Cope, A. P. | Search bioRxiv

bioRxiv2022

Subcapsular sinus macrophage sensing of extracellular matrix rigidity alters membrane topography and immune complex mobility

Subcapsular sinus macrophages (SSMs) play a key role in immune defence by forming immunological barriers that control the transport of pathogens 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 nodederived 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 immune complexes 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. Our results reveal a new mechanism regulating physical aspects of antigen presentation by antigen-presenting cells, which may have implications for B cell activation and antibody responses.

immunology↗

bioRxiv2019

Multi-colour DNA-qPAINT reveals how Csk nano-clusters regulate T-cell receptor signalling

Phosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk down-modulates T-cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association - PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and re-recruited at later time points. Unexpectedly, these clusters do not directly co-localise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T-cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.

immunology↗