Volume 33, Issue 5, Pages (June 2015)

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1 Volume 33, Issue 5, Pages 611-621 (June 2015)
Decrease in Cell Volume Generates Contractile Forces Driving Dorsal Closure  Laure Saias, Jim Swoger, Arturo D’Angelo, Peran Hayes, Julien Colombelli, James Sharpe, Guillaume Salbreux, Jérôme Solon  Developmental Cell  Volume 33, Issue 5, Pages (June 2015) DOI: /j.devcel Copyright © 2015 Elsevier Inc. Terms and Conditions

2 Developmental Cell 2015 33, 611-621DOI: (10.1016/j.devcel.2015.03.016)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3 Figure 1 DC Proceeds without Apical Constriction and Apico-Basal Elongation but with Cell Volume Decrease (A) Schematics depicting the dorsal (top) and transverse views of AS and epidermal tissues during DC. Before DC completion, the dorsal pole of a Drosophila embryo is covered by the AS tissue, surrounded by epidermis. The AS tissue is described by the size of the opening L and the thickness of the cells along the apico-basal axis h. (B) Transversal (left) and dorsal (right) views of the AS tissue during DC imaged on a sqh-Moe GFP expressing embryo using SPIM (left) and on sqh-GFP expressing embryo using confocal microscopy (right). Time stamps indicate the time after the onset of closure defined by the increase in myosin intensity within the actin cable. The transversal view shows an absence of apico-basal elongation during most of DC, while the dorsal view shows an absence of myosin enrichment. The red rectangle represents the central area of the AS tissue, where SPIM transverse sections were performed. The arrows indicate the position of the leading edge. The scale bar represents 40 μm. (C) Average initial recoil velocity, proportional to the ratio tension over friction T/μ, after laser dissection of AS tissue junctions at different time points of DC. Averaging was performed from the individual measurements shown in Figure S2C, and time was estimated from the opening length (L in [A]) of the AS tissue at which the dissection was performed (from Figure S1C). N = 53 embryos. Error bars are SDs. (D) (Left) Average opening size L and average thickness h over time of the AS cells measured on SPIM imaging of sqh-Moe-GFP embryos (n = 15 sections on 5 embryos, bars are SDs). Onset of closure is defined by the time at which myosin intensity starts to increase within the actin cable (Figure 3C). AS cell height does not significantly change during the first 80 min after the onset of closure. The arrow indicates the approximate time of the onset of cell apico-basal elongation. The shaded area defines the time interval where volume estimation was not performed because of the tissue invagination. (Right) Average volume of transverse AS tissue strips in the central area determined by the product of the lateral area, Alat, by the average cellular anterior-posterior distance, lAP; n = 15 strips on 5 embryos. Bars are SDs. The dashed line labels the onset of DC. The AS tissue strips volume shows a decrease of about 40% in 100 min. (E) (Left) Schematics showing the two different methods of single-cell volume calculation. The cell volume is estimated on one hand by the product of the apical cell surface Aa with the cell height h and by the sum of segmented areas of single AS cells measured on individual confocal sections with the Brainbow system on the other hand. (Right) AS cell volume variation during DC. The volume is measured by the product Aa · h (green, n = 17 cells on 3 embryos), where Aa was measured from confocal data and h from SPIM, or by the sum of the segmented area performed on confocal sections of AS cells expressing the Brainbow system (red, n = 10 cells on 5 embryos). Both measurements show a similar decrease in volume in the first 100 min of closure. Bars are SDs. Developmental Cell  , DOI: ( /j.devcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions

