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Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography

Abstract

The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate ‘roof tile’-like overlaps. These shorten to produce the force, ‘zipping’ the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.

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Figure 1: DC with electron microscopy resolution.
Figure 2: 3D reconstruction of zipping.
Figure 3: Opposing LE cells generate a single membrane overlap.
Figure 4: Cytoskeleton organization within LE cell before zipping.
Figure 5: MTs as zipping force generators.
Figure 6: MTs orient cellular extensions.
Figure 7: Proposed model of zipping.

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Acknowledgements

We are particularly grateful to the EMBL electron microscopy facility and S. Pruggnaller, A. Habermann, V. Rybin, Y. Belyaev and R. Gibeaux for help with embryo processing, image acquisition and image analysis. We are grateful to J. Ellenberg, M. Vabulas and C. Pohl for critical reading and discussion of the manuscript. We thank I. Simeonova, A. Schmidkunz, N. Wollf, S. Wali and V. Eltsova for tracing. M.E. was supported by an EIPOD fellowship; N.D. was supported in part by a Fellowship from the Canadian Cancer Society Research Institute (Terry Fox Foundation, award no. 018608). Core funding (EMBL, University of Zurich, CEF and CEFII) and an ERC starting grant to A.S.F. provided further support.

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M.E. designed and carried out experiments, analysed data and wrote the manuscript. N.D., Z.Y., L.P. and U.H-W. carried out experiments and analysed data. D.B. and A.S.F. designed experiments, analysed data and wrote the manuscript. All authors have proofread the manuscript.

Corresponding authors

Correspondence to Damian Brunner or Achilleas S. Frangakis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 AS and LE cell organization during dorsal closure.

(A) Apical surfaces of AS cells extend filopodia into the perivitelline space. The inset shows a magnification of bundled actin filaments (black arrow) in the selected filopodium. (B) Examples of cavities formed by LE cells around AS cell protrusions (black arrows). (C) Tomographic slices through the LE/AS adherens junction. The cross section is perpendicular and the longitudinal section parallel to the anterior/posterior embryonic axis. (D) Tomographic slice and rendered view showing lateral lamellipodia overlaps of neighbouring LE cells before zipping. Blue dots mark the dorsal most position of LE/AS adherens junctions (B and D).

Supplementary Figure 2 Stage-specific zipping architecture.

(A) Characteristic view of initial adhesion junctions. The insert shows a magnified view of putative cadherin densities, which are obliquely oriented and visible as thin lines (white arrows) lying in the extracellular space of the contact areas. (B) Schematic representation of cell membrane organization and cytoskeletal remodelling of the LE cells during early (yellow), mid (green) and late zipping (blue), and after zipping is completed (grey). The respective number of LE cells occupying the different zipping phases of three anterior (ant 1–3) and one posterior (post 1) zipping event, are shown in the lower halve.

Supplementary Figure 3 Actin-MyoII cables in LE cells.

(A) Tomographic slice showing a cross-section of an LE and an AS cell. The actin/MyoII purse string is visible as the textured area with higher density outlined with a red line. (B) Tomographic slice parallel to the anterior/posterior axis showing areas of three neighbouring LE cells and an AS cell. The actin/MyoII cable (outlined in red) in cell 2 (colored in blue) connects pockets formed by lateral protrusions (LP) of cell 1 and cell 3 respectively (both colored in green). The inserts in (A) and (B) are magnifications of the according boxed areas showing that the actin filaments form a mesh with electron dense nodes. (C) Tomographic slice through the long axis of a lateral protrusion. The actin/MyoII cable can be seen within the protrusion attached to the AJs. The protrusion also contains microtubules (MTs) reaching the protrusion tip.

Supplementary Figure 4 Immuno-gold labeling of actin and MyoII.

Electron micrographs of immuno-gold labelled 70 nm sections of high-pressure frozen and freeze-substituted embryos. (A) Anti-actin labeling of actin bundles in LE cell protrusions. The bundle in the lamellipodium is cut obliquely, while in the overlaying filopodium (red arrows) it is cut across. Arrows point out gold particles. The insert shows a magnification of the lamellipodial actin bundle boxed in white. (B) Anti-actin (C) and anti-MyoII labeling of the actin/MyoII purse string and the respective magnification of the boxed areas in the insert. The characteristic texture of the actin/Myo2 cable cannot be seen in the projection images. However, it can be distinguished due to its higher electron density.

Supplementary Figure 5 Effects of LatB treatment on DC.

