Apical oscillations in amnioserosa cells: basolateral coupling and mechanical autonomy

Abstract

Holographic laser microsurgery is used to isolate single amnioserosa cells in vivo during early dorsal closure. During this stage of Drosophila embryogenesis, amnioserosa cells undergo oscillations in apical surface area. The postisolation behavior of individual cells depends on their preisolation phase in these contraction/expansion cycles: cells that were contracting tend to collapse quickly after isolation; cells that were expanding do not immediately collapse, but instead pause or even continue to expand for ∼40 s. In either case, the postisolation apical collapse can be prevented by prior anesthetization of the embryos with CO2. These results suggest that although the amnioserosa is under tension, its cells are subjected to only small elastic strains. Furthermore, their postisolation apical collapse is not a passive elastic relaxation, and both the contraction and expansion phases of their oscillations are driven by intracellular forces. All of the above require significant changes to existing computational models.

Figure 1

An example cell-isolation experiment. (A–E) Pre- and postablation confocal images (inverted grayscale) showing retraction of the wound and eventual contraction of the isolated amnioserosa cell in a Drosophila embryo expressing eCadherin::GFP. (Upper left) Times relative to ablation. Overlays denote preablation shapes of the isolated cell (blue dashed) and the outer boundary of the wound (red dotted). A common scale bar is shown in panel E. (F) Comparison of cell shape dynamics for the total area inside the outer wound margins (red) and the apical area of the isolated cell (blue dashed). (G) Two-color confocal scan of an isolated cell imaged 80 s after separation from the surrounding tissue. (Green) E-Cad::GFP and (red) Sqh::mCherry. Note the accumulation of myosin along the outer margin of the wound. (H) Dynamics of myosin signal intensity. Each line shows the radial profile of myosin signal intensity about the centroid of the isolated cell (upper) or outer wound (lower). Separate graphs are necessary because the isolated cell and wound are not exactly concentric. The time at which each profile was measured is color-coded (blue to red from 10 s before to 200 s after ablation). The selected profiles are from the imaging planes that showed the most dynamic myosin profiles: close to the apical surface for the isolated cell; more basal (3-μm deeper) for the outer wound margin. See also Movie S1 in the Supporting Material.

Figure 2

Dynamic changes in apical area after isolation of pulsating amnioserosa cells. (A) Normalized apical area versus time for cells that were expanding just before ablation (red, N = 25) or those that were contracting (blue dashed, N = 16). (Lines) Mean behavior of each group. (Shaded areas) ±1 standard deviation. Cell areas were individually normalized to each cell’s mean area before ablation and then averaged to generate the group curves. (B) Initial rate of normalized area change, $\dot{A}/\overline{A}$ – where $\dot{A}$ is the rate of change of area and $\overline{A}$ is average preablation area of each cell—for cells isolated at different phases of their respective oscillation cycles. Results are grouped into 12 equal-width bins from –π to +π. Cells that were expanding have a negative phase; contracting cells have a positive phase. A phase of zero represents a cell at a temporally local maximum area. (Horizontal lines) Means for each bin. (Error bars) ±1 standard error of the mean. Two bins had no data and one bin (#) had only one data point. (C) Heat-map plot showing variation in the rate of area change, $\dot{A}/\overline{A}$, as a function of time after ablation and preablation oscillation phase. Rates of area change are shaded according to the legend. The strongest contractions correspond to the most negative rates of area change. The entire set of individual area versus time curves is compiled in Fig. S1 in the Supporting Material.

Figure 3

Comparison of high elastic strain and low elastic strain models. (A and B) Simulation of a cell-isolation experiment using a high strain model. (Dashed blue outline) Preablation shape and size of the isolated cell. (C) Normalized apical area versus time for cells that were expanding just before ablation (red, N = 48) or those that were contracting (blue dashed, N = 45). (Lines) Mean behavior of each group. (Shaded areas) ±1 standard deviation. Cell areas were individually normalized to each cell’s mean area before ablation and then averaged to generate the group curves. (D) Initial rate of normalized area change, $\dot{A}/\overline{A}$, for cells isolated at different phases of the oscillation cycle. Results are grouped into 12 equal-width bins from –π to +π. (Horizontal lines) Means for each bin. (Error bars) ± standard error of the mean. (E) Heat-map plot showing variation in the rate of area change, $\dot{A}/\overline{A}$, as a function of time after ablation and preablation oscillation phase. Rates of area change are shaded according to the legend. The strongest contractions correspond to the most negative rates of area change. (F–J) Matching results for simulations using a modified low-elastic-strain model with active wound-induced contraction. See also Movie S2, Fig. S3, Fig. S4, Fig. S5, and Table S1.

Figure 4

Cell isolation experiment in a CO₂-anesthetized embryo. (A–E) Pre- and postablation confocal images (inverted grayscale) showing slow retraction of the wound and almost no contraction of the isolated cell. (Upper-left) Times relative to ablation. Overlays denote preablation shapes of the isolated cell (blue dashed) and the outer boundary of the wound (red dotted). A common scale bar is shown in panel E. (F) Comparison of cell shape dynamics for the total area inside the outer wound margins (red) and the apical area of the isolated cell (blue dashed). (Uppermost curve) A cell in a different embryo exposed to CO₂ for the same length of time, but not ablated (black). (Shaded region) Duration of CO₂ exposure. See also Movie S3 and Movie S4.

Figure 5

Simulations of cell-isolation experiments in CO₂-anesthetized embryos using the high-elastic-strain (A–E) or low-elastic-strain models (F–J). CO₂ exposure was simulated by transiently suppressing all active contractions from −500 to +500 s. Overlays denote preablation shapes of the isolated cell (blue dashed) and the outer boundary of the wound (red dotted). (E and J) Area versus time for the wound (red) and the isolated cell (blue dashed) using each model. (Shaded area) Time during which active contractions were suppressed.

Figure 6

Three-dimensional dynamic changes in amnioserosa cell shape. (A–C) Three views are shown for each time point: an xy view of the apical area (bottom right); an xz cross-section (top); and a yz cross-section (left). (Darker/red shading) Extent of one cell. The rougher, outermost surface in each cross-section corresponds to the basal surface. A common scale bar is shown in panel C. (D) Changes in apical area and average cell thickness are anticorrelated: mean area versus thickness cross-correlation (green, solid, N = 6); mean autocorrelation of apical area (purple, dotted). The full set of apical area and thickness versus time graphs is compiled in Fig. S2B. One of the cells in Fig. S2B was excluded from the mean correlation functions because its autocorrelation function showed no evidence of oscillation. See also Movie S5.