Real-time monitoring of cell elasticity reveals oscillating myosin activity

Abstract

The cytoskeleton is the physical and biochemical interface for a large variety of cellular processes. Its complex regulation machinery is involved upstream and downstream in various signaling pathways. The cytoskeleton determines the mechanical properties of a cell. Thus, cell elasticity could serve as a parameter reflecting the behavior of the system rather than reflecting the specific properties of isolated components. In this study, we used atomic force microscopy to perform real-time monitoring of cell elasticity unveiling cytoskeletal dynamics of living bronchial epithelial cells. In resting cells, we found a periodic activity of the cytoskeleton. Amplitude and frequency of this spontaneous oscillation were strongly affected by intracellular calcium. Experiments reveal that basal cell elasticity and superimposed elasticity oscillations are caused by the collective action of myosin motor proteins. We characterized the cell as a mechanically multilayered structure, and followed cytoskeletal dynamics in the different layers with high time resolution. In conclusion, the collective activities of the myosin motor proteins define overall mechanical cell dynamics, reflecting specific changes of the chemical and mechanical environment.

Figure 1

Phase micrograph of living 16HBE14o⁻ cells. Glass-cultured bronchial epithelial cells show an almost homogenous distribution of size and shape. Scale bar is 50 μm.

Figure 2

Real-time monitoring of cell elasticity reveals an oscillation with a period of 200 s. Each point represents the result calculated from a force-indentation cycle. Adjacent-averaging with a 20-points window was performed to smooth out short-term fluctuations and highlight periodic elasticity changes.

Figure 3

Time course of elasticity changes while blocking actin polymerization (A), lowering [Ca²⁺]_i (B), increasing [Ca²⁺]_i (C), and blocking myosin II activity (D). Adjacent-averaging with a 20-points window was performed to smooth out short-term fluctuations and highlight periodic elasticity changes.

Figure 4

Relative elasticity changes observed 500 s after application of substances known to change intracellular calcium concentration (light shaded bars) and to influence cytoskeletal dynamics (dark shaded bars) (mean ± SE, n = 5–8).

Figure 5

Dependency of the elasticity from the indentation depth. (A) Force at the power 2/3 versus indentation curve. Line a represents elasticity below 300 nm and line b indicates elasticity measured at an indentation above 500 nm. (B) Comparison of elasticities calculated from indentations below 300 nm and above 500 nm from the same dataset. Adjacent-averaging with a 20-points window were shown for indentations of <300 nm and >500 nm.

Figure 6

Effect of blocking myosin II on elasticities measured at indentations below 300 nm and above 500 nm.