1.1 Macropinocytosis – evolutionarily conserved and functionally multi-faceted

In eukaryotic cells, elements of the extracellular environment are constantly internalised through a variety of endocytic processes. Among them, macropinocytosis, named from Greek 'pino' meaning 'to drink', allows the cells to take up a big amount of extracellular fluids and soluble macromolecules. First described by Warren Lewis in 1931 (Lewis, 1931), this pathway is initiated through the formation of dynamic protrusions of actin-rich plasma membrane folds called ruffles. When the ruffles close at their distal margins, they enclose the extracellular content within membrane-bound organelles called macropinosomes (Swanson & Watts, 1995). These compartments present a heterogeneous size range and are on average significantly larger (>200 nm) than other endocytic compartments such as clathrin-coated vesicles, caveolae and other clathrin-independent carriers (Doherty & McMahon, 2009). The nascent macropinosomes mature and follow different paths for their recycling or degradation. These processes are tightly controlled by a plethora of molecular regulators, including growth factor receptors, kinases, small GTPases and phospholipids (Kerr & Teasdale, 2009). Substantially different and often partly redundant pathways govern the formation and maturation of macropinosomes depending on the cell type, stimulation and macropinosome function (reviewed in Lin et al., 2020; Buckley & King, 2017; Bloomfield & Kay, 2016; Amyere et al., 2001).

From an evolutionary point of view, macropinocytosis is thought to have first fulfilled a nutritive function, as it takes place in amoeba. Later in time, macropinocytosis is related to specialised roles in vertebrates. For example, it is crucial in immune cells for the sampling of environmental material processed for antigen presentation (Lanzavecchia, 1996; Norbury, 2006) and clearance of apoptotic cells (Henson et al., 2001; Krysko et al., 2006), and it eases dendritic cell migration (Moreau et al., 2019; Stow et al., 2020). In neurons, macropinocytosis allows bulk endocytosis during intense synaptic activity and enables modulation of synapse signalling by regulating the amounts of cell surface receptors (Clayton & Cousin, 2009). In addition to their physiological roles, macropinosomes have been associated with many pathologic processes, ranging from neurodegenerative disease and tumour growth to infections. Our knowledge on the overarching paradigms of this puzzling diversity has remained limited, underpinning the need for tools to scrutinise this diversity at the molecular level.

1.2 Naissance, maturation and recycling of a macropinosome

Macropinocytosis starts with actin-driven membrane ruffles (Quinn et al., 2020). These membrane protrusions can take the cup-shaped form of circular dorsal ruffles in a large number of cell types such as fibroblasts, epithelial cells, macrophages or glial cells (Bernitt et al., 2015). Otherwise, flat sheet-like projections called lamellipodia can be formed at protrusive regions of motile cells (Swanson, 2008). These processes either occur constitutively – as observed in dendritic cells – or they are induced by extracellular signals such as the binding of growth factors (e.g. epidermal growth factor [EGF]) to their specific receptor tyrosine kinases (RTK) (as detailed in Box 1). Alternatively, macropinocytosis is triggered by signals generated inside the cell that activate the signalling cascade downstream of RTK activation. This is the case for the oncogenic v-Src and K-Ras (Hobbs & Der, 2020; Veithen et al., 1996). Similarly, a variety of particles including apoptotic bodies, necrotic cells, bacteria and viruses bypass RTK activation and induce ruffle formations independent of growth factors to trigger their internalisation (Hoffmann et al., 2001; Mercer & Helenius, 2009). Following the induction signal, actin polymerisation is initiated by the activated small GTPases acting in concert with the phosphatidylinositol phosphates (PIPs) on the plasma membrane (reviewed by Buckley & King, 2017). Together, they activate actin nucleation–promoting factors allowing the formation and growth of new actin branches. As the actin microfilaments grow, the plasma membrane locally extends up to dozen micrometre-long protruding ruffles. The need of additional membrane for the expanding ruffles is fuelled to varying degrees by different intracellular organelles (Huynh et al., 2007). The membrane protrusions usually recede spontaneously. However, they sometimes curve at their base into open crater-like cups, while the protrusions apex can either fuse together (circular dorsal ruffle) or fold back and fuse with the flat cell-surface membrane (lamellipodia). Following these fusion events, the crater-like cups (called macropinocytic cups) turn into intracellular organelles called macropinosomes and encapsulate a large volume of extracellular fluid into their lumen. Some molecular players such as the actin-associated motor myosin (Buss et al., 1998; Jiang et al., 2010), and the CtBP1/BARS-dependent fission machinery (Liberali et al., 2008) have been reported to be involved in macropinosome fission.

After closure of the macropinocytic cup and fission from the plasma membrane, the newly formed macropinosomes obtain a specific identity. The small GTPase Rab5 is recruited to the closing macropinosomes and recruits the PI-3-kinase Vps34 that converts PI to PI(3)P (Bohdanowicz & Grinstein, 2013; Porat-Shliom et al., 2008). This shift in the macropinosome lipid composition allows the organelle maturation through the temporally dependent recruitment of a suite of membrane tethering and coat proteins (Feliciano et al., 2011; Schnatwinkel et al., 2004). In addition, these early macropinosomes strip off their dense actin coat and escape from the cortical actin meshwork (Schink et al., 2017). They are further partly deflated by the extrusion of ions and the osmotically coupled release of H₂O (Freeman et al., 2020). Concomitantly, BAR-domain containing sorting nexin (SNX) proteins are recruited to discrete subdomains of the membrane (Kerr et al., 2006). This leads to the formation of extensive tubules for membrane removal. After scission, these tubulations communicate via the retromer protein complex with the Golgi network allowing the recycling of key surface proteins (Seaman, 2012).

