Extracellular matrix rigidity modulates physical properties of SCS macrophage-B cell immune synapses.

Subcapsular sinus (SCS) macrophages (SSMs) play a key role in immune defence by forming immunological barriers that control the transport of antigens from lymph into lymph node follicles. SSMs participate in antibody responses by presenting antigens directly to naive B cells and by supplying antigens to follicular dendritic cells to propagate germinal centre reactions. Despite the prominent roles that SSMs play during immune responses, little is known about their cell biology because they are technically challenging to isolate and study in vitro. Here, we used multi-colour fluorescence microscopy to identify lymph node-derived SSMs in culture. We focused on the role of SSMs as antigen-presenting cells, and found that their actin cytoskeleton regulates the spatial organisation and mobility of multivalent antigens (immune complexes, ICs) displayed on the cell surface. Moreover, we determined that SSMs are mechanosensitive cells that respond to changes in extracellular matrix (ECM) rigidity by altering the architecture of the actin cytoskeleton, leading to changes in cell morphology, membrane topography, and immune complex mobility. Changes to ECM rigidity also modulate actin remodelling by both SSMs and B cells when they form an immune synapse. This alters synapse duration but not IC internalisation nor NF-κB activation in the B cell. Taken together, our data reveal that the mechanical microenvironment may influence B cell responses by modulating physical characteristics of antigen presentation by SSMs.

Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription.

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear functions for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We find that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover nuclear roles for NDP52 in gene expression and DNA structure regulation.

A practical field extraction method for non-invasive monitoring of hormone activity in the black rhinoceros.

Non-invasive hormone analysis is a vital tool in assessing an animal's adrenal and reproductive status, which can be beneficial to in situ and ex situ conservation. However, it can be difficult to employ these techniques when monitoring in situ populations away from controlled laboratory conditions, when electricity is not readily available. A practical method for processing faecal samples in the field, which enables samples to be extracted soon after defaecation and stored in field conditions for prolonged periods prior to hormone analysis, is therefore warranted. This study describes the development of an optimal field extraction method, which includes hand-shaking faecal material in 90% methanol, before loading this extract in a 40% solvent onto HyperSep™ C8 solid-phase extraction cartridges, stored at ambient temperatures. This method was successfully validated for measurement of adrenal and reproductive hormone metabolites in faeces of male and female black rhinoceros (Diceros bicornis) and was rigorously tested in controlled laboratory and simulated field conditions. All the hormones tested demonstrated between 83 and 94% and between 42 and 89% recovery of synthetic and endogenous hormone metabolites, respectively, with high precision of replication. Furthermore, results obtained following the developed optimal field extraction method were highly correlated with the control laboratory method. Cartridges can be stored at ambient (cool, dry or warm, humid) conditions for periods of up to 6 months without degradation, before re-extraction of hormone metabolites for analysis by enzyme immunoassay. The described method has great potential to be applied to monitor faecal reproductive and adrenal hormone metabolites in a wide variety of species and allows samples to be stored in the field for up to 6 months prior to analysis. This provides the opportunity to investigate hormone relationships within in situ populations, where equipment and facilities may previously have been limiting.