Mechanical Causation of Biological Structure: Productive Pulls Produce Persistent Filaments in a Human Fibroblast Model of Matrix Development

During development, mesenchymal cells direct the elaboration of extracellular matrix that shapes the initial animal bauplan which subsequently grows to produce mechanically-competent structure. To gain insight into the processes that initiate matrix formation at the cellular level, high temporal and spatial resolution videos were obtained from a primary human corneal fibroblast (PHCF) cell culture system known to produce an organized, collagenous stroma similar to a human cornea. The images were taken over a 4-day period prior to culture confluency which permitted a clear view of the cell kinematics and any elaborated filaments. The movies reveal an active cellular system in which the PHCFs execute five types of high-velocity and high extensional strain-rate 'pulls' that produce persistent filaments. In four of the pull types, average maximum strain rates (~0.1-0.33s-1) were adequate to induce aggregation and/or crystallization in crowded biopolymer systems. The results demonstrate that PHCFs have the capacity to mechanically induce the formation of biopolymer structures intercellularly and in the path of force.

Newly synthesized claudins but not occludin are added to the basal side of the tight junction

A network of claudin strands creates continuous cell–cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse–block–pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin–Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

Listeria monocytogenes and Shigella flexneri Activate the NLRP1B Inflammasome

ABSTRACT Activation of the innate immune receptor NLRP1B leads to the formation of an inflammasome, which induces autoproteolytic processing of pro-caspase-1, and ultimately to the release of inflammatory cytokines and to the execution of pyroptosis. One of the signals to which NLRP1B responds is metabolic stress that occurs in cells deprived of glucose or treated with metabolic inhibitors. NLRP1B might therefore sense microbial infection, as intracellular pathogens such as Listeria monocytogenes and Shigella flexneri cause metabolic stress as a result of nutrient scavenging and host cell damage. Here we addressed whether these pathogens activate the NLRP1B inflammasome. We found that Listeria infection activated the NLRP1B inflammasome in a reconstituted fibroblast model. Activation of NLRP1B by Listeria was diminished in an NLRP1B mutant shown previously to be defective at detecting energy stress and was dependent on the expression of listeriolysin O (LLO), a protein required for vacuolar escape. Infections of either Listeria or Shigella activated NLRP1B in the RAW264.7 murine macrophage line, which expresses endogenous NLRP1B. We conclude that NLRP1B senses cellular infection by distinct invasive pathogens.