ZnUMBA - a live imaging method to detect local barrier breaches.

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance, which measures ion flux across a monolayer, or by adding traceable macromolecules and monitoring their passage across the monolayer. Although these methods measure changes in global barrier function, they lack the sensitivity needed to detect local or transient barrier breaches, and they do not reveal the location of barrier leaks. Therefore, we previously developed a method that we named the zinc-based ultrasensitive microscopic barrier assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction leaks with high spatiotemporal resolution. Here, we present expanded applications for ZnUMBA. ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin accumulation following laser injury. ZnUMBA can also be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby canine kidney (MDCK) II epithelial cells. ZnUMBA is a powerful and flexible method that, with minimal optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

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.

Multiscale dynamics of tight junction remodeling.

Epithelial cells form tissues that generate biological barriers in the body. Tight junctions (TJs) are responsible for maintaining a selectively permeable seal between epithelial cells, but little is known about how TJs dynamically remodel in response to physiological forces that challenge epithelial barrier function, such as cell shape changes (e.g. during cell division) or tissue stretching (e.g. during developmental morphogenesis). In this Review, we first introduce a framework to think about TJ remodeling across multiple scales: from molecular dynamics, to strand dynamics, to cell- and tissue-scale dynamics. We then relate knowledge gained from global perturbations of TJs to emerging information about local TJ remodeling events, where transient localized Rho activation and actomyosin-mediated contraction promote TJ remodeling to repair local leaks in barrier function. We conclude by identifying emerging areas in the field and propose ideas for future studies that address unanswered questions about the mechanisms that drive TJ remodeling.

Rho Flares Repair Local Tight Junction Leaks.

Tight junctions contribute to epithelial barrier function by selectively regulating the quantity and type of molecules that cross the paracellular barrier. Experimental approaches to evaluate the effectiveness of tight junctions are typically global, tissue-scale measures. Here, we introduce Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which we used in Xenopus laevis embryos to visualize short-lived, local breaches in epithelial barrier function. These breaches, or leaks, occur as cell boundaries elongate, correspond to visible breaks in the tight junction, and are followed by transient localized Rho activation, or Rho flares. We discovered that Rho flares restore barrier function by driving concentration of tight junction proteins through actin polymerization and ROCK-mediated localized contraction of the cell boundary. We conclude that Rho flares constitute a damage control mechanism that reinstates barrier function when tight junctions become locally compromised because of normally occurring changes in cell shape and tissue tension.

Anillin regulates epithelial cell mechanics by structuring the medial-apical actomyosin network.

Cellular forces sculpt organisms during development, while misregulation of cellular mechanics can promote disease. Here, we investigate how the actomyosin scaffold protein anillin contributes to epithelial mechanics in Xenopus laevis embryos. Increased mechanosensitive recruitment of vinculin to cell-cell junctions when anillin is overexpressed suggested that anillin promotes junctional tension. However, junctional laser ablation unexpectedly showed that junctions recoil faster when anillin is depleted and slower when anillin is overexpressed. Unifying these findings, we demonstrate that anillin regulates medial-apical actomyosin. Medial-apical laser ablation supports the conclusion that that tensile forces are stored across the apical surface of epithelial cells, and anillin promotes the tensile forces stored in this network. Finally, we show that anillin's effects on cellular mechanics impact tissue-wide mechanics. These results reveal anillin as a key regulator of epithelial mechanics and lay the groundwork for future studies on how anillin may contribute to mechanical events in development and disease.

Comprehensive analysis of formin localization in Xenopus epithelial cells.

Reorganization of the actin cytoskeleton is crucial for cellular processes, including cytokinesis and cell-cell junction remodeling. Formins are conserved processive actin-polymerizing machines that regulate actin dynamics by nucleating, elongating, and bundling linear actin filaments. Because the formin family is large, with at least 15 members in vertebrates, there have not been any comprehensive studies examining formin localization and function within a common cell type. Here, we characterized the localization of all 15 formins in epithelial cells of Xenopus laevis gastrula-stage embryos. Dia1 and Dia2 localized to tight junctions, while Fhod1 and Fhod3 localized to adherens junctions. Only Dia3 strongly localized at the cytokinetic contractile ring. The Diaphanous inhibitory domain-dimerization domain (DID-DD) region of Dia1 was sufficient for Dia1 localization, and overexpression of a Dia1 DID-DD fragment competitively removed Dia1 and Dia2 from cell-cell junctions. In Dia1 DID-DD-overexpressing cells, Dia1 and Dia2 were mislocalized to the contractile ring, and cells exhibited increased cytokinesis failure. This work provides a comprehensive analysis of the localization of all 15 vertebrate formins in epithelial cells and suggests that misregulated formin localization results in epithelial cytokinesis failure.

