Study of cell-matrix adhesion dynamics using surface plasmon resonance imaging ellipsometry

Abstract

The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.

Figure 1

(A) Schematic of the surface plasmon resonance imaging ellipsometry (SPRIE) system. (B) Image of the cell-matrix interface by SPRIE. The bright area indicates the cell-matrix adhesion area obtained by off-nulling. The dark area indicates the cell-free matrix area obtained by nulling. (C) Normalized intensity as a function of distance between the cell membrane and a gold film when nulled at a cell-free medium region. ATR coupler configuration: prism (n_P = 1.7367), gold (n_G = 0.467 – i 2.4075, d_G = 30 nm), ECM (n_E = 1.45, d_E = 20 nm), media (n_M = 1.333), cell (n_C = 1.36), wavelength = 532 nm, angle of incidence = 63°. All parameters are indicated in Table S1. (D) Schematic of fluidic chamber integrated in the SPRIE system.

Figure 2

Cell-matrix interfaces imaged by SPRIE. (A–C) Cell-matrix interfaces were imaged for human umbilical vein endothelial cells (HUVECs), human coronary artery smooth muscle cells (CASMCs) and a rat vascular smooth muscle cell line (A10). SPRIE images (left), corresponding three-dimensional intensity topography (middle frame), and X-Z surface map of the black line in topography (right) were compared at identical sites. The leading edge of the migratory cells is indicated by the arrow in B. The fibrillar structure is indicated by a dotted oval in C. Scale bar = 50 μm. (D) Quantification of the adhesion area of three cell types imaged by SPRIE. The adhesion area was quantified at the single cell level after normalization by cell area.

Figure 3

Comparative validation between SPRIE and CLSM. (A) The same cell site was consecutively imaged by SPRIE and CLSM. Cell-matrix adhesions in SPRIE are shown in pseudo-red, and paxillin is shown in green in CLSM. The merged area is shown in yellow. (B) To obtain a functional mapping of adhesion molecules, two types of adhesion molecules were compared using SPRIE and CLSM in the same manner. Green indicates paxillin, and red indicates tensin. In CASMC and A10, the nuclei are stained blue. Scale bar = 50 μm.

Figure 4

Cell-matrix adhesion dynamics during cell division. The red arrows show one mitotic cell progressing from one mother cell to two daughter cells. The elapsed time is indicated by hour:minute. Scale bar = 50 μm.

Figure 5

Cell-cell interactions imaged by SPRIE and validated by CLSM. (A) Cell-cell interactions of HUVECs imaged by SPRIE. (B and C) Intensity topography of inserted dotted rectangles in A. The arrows indicate fibril-like cell-cell connections. (D) CLSM image for VE-cadherin (red), actin (green), and nuclei (blue) on the same cells shown in A. (E) Histogram of the fluorescence intensity and SPRIE signal of line E inserted in A and D. The green line indicates actin, and the red line indicates VE-cadherin. The black line indicates SPRIE signal. Note that VE-cadherin and actin overlap, and the length of the peak indicates the thickness of the fibril-like cell-cell connections. Scale bar = 50 μm.

Figure 6

Cell adhesion dynamics modulated by shear stress (1.2 Pa). (A and B) Trajectory maps of cells in both static and shear stress conditions. (C) Migration velocity in both static and shear stress conditions. (D) Cell-matrix adhesion images obtained during modulation of shear stress; static (4 h), shear stress (18 h), static (18 h). The total elapsed time is 40 h. Scale bar = 50 μm.