Sorting nexin 5 (SNX5) selectively regulates dorsal ruffle-mediated macropinocytosis in primary macrophages

Abstract

The regulation of macropinocytosis, a specialised endocytosis pathway, is important for immune cell function. However, it is not known whether the biogenesis of macropinosomes involves one or more distinct pathways. We previously identified sorting nexin 5 (SNX5) as a regulator of macropinocytosis in macrophages. Here, we show that bone-marrow-derived macrophages from SNX5-knockout mice had a 60-70% reduction in macropinocytic uptake of dextran or ovalbumin, whereas phagocytosis and retrograde transport from the plasma membrane to the Golgi was unaffected. In contrast, deficiency of SNX5 had no effect on macropinocytosis or antigen presentation by dendritic cells. Activation of macrophages with CSF-1 resulted in a localisation of SNX5 to actin-rich ruffles in a manner dependent on receptor tyrosine kinases. SNX5-deficient macrophages showed a dramatic reduction in ruffling on the dorsal surface following CSF-1 receptor activation, whereas peripheral ruffling and cell migration were unaffected. We demonstrate that SNX5 is acting upstream of actin polymerisation following CSF-1 receptor activation. Overall, our findings reveal the important contribution of dorsal ruffing to receptoractivated macropinocytosis in primary macrophages and show that SNX5 selectively regulates macropinosomes derived from the dorsal ruffles.

Fluorescence labelling of extracellular vesicles using a novel thiol-based strategy for quantitative analysis of cellular delivery and intracellular traffic

“…Cells were successfully transfected with siRNA sequences targeting PAK-1 and Cdc42 (Fig. 6A) to investigate the roles of these proteins on initially the uptake of dextran that represents in the absence of growth factor activation, a constitutive fluid phase probe 60,61 . Our previous studies indicated that PAK-1 was involved in the cellular uptake of cationic cell penetrating peptides that may be inducing a form of macropinocytosis for cell entry 34,62 .…”

FcRn mediates fast recycling of endocytosed albumin and IgG from early macropinosomes in primary macrophages

“…The pH dependence of FcRn binding to ligand defines the intracellular location where the interaction takes place. Albumin is internalised by fluid phase endocytosis, possibly by the efficient macropinocytosis pathway, a pathway highly active in endothelial cells, macrophages and dendritic cells (Lim and Gleeson, 2011;Lim et al, 2015). Cell surface receptors may also be involved in the endocytosis of the FcRn ligands, especially for IgG via Fcγ receptors on haematopoietic cells.…”

The cell surface environment for pathogen recognition and entry

“…At the behest of EGFR activation, SNX5 is recruited to PI(3,4)P2‐enriched membranes on early macropinosomes and it is present on tubulating macropinosomes, where in both cases, the SNX5 BAR domain may contribute to membrane curvature 41 , 42 . Macrophages, but interestingly not immature dendritic cells, from SNX5 knockout mice have significantly reduced macropinocytosis and dorsal ruffling 51 …”

“…41,42 Macrophages, but interestingly not immature dendritic cells, from SNX5 knockout mice have significantly reduced macropinocytosis and dorsal ruffling. 51 A diagnostic feature of macropinocytosis, used to distinguish this pathway from clathrin-mediated endocytosis, is its sensitivity to inhibitors of Na + /H + ion exchangers, such as amiloride. 52,53 Although the mode of action of amiloride is controversial, it remains a useful experimental tool for blocking macopinocytic uptake of markers or pathogens.…”