Macropinosome formation, maturation and membrane recycling: lessons from clathrin-independent endosomal membrane systems

Abstract

Macropinocytosis is a form of endocytosis that brings large fluid-filled endosomes into the cell interior. Macrophages and dendritic cells are especially active in this process, but all cells can be stimulated to initiate this remarkable form of endocytosis. Although much is known about the membrane lipid and actin requirements for initiating macropinocytosis, less is known about the membrane that forms the macropinosome and the fate of that membrane once the macropinosome enters the cell interior. Since macropinocytosis is a specialized form of clathrin-independent endocytosis (CIE), studies of the constitutive internalization and trafficking of cargo proteins and membrane that enter cells independently of clathrin could reveal the types of membrane that form the macropinosome and the machinery that handles cargo sorting and recycling during the maturation of the macropinosome.

This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

1. Introduction

Macropinocytosis is a specialized form of endocytosis that is dependent upon cortical actin ruffling and results in the internalization of large amounts of fluid contained in an enlarged endosome. The process was first observed almost 90 years ago and has fascinated cell biologists ever since [1–3]. Most of the research has been conducted on professional macropinocytosing cells such as Dictyostelium and in mammals, macrophages and dendritic cells. In Dictyostelium, the contents of the fluid taken up in this manner get degraded in lysosomes and thus provide nutrients to the amoeba. In dendritic cells, fluid uptake provides a sampling of extracellular antigens that are degraded for peptide presentation on major histocompatibility complex (MHC) Class II molecules. Macrophages exhibit both constitutive and stimulated macropinocytosis [4] for internalizing antigens but also perform phagocytosis, a distinctly different mechanism of internalization of large particles (cells, bacteria) that involves a tight zippering of the plasma membrane (PM) around the particle during entry [3]. Renewed interest in macropinocytosis surfaced with the finding that Ras-driven tumor cells use macropinocytosis as a source of nutrients for cells residing in the interior regions of solid tumors [5]. Macropinoctyosis is also of interest to the pharmaceutical industry as it can be used for the delivery of large particles into cells for therapeutic purposes [6].

The drivers or initiators of macropinocytosis include low molecular weight GTP-binding proteins Ras, Rac, Arf6 and src family kinases [3]. These initiators can be activated through growth factor receptor signalling or interaction with bacterial or viral pathogens that induce this process as a mode of entry [2]. They then stimulate actin polymerization at the PM by themselves or in collaboration with the other initiators. Increased levels of polyphosphoinositides—both phosphatidylinositol 4,5 bis-phosphate (referred to here as PIP2) and phosphatidylinositol 3,4,5 tris-phosphate (referred to here as PIP3)—at the cell surface are also required for actin polymerization and to initiate the process of macropinocytosis [1]. Ras and Src can stimulate PI-3-kinases to generate PIP3, and Arf6 is an activator of PI4P-5-kinase to generate PIP2 [7,8]. Rac and Cdc42 can promote the recruitment and activation of Arp2/3 to promote actin polymerization. The details of how membrane-specific lipids and actin filaments work together to form macropinosomes and bring them into the cell interior have been largely elucidated [1,3]. What is less clear is what types of membrane comprise the macropinocytic container, what regulates closure or sealing and what happens to macropinosomal membrane once in the cell interior.

2. Endocytosis and endosomal membrane systems

Endocytosis occurs in all nucleated cells and involves the internalization of the PM and extracellular material into the cell interior. The resulting endosome can be small (50–100 µm) pinocytic vesicles or large (0.2–2.0 µm) macropinoctyic or phagocytic structures. Clathrin-mediated endocytosis (CME) is a selective form of endocytosis that brings in PM proteins that contain specific cytoplasmic sequences that bind to the clathrin adaptor proteins, concentrating them into forming clathrin-coated pits that then pinch off from the PM. While CME is dependent upon clathrin, most other forms of endocytosis occur independently of clathrin. However, clathrin-independent endocytosis (CIE) is the term generally used for pinocytic uptake observed in all cells. Macropinocytosis and phagocytosis can be thought of as specialized forms of endocytosis not involving clathrin.

