Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.

How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in G1, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. Our quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1ß release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage.

Influence of Nanopillar Arrays on Fibroblast Motility, Adhesion, and Migration Mechanisms.

Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behavior. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading, and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, fibroblast behavior is investigated on regular arrays of 1 µm high polymer nanopillars with varying pillar to pillar distance. Embryonic mouse fibroblasts (NIH-3T3) spread on all arrays, and on contact with the substrate engulf nanopillars independently of the array pitch. As the cells start to spread, different behavior is observed. On dense arrays which have a pitch equal or below 1 µm, cells are suspended on top of the nanopillars, making only sporadic contact with the glass support. Cells stay attached to the glass support and fully engulf nanopillars during spreading and migration on the sparse arrays which have a pitch of 2 µm and above. These alternate states have a profound effect on cell migration rates. Dynamic F-actin puncta colocalize with nanopillars during cell spreading and migration. Strong membrane association with engulfed nanopillars might explain the reduced migration rates on sparse arrays.

Human Toll-like Receptor 8 (TLR8) Is an Important Sensor of Pyogenic Bacteria, and Is Attenuated by Cell Surface TLR Signaling.

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus, GBS), resulting in the activation of IRF5 and production of IFNß, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus, GBS, Streptococcus pneumonia, Pseudomonas aeruginosa, and E. coli. The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus, GBS, and S. pneumonia, the TLR8 inhibitor almost eliminated the production of IL-1ß and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1ß, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNß and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1ß and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

Dissolution of copper mineral phases in biological fluids and the controlled release of copper ions from mineralized alginate hydrogels.

Here we investigate the dissolution behaviour of copper minerals contained within biocompatible alginate hydrogels. Copper has a number of biological effects and has most recently been evaluated as an alternative to expensive and controversial growth factors for applications in tissue engineering. Precise control and sustained release of copper ions are important due to a narrow therapeutic window of this potentially toxic ion, and alginate would appear to be a good material of choice for this purpose. We found that aqueously insoluble copper minerals could be precipitated during gelling within or mixed into alginate hydrogels in the form of microbeads prior to gelling to serve as depots of copper. These minerals were found to be soluble in a variety of biological fluids relevant to in vitro and in vivo investigations, and the alginate carrier served as a barrier to diffusion of these ions and therefore offered control over the rate and duration of release (Cu(2+) release rates observed between 10-750 µMol g(-1) h(-1) and duration for up to 32 d). Copper mineral and copper mineralized alginate microbeads were characterized using powder x-ray diffraction, FTIR, thermogravimetric analysis and scanning electron microscopy. Dissolution kinetics were studied based on measurements of copper ion concentrations using colourimetric methods. In addition we characterized the complexes formed between released copper ions and biological fluids by electron paramagnetic spectroscopy which offers an insight into the behaviour of these materials in the body.