Analysis of Morphological Changes in Focal Adhesion during Cell Adhesion and Spreading on Glass Substrate

Abstract

When trypsinized cells are seeded on a substrate, the initially spherical cells adhere to the substrate and spread extensively over time. During this process, focal adhesions (FAs), which are large protein complexes organized at the basal surface of cells, physically connect actin filaments to the extracellular matrix and play crucial roles in cellular morphology and signaling. However, the dynamics of substrate adhesion and morphological changes of the spherical cells remain unknown. In this study, we plated MC3T3-E1 osteoblast-like cells, which had been cell cycle-synchronized by serum starvation, to fibronectin-coated glass bottom dishes, and cultured for 10 min to 24 h to observe changes in FA morphology by measuring the cell area (A_cell) and FA morphological parameters including mean area of each FA (A_FA), number of FAs per cell (N_FA), and total area of FAs per cell (TA_FA). Subsequently, we investigated the size dependence of FA dynamics. We also analyzed FA density (D_FA), size (S_FA) and shape index (SI_FA) to compare the morphology between FAs that are underneath the nucleus to the morphology of FAs outside the nucleus. We found that A_cell, A_FA, N_FA and TA_FA increased continuously until 60 min post-plating. A_cell continued to increase after 60 min, while A_FA, N_FA and TA_FA showed complex changes over time. The percentage of small FAs was the highest early during adhesion (20 min) and decreased over time, while the percentage of moderate-to-large FAs increased until 1 h. After 1h, the percentages of small and middle FAs showed little changes, while the percentage of large FAs decreased gradually. These complex changes may reflect cellular demand for FAs. FAs underneath the nuclei were generally smaller than those outside the nuclei during the observed period. The density of FAs increased after 6 h, and FA shape became rounder after 3 h. These differences may be caused by regional differences in FA function: FAs underneath the nucleus may connect the nucleus to the substrate, while those outside the nucleus may connect the whole cell body to the substrate. We confirmed that FAs underwent time-dependent morphological changes during the adhesion process, including changes in size and position.