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Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

ABSTRACT: Maintenance of epithelial tissue integrity requires coordination between cell-cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. Here we addressed the hypothesis that alterations in TJ structure and remodeling of the actomyosin cytoskeleton modify epithelial mechanics. Current methods to measure supracellular mechanical properties disrupt intact monolayers, therefore, we developed a novel method using noncontact acoustic frequency-modulation atomic force microscopy (FM-AFM) and tested it on MDCK polarized monolayers. Our results show that double knockdown (dKD) of ZO-1/ZO-2 elevates the apical epithelial tension and effective viscosity. Interestingly, epithelial tension is more sensitive to inhibition of myosin II ATPase activity than to inhibition of ROCK activity, but viscosity is highly sensitive to both. Additionally, we showed epithelial intercellular pulling forces at tricellular junctions and adhesion forces in dKD cells are elevated with an increase in contractility. In conclusion, FM-AFM enables the physiological and quantitative investigation of mechanics in intact epithelium.

SUBMITTER: Cartagena-Rivera AX

PROVIDER: S-EPMC5715111 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

Cartagena-Rivera Alexander X AX Van Itallie Christina M CM Anderson James M JM Chadwick Richard S RS

Nature communications 20171018 1

Maintenance of epithelial tissue integrity requires coordination between cell-cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. Here we addressed the hypothesis that alterations in TJ structure and remodeling of the actomyosin cytoskeleton modify epithelial mechanics. Current methods to measure supracellular mechanical properties disrupt intact monolayers, therefore, we developed a novel method using noncontact acoustic frequency-modulation atomic for ...[more]

PMID: 29044161

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Project description:UnlabelledBloodstream spread is a critical step in the pathogenesis of many viruses. However, mechanisms that promote viremia are not well understood. Reoviruses are neurotropic viruses that disseminate hematogenously to the central nervous system. Junctional adhesion molecule A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia in infected newborn mice and viral spread to sites of secondary replication. To determine how viruses gain access to the circulatory system, we examined reovirus infection of polarized human brain microvascular endothelial cells (HBMECs). Reovirus productively infects polarized HBMECs, but infection does not alter tight junction integrity. Apical infection of polarized HBMECs is more efficient than basolateral infection, which is attributable to viral engagement of sialic acid and JAM-A. Viral release occurs exclusively from the apical surface via a mechanism that is not associated with lysis or apoptosis of infected cells. These data suggest that infection of endothelial cells routes reovirus apically into the bloodstream for systemic dissemination in the host. Understanding how viruses invade the bloodstream may aid in the development of therapeutics that block this step in viral pathogenesis.ImportanceBloodstream spread of viruses within infected hosts is a critical but poorly understood step in viral disease. Reoviruses first enter the host through the oral or respiratory route and infect cells in the central nervous system. Spread of reoviruses to the brain occurs by blood or nerves, which makes reoviruses useful models for studies of systemic viral dissemination. In this study, we examined how reoviruses infect endothelial cells, which form the walls of blood vessels. We found that reovirus infection of endothelial cells allows the virus to enter blood vessels and serves as a means for the virus to reach high titers in the circulation. Understanding how reovirus is routed through endothelial cells may aid in the design of antiviral drugs that target this important step in systemic viral infections.

Project description:Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

Dataset Information

Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

Publications

Apical surface supracellular mechanical properties in polarized epithelium using noninvasive acoustic force spectroscopy.

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