In contrast to the high cargo selectivity of CME, CIE affords a wide variety of PM proteins a mechanism for entry into the cell interior. The characteristics and appearance of CIE in different cells and with different cargo proteins vary widely [9–11]. This heterogeneity may be a key feature of CIE that allows cells to tailor endocytosis for specific functions. There is the clathrin-independent carriers/GPI-AP-enriched endosomal compartments (CLIC/GEEC) pathway (for clathrin-independent carriers and glycerylphosphatidyl inositol (GPI)-anchor protein (AP)-enriched early endosomal compartments) that is described as a high capacity form of CIE observed in fibroblasts and other cells types [12]. CLIC/GEEC entry is dependent upon Cdc42 and Arf1 [13] and cargo includes GPI-APs and CD44 [14]. Another CIE pathway studied is one associated with Arf6 that we have focused on, with emphasis on following the trafficking itinerary of a number of endogenous PM cargo proteins that enter cells through this pathway. Both the CLIC/GEEC- and Arf6-associated forms of CIE share some cargo proteins—including GPI-APs like CD59 and CD44, suggesting that they may be related. Recently, a new form of CIE has been described called FEME for fast endophilin-mediated endocytosis [15]. FEME is distinctive in that it is only activated by ligand-receptor signalling and is selective for internalization of the receptor [15,16], which sets it apart from other types of constitutive CIE.

While it is important to define the mechanism of entry for CIE (whether it be CLIC/GEEC- or Arf6-associated), our research has been directed more to understanding the itinerary taken by cargo proteins after entry (figure 1). Do they traffic to lysosomes for degradation or do they recycle back to the cell surface and by what mechanism? We also follow the itinerary of specific, endogenous PM proteins and in this way, our studies on clathrin-independent endosomal membrane systems differ from those examining CLIC/GEEC, where either expressed green fluorescent protein (GFP)-GPI-APs or fluid is quantified. We also found that this endosomal membrane system that is fed by CIE can be converted into one supporting macropinocytosis in cells expressing active forms of Ras [17] (see below). It is this Arf6-associated form of CIE that we will discuss further below.

Figure 1.

Model of CME and CIE and the resultant trafficking of membrane and cargo that enter the cells. CIE is associated with Arf6 and cargo enters cells in endosomes devoid of transferrin receptor (TfR), a CME cargo protein. Arf6 gets inactivated (to Arf6-GDP) prior to fusion of the endosome with the sorting endosome (SE). Here B-cargo proteins (for bulk pathway) (MHCI and CD59) are either routed to lysosomes or into recycling endosomes (via the endocytic recycling compartment (ERC)) for recycling to the PM. By contrast, A-cargo proteins (for alternative pathway) are sorted out of this compartment through interactions with Hook1, microtubules and Rab22 and returned to the PM, thus escaping transport to lysosomes. Arf6-GTP is present at the PM but Arf6 inactivation is required shortly after internalization because expression of an active mutant, Arf6Q67 L, leads to accumulation of PIP2 and actin-coated vacuoles that contain CIE cargo proteins. Recycling of membrane back to the PM requires Arf6, Rab11, Rab22 and actin. Endosomal membranes fed by CIE contain H-Ras, Rac1, Src and Arf6 and acute activation of any of these stimulates ruffling and macropinocytosis (*), which takes in CIE cargo in large macropinosomes (figure 2).

Much work has focused on CIE that is associated with Arf6, a GTP-binding protein that regulates membrane traffic and the actin cytoskeleton [18,19]. Arf6 functions at the PM and on endosomal membranes fed by CIE, and Arf6 activation is required for recycling of this membrane back to the cell surface. This endosomal membrane system also connects with endosomes entering through CME (figure 1). That is to say that in many cases cargo entering by CIE and by CME meet up in the early endosome, which is defined by the presence of Rab5. Recycling endosomal membrane systems may be complex and there is evidence of separate pathways for the recycling of CME versus CIE cargo proteins (note the separate returning endosomes in figure 1), but the recycling of membrane is critical for membrane remodeling, cell migration, wound healing and polarization [20]. The importance of this PM–endosomal trafficking loop is often overlooked as scientists focus on the recycling of specific receptors (e.g. growth factor or transferrin receptors), but the return of bulk membrane back to the surface is critical to maintain the cell surface composition.