Thiol Starvation Induces Redox-Mediated Dysregulation of Escherichia coli Biofilm Components.

A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function.

Epithelial tissues are defined by polarized epithelial cells that are integrated into tissues and exhibit barrier function in order to regulate what is allowed to pass between cells. Cell-cell junctions must be stable enough to promote barrier function and tissue integrity, yet plastic enough to remodel when necessary. This remarkable ability to dynamically sense and respond to changes in cell shape and tissue tension allows cell-cell junctions to remain functional during events that disrupt epithelial homeostasis including morphogenesis, wound healing, and cell division. In order to achieve this plasticity, both tight junctions and adherens junctions are coupled to the underlying actomyosin cytoskeleton. Here, we discuss the importance of the junctional linkage to actomyosin and how a localized zone of active RhoA along with other Rho GTPases work together to orchestrate junctional actomyosin dynamics. We focus on how scaffold proteins help coordinate Rho GTPases, their upstream regulators, and their downstream effectors for efficient, localized Rho GTPase signaling output. Additionally, we highlight important roles junctional actin-binding proteins play in addition to their traditional roles in organizing actin. Together, Rho GTPases, their regulators, and effectors form compartmentalized signaling modules that regulate actomyosin structure and contractility to achieve proper cell-cell adhesion and tissue barriers.

Tools for live imaging of active Rho GTPases in Xenopus.

Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP-bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well-suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.

Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis.

Epithelial integrity and barrier function must be maintained during the complex cell shape changes that occur during cytokinesis in vertebrate epithelial tissue. Here, we investigate how adherens junctions and bicellular and tricellular tight junctions are maintained and remodeled during cell division in the Xenopus laevis embryo. We find that epithelial barrier function is not disrupted during cytokinesis and is mediated by sustained tight junctions. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that adherens junction proteins are stabilized at the cleavage furrow by increased tension. We find that Vinculin is recruited to the adherens junction at the cleavage furrow, and that inhibiting recruitment of Vinculin by expressing a dominant-negative mutant increases the rate of furrow ingression. Furthermore, we show that cells neighboring the cleavage plane are pulled between the daughter cells, making a new interface between neighbors, and two new tricellular tight junctions flank the midbody following cytokinesis. Our data provide new insight into how epithelial integrity and barrier function are maintained throughout cytokinesis in vertebrate epithelial tissue.

Elucidation of bacteria found in car interiors and strategies to reduce the presence of potential pathogens.

The human microbiome is influenced by a number of factors, including environmental exposure to microbes. Because many humans spend a large amount of time in built environments, it can be expected that the microbial ecology of these environments will influence the human microbiome. In an attempt to further understand the microbial ecology of built environments, the microbiota of car interiors was analyzed using culture dependent and culture independent methods. While it was found that the number and type of bacteria varied widely among the cars and sites tested, Staphylococcus and Propionibacterium were nearly always the dominant genera found at the locations sampled. Because Staphylococcus is of particular concern to human health, the characteristics of this genus found in car interiors were investigated. Staphylococcus epidermidis, S. aureus, and S. warnerii were the most prevalent staphylococcal species found, and 22.6% of S. aureus strains isolated from shared community vehicles were resistant to methicillin. The reduction in the prevalence of pathogenic bacteria in cars by using silver-based antimicrobial surface coatings was also evaluated. Coatings containing 5% silver ion additives were applied to steering wheels, placed in cars for five months and were found to eliminate the presence of culturable pathogenic bacteria recovered from these sites relative to controls. Together, these results provide new insight into the microbiota found in an important built environment, the automobile, and potential strategies for controlling the presence of human pathogens.

Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms.

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

Hemoglobin promotes Staphylococcus aureus nasal colonization.

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.