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. 2021 Jan 15;10(1):157.
doi: 10.3390/cells10010157.

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils

Kiho Son  1 , Amer Hussain  1 , Roma Sehmi  1 , Luke Janssen  1
Affiliations

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils

Kiho Son et al. Cells. .

Abstract

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell-substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

Keywords: actin; calcium signaling; confocal microscopy; integrin; mechanosensing; membrane ruffles; pseudopodia; shear stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterizing changes in cell–substrate contact area in response to pharmacological inhibitors. Each data point represents an individual eosinophil. Control-normalized cell flattening (see Section 2) in cells treated with vehicle (control; n = 20), CPA (n = 12), ryanodine (n = 10), CK-666 (n = 12), or Y-27632 (n = 11). * p < 0.05 by Kruskal–Wallis test.
Figure 2
Figure 2
Characterizing changes in perfusion-induced calcium response (PICR) in response to pharmacological inhibitors. (A) Numbers of cells exhibiting (blue bars) or not exhibiting (red bars) a PICR under control conditions or in the presence of cyclopiazonic acid (CPA), ryanodine, CK-666, or Y-27632, as indicated. * p = 0.037 (Fischer’s exact test). The control-normalized fluorescence response and latency for onset of the calcium-response under those same experimental conditions are given in (B,C), respectively. Each data point represents an individual eosinophil. * p = 0.022 and p = 0.002, respectively (Kruskal–Wallis test).
Figure 3
Figure 3
Analyzing the relationship between loss of circularity and the PICR in eosinophils. Each data point represents an individual eosinophil. (A) The peak elongation ratio for eosinophils under control conditions or in the presence of CPA, ryanodine, CK-666, or Y-27632, as indicated. (B) The time it took for the eosinophil to begin losing circularity after the calcium spike was measured across all treatment groups and compared against the untreated controls (*** p < 0.001; ** p < 0.01; p < 0.0001, Kruskal–Wallis test).
Figure 4
Figure 4
Assessing morphological changes in response to fluid shear stress. Eosinophils may exhibit distinct actin cytoskeletal protrusions in response to fluid shear stress. (A) Pseudopodia extension generally precedes cellular movement in the matching direction, whereas (B) membrane ruffles maintain membrane circularity and do not associate with cell motility. The cell tracker image (right) traces the cell membrane over the course of the experiment with a green-to-yellow color transition indicator. The capacity of eosinophils to develop pseudopodia (C) or membrane ruffling (D) was observed under control or treated conditions with CPA, ryanodine, CK-666, or Y-27632. Significance was calculated at (C) ** p = 0.0017 (Y-27632), (D) ** p = 0.0001 (ryanodine), and * p = 0.012 (CK-666), respectively (Fischer’s exact test).
Figure 5
Figure 5
Evaluating eosinophil motility response and its relationship with the PICR. (A) The centroid of analyzed eosinophils was tracked over the course of the experiment to determine the total distance traveled (µm) for control and treated (CPA, ryanodine, CK-666, and Y-27632) eosinophils. (*** p < 0.001; **** p < 0.0001) (B) A number of eosinophils changed their direction of movement subsequent to the calcium flash. The time in between the events was measured across treated eosinophils and compared with control eosinophils (* p < 0.05; ** p < 0.01; p = 0.008, Kruskal–Wallis test). (C) Percentage of cells in each group that altered their movement path towards one of four different directions (perfusion moves from left to right in the field of view (FOV)).
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