Time point 0 sec is within 5–10 sec of injection with the various LatB concentrations (2 independent experiments, 18 embryos were injected with 100 mM LatB, 18 embryos with 10 mM, and 5 embryos with 1 mM). In each experiment, the second frame was chosen to depict the time point when the first defects are evident meaning that the known actin-based force generating systems, the AS tissue and the actin/MyoII purse string begin losing tension and integrity. Red arrows depict rupturing points. The following frames show the timeline of the resulting DC area disintegration. The third frame depicts the time point right before first defects of the zipping area become evident. The fourth frame the time point when also the zipping area is clearly disrupted. The LatB effect is scalable—the higher the concentration, the faster rupturing of the tissues occurs. However, common to all LatB concentrations is that the zipping area only starts showing defects with a considerable time delay, suggesting that this force generating process does not react acutely to actin filament depolymerization, unless for some reason LatB mediated actin depolymerization is less efficient in zipping LE cells as compared to LE cells before or post zipping.

Supplementary Figure 6 Arrangement of the MT cytoskeleton during zipping.

(A) Representative tomographic slices and schematic drawings of MT ends, defining how they were linked to their polarity and growth status. (B) Distribution and number of growing and shrinking MT plus ends in overlapping extensions during early zipping as color coded in (A). The blue tubes depict the dorsal-most borders of LE/AS adherens junctions. (C) Table showing the orientation and growth state of individual MTs in LE cell protrusions. The MTs within the protrusions were classified on the basis of their end morphology and are presented as the median value per cell protrusion before zipping (84 MTs from 3 cells), during early zipping (160 MTs from 6 cells) and in mid zipping (141 MTs from 5 cells). The first three categories describe MTs oriented with their plus ends towards the protrusion tips. Flared or sheet-like ends were classified as growing-, curled plus ends as shrinking. MT with blunt ends were considered being in a transitional state. The next two categories represent MTs oriented towards the protrusion tips with their capped minus ends or with ends which could not be identified. Accidentally we found short MTs which were oriented perpendicularly to the MTs of the aforementioned categories. They constituted below 4% of all identified MTs per cell and were omitted in our analysis. The median percentage of MTs with located ends was approximately 57% of the total. The remaining MTs and microtubule fragments ended at tomogram borders and could not be interpreted.

Supplementary Figure 7 MTs in Spastin expressing LE cells.

(A) Microtubule organization in the LE cells shown in Fig. 6. The wild type cell (green) contains MTs, while the other cell (brown) expresses Spastin-EGFP to eliminate the MTs. The blue tubes depict the dorsal- most borders of LE/AS adherens junctions. (B) Histograms of the lobe lengths measured in sections of wild type and Spastin expressing cells. Spastin expressing cells have a much shorter lobe as compared to the wild type (131 measurements from 17 Spastin-expressing cells from 3 zipping events, 2 embryos; 136 measurements from 16 WT cells from 2 zipping events, 2 embryos).

Supplementary information

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Supplementary Information (PDF 2749 kb)

Surface representation of LE and AS cells in the zipping area as shown in Fig. 2B and C.

The model is tilting around the central vertical axis. LE cells are sequentially removed to reveal the underlying AS cell surfaces. (MOV 7250 kb)

Surface representation of the LE cells shown in Fig. 3B.

The model is tilting around the central horizontal axis. The LE cells LE#1 and LE#2 from one epidermis front (red/brown) are subsequently removed to reveal the contact areas (dark red and purple spheres) with the opposing LE cell (green). (MOV 3376 kb)

Live fluorescence imaging of α-catenin-GFP in a zipping embryo.

α-catenin-GFP (expressed in paired-expressing cell stripes) marks LE/LE adherens junctions and visualizes amongst others the formation of nascent AJs between opposing LE cells as shown in Fig. 3F. Lateral views (Z-stack maximal projections) were acquired at 1 min intervals. (MOV 4380 kb)

Live fluorescence imaging of EB1-GFP in LE cells visualizing growing MT plus ends.

Whereas LE cell bodies show bidirectional EB1 movement, in cell protrusions, it is predominantly (and almost exclusively) moving towards the protrusion tips at all zipping stages, revealing that MTs are mainly oriented such that their plus ends are growing into LE cell protrusions. (MOV 3412 kb)

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Eltsov, M., Dubé, N., Yu, Z. et al. Quantitative analysis of cytoskeletal reorganization during epithelial tissue sealing by large-volume electron tomography. Nat Cell Biol 17, 605–614 (2015). https://doi.org/10.1038/ncb3159

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