The origins and evolution of macropinocytosis

Abstract

In macropinocytosis, cells take up micrometre-sized droplets of medium into internal vesicles. These vesicles are acidified and fused to lysosomes, their contents digested and useful compounds extracted. Indigestible contents can be exocytosed. Macropinocytosis has been known for approaching 100 years and is described in both metazoa and amoebae, but not in plants or fungi. Its evolutionary origin goes back to at least the common ancestor of the amoebozoa and opisthokonts, with apparent secondary loss from fungi. The primary function of macropinocytosis in amoebae and some cancer cells is feeding, but the conserved processing pathway for macropinosomes, which involves shrinkage and the retrieval of membrane to the cell surface, has been adapted in immune cells for antigen presentation. Macropinocytic cups are large actin-driven processes, closely related to phagocytic cups and pseudopods and appear to be organized around a conserved signalling patch of PIP3, active Ras and active Rac that directs actin polymerization to its periphery. Patches can form spontaneously and must be sustained by excitable kinetics with strong cooperation from the actin cytoskeleton. Growth-factor signalling shares core components with macropinocytosis, based around phosphatidylinositol 3-kinase (PI3-kinase), and we suggest that it evolved to take control of ancient feeding structures through a coupled growth factor receptor. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Keywords: Dictyostelium; PI3-kinase; Ras; macropinocytosis.

Figure 1.

Examples of macropinocytosis and its evolution. (a) Macropinocytosis in macrophages. A still taken from a time-lapse movie made by Warren Lewis, who first described macropinocytosis in mammalian cells in 1931 [1]. Vigorous ruffling and macropinosome formation can be seen in the movie—newly formed macropinosomes are indicated by arrows in the figure (added by the authors). The movie was recovered by Dr Joel Swanson, to whom we express our gratitude. (b) Macropinocytic cups in a Dictyostelium amoeba. The cell is expressing a fluorescent reporter for F-actin and is viewed by lattice light sheet microscopy [3]. The cups are several microns in diameter and are produced at a rate of 1–2 per minute. An axenic strain, Ax2, was used in which neurofibromin (NF1) is deleted and macropinocytosis is much higher than in wild-type cells. Taken from [4]. (c) Evolutionary origin of macropinocytosis. Macropinocytic organisms were identified from the literature. The plants and fungi taken as negative are well studied, making it unlikely that macropinocytosis could have been overlooked. Homologous genes were identified by reciprocal BLAST searches and the expected domain structure confirmed using Pfam. The negative organisms have well-annotated genomes, making it unlikely that a homologue would be missed. Note that PI3K orthologues found in Physocomitrella and other plants lack Ras-binding domains and thus are not functionally equivalent. The evolutionary relationship among animals, fungi, amoebozoa and plants is shown, with the amoebozoa as a sister clade to the opisthokonts [5]. (d) Organization of macropinocytic cups in a Dictyostelium amoeba. The macropinocytic patch is revealed by a reporter for PIP3 and the irregular necklace of the SCAR/WAVE reporter around it by HSPC300-GFP. As SCAR/WAVE activates the Arp2/3 complex and is always recruited to the edge of patches, this arrangement should trigger a ring of actin polymerization to form the walls of the macropinocytic cup. Taken from [4].

Figure 2.

The relationship between eating and migration mechanisms. A shared machinery is used to generate both the cup-shaped protrusions required for macropinocytosis and phagocytosis and pseudopodia that drive migration. This involves small GTPases of the Ras and Rac family, as well as local activation of actin polymerization by the SCAR/WAVE complex. Both (a) macropinocytic cups and (c) pseudopods form from the spontaneous excitability of the cytoskeleton, and can split. In contrast, phagocytic cups (b) are initiated by localized signalling owing to contact with the prey. Whilst each protrusion is driven by SCAR/WAVE activation, cups differ from pseudopods by the presence of a static interior domain, corresponding to the presence of PIP3_. This self-organizes within a macropinocytic cup, but may be driven by interactions with the target during phagocytosis.

Figure 3.

Macropinosome shrinkage and concentration during early maturation. (a) Dictyostelium amoebae (Ax3) expressing the PI(3)P reporter GFP-2xFYVE [85] were given a 2 min pulse of 0.2 mg ml⁻¹ TRITC-dextran (red) before washing and imaging by confocal microscopy. Arrows indicate tubulation of macropinosomes, which occurs while they shrink. The size, fluorescence intensity and degree of colocalization with PI(3)P over time are quantified in (b). N > 200 vesicles per time point, quantified by automated image analysis (ImageJ). Error bars denote standard deviation. These data are comparable to previous reports in mammalian cells [86,87], indicating that shrinkage and concentration are evolutionarily conserved features of macropinosome maturation.

Figure 4.

Comparison of macropinosome fate in different cells. The early maturation of macropinosomes and their ability to fuse with each other appears to be universal, but later maturation is more diverse and cell-type specific. In amoebae, macropinosomes appear be independent of other endocytic pathways and undergo a unique post-lysosomal neutralization step prior to constitutive exocytosis. Similar isolation from other pathways has been reported in mammalian epidermal and fibroblast cells, although how the insoluble material is eventually released is not known. In contrast with macrophages and dendritic cells, which use macropinocytosis for antigen presentation, macropinosomes can interact with both early and late endosomes.