Collective movement of epithelial cells on a collagen gel substrate

Abstract

Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader" cells, which extended large lamellae and were accompanied by many "follower" cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.

FIGURE 1

(A) Typical phase contrast micrograph of the collagen gel surface taken just before the time-lapse measurement. The bar represents 20 μm. (B) A histogram of the 2-D power spectrum calculated from the phase contrast micrograph shown in panel A as a function of orientation. The spectrum showed a good fit with the Gaussian distribution function (solid curve). Φ₀ and σ represent mean angle and standard deviation, respectively.

FIGURE 2

Temporal sequence of phase contrast micrographs of an epithelial colony on the collagen gel surface. Numbers in the images represent relative time from the start of observation. The direction of each cell movement is marked by colored dots in the micrographs. Arrows in the image at 60 h represent the direction of massive cell streams. Note that not all cell movements are marked, especially in the images after 70 h, because in some cells, the distance of cell movement was too small to determine the direction of movement from the captured images. A hue circle is shown as a legend for the angular displacement. Bar is 100 μm.

FIGURE 3

Time-lapse micrographs of an epithelial colony on the glass substrate. Representative actual images taken every 5 min are shown. Numbers in the micrographs denote the observation time. Colored dots represent the directions of cell movements, which are defined by the hue circle shown in the figure. Bar is 100 μm.

FIGURE 4

(A) Typical mean-squared displacements calculated as a function of time interval, Δt. The asymptotic power-law function (Eq. 2) was fitted by the linear least-squares method (solid lines). Both data were linear on a log-log scale, implying anomalous diffusion due to temporal and/or spatial correlations. (B) Exponents of the asymptotic power-law behavior of the MSD as a function of the actual observation time. Twenty cell trajectories on each substrate were chosen at random and analyzed. Error bars denote 95% confidential limits calculated by Student's t-test.

FIGURE 5

Spatial correlation functions of the cell velocity on glass (A) and gel (B) substrates as a function of distance between cells. Different symbols represent the different observation times corresponding to Figs. 2 and 3.

FIGURE 6

Average cosine of the angle between the direction of cell movement, θ, and the mean polarization of the gel substrate, Φ₀, plotted as a function of observation time. Averages were determined from 50 to 300 cell trajectories on each gel substrate. σ denotes the standard deviation of the substrate polarization; large values of σ represent low polarity of the substrate. Even in the poorly oriented substrate, the difference in angle between cell movement and collagen orientation decreased after 60 h, which is represented in the figure as interpolating lines.

FIGURE 7

Time-lapse phase contrast images of a portion of an epithelial colony on the collagen gel surface. Contrast on the micrographs was intensified to allow visualization of the thick collagen fibers. Numbers in the images represent relative times from the start of observation. The “leader” cells are marked L in the micrographs. Arrowheads in the image indicate the collagen fibers contracted by the cells. The bar represents 50 μm.