Mechanosensitive calcium flashes promote sustained RhoA activation during tight junction remodeling

Epithelial cell–cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed “Rho flares,” but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli

The Role of Mechanosensitive Calcium Signaling in Remodeling of Epithelial Cell-Cell Junctions

Epithelial cells line most of our body cavities, separating different compartments in our bodies, creating selective barriers, and protecting us from external pathogens. In epithelial tissues, cells experience a range of mechanical forces like fluid flow, tissue distention, and tissue compression or contraction, which occur both during development and normal organ function. All of these events require cells to sense the mechanical force and remodel their cell-cell junctions accordingly in order to maintain barrier function and structural integrity of the tissue. However, the precise mechanism by which epithelial barrier function is regulated in response to mechanical stimuli is unknown. In the vertebrate epithelium, the most apically localized cell-cell junctions, called tight junctions (TJs), regulate barrier function by selectively allowing solutes and ions to pass through the space between cells, while the more basally localized adherens junctions (AJs) mechanically couple the cells together to maintain the structural integrity of the tissue. Using gastrula-stage Xenopus laevis embryos as a model for the vertebrate epithelium, previous work from our lab identified local, short-lived leaks in the epithelial TJ barrier, which occur in response to elongating cell-cell junctions. These leaks were rapidly repaired by reinforcement of TJ proteins, mediated by localized, transient activation of the small GTPase RhoA – termed “Rho flares”. But the mechanism by which cells sense the leak in the barrier and activate Rho flares locally at the site of the barrier leak remained unclear. In this dissertation, I investigate how a mechanical cue is converted into biochemical signals to regulate barrier function in the vertebrate epithelium. Here, I found that a local calcium increase occurs at the site of barrier leaks in response to loss of the TJ protein ZO-1. Using intracellular calcium chelation and a mechanosensitive calcium channel (MSC) blocker, I show that a local calcium increase is required for robust activation of Rho flares to efficiently reinforce TJs at sites of local barrier breaches. Further, I show that MSC-dependent calcium influx is required to maintain global barrier function by regulating efficient repair of local barrier leaks through robust contraction of junctions. Thus, we propose that MSC-mediated calcium influx is a mechanism by which epithelial cells detect local leaks and regulate barrier function by activating Rho flares. Additionally, I explored the localization and function of a eukaryotic MSC, Piezo1, at cell-cell junctions. I found that Piezo1 localizes to AJs and regulates TJ barrier function during cell-generated tensile stress, possibly by strengthening cell-cell adhesion. Together, my work reveals MSC-mediated calcium signaling as part of a mechanotransduction pathway working at apical cell-cell junctions to regulate barrier function. In addition to revealing a novel role for intracellular calcium signaling in regulating TJ barrier function in a mature epithelium, application of my research could help identify new targets to treat chronic inflammation associated with impaired cell-cell junctions in response to aberrant mechanotransduction.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/171440/1/varadars_1.pd

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-α promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-α (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other

Gut Microbiota-Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens

The gut microbiota is compartmentalized in the intestinal lumen and induces local immune responses, but it remains unknown whether the gut microbiota can induce systemic response and contribute to systemic immunity. We report that selective gut symbiotic gram-negative bacteria were able to disseminate systemically to induce immunoglobulin G (IgG) response, which primarily targeted gram-negative bacterial antigens and conferred protection against systemic infections by E. coli and Salmonella by directly coating bacteria to promote killing by phagocytes. T cells and Toll-like receptor 4 on B cells were important in the generation of microbiota-specific IgG. We identified murein lipoprotein (MLP), a highly conserved gram-negative outer membrane protein, as a major antigen that induced systemic IgG homeostatically in both mice and humans. Administration of anti-MLP IgG conferred crucial protection against systemic Salmonella infection. Thus, our findings reveal an important function for the gut microbiota in combating systemic infection through the induction of protective IgG