The remaining macropinosomes presenting early endosome markers mature and participate in homo- and heterotypic fusions (Kerr & Teasdale, 2009). While some macropinosomes undergo acidification, acquire late endosome markers such as Rab7 and Rab9, migrate to the centre of the cells and eventually fuse with the lysosome (Racoosin & Swanson, 1993), others recycle back to the plasma membrane via a fast-recycling process involving Rab4 and Rab35 or through a slow recycling process involving Rab8 and Rab11.

2.1 Viral induction of macropinocytosis

As viruses need to enter host cells for their replication and propagation, macropinocytosis has been known to be subverted by viruses for a long time. When infecting non-phagocytic cells, viruses must mimic a physiological extracellular stimulus to induce macropinocytosis. Extracellular viral particles interact with receptors on the cell surface for the triggering of membrane ruffling. Usually, this is followed by the formation of macropinosomes and virus internalisation. The virus or their capsids can subsequently penetrate into the cytosol breaching the macropinosome membrane. Even though many viruses hijack the macropinocytosis process, the subverted molecular mechanisms differ profoundly among them (reviewed by Mercer & Helenius, 2009, 2012).

Vaccinia virus, influenza A virus or reovirus induce macropinocytosis by activating RTK (Aravamudhan et al., 2020). This activation is triggered either through direct binding or via glycans acting as bridging molecules. Alternatively, some adenoviruses, echovirus and herpes virus bind to integrin receptors to induce macropinocytosis through a molecular mechanism similar to RTK-induced macropinocytosis. Finally, apoptotic mimicry is a typical strategy of enveloped viruses to be taken up into macrophages (reviewed by Amara & Mercer, 2015). Here, viruses such as vaccinia virus, dengue virus, Ebola virus and pseudotyped lentiviruses expose the apoptosis marker phosphatidylserine (PS) on their surface to mimic apoptotic bodies. As they bind to PS receptors on the surface of macrophages, they elicit a macropinocytic response similar to necroptosis.

2.2 Bacterial induction of macropinocytosis

3.1 Candidate-based approaches to trace macropinosomes

For candidate-based approaches, specific proteins known to be present on macropinosomes are labelled, for example, by genetically encoded fluorescent probes to monitor them by standard fluorescence microscopy (Buckley et al., 2016; Morishita et al., 2019). Commonly employed macropinosome markers include regulatory proteins, such as GTPases Rab5 and Rab11 or certain lipids, such as PI3P that is recognised by the FYVE domain (reviewed by Kühn et al., 2017). Besides, introduction of advanced microscopic techniques, such as lattice light sheet microscopy (LLSM), structured illumination microscopy (SIM) and correlative light electron microscopy (CLEM) have contributed to the broadening of knowledge on macropinosome dynamics and identity (Condon et al., 2018;Fredlund et al., 2018; Weiner et al., 2016). CLEM, coupled with fluorescent markers, such as labelled dextran or probes recognising PI3P, has been instrumental for investigating macropinosomes during Salmonella and Shigella invasion. Performing focused ion beam milling combined with scanning electron microscopy (FIB-SEM) revealed that the nascent bacteria-containing vacuoles (BCVs) and the surrounding macropinosomes are discrete compartments, which acquired strikingly different molecular identities (Fredlund et al., 2018; Weiner et al., 2016). For the sake of clarity, we named these latter compartments 'infection-associated macropinosomes' (IAMs). These studies also substantiated that Salmonella and Shigella engulfment within enterocytes is distinct from and does not rely on macropinocytosis. Consequently, this finding calls for revisiting the trigger entry paradigm and for scrutinising the identities of the nascent compartments of other ruffle-inducing bacteria.

3.2 Toward an unbiased understanding of macropinosomes

While candidate-based approaches illuminated IAMs as novel distinct pathogenic compartments, unbiased approaches such as proteomic analysis provides the means to address their whole molecular identity. Conventional cell fractionation of vesicular compartments is markedly dependent on the equilibrium states of the density separation, whereas IAMs, by nature, are very similar to other endosomal compartments (Walker et al., 2016). Due to the rapid maturation of macropinosomes and dynamics of host–pathogen interactions, methodologies were thus under-equipped to elucidate the molecular players on IAMs. These hurdles have been overcome by a novel method to purify IAMs inspired by a reported magnetic cell fractionation method (Chang et al., 2020; Steinhäuser et al., 2014; Stévenin et al., 2019). This magnetic purification utilises magnetic beads of moderate size (100 nm in diameter), which are readily englobed within IAMs during Salmonella and Shigella invasion when administered to the extracellular medium (Figure 1). Infected cells are then lysed and the magnetic bead-containing IAMs are immobilised on a table-top magnet. The non-magnetic material is eluted, whereas the purified IAMs are recovered within the magnetic fraction. The isolated IAMs are of high purity and can be analysed by mass spectrometry without any additional purification procedure. This method thus minimises the contamination of other endosomal compartments and specifically isolates IAMs. The proteome of the magnetic and non-magnetic fractions extracted from both infected and non-infected samples can be comparatively analysed to determine protein candidates that are significantly enriched on the IAMs.