Cell surface-localized CsgF condensate is a gatekeeper in bacterial curli subunit secretion.

Curli are functional amyloids present on the outer membrane of E. coli. CsgF is required for the proper assembly of curli. Here, we found that the CsgF phase separates in vitro and that the ability of CsgF variants to phase-separate is tightly correlated with CsgF function during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli assembly. Exogenous addition of purified CsgF complemented csgF - cells. This exogenous addition assay was used to assess the ability of CsgF variants to complement csgF ‒ cells. CsgF on the cell surface modulated the secretion of CsgA, the curli major subunit, to the cell surface. We also found that the CsgB nucleator protein can form SDS-insoluble aggregates within the dynamic CsgF condensate. We propose that these multicomponent CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface.

SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos.

Traditional fluorescent proteins exhibit limitations in brightness and photostability that hinder optimal characterization of the dynamic cellular behavior of proteins of interest. SNAP- and Halo-tagging are alternatives to traditional fluorescent protein tagging utilizing bright, stable chemical dyes, which may improve signal-to-noise ratio. However, there has been limited use of this approach in vivo in developing organisms. Here, we present a protocol for implementing SNAP- and Halo-tagging in gastrula-stage Xenopus laevis embryos for live confocal microscopy. For complete details on the use and execution of this protocol, please refer to Varadarajan et al. (2022).

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.

"What I thought was so important isn't really that important": international perspectives on making meaning during the first wave of the COVID-19 pandemic.

BACKGROUND: The global COVID-19 pandemic has had a significant impact on the physical and mental health of people everywhere. The aim of the study is to understand how people living in 15 countries around the globe experience an unexpected crisis which threatens their health and that of loved ones, and how they make meaning of this disruption in their narratives. METHODS: Data were collected through an anonymous online survey during May-September 2020, which was during or just after the first wave of the COVID-19 pandemic, depending on the country. The questionnaire included demographic and three open-ended questions as prompts for stories about experiences during the initial months of the pandemic. The text was analyzed through inductive thematic content analysis and quantified for full sample description, demographic and subsequently international comparisons. RESULTS: The final qualitative dataset included stories from n = 1685 respondents. The sample was 73.6% women and 26.4% men. The mean age of participants was 39.55 years (SD = 14.71). The identified four groups of overarching themes were: The presence and absence of others; Rediscovering oneself; The meaning of daily life; Rethinking societal and environmental values. We discuss the prevalence of each theme for the sample as a whole and differences by demographic groups. The most prevalent theme referred to disruptions in interpersonal contacts, made meaningful by the increased appreciation of the value of relationships, present in (45.6%) of stories. It was more prevalent in the stories of women compared to men (χ² = 24.88, p = .001). CONCLUSIONS: The paper provides a detailed overview of the methodology, the main themes identified inductively in the stories and differences according to select demographic variables. We identify several major ways of making meaning of the pandemic. The pandemic has impacted many aspects of people's lives which give it meaning, no matter where they live.