keywords
Type of access: Restricted, focal adhesions, cell morphology, cancer cells, metastasis, epithelial-to-mesenchymal transition
Citation
Nurmagambetova, A. (2024). Cytoskeleton dynamics and spatial organization during epithelial-to-mesenchymal transition. Nazarbayev University School of Engineering and Digital Sciences