Observations of intracellular tension dynamics of MC3T3-E1 cells during substrate adhesion using a FRET-based actinin tension sensor

Abstract

Tension in actin filaments plays crucial roles in multiple cellular functions, although little is known about tension dynamics of cells during adhesion to substrates. In this study, we visualized intracellular tension in actin filaments using a newly developed Förster resonance energy transfer (FRET)-based tension sensor (Actinin-sstFRET-GR). Tension dynamics were monitored during adhesion in MC3T3-E1 mouse osteoblastic cells after introduction of the sensor. Whole cell areas continued to increase from 10 to 180 min after plating and tension was monitored in a typical section close to the bottom, where the mean fluorescence was the largest. Tension on the bottom side was negatively correlated with cell area from 10 to 110 min. Thereafter, this correlation was positive until 180 min. We then analyzed central-peripheral differences in tension close to the bottom. In these experiments, tension in the cell periphery increased during expansion of the area and decreased during contraction. As a result, fluctuations of tension in this area were much larger than those in the central area of the cell. Finally, we analyzed upper-lower differences in tension development during initial adhesion and showed continuous decreases in the lower side of the cell from 10 to 110 min after plating, and decreases in the upper side until 70 min, followed by increases. In this study, we successfully visualized dynamic changes in intracellular tension at the sub-cellular level and found that development of tension during initial adhesion processes is time-dependent and has central-peripheral and upper-lower differences. The present FRET tension sensor may become a powerful tool in studies of cell biomechanics.

Keywords

• fret
• tension sensor
• cytoskeleton
• actin filament
• α-actinin
• mc3t3-e1
• adhesion