CYTOSKELETON DYNAMICS AND SPATIAL ORGANIZATION DURING EPITHELIAL-TO-MESENCHYMAL TRANSITION

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 v 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 vi 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.