Recoil Measurements in Drosophila Embryos: from Mounting to Image Analysis.

Tension and force propagation play a central role in tissue morphogenesis, as they enable sub- and supra-cellular shape changes required for the generation of new structures. Force is often generated by the cytoskeleton, which forms complex meshworks that reach cell-cell or cell-extracellular matrix junctions to induce cellular rearrangements. These mechanical properties can be measured through laser microdissection, which concentrates energy in the tissue of interest, disrupting its cytoskeleton. If the tissue is undergoing tension, this cut will induce a recoil in the surrounding regions of the cut. This protocol describes how one can perform laser microdissection experiments and subsequently measure the recoil speed of the sample of interest. While we explain how to carry out these experiments in Drosophila embryos, the recoil calibration and downstream analyses can be applied to other types of preparations. Key features Allows measuring tension in live Drosophila embryos with a relatively simple approach. Describes a quick way to mount a high number of embryos. Includes a segmentation-free recoil quantification that reduces bias and speeds up analysis. Graphical overview.

Autophagy-mediated plasma membrane removal promotes the formation of epithelial syncytia.

Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo-membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub-cellular membranes, as shown by its suppression of experimentally induced laminopathy-like nuclear defects. Our findings reveal a function for TORC1-mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.

Mechanical competition alters the cellular interpretation of an endogenous genetic program.

The intrinsic genetic program of a cell is not sufficient to explain all of the cell's activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. Our models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with our experimental observations of actin reorganization within stretching cells.

Myosin Vb mediated plasma membrane homeostasis regulates peridermal cell size and maintains tissue homeostasis in the zebrafish epidermis.

The epidermis is a stratified epithelium, which forms a barrier to maintain the internal milieu in metazoans. Being the outermost tissue, growth of the epidermis has to be strictly coordinated with the growth of the embryo. The key parameters that determine tissue growth are cell number and cell size. So far, it has remained unclear how the size of epidermal cells is maintained and whether it contributes towards epidermal homeostasis. We have used genetic analysis in combination with cellular imaging to show that zebrafish goosepimples/myosin Vb regulates plasma membrane homeostasis and is involved in maintenance of cell size in the periderm, the outermost epidermal layer. The decrease in peridermal cell size in Myosin Vb deficient embryos is compensated by an increase in cell number whereas decrease in cell number results in the expansion of peridermal cells, which requires myosin Vb (myoVb) function. Inhibition of cell proliferation as well as cell size expansion results in increased lethality in larval stages suggesting that this two-way compensatory mechanism is essential for growing larvae. Our analyses unravel the importance of Myosin Vb dependent cell size regulation in epidermal homeostasis and demonstrate that the epidermis has the ability to maintain a dynamic balance between cell size and cell number.