Actin-ring segment switching drives nonadhesive gap closure

Abstract

Gap closure to eliminate physical discontinuities and restore tissue integrity is a fundamental process in normal development and repair of damaged tissues and organs. Here, we demonstrate a nonadhesive gap closure model in which collective cell migration, large-scale actin-network fusion, and purse-string contraction orchestrate to restore the gap. Proliferative pressure drives migrating cells to attach onto the gap front at which a pluricellular actin ring is already assembled. An actin-ring segment switching process then occurs by fusion of actin fibers from the newly attached cells into the actin cable and defusion from the previously lined cells, thereby narrowing the gap. Such actin-cable segment switching occurs favorably at high curvature edges of the gap, yielding size-dependent gap closure. Cellular force microscopies evidence that a persistent rise in the radial component of inward traction force signifies successful actin-cable segment switching. A kinetic model that integrates cell proliferation, actin fiber fusion, and purse-string contraction is formulated to quantitatively account for the gap-closure dynamics. Our data reveal a previously unexplored mechanism in which cells exploit multifaceted strategies in a highly cooperative manner to close nonadhesive gaps.

Keywords: actin ring; cell patterning; gap closure; traction force microscopy.

Fig. 1.

Gap-size–dependent closure of the MDCK cell monolayers over nonadhesive gaps. (A) Representative gap-closure behaviors at day 5 of culture for different initial gap sizes (denoted by the white dashed lines, elliptical gaps: major/minor radii, 75/50 μm for $R_{eq}=61.2\hspace{0.17em}\mathrm{\mu m}$, and 100/50 μm for $R_{eq}=70.7\hspace{0.17em}\mathrm{\mu m}$, respectively) and different monolayer sizes on 6 kPa hydrogel. Scale bar, 100 μm. (B) Closure probability for different gap sizes and different monolayer sizes. For each gap type, data were collected from n = 48 gaps. (C) Gap closure kinetics (starting at 40 h after cell seeding). The sample size ranges from 5 (for $R_{eq}=61.2\hspace{0.17em}\mathrm{\mu m}$ and $R_{eq}=70.7\hspace{0.17em}\mathrm{\mu m}$ gaps) to 13 (for $R=50\hspace{0.17em}\mathrm{and}\hspace{0.17em}100\hspace{0.17em}\mathrm{\mu m}$ gaps) at each time point for different initial gap sizes.

Fig. 2.

The influence of cell contractility and cell proliferation on the nonadhesive gap closure. (A) With increasing gel stiffness, cell contractility increases, and so does the gap-closure probability. The white arrows point to the actin ring segments. (B) Blebbistatin inhibits cell contractility and hence the actin-ring formation, leading to decreased gap-closure probability. (C) Cell contractility and substrate stiffness-dependent gap-closure probability at day 4 of culture. Increasing hydrogel stiffness increases the closure probability of the 50-μm-radius gaps at day 4 of culture but has negligible effects on the closure probability at day 5 of culture, beyond which the closure probability of the gaps remains nearly unchanged. Sample size: n = 48 gaps. *P < 0.05. (D) Adding mitomycin C inhibits cell proliferation and cell attachment to the gap front, but the formation of the actin ring at the gap edge is nearly unaffected. The white arrows point to cells attached onto the gap front in the absence of mitomycin C. Scale bar, 50 μm.

Fig. 3.

The time evolution of F-actin and cell morphologies at the gap boundary is determined by the nonadhesive gap size. (A) A circular gap with $R_{eq}=50\hspace{0.17em}\mathrm{\mu m}$. (B) A circular gap with $R_{eq}=100\hspace{0.17em}\mathrm{\mu m}$. (C) An elliptical gap with $R_{eq}=61.2\hspace{0.17em}\mathrm{\mu m}$ (major/minor radii, 75/50 μm). The white dashed lines indicate the initial gap regions. Scale bar, 50 μm. Green/blue: F-actin/nuclei.

Fig. 4.

Live-cell imaging of the gap-closure process. (A, Top) Time-lapse phase-contrast images showing the swapping in positions of two MDCK cells at the gap front. (Bottom) Time-lapse images using LifeAct-GFP-expressing MDCK cells, showing the updating actin network due to the swap of the cells at the gap front. Initial gap radius: 50 µm. The manually highlighted cells, in red and blue, swapped their locations. Scale bar, 50 µm. (B) Confocal fluorescent imaging of the leading front of MDCK cells tagged with LifeAct-RFP when crossing the nonadhesive gap (dashed green circle; diameter of 40 µm). Three events of one follower cell (denoted with the magenta, cyan, and yellow stars in sequence) fusing into the leading front were observed, contributing to the formation of updated and reinforced actin cable (denoted with magenta, cyan, and yellow arrowheads in sequence) over the gap region. The dashed magenta line outlines one follower cell that gradually moved to the leading front (30 min), fused into the leading front (40 min), and finally reinforced the actin-cable segment that formed a prominent purse string over the gap region (50 min; magenta arrowheads). Subsequently, the reinforced purse string at the lead front depolymerized as another follower cell fused into the leading front (80 min; marked with blue manually), which allowed for the recovery of the purse string over the gap (90 min; cyan arrowheads). Finally, the third fusion with actin segment update (yellow star) was observed for the same cell (marked with blue). Scale bar, 10 µm. (C) Color-coded montage of the region of interest (dashed yellow box in B). The dashed white line shows the flow of actin from the follower into the leading front. For A and B, the original time-lapse images without the manually colored marks can be found in Movies S1, S2, and S3.

Fig. 5.

Traction force microscopy (TFM) of the gap-closure process. (A and B) Phase-contrast images and vector plot of traction force at different gap-closure stages. (A) $R_{eq}=50\hspace{0.17em}\mathrm{\mu m}$. (B) $R_{eq}=100\hspace{0.17em}\mathrm{\mu m}$. Scale bar for phase-contrast images, 100 μm. The size of the traction force maps is $300\times 300\hspace{0.17em}\mathrm{\mu }{\mathrm{m}}^2$, with the center the same as the initial gap center. (C and D) The average radial and tangential traction forces as a function of distance from the gap center $\left(r=0\right)$. (C) $R_{eq}=50\hspace{0.17em}\mathrm{\mu m}$. (D) $R_{eq}=100\hspace{0.17em}\mathrm{\mu m}$. TFM was performed on eight parallel samples. Each data point was averaged over n = 16 gaps.

Fig. 6.

The change of the radial component of the RCFP stress during the gap closure process as a function of distance from the gap center $\left(r=0\right)$. (A) The change of RCFP for the 50-μm-radius gap. (B) The change of RCFP for the 100-μm-radius gap.

Fig. 7.

Modeling of the gap closure kinetics. (A) A model prediction of the gap perimeter changes with time in comparison with experimental data (n = 5 for the elliptical gap; n = 13 for the circular gap). The experimental data are from three parallel experiments. (B) The predicted morphological change of an elliptical gap with a semimajor axis of 75 μm and a semiminor axis of 50 μm at different time points.