Posted on January 28, 2022
These primary cell sheet explants continued to migrate away from their scale of origin while remaining attached to the scale
These primary cell sheet explants continued to migrate away from their scale of origin while remaining attached to the scale. retraction events. (Epithelial Cell Sheet-PAH-PEMU +5 m Blebbistatin) Addition of Blebbistatin promotes progressive migration by attenuating the leading edge retraction events, but Blebbistatin treatment causes disruption of connections between leading edge cells and internal cells behind the leading edge forming defects in the sheet. The Blebbistatin treatment has little detectable effect on individual edge cell lamellipodia but causes some cells to lose distinctive polarity and become highly elongated. Motility of cell sheets was recorded at a rate of one image/30 sec; playback acquisition time, as indicated. NIHMS801023-supplement-4.mp4 (23M) GUID:?2312CA24-C57C-4127-9B45-0377EACA93D9 5. NIHMS801023-supplement-5.docx (14K) NAV-2729 GUID:?B566E182-AF68-469B-B355-2CB3FC4C0CFE Abstract Polyelectrolyte multilayers (PEMUs) are tunable thin films that could serve as coatings for biomedical implants. PEMUs built layer by layer with the polyanion poly(acrylic acid) (PAA) modified with a photosensitive 4-(2-hydroxyethoxy) benzophenone (PAABp) group and the polycation poly(allylamine hydrochloride) (PAH) are mechanically tunable by UV irradiation, which forms covalent bonds between the layers and increases PEMU stiffness. PAH-terminated PEMUs (PAH-PEMUs) that were NAV-2729 uncrosslinked, UV-crosslinked to a uniform stiffness, or UV-crosslinked with an edge mask or through a neutral density optical gradient filter to form continuous compliance gradients were used to investigate how differences in PEMU stiffness affect the adhesion and migration of epithelial cell sheets from scales of the fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish). During the progressive collective cell migration, the edge cells (also known as leader cells) in the sheets on softer uncrosslinked PEMUs and less crosslinked regions of the gradient formed more actin filaments and vinculin-containing adherens junctions and focal adhesions than formed in the sheet cells on stiffer PEMUs or glass. During sheet migration, the ratio of edge cell to internal cell (also known as follower cells) motilities were greater on the softer PEMUs than on the stiffer PEMUs or glass, causing tension to develop across the sheet and periods of retraction, during which the edge cells lost adhesion to the substrate and regions of the sheet retracted toward Snca the more adherent internal cell region. These retraction events were inhibited by the myosin II inhibitor Blebbistatin, which reduced the motility velocity ratios NAV-2729 to those for sheets on the stiffer PEMUs. Blebbistatin also caused disassembly of actin filaments, reorganization of focal adhesions, increased cell spreading at the leading edge, as well as loss of edge cell-cell connections in epithelial cell sheets on all surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked NAV-2729 PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization. strong class=”kwd-title” Keywords: Polyelectrolyte Multilayer (PEMU), Collective Cell Migration, Durotaxis, Poly(acrylic acid) (PAA), Poly(allylamine hydrochloride) (PAH), Myosin II, Modulus Gradient, Photocrosslinking Graphical abstract 1. Introduction Collective cell migration is crucial for normal tissue development and wound healing. Injury to skin, for example, triggers activation of various cells that release cytokines, remodel ECM, sprout blood vessels, and close the wound through epithelial cell sheet migration.  As epithelial cell sheets migrate NAV-2729 to close the wound, unified contractile forces within the sheet help pull the skin tissue together. [1C3] Cells in these migrating multilayer sheets remain connected to each other through cadherin-containing cell-cell adhesions, which are stabilized by the cortical actin cytoskeleton and intermediate filaments. The interconnectedness of the cells and their.