Macropinocytosis is an endocytic process that enables cells to internalize large amounts of solutes from the external environment. Macropinosomes are generated from the base of actin-mediated membrane ruffling when the lamellipodia folds back onto itself thereby forming very large endocytic structures. Macropinosomes are heterogeneous in size and generally considered to be > 0.2 μm in diameter [1, 2], a size considerably larger than clathrin-coated vesicles. The formation of macropinosomes is largely a signal dependent process that is transiently induced by growth factors such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) or tumour promoting factors such as phorbol myristate acetate (PMA) [3–6]. Given the large size of macropinosomes, this unique organelle provides an efficient route for non-selective entry of solute macromolecules as well as considerable amounts of plasma membrane into the cell .
Macropinocytosis is important in a range of physiological processes. For example, macropinocytosis has a role in the down-regulation of signalling from the plasma membrane  and, because of its dependence upon membrane ruffling, in cell motility . Consequently macropinocytosis is very relevant to tumour progression and metastasis. In addition, this endocytic pathway is the primary mechanism by which macrophages and dendritic cells sample their immediate environment for circulating antigens . Indeed, the major antigen presenting cells, namely macrophages and dendritic cells, are highly active in macropinocytosis . For example, macrophages undergo extensive constitutive macropinocytosis, internalizing up to 200% of their surface area every hour , patrolling and sampling the environment for their role as antigen presenting cells of the immune system. Also immature dendritic cells are able to macropinocytose large quantities of exogenous solute as part of their sentinel function . On the other hand, maturation of dendritic cells is associated with down-regulation of macropinocytosis to maximise the presentation of captured antigen . In addition to antigen uptake, macropinocytosis is also considered important in the chemotactic response of neutrophils and macrophages . This endocytic pathway is also utilised by various pathogens such as Salmonella and Shigella to gain entry into host cells .
Despite the physiological relevance of macropinocytosis, the molecular basis for the regulated formation and maturation of macropinosomes is very poorly understood. Macropinosome formation in a range of cell types has been shown to be phosphoinositide-3 kinase dependent  and unlike the relatively well-characterised phagosome, its regulation is receptor-mediated. As endocytic compartments mature, the bulk of their protein constituents are maintained . Peripheral membrane proteins are differentially recruited in a temporally dependent manner in response to a shift in the organelle's phosphoinositide composition. The phosphoinositides (PtdIns) have become the focus of intense interest as they are linked to a range of cell signalling events and are key regulators of intracellular membrane trafficking. Whilst PtdIns(3,4)P2 , PtdIns(4,5)P2  and PtdIns(3,4,5)P3  are traditionally considered to be associated with signalling at the plasma membrane in response to extracellular stimuli, the monophosphorylated phosphoinositide PtdIns(3)P, is implicated in the membrane trafficking of the endosomal system.
Sorting nexins are a large family of proteins characterised by the presence of a phox (PX) domain at the amino terminus. The modestly conserved PX domain is a sequence of 70 to 120 residues that has been shown to bind to various phosphoinositides hence the PX domain confers phosphoinositide specificity to the protein . Sorting nexins have roles in endocytic trafficking events [19–21]. One such sorting nexin is SNX5, a 404 residue protein that contains a central PX domain and large C-terminal domain predicted to include a BAR domain [22, 23], a domain believed to be involved in membrane curvature and tubulation. SNX5 was first discovered based on its homology to SNX1 and subsequently shown to associate with early endosomes. We have previously reported that the PX domain of SNX5 binds specifically to PtdIns(3)P and PtdIns(3,4)P2  and SNX5 is associated with newly-formed macropinosomes following EGF stimulation . After EGF stimulation of HEK293 cells, large EEA1- and SNX5-positive structures were detected in close proximity to membrane ruffles which labelled with fluid phase markers , characteristics that define macropinosomes. We have also exploited the association of SNX5 with macropinosomes to track their maturation in live cells . Our previous work showed that the association of SNX5 with macropinosomes is highly dynamic and SNX5 is localised not only to the macropinosome body but also tubules that extend and depart from the newly-formed macropinosomes . Here, we present data showing that SNX5 modulates macropinocytic activity as well as evidence that the generation of phosphoinositides is involved in the recruitment of SNX5 to the plasma membrane prior to macropinosome formation.