Shock wave induced cytoskeletal and morphological deformations in a human renal carcinoma cell line

Abstract

Effects of shock waves on the morphology and cytoskeleton of a human renal carcinoma cell line (ACHN) were investigated in vitro. ACHN monolayer cultured on a cover slide glass was treated with 10 shots of focused underwater shock waves, with 16 MPa peak pressure at the focal area of a piezoceramic shock wave generator. After exposure to the shock wave, based on the severity of morphological deformations of the treated cells, the monolayer was divided into three morphological areas; focal, marginal and intact. Morphological deformations were found to be associated with disorganization of the intracellular cytoskeletal filaments. Deformation of the cytoskeletal proteins in the treated cells were separately studied with respect to the location of the cells within the three morphological areas. Among three major cytoskeletal proteins, actin and tubulin, but not vimentin, were affected by the shock waves. The deformed cells reorganized their cytoskeletal network within 3 h with a pattern similar to the control, indicating the transient characteristic of the shock wave induced cytoskeletal damage in the surviving cells. The remaining cell fragments on the slide glass, which contained short actin filaments, indicated the important role of shear stress in damaging the cytoskeletal fibers by shock waves.

Figure 1

Schematic diagram of the experimental set‐up with a piezoceramic generator placed at the bottom of a degassed water bath. Cell monolayer cultured on cover slide glass positioned at the focal area of the piezoceramic dish was treated with 10 shock waves with P⁺ _max = 16 MPa.

Figure 2

Measured pressure history at the focal point for a discharge voltage of 0.1 kV.

Figure 3

(a) Measured pressure history at the focal point; (b) the lateral peak pressure distribution of the focal area after a maximum voltage discharge of 1.5 kV.

Figure 4

Level of monolayer damage associated with the applied peak pressure observed by light microscopy. (a) Control; (b) 1.2 MPa; (c) 4.0 MPa; (d) 7.0 MPa; (e) 13 MPa; (f) 16 MPa. (hematoxylin and eosin staining, original magnification 40×).

Figure 5

Morphological deformations in the shock wave treated monolayer observed by scanning electron microscopy: (a) Three morphological areas in the monolayer: focal, marginal and intact area (scale bar 2.0 mm); (b) the remaining cell fragments (arrows) on the slide glass within the focal area (scale bar 100 µm); (c) blebs (arrows) and wrinkles on the surfaces of the rounded cells within the marginal area (scale bar 10 µm).

Figure 6

Immunofluorescence staining of a control monolayer with phalloidin‐fluorescein (FITC) antibody to actin filaments. (a) Cell cortex at the cell periphery contains short actin filaments. Arrow indicates the network of cytoplasmic actin filaments. (b) Long actin bundles of stress fibers at the lower surface of the cells (original magnification 1000×).

Figure 7

Immunofluorescence staining of the focal area with phalloidin‐fluorescein (FITC) antibody to actin filaments. (a) Remaining cell fragments on the slide glass (indicated by arrows) containing fragments of actin filaments. (b) Depolymerized actin filaments were aggregated at the periphery of spherical‐shaped cells detached from the substratum (original magnification 1000×).

Figure 8

Immunofluorescence staining of the marginal area with phalloidin‐fluorescein (FITC) antibody to actin filaments. (a,b) Disrupted cell cortex at the cell periphery indicated by arrows. (c) Stress fibers were thickened, aggregated and shortened in the retracted cells. (d) Retraction of cytoplasmic actin filaments toward the cell periphery produced a fluorescence‐negative zone at the center of the cells (original magnification 1000 ×).

Figure 9

Development of actin filaments during spreading of shock wave treated ACHN cells (a human renal carcinoma cell line) fixed and stained (a) after 3 h; (b) after 20 h (original magnification 1000×).

Figure 10

Immunofluorescence staining with anti β‐tubulin antibody: (a) control ACHN cell (a human renal carcinoma cell line); (b) shock wave treated cell (original magnification 1000×).

Figure 11

Immunofluorescence staining with antivimentin antibody: (a) control ACHN cell (a human renal carcinoma cell line); (b) shock wave treated cell (original magnification 1000×).