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.

Microtubules in 3D cell motility

“…Microtubule organization has been the subject of early electron microscopy studies in migrating myogenic cells (Jacob et al, 1978), epiblasts that acquire invasive properties due to epithelialmesenchymal transition (EMT) during gastrulation (Granholm and Baker, 1970) and cardiac cushion cells grown in collagen gels (Bernanke and Markwald, 1982). A recent example of the effect of microtubule depolymerization on developmental processes is a study of protrusion formation during epithelial tissue sealing (Eltsov et al, 2015).…”

Myosin II is not required for Drosophila tracheal branch elongation and cell intercalation

“…Along the leading edge of the closing epidermis, an actomyosin cable forms ( (Fig. S3A, right) (Eltsov et al, 2015). To perturb Sqh function in the embryonic epidermis, we used engrailed-Gal4 (en-Gal4) (Tabata et al, 1992) to restrict expression of deGradFP to the posterior compartment of each segment in sqhAX3; sqh-Sqh-GFP embryos.…”

Quantifying dorsal closure in three dimensions

“…Purse-string disassembly and the softening of purse string ends may support the formation of filopodia and lamellipodia and the junctional remodeling of apposing leading-edge cells during zipping (Jacinto et al. , 2002a; Eltsov et al. , 2015).…”

“…, 2015) and electron microscope (Turner and Mahowald, 1977; Underwood et al. , 1980; Eltsov et al. , 2015) studies.…”

“…Apposing leading-edge cells first come together at their apical ends and then square off basally to form a lateral junction (Lu et al. , 2015), where filopodia mediate the initial contact, and the seam includes overlapping single lamella (Eltsov et al. , 2015).…”