As CIE is potentially an entry mode for many different kinds of PM proteins, especially those lacking adaptor proteins/clathrin sorting sequences, the resultant endosome largely reflects the composition of the PM [21]. Among the cargo entering cells by CIE are nutrient transporters (Glut1, CD98/Lat-1, CD147/Mct1), cell–cell and cell–matrix adhesion molecules (cadherins, integrins, CD44), molecules important for immune response (MHC Class I and peptide-loaded Class II, CD59, CD1a) and G protein-coupled receptors (GPCRs) in the absence of activation [22]. In addition to these integral membrane proteins, peripheral membrane proteins involved in cell signalling also enter cells associated with these endosomes. These include the initiators implicated in macropinocytosis—Ras [17], Src [7], Rac [23] and Arf6 [24]. This bulk entry of PM proteins and lipids is then counter-balanced by sorting of cargo in endosomes [25] towards PM recycling or trafficking to degradation in lysosomes (figure 1). As Arf6 regulates the recycling of these endosomal membranes, Arf6 regulates the trafficking of H-Ras, Src, Rac and Cdc42 back to the cell surface where they can influence signalling and actin rearrangements [7,17,23,26,27]. Indeed, H-Ras-stimulated macropinocytosis requires Arf6 [17] and Rac-mediated alterations of surface actin also require Arf6 [23] for at least the trafficking of these regulators to the cell surface but also likely for the activities of Arf6 on membrane lipid composition (see below).

3. Arf6 influences membrane lipid composition through activation of PIP5-kinase and phospholipase D (PLD)

Arf6 is present at the PM and on endosomes derived from CIE [24] and although Arf6 activation (GTP binding) is not required in most cases for endocytosis, it is required for membrane recycling [24,28]. Furthermore, Arf6 inactivation (GTP hydrolysis) is required for subsequent trafficking of endosomes, i.e. for fusion with the Rab5 sorting endosome (SE) and recycling back out to the PM [29,30] (figure 1). Arf6 activates PIP 5-kinase leading to the generation of PIP2 and this activity of Arf6 can explain its role in CIE endosomal membrane trafficking [7]. Acute, but reversible, activation of Arf6 and PIP2 generation leads to cell surface protrusions powered by actin polymerization. Prolonged activation of Arf6, however, induced by expression of a constitutively active mutant of Arf6, Arf6Q67 L, leads to the accumulation of early CIE endosomes that fuse and build up as vacuoles in the cell interior (figure 1) [7]. These vacuoles contain PIP2, are coated with actin and trap CIE cargo proteins and peripheral membrane proteins (Src, H-Ras and Rac) [7,17]. These vacuoles also form upon over-expression of PIP 5-kinase, suggesting that Arf6 regulates CIE membrane traffic through the activities of PIP 5-kinase [7]. Arf6 also activates phospholipase D (PLD) [31,32] and this specific activity is important for membrane recycling [33]. Phosphatidic acid (PA), the product of PLD, can be converted to diacylglycerol (DAG), a lipid that promotes the closure step during macropinocytosis [34].

Acute activation of Arf6 alone in HeLa cells stimulates the formation of actin protrusions and the generation of macropinosomes [35]. Interestingly, these macropinosomes that are formed contain CIE cargo proteins, but are short lived as they are all recycled back to the cell surface [7,35]. This is in contrast to macropinosomes formed by activation of H-Ras and/or Rac [17], which also contain CIE cargo proteins and deliver lumenal content to the lysosome. H-Ras-stimulated macropinosomes recruit Akt, a downstream effector of Ras activation, whereas Arf6-stimulated macropinosomes do not recruit Akt [17]. Despite these signalling differences, macropinosomes generated through acute activation of either H-Ras or Arf6 led to a progressive change in phosphoinositide composition from PIP3 and PIP2 positive at the cell surface and on the incoming macropinosome, followed by a loss of PIP2 and then loss of PIP3, acquisition of Rab5 [17] and then recruitment of APPL, a Rab5 effector [36] (figure 2). This progression or maturation was dependent upon Arf6 inactivation on the newly formed macropinosome because expression of Arf6Q67 L led to the accumulation of stalled macropinosomes in the form of vacuoles [17]. Arf6 can also set in motion a relay and amplification process to activate Arf1 at the PM [37,38]. This Arf cascade is observed during phagocytosis [39] and may also be occurring during macropinocytosis.

Figure 2.

Maturation of macropinosomes. Changes in the membrane during macropinosome maturation in HeLa cells expressing active forms of Ras [17,36]. Arf6 inactivation coincides with the loss of PIP2 and actin from the macropinosome. Acquisition of Rab5 and APPL, a Rab5 effector [36], signals the initial round of membrane recycling while the macropinosome further transits towards lysosomal degradation.

4. Endosomal membrane systems fed by CIE provide membrane and lipid for signalling and machinery for cargo sorting

As mentioned earlier, CIE membranes can provide a good container for forming macropinosomes. Nutrient transporters present on these containers may continue to transport nutrients from the macropinosome into the cytosol until they (the nutrient transporters) are recycled (see below). After macropinosome formation and during the maturation phase, phosphoinositides are altered and actin is shed as the macropinosome moves inward to deliver content to lysosomes (figure 2).

During this maturation phase, PM proteins must be recycled back to the cell surface to maintain the surface area for continued macropinocytosis. The presence of H-Ras and Rac1 on these membranes would position them, upon recycling, to contribute to macropinosome formation. Both PIP 5-kinase [7] and PLD2 [17] are associated with these endosomal membranes along with their activator Arf6. In addition to recycling of these initiators, bulk PM proteins are also recycled back to the cell surface.

Much is known about the regulation of these endosomal recycling systems from work in HeLa cells, which is remarkably similar to endosomal trafficking studies performed in C. elegans [20], suggesting a highly conserved process. In addition to an Arf6 requirement, Rab8, Rab11 and Rab22a are also required for recycling. Rab8 likely functions with Arf6 as they both induce cellular protrusions in connection with membrane recycling [40]. There are multiple routes for endosomal recycling for both CIE and CME cargo (figure 1). There are rapid or direct recycling pathways and a ‘slow’ recycling pathway that arises from the juxtanuclear endocytic recycling compartment (ERC). A direct route of recycling for specific CIE cargo proteins (CD98, CD147) has been identified whereby they are rapidly sorted away from endosomes after entry [41], into recycling tubules and thus do not move on to lysosomes [42] (figure 1). This sorting out of ‘A’ (for alternative route) cargo proteins is owing to specific binding of the cytoplasmic tails of CD98 and CD147 to Hook1, a microtubule and cargo adaptor protein. Hook1 works in conjunction with microtubules and Rab22a to facilitate sorting of CD98 and CD147 out of the common endosome and into recycling tubules. Loss of Hook1, Rab22a or microtubules impaired this sorting and recycling pathway [43]. This differential sorting is observed in other cell lines and in human peripheral blood lymphocytes [44] and is likely to be an important process during macropinosome maturation. Indeed, we have observed sorting out of CD98 and CD147 from newly formed macropinosomes in HT1080 cells, a human fibrosarcoma cell line expressing a constitutively active form of N-Ras. Studying this cargo sorting in cells undergoing macropinocytosis should be especially conducive to analysis using live cell imaging. The information gained in these macropinocytosing cells will inform our understanding of how cargo is sorted in HeLa cells.

5. Closing thoughts

Perhaps the most puzzling aspect of macropinocytosis is how the cell organizes ruffles and protrusions into forming a closed, sealed structure. Not all ruffling membranes produce macropinosomes. Indeed, ventral actin waves induced by activation of protein kinase C do not result in macropinocytosis [45]. There appear to be distinct mechanisms for closure of macropinosomes formed by circular dorsal ruffles, which close using a purse-string contraction mechanism versus the membrane fusion and sealing that are required for macropinocytosis formed at ruffling, protrusive regions, where specific lipid requirements (PA, DAG) need to be met [3]. Further understanding of this closure will likely be relevant to the scission step of CIE, which in most cases occurs independently of dynamin.

Macropinocytosis is a stimulated, specialized form of CIE. Instead of small pinosomes entering cells, membrane is internalized in large gulps, increasing the amount of fluid entering cells. The entry mechanism is fundamentally different as macropinocytosis so clearly requires actin-generated membrane ruffles and the machinery that goes with this. Pinocytosis (i.e. regular CIE) does not have such a requirement, although the same cargo proteins can enter cells either way. The cargo sorting and membrane recycling systems and mechanisms, however, will likely be shared. Cargo sorting from macropinosomes should provide an excellent model system to study the complex iterative process of endosomal sorting observed in mammalian cells.

Data accessibility

This article has no additional data.

Competing interests

I declare I have no competing interests.

Funding

I received no funding for this study.