Post-injury hydraulic fracturing drives fissure formation in the zebrafish basal epidermal cell layer.

The skin epithelium acts as the barrier between an organism's internal and external environments. In zebrafish and other freshwater organisms, this barrier function requires withstanding a large osmotic gradient across the epidermis. Wounds breach this epithelium, causing a large disruption to the tissue microenvironment due to the mixing of isotonic interstitial fluid with the external hypotonic fresh water. Here, we show that, following acute injury, the larval zebrafish epidermis undergoes a dramatic fissuring process that resembles hydraulic fracturing, driven by the influx of external fluid. After the wound has sealed-preventing efflux of this external fluid-fissuring starts in the basal epidermal layer at the location nearest to the wound and then propagates at a constant rate through the tissue, spanning over 100 µm. During this process, the outermost superficial epidermal layer remains intact. Fissuring is completely inhibited when larvae are wounded in isotonic external media, suggesting that osmotic gradients are required for fissure formation. Additionally, fissuring partially depends on myosin II activity, as myosin II inhibition reduces the distance of fissure propagation away from the wound. During and after fissuring, the basal layer forms large macropinosomes (with cross-sectional areas ranging from 1 to 10 µm2). We conclude that excess external fluid entry through the wound and subsequent closure of the wound through actomyosin purse-string contraction in the superficial cell layer causes fluid pressure buildup in the extracellular space of the zebrafish epidermis. This excess fluid pressure causes tissue to fissure, and eventually the fluid is cleared through macropinocytosis.

Mechanical Forces Govern Interactions of Host Cells with Intracellular Bacterial Pathogens.

To combat infectious diseases, it is important to understand how host cells interact with bacterial pathogens. Signals conveyed from pathogen to host, and vice versa, may be either chemical or mechanical. While the molecular and biochemical basis of host-pathogen interactions has been extensively explored, relatively less is known about mechanical signals and responses in the context of those interactions. Nevertheless, a wide variety of bacterial pathogens appear to have developed mechanisms to alter the cellular biomechanics of their hosts in order to promote their survival and dissemination, and in turn many host responses to infection rely on mechanical alterations in host cells and tissues to limit the spread of infection. In this review, we present recent findings on how mechanical forces generated by host cells can promote or obstruct the dissemination of intracellular bacterial pathogens. In addition, we discuss how in vivo extracellular mechanical signals influence interactions between host cells and intracellular bacterial pathogens. Examples of such signals include shear stresses caused by fluid flow over the surface of cells and variable stiffness of the extracellular matrix on which cells are anchored. We highlight bioengineering-inspired tools and techniques that can be used to measure host cell mechanics during infection. These allow for the interrogation of how mechanical signals can modulate infection alongside biochemical signals. We hope that this review will inspire the microbiology community to embrace those tools in future studies so that host cell biomechanics can be more readily explored in the context of infection studies.

Volume measurement and biophysical characterization of mounds in epithelial monolayers after intracellular bacterial infection.

Mechanical forces are important in (patho)physiological processes, including how host epithelial cells interact with intracellular bacterial pathogens. As these pathogens disseminate within host epithelial monolayers, large mounds of infected cells are formed due to the forceful action of surrounding uninfected cells, limiting bacterial spread across the basal cell monolayer. Here, we present a protocol for mound volume measurement and biophysical characterization of mound formation. Modifications to this protocol may be necessary for studying different host cell types or pathogenic organisms. For complete details on the use and execution of this protocol, please refer to Bastounis et al. (2021).

Wounding Zebrafish Larval Epidermis by Laceration.

Wound healing is a critical process for maintaining the integrity of tissues, driven in large part by the active migration of cells to cover damaged regions. While the long-term tissue injury response over hours and days has been extensively studied, the rapid early migratory response of cells to injury in vivo is still being uncovered, especially in model systems such as zebrafish larvae, which are ideal for live imaging with high spatiotemporal resolution. Observing these dynamics requires a wounding method that prompts a robust wound response and is compatible with immediate live imaging or other downstream applications. We have developed a procedure for wounding the epidermis in the tailfin of larval zebrafish, which we term "tissue laceration". In this procedure, the tailfin is impaled with a glass needle that is then dragged through the tissue, which generates a full-thickness wound that elicits a dramatic migratory wound response within seconds from cells up to several hundred micrometers away from the wound. Laceration generates a larger wound response in the first few minutes following wounding compared to other mechanical wounds such as tail transection, and laceration does not require specialized equipment compared to laser wounding methods. This procedure can be used to interrogate the processes by which epidermal cells far away from the wound are able to rapidly detect injury and respond to the wound.