Distribution of intracellular calcium during flow-induced migration of RAW264.7 cells

Abstract

Cell migration is an important biological process regulated by mechanical stimulation, which leads to intracellular calcium response. Cell migration are dependent on the distribution and dynamic changes of intracellular calcium concentration. However, the temporal relation among mechanical stimulation, cell migration, and intracellular calcium distribution remains unclear. In this study, unidirectional flow and oscillatory flow were applied on osteoclast precursor RAW264.7 cells. The parameters of cell migration under fluid flow and intracellular calcium distribution along the migration or flow direction were calculated. Experimental results suggest the cells to adjust the [Ca²⁺]_i distribution in the migration direction is independent of flow application or the reverse of flow direction, but the [Ca²⁺]_i distribution in the flow direction is determined by the [Ca²⁺]_i distribution-adjusting ability of cells and flow stimulation. Blocking calcium signaling pathways, namely, mechanosensitive cation-selective channels, phospholipase C, and endoplasmic reticulum, and removing extracellular calcium inhibited cell migration along the flow direction and the gradient distribution of intracellular calcium. This study provided insights into the mechanism of flow-induced cell migration and quantitative data for the recruitment of osteoclast precursors targeting the location of bone resorption.

Keywords: Calcium signaling; Cell migration; Fluid shear stress; Intracellular calcium distribution; Osteoclast precursor.

Fig. 1

Flow chamber and image processing. (a) Custom-made parallel flow chamber. (b) Schematic graph of experiment setup. (c) Schematic of flow chamber. (d) Definition of coordination systems for image processing, including local coordinate system x_mO₂y_m along the migration direction and x_fO₂y_f along the flow direction. The dotted and solid lines indicate the contours of a cell at its former and later location during migration. (e) Image processing for determining the parameters of cell migration and [Ca²⁺]_i distribution under fluid flow. In the [Ca²⁺]_i distribution graph, the red or green dots denote the points in the rear or front side along the migration direction and in the upstream or downstream side along the flow direction.

Fig. 2

Effect of unidirectional flow (5 ​dyne/cm²) on [Ca²⁺]_i distribution and its regulatory signaling pathways. (a) Time-lapsed pseudo-color images of RAW264.7 cells under unidirectional flow with different treatments. Scale bar, 10 ​μm. (b) Normalized ratio of fluorescence intensity under no-flow or unidirectional flow with different treatments along the migration direction ($R_M$) and flow direction ($R_F$), respectively. Plus (+) represents significant difference between non-treated and corresponding no-flow group. Asterisk (∗) means significant difference with corresponding non-treated group, p ​< ​0.05 (+, ∗, #).

Fig. 3

Statistical analysis on the normalized ratio of fluorescence intensity, migration speed, and migration orientation of RAW264.7 cells under different treatments. (a) No-flow group. (b) Non-treated group, (c) TG group, (d) U73122 group, (e) Gd³⁺ group, and (f) Ca²⁺-free group under oscillatory flow (5 ​dyne/cm²). Each data point was obtained from at least 12 cells for each group every 1 ​min during 20 ​min unidirectional flow stimulation.

Fig. 4

Effect of oscillatory flow (5 ​dyne/cm²) on [Ca²⁺]_i distribution and its regulatory signaling pathways. (a) Time-lapsed pseudo-color [Ca²⁺]_i images of RAW264.7 cells under oscillatory flow with different treatments. Scale bar, 10 ​μm. (b) Normalized ratio of fluorescence intensity under no-flow or oscillatory flow with different treatments along the migration direction ($R_M$) and flow direction ($R_F$), respectively. Plus (+) represents significant difference between non-treated and corresponding no-flow group. Asterisk (∗) means significant difference with corresponding non-treated group, p ​< ​0.05 (+, ∗, #).

Fig. 5

Statistical analysis on the normalized ratio of fluorescence intensity, migration speed, and migration orientation of RAW264.7 cells under different treatments. (a) No-flow group, (b) Non-treated group, (c) TG group, (d) U73122 group, (e) Gd³⁺ group, and (f) Ca²⁺-free group under oscillatory flow (5 ​dyne/cm²). Each data point was obtained from at least 12 cells for each group in every 1 ​min during 20 ​min oscillatory flow stimulation.

Fig. 6

Schematic of the FSS-induced migration of RAW264.7 cells and calcium signaling pathways. Osteoclast precursors move after being exposed to Unidirectional or oscillatory fluid flow and then the gradient distribution of [Ca²⁺]_i can be found in the cells although long-lasting oscillatory flow may reduce the adjustment of [Ca²⁺]_i distribution. This flow-induced [Ca²⁺]_i distribution and cell migration is regulated by MSCC-PLC-ER pathway.