4 Figure 2 AS Cell Volume Decrease Is Triggered by the Activation of Caspases at the Onset of DC (A) Time lapses of ECadh-GFP C381-Gal4-UAS Apoliner5 expressing embryos during DC with close-up views of a group of cells. The onset of DC and actin cable formation coincides with the activation of caspases in all AS cells (appearance of the GFP within AS cell nucleus). The dashed red lines highlight a cell nucleus before and after the activation of caspases. The scale bar represents 40 μm. (B) Average nuclear fluorescence intensity, normalized to the epidermis intensity, of AS cells expressing the marker Apoliner-5. We observe an increase of intensity within the AS cells nuclei at the onset of closure indicating an early activation of caspases (n = 27 cells on three embryos). Error bars are SDs. (C) Time lapses of sqh-Moe-GFP expressing embryos during DC with close-up views on the actin cable. Actin cable forms at the onset of DC, together with the activation of caspases (Figure 2A). The scale bar represents 40 μm. (D) Fluorescence intensity along the actin cable of sqh-Moe-GFP expressing embryos over time (n = 3 embryos; bars are SDs). The dashed line represents the onset of DC. (E) Average opening size L and thickness h of the AS tissue during DC for C381-Gal4 UAS-p35 expressing embryos (light and dark green lines, n = 19 sections on nine embryos; bars are SDs) compared with WT (light and dark blue lines). The dashed line indicates the onset of DC. The shaded area defines the time interval during which volume estimation of the p35 expressing tissue was not performed because of the tissue invagination. p35 AS tissue shows an earlier cell elongation and a slower closure speed than in the WT. (F) Time sequence of sqh-GFP and sqh-GFP C381-Gal4 UAS-p35 expressing embryos by SPIM imaging. The tissue in UAS p35 embryos undergoes an early elongation compared with WT. The arrows show the actin cable surrounding the AS tissue. The red dashed line surrounds the AS tissue at the end of the process. The scale bar represents 20 μm. (G) Normalized volume variation of AS tissue strips for C381 Gal4-UAS-p35 (green line) compared with WT during DC (n = 16 strips on seven embryos; bars are SDs). Decrease of the volume of the tissue strip is impaired in p35-expressing embryos. (H) Transverse views of confocal sections of WT (1 and 2) and TEA-treated (3 and 4) embryos expressing sqh-GFP. AS cells do not elongate before L = 60 μm in WT and show a height of approximately 5 μm (in 1, the red bar represents 5.3 μm, and the arrows indicate the actin cable position). The TEA-treated AS cells show a similar height at the onset of closure (in 3, the red bar represents 5.3 μm, and the arrows indicate the actin cable position), but show a larger height (10 μm) than WT at L = 60 μm (in 4, the red bar represents 10.3 μm, and the arrows indicate the actin cable position). WT AS cells reach a height of 10 μm for L = 20 μm (in 2, the red bar represents 10.3 μm, and the arrows indicate the actin cable position). TEA-treated AS tissue show an early apico-basal elongation compared with WT, consistent with an absence of cell volume decrease. The arrows indicate the actin cable position. The scale bar represents 10 μm. (I) Single-cell volume variation measured with the Brainbow system on WT AS cells (red, n = 10 cells on five embryos) and for AS cells treated with the potassium channel inhibitor TEA (blue, n = 9 cells on five embryos). Although AS cell volume decreases by about 40% in 100 min in WT embryos, cell volume variation is strongly reduced in AS cells treated with TEA (variation is <20%) over the same period. Bars are SDs. Developmental Cell  , DOI: ( /j.devcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions

5 Figure 3 Three-Dimensional Model of DC Reveals that Cell Volume Decrease and Actin Cable Tension Govern Closure Progression (A) Schematics representing the 3D model of DC. AS cells shapes are determined by the balance of tensions in the epithelium. The lengths l, lAP, and h characterize the shape of an AS cell, γcc is the lateral cellular interfacial tension, γa the apical cellular interfacial tension, Te the epidermal tissue tension, and Λ the line tension of the actin cable. The internal pressure P in the AS cells contributes to the total AS planar tension T by counteracting the apical tension γa. (B) Snapshots showing laser ablation and the resulting recoil of the actin cable on a sqh-Moe-GFP expressing embryo. The scale bar represents 10 μm. (C) Normalized recoil velocities in the actin cable following laser ablation in WT (n = 68 embryos) and normalized cable myosin intensity in WT, p35, and CtMLCK expressing embryos, at successive stages of DC, relative to their average values before DC (nWT = 6, np35 = 3, nMLCK = 5). Both laser recoil velocities and myosin intensities increase linearly in the cable over time. The cable tension Λ is therefore also assumed to increase linearly after DC onset. Error bars are SDs. (D) Experimental (dots) and theoretical (dashed lines) plots of the time evolution of the DV tissue length L and height h (nWT = 15 sections, np35 = 19 sections, nMLCK = 11 sections, nTEA = 9 sections). Theoretical plots are obtained by integration of Equation 1, with parameters determined from the fitting procedure described in the Supplemental Information. Error bars are SDs. Developmental Cell  , DOI: ( /j.devcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions

6 Figure 4 Acto-Myosin-Based Interfacial Cell Tension Resists DC
(A) (Top) Transversal views of a sqh-GFP C381-Gal4 UAS-Ct-MLCK expressing embryo and (bottom) dorsal views of a Ecadh-GFP C381-Gal4 UAS-Ct-MLCK expressing embryo. The scale bar represents 20 μm. (B) (Top) Average L and h over time for CtMLCK expressing embryos (red and orange) compared with WT (light and dark blue) (nMLCK = 11 sections on four embryos; bars are SDs). (Bottom) Average normalized volume of tissue strips for WT and C381-Gal4 UAS-Ct-MLCK embryos over time (nWT = 15 strips on five embryos, nMLCK = 11 strips on four embryos; bars are SDs). (C) Schematic illustrating tissue contraction by cell volume decrease during DC. (i) AS tissue tension is initially balanced by the epidermis tissue tension. (ii) Reduction in cell volume increases the total AS tissue tension, favoring closure in conjunction with actin cable contraction pushing on the AS. In the first part of the closure, contraction occurs without apico-basal elongation due to cell volume decrease. (iii) As closure proceeds, the increase in cable line tension promotes apico-basal cell elongation. Developmental Cell  , DOI: ( /j.devcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions