Origin, originality, functions, subversions and molecular signalling of macropinocytosis

Int. J. Med. Microbiol. 291, 487-494 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm

IJMM IJ

Origin, originality, functions, subversions and molecular signalling of macropinocytosis Mustapha Amyere1, Marcel Mettlen1, Patrick Van Der Smissen1, Anna Platek1, Bernard Payrastre2, Alex Veithen1, Pierre J. Courtoy1 1 2

Cell biology Unit, UCL and ICP, 75 av. Hippocrate, B-1200 Brussels, Belgium INSERM U326, Hôpital Purpan, F-31059 Toulouse, France

Abstract Macropinocytosis refers to the formation of primary large endocytic vesicles of irregular size and shape, generated by actin-driven evaginations of the plasma membrane, whereby cells avidly incorporate extracellular fluid. Macropinosomes resemble “empty” phagosomes and show no difference with the “spacious phagosomes” triggered by the enteropathogenic bacteria Salmonella and Shigella. Macropinosomes may fuse with lysosomes or regurgitate their content back to the extracellular space. In multiple cell types, macropinocytosis is a transient response to growth factors. When amoebas are cultured under axenic conditions, macropinocytosis is induced so as to fulfil nutritional requirements. In immature dendritic cells, macropinocytosis allows for extensive sampling of soluble antigens; after a few days of maturation, this activity vanishes as processed peptides are being presented. Macropinosomes are also formed at the leading edge of motile leukocytes or neurons. In all these examples, macropinocytosis appears tightly regulated. Transformation of fibroblasts by Src or Ras also results in constitutive formation of macropinosomes at “ruffling” zones, that could be related to accelerated cell motility. Like phagocytosis, macropinocytosis depends on signalling to the actin cytoskeleton. We have explored this signalling in transformed cells. v-Src and K-Ras activate PI3K and PLC, as demonstrated by in situ production of the corresponding lipid products. Pharmacological inhibitors of PI3K and PLC and stable transfection leading to a dominant-negative PI3-kinase construct in transformed fibroblasts abolish macropinocytosis, demonstrating that both enzyme activities are essential. Conversely, stable transfection leading to a dominant-positive PI3K in non-transformed fibroblasts is sufficient to induce macropinocytosis. Combination of experiments allows to conclude that PI3K and PLC act in sequential order. In non-polarized cells expressing a thermosensitive v-Src mutant, v-Src kinase activation accelerates fluid-phase endocytosis. In polarized MDCK cells, this stimulation occurs selectively at the apical domain and the response is selectively abrogated by pharmacological inhibitors of PI3K and PLC. Thus, two paradigmatic oncogenes cause constitutive macropinocytosis. For v-Src, this response is polarized at the apical membrane. It is suggested that, in enterocytes that do not normally phagocytose, the PI3K-PLC signalling pathway leading to selective induction of macropinocytosis at the luminal surface has been subverted by enteropathogenic bacteria to penetrate via “spacious phagosomes”. Key words: Endocytosis – actin – PI3 kinase – PLC – Src – Ras

Corresponding author: Prof. Pierre J. Courtoy, ICP and UCL, CELL Unit, UCL 75.41, 75, avenue Hippocrate, B-1200 Bruxelles, Belgium, Phone: +32 2 764 7569, Fax: +32 2 764 7543, E-mail: [email protected] 1438-4221/01/291/6-7-487 $ 15.00/0

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Definitions: kinetic modes and structures of endocytosis A striking difference between prokaryotes and eukaryotes is endocytosis, i. e. the vesicular uptake of extracellular macromolecules. Different forms of endocytosis can be distinguished on kinetic, structural, and physiological criteria. Kinetic modes Kinetically, three modes of increasing complexities can be defined. Fluid-phase endocytosis refers to the bulk uptake of solutes in exact proportion of their concentration in the extracellular fluids. This is a low-efficiency, non-specific process. Typically, non-specialized cells drink up by fluid-phase endocytosis about the equivalent of their volume every day. By analogy to the role of whalebone in plankton retention, endocytosed nutrients are somehow sequestered and water is secondarily expelled, by a still poorly understood mechanism of lysosomal concentration and transmembrane expulsion. In adsorptive endocytosis, families of constituents preferentially interact with generic groups of complementary binding sites (e. g. by lectin or charged interaction). By this process, bound macromolecules follow the fate of plasma membrane as a whole. Typically, non-specialized cells internalise the equivalent of their surface every hour. By receptor-mediated endocytosis, individual ligands recognize specific receptors that can be strictly regulated in terms of genetic expression, surface translocation and ligand affinity. The efficiency of receptor-mediated endocytosis reflects both the affinity of ligand-receptor interaction and the concentration of these complexes in clathrin-coated pits. Up to 30 % of surface-bound ligands can be internalised per minute. Endocytic structures Structurally, three types of primary endocytic vesicles have been demonstrated at the electron microscope, each serving distinct functions and depending on different regulatory and/or effector machineries. There is no strict correlation between a given kinetic mode of entry and a particular endocytic structure. Micropinocytosis, exemplified by clathrin-coated pits and vesicles, is a constitutive process of all interphase eukaryotic cells, involving small invaginations of regular size ( 100 nm). The polymerised clathrin coat is associated with multiple proteins including adaptin complexes, that allow to concentrate therein a variety of receptors. Among these are receptors for carrier proteins for essential nutrients, such as iron-bound transferrin.

Clathrin-coated pits are not only essential for receptormediated endocytosis, including of some viruses, but also support a major inflow of membrane (i. e. adsorptive endocytosis) and in several cases may fully account for fluid-phase endocytosis (Cupers et al., 1994). The role of actin in micropinocytosis is still controversial. Macropinocytosis (illustrated in Figure 1) defines the formation of large vacuoles of irregular size, generated by actin-driven evaginations of the plasma membrane in active zones, called “ruffling” domains (for a review, see (Swanson and Watts, 1995)). Historically, macropinocytosis was the first form of pinocytosis to be reported (Lewis, 1931). The fate of macropinosomes varies: in the macrophage lineage, they fuse with lysosomes while they may be prevented to do so in other cells (Hewlett et al., 1994). In higher eukaryotes, “permanent”, i. e. constitutive macropinocytosis is typically restricted to specialized conditions such as immature dendritic cells (Steinman and Swanson, 1995), activated macrophages (Lewis, 1931) or transformed cells (Veithen et al., 1996, 1998; Amyere et al., 2000). In most other cells, macropinocytosis can also be transiently induced (for  5–10 min) by growth factors or tumour-promoting factors such as PMA. In Dictyostelium, macropinocytosis and phagocytosis are inversely regulated (Table 1): when bacteria are abundantly available for phagocytosis, macropinocytosis is suppressed; conversely, when amoebas are grown under axenic conditions, macropinocytosis is induced to reach a level sufficient for nutrition (Clarke and Kayman, 1987). Phagocytosis defines the uptake of particles too large to be accommodated by clathrin-coated pits. This process generally involves multiple interactions between surface receptors and cognate ligands called opsonins, deposited by the host to tag particles for engulfment. The close membrane apposition that progressively wraps particles precludes a significant concomitant fluid-phase endocytosis, and phagosomes are almost entirely defined by their particulate content. Since phagocytosis also depends on cortical actin, macropinosomes resemble “empty phagosomes”, yet both processes obey different regulations and serve different purposes (see below). An evolutionary view of endocytosis Vesicular uptake by endocytosis requires only three acquisitions: (i) membrane deformability (e. g. by inclusion of cholesterol); (ii) a mechanical device providing deformation forces (i. e. the actin cytoskeleton); and (iii) budding and fusion machineries, allowing for intracellular mixing of substrate and digestive enzymes. These properties combined arguably provided three major evolutionary advantages, that can be considered

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to represent three stages in the eukaryotic history (de Duve, 1992). In a first stage, co-existing prokaryotes and ancestral eukaryotes were forced to share a nutritive macromolecular solution with limited renewal potential. When competing for this “primitive soup”, endocytosis followed by intracellular digestion in a limited degradative (lysosomal) compartment should have allowed to concentrate hydrolytic enzymes, to control digestive conditions, and to fully recover digestion products. In such a context, the invention of massive fluid-phase endocytosis by macropinocytosis must have favoured cell survival, as exemplified by amoebas grown under axenic conditions. In a second stage, with further expansion of the biosphere, nutrition may have becoming increasingly problematic because of almost exhaustion of the nutritive environment. Acquisition of phagocytosis demands no more than the generation of surface binding sites to capture extracellular particles such as prokaryotes. Considering bacteria as a merely concentrated meal, the simple invention of phagocytosis dramatically increases the efficacy of nutrition. In a third stage, the success of blue algae completely changed the earth atmospheric conditions about 1.5 billion years ago, from a reducing to an oxidative environment. By a further refinement of the phagocytic machinery, ancestral eukaryotic cells presumably realized the lasting benefit of preferring to a single meal the almost indefinite sequestration of enslaved bacteria able to domesticate oxygen (to become mitochondria), or light (to become chloroplasts). However, endosymbiosis is but a pact of mutual assistance, and like every peace treaty, it can be denied when necessary, ending-up in autophagy. In this evolutionary perspective, macropinocytosis can thus be viewed as the non-specific prototype of endocytosis, on which more refined variations had been developed, micropinocytosis placing emphasis on the qualitative aspects of membrane composition and selection of rare solutes, phagocytosis on content, and endosymbiosis on the control of intracellular trafficking, respectively.

Functions and subversions of macropinocytosis As life keeps track of its previous successes, macropinocytosis can be reactivated in higher eukaryotes. Macropinocytosis remains the most elegant solution when massive fluid-phase endocytosis is necessary, e. g. for bulk uptake of soluble antigens by immature dendritic cells; it also supports a major membrane traffic and allows for displacement of rigid membrane domains; and it can be subverted by some enteropathogens and in cancer.

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Fig. 1. Formation of a macropinosome in a v-Src transformed fibroblast. These two images, taken at 60 s interval, show the dynamics at the tip of a cellular extension that includes a series of pre-existing light-lucent vesicles (the distal one is marked by an asterisk). At A, a prominent lamellar extension (arrow) emerges at the “ruffling” zone and folds back towards the base of the extension. At B, a new macropinosome has been generated (arrow). Bar, 10 mm. (Reproduced from (Amyere et al., 2000)).

Table 1. Some differences in the regulation of macropinocytosis and phagocytosis in Dictyostelium discoideum. Effector

Macropinocytosis

Phagocytosis

References

PI3K

+ –

Buczynski et al., 1997; Zhou et al., 1998; Seastone et al., 1999

RacC

–

+

Seastone et al., 1998

Rap1

–

+

Seastone et al., 1999

profilin

+

–

Temesvari et al., 2000

For more details, see (Cardelli, 2001).

Massive fluid-phase endocytosis is a hallmark of immature dendritic cells, these specialized macrophages that play a key role in the immune response. At the immature stage, peripheral dendritic cells show a prominent macropinocytotic activity that allows for efficient “sampling” of soluble antigens. Upon maturation, dendritic cells stop migrating and settle in lymph nodes. At this stage, motility and macropinocytosis have both essentially disappeared and dendritic cells present instead antigen-derived peptides to naive T cells (Sallusto et al., 1995). Thyrocytes are polarized epithelial cells specialized in the production of thyroid hormones. This is achieved by endocytosis of thyroglobulin concentrated in the thyroid follicle at the apical surface, followed by hydrolysis by cathepsins in late endocytic compartments. Since both substrate (thyroglobulin) and hydrolases (cathepsins) are in vast excess, endocytosis is a key regulator of thyroid hormone production, controlling the encounter between substrate and enzymes. In steady-state conditions, thyroglobulin is taken up by micropinocytosis. However, upon acute TSH stimulation, macropinocytosis is selectively induced at the

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apical surface resulting in a major wave of substrate import (Ketelbant-Balasse and Neve, 1973). Macropinocytosis appears to be subverted by some pathogenic bacteria such as Salmonella and Shigella which delude cells by inducing macropinocytosis (the so-called “triggered” phagocytosis), used as a Trojan horse to penetrate the cell and prepare breaking into the cytosol upon secretion of pore-forming toxins (Alpuche-Aranda et al., 1994; Finlay and Cossart, 1997). More recently, macropinocytosis has also been reported to be involved in the entry of Chlamydia into dendritic cells (Ojcius et al., 1998). Macropinocytosis is intimately linked to actindriven membrane movement, manifested in “ruffling”, that is also a characteristics of cell motility. At the elongating tip of neuronal axons called “growth cones”, plasma membrane “ruffling” produces a large number of light-lucent vesicles (i. e. macropinosomes) that migrate towards the cell body (Nakai, 1956). During the chemotactic response of neutrophils and macrophages, “ruffling” and macropinocytosis are also observed at the leading edge (Carpentier et al., 1991). Using two

cell lines harbouring thermosensitive v-Src mutants, we have observed in a variety of experimental conditions a systematic correlation between induction or suppression of macropinocytosis and of accelerated motility (A. Platek, unpublished observations). Whether association of macropinocytosis and motility is merely coincidental or reflects a necessary mutual interaction remains unknown. However, it is conceivable that actindriven macropinocytosis is a necessary component of the endocytic membrane traffic during cell locomotion (Bretscher and Aguado-Velasco, 1998). A particular case is displacement of epithelial cells. Cells can be linked to their substrate not only by focal adhesions, that can be dissociated under the control of focal adhesion kinase itself supervised by Src, but also by hemi-desmosomes, these structured rigid membrane domains that are too large to be taken care of by clathrin-coated vesicles. Hemi-desmosomes are instead retrieved inside the cell by large, non-degradative electron-lucent vesicles, that correspond to macropinosomes (Holm et al., 1993; Poumay et al., 1994).

Regulation of macropinocytosis in non-polarized mammalian cells

Fig. 2. Multiple stages of macropinocytosis regulation in mammalian cells. For the explanations, see text.

Like the induction of phagosomes triggered by opsonised particles (for a review, see (Aderem and Underhill, 1999)), the signalling cascade leading to macropinosome formation involves several steps (see Fig. 2). In a first stage, plasma membrane signals are generated. This occurs upon binding of growth factors such as PDGF and GM-CSF, or upon plasma membrane recruitment of activated oncogenes in transformed cells such as K-Ras and v-Src (Fincham et al., 1996). The second stage involves signal amplification by activation of early enzymatic relays: phosphoinositide 3 kinase (PI3K) and phosphoinositide-specific phospholipase C (PLC; Fig. 3). Class IA PI3K are heterodimeric bifunctional enzymes made of a regulatory subunit of 85 kDa (p85) and a catalytic subunit of 110 kDa (p110). PI3K is activated either indirectly by recruitment with p85 or directly by Ras. p85 is translocated to the plasma membrane by interaction with activated growth factor receptors or by Src and, in turn, recruits p110 at the plasma membrane, i. e. in close proximity of its phosphoinositide substrates. PI3K catalyses the addition of a bulky negative charge (phosphate) on the hydroxyl group of the third carbon in the inositide ring of phosphoinositides, generating the so-called D3 products (Fruman et al., 1998). D3 serves as a docking site for multiple targets, including phospholipase C, exchange factors activating small GTPases, and actin-regulatory proteins (Martin, 1998). PI3K activation is an almost immediate response in multiple processes such as plate-

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Fig. 3. Relation between macropinocytosis, phosphoinositide 3 kinase (PI3K) and phospholipase C (PLC) activities. Left panel. Fluid-phase endocytosis is measured by the accumulation of horseradish peroxidase. Macropinocytosis is demonstrated by the difference in the level of accumulation of the tracer in control, transformed (v-Src) and transfected cells (Wp85α, dominant-positive PI3K construct in a non-transformed background; v-Src/Dp85α, dominant-negative PI3K construct in a v-Src-transformed background). PI3K and PLC were inhibited, as indicated, by 50 mM wortmannin or 50 µM NCDC, respectively. Values are means  SEM of 16–39 dishes. Middle panel. Measurement of PI3K activity. After 32P incorporation, phosphoinositides were analysed by HPLC. In an attempt to correct for variation in 32P uptake, values are normalised as PtdIns3,4P2/PtdIns4,5P2 ratios. Values are from a typical experiment representative of 2 to 5 experiments. Right panel. PLC activity was estimated by inositol 1,4,5 trisphosphate level, measured by a competitive radioligand binding assay. Values are  SEM of 4 dishes. (Adapted from (Amyere et al., 2000)).

let aggregation, phagocytosis of opsonised particles, and penetration of enteropathogens such as Listeria (Ireton et al., 1996). It is also involved in the increased motility and metastasis of cancer cells. These various processes are indeed abrogated by the irreversible inhibitor wortmannin and the reversible inhibitor LY290042 (Stein and Waterfield, 2000). PI3K activation can be mimicked, in some circumstances, by stable transfection with an expression vector for wild-type p85α, generating “dominant-positive” PI3K constructs. Conversely, expression vectors for a truncated p85α, deprived of its interacting domain with p110, can presumably compete with endogenous p85 for plasma membrane docking sites and act as “dominant-negative” PI3K construct. Macropinocytosis is inhibited by wortmannin, LY290042 and the “dominant-negative” PI3K construct and is induced by a “dominant-positive” PI3K construct (Amyere et al., 2000). Among targets of D3 phosphoinositide is PLC, that catalyses the hydrolysis of PIP2 into diacylglycerol, thereby activating protein kinase C (PKC), and inositol 1,4,5 trisphosphate, thereby increasing calcium release from regulated intracellular stores. PLC is activated upon transformation by Src and Ras and upon contact of cells bearing human E-cadherin with virulent Listeria; macropinocytosis (Amyere et al., 2000),

but not entry of Listeria (Bierne et al., 2000), is abrogated by pharmacological inhibitors of PLC. Downstream of the early enzymatic relays, the actin cytoskeleton is coordinated by the three Rho-GTPases (Hall, 1998). Rac and Cdc42 are generally activated together and trigger cortical actin remodelling, resulting into “ruffling” and filopodia formation, respectively, whereas Rho stimulates instead the formation of transcellular stress fibres. These are antagonistic responses, since stress fibres immobilize cells and rigidify the plasma membrane, whereas “ruffling” requires a decreased tension of the plasma membrane (Raucher and Sheetz, 2000) and is generally associated with increased cell motility. Accordingly, when Rac and Cdc42 are stimulated, Rho is generally suppressed. Rac is activated by guanyl nucleotide exchange factors such as Vav that can be recruited on D3 phosphoinositides and even on p85 itself; Rho is inactivated by the p190RhoGAP/p120RasGAP complex, that can be activated by Src and Ras. Ras activates Cdc42, that activates Rac, resulting in Rho inactivation (Kjøller and Hall, 1999; Sander et al., 1999). When bone marrow-derived immature dendritic cells are microinjected with an expression vector for “dominant-negative” Rac expression (N17Rac1) or treated with Clostridium difficile toxin B that inacti-

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vates all Rho-GTPases, macropinocytosis is abrogated. In contrast, microinjection of the specific Rho inhibitor C3-transferase has no effect. These observations indicate that Rac is a key regulator of constitutive macropinocytosis in mammalian cells. However, regulation varies according to the origin of dendritic cells. In bone marrow-derived dendritic cells, “dominant-positive” Cdc42 is sufficient to restore macropinocytosis in the mature stage (Garrett et al., 2000). In contrast, in spleen-derived dendritic cells, “dominant-negative” Cdc42 does not affect macropinocytosis at the immature stage, and “dominant-positive” Rac fails to restore this activity at the mature stage (West et al., 2000). The involvement of Rho-GTPases in the various forms of macropinocytosis deserves further studies. Several actin regulatory proteins have been associated with macropinosomes, including cortactin (Kaksonen et al., 2000). Cortactin, a key target downstream of Rho-GTPases (Weed et al., 1998), can either bundle stress fibres in the non-phosphorylated state, or act at the cytocortex when tyrosine-phosphorylated by c-Src, e. g. in response to growth factors (Huang et al., 1997). Interestingly, invasion of epithelial cells by Shigella somehow triggers tyrosine phosphorylation of cortactin by c-Src and results in its recruitment at “ruffling” domains and on the spacious phagosomes containing this bacteria (Dehio et al., 1995).

Regulation of macropinocytosis in polarized mammalian cells Apical endocytosis is distinctive in its clear dependence of actin (Gottlieb et al., 1993) and by its unique sensitivity to a variety of pharmacological and empirical treatments ((Sandvig et al., 2000) and references therein). There are three examples of polarized apical macropinocytosis in epithelial cells. First, acute TSH stimulation induces macropinocytosis at the apical surface of thyrocytes (Ketelbant-Balasse and Neve, 1973). Second, the enteropathogenic bacteria Salmonella and Shigella force entry into the luminal surface of epithelial cells, that do not normally phagocytose, by triggering micropinocytosis so as to become engulfed in “spacious phagosomes” (Alpuche-Aranda et al., 1994; Mengaud et al., 1996). Third, we have recently observed that in MDCK cells infected with a thermosensitive variant of v-Src, a shift from the non-permissive to the permissive temperature for Src kinase activity selectively triggers fluid-phase endocytosis at the apical surface, without affecting endocytosis at the basolateral surface. This is accompanied by a reorganization of apical microvilli, that become branched and irregular, and by the appearance of large endocytic vesicles in the apical cytoplasm, and is abolished by wortmannin and

NCDC, implying PI3K and PLC dependence (Mettlen et al., in preparation).

Conclusions and perspectives Historically, macropinocytosis led to the discovery (Lewis, 1931) if not the invention of pinocytosis. This original mode of fluid-phase endocytosis can be induced in several circumstances so as to fulfil nutrition requirements of Dictyostelium or support massive uptake of soluble antigens by antigen-presenting cells, and it can be subverted by enteropathogens and by cancer cells. Its signalling cascade involves several relays. A key early response is production of D3 phosphoinositides, that can be concentrated on specific plasma membrane domains (Rickert et al., 2000) including “ruffling” zones; it is also suggested that there is a concomitant increase in local calcium concentration. It is likely that D3, possibly assisted by calcium, provides recruiting platforms for multiple effectors, including PLC, regulators of Rho-GTPases and of the actin cytoskeleton. Several of these components have been demonstrated to be essential for macropinocytosis in Dictyostelium. A delineation of the hierarchy between these various factors and the reconstitution of a minimum platform are the next goals. Available studies on phagocytosis will provide some ideas on how to proceed but do not provide direct clues. Indeed, these two activities share the same tools, yet differ in their rules that remain to be elucidated. Acknowledgements. This work was supported by grants from the UCL, FNRS, Télévie, ARC and IUAP. The outstanding secretarial assistance of Mr. Y. Marchand is gratefully acknowledged.

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