Pathways of Clathrin-Independent Endocytosis

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Pathways of clathrin-independent endocytosis

Satyajit Mayor* and Richard E. Pagano‡ Abstract | There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin- independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

6 Rab GTPase Endocytosis is a basic cellular process that is used by patches of membrane . However, these pathways may A Ras family GTPase that cells to internalize a variety of molecules. Because these share some of the same molecular machinery, especially regulates membrane-trafficking molecules can be quite diverse, understanding the differ- that related to the polymerization of actin in membrane events in eukaryotic cells. ent pathways that mediate their internalization and how remodelling. Different Rab proteins are specific for different transport these pathways are regulated is important to many areas pathways and different of cell and developmental biology. Any endocytic pathway Endocytosis: so many choices! subcellular compartments. that mediates the transport of a specific cargo will first Many entry pathways into cells have been identified, require mechanisms for selection at the cell surface. Next, which vary in the cargoes they transport and in the Macropinocytosis the plasma membrane must be induced to bud and pinch protein machinery that facilitates the endocytic process. A mainly actin-dependent endocytic mechanism that is off. Finally, mechanisms are required for tethering these The large GTPase dynamin was originally noted for its used to internalize large vesicles to the next stop on their itinerary and for inducing role in severing clathrin-coated vesicles from the plasma amounts of fluid and growth their subsequent fusion to this target membrane. membrane and was subsequently found to be involved in factors; vesicles mediating this The discovery of clathrin-mediated endocytosis a CI pathway that was mediated by caveolae (see below). form of endocytosis are usually 1 uncoated and >500 nm in of specialized cargo and the subsequent identification of Because vesicle scission at the plasma membrane is diameter. many of the players involved in this complex process required for subsequent internalization, it was initially (for example, adaptor complexes and Rab GTPases) has assumed that dynamin might have a role in all forms Phagocytosis provided a fundamental paradigm for the analysis of of endocytosis. However, it was later discovered that An actin-based endocytic membrane trafficking in cells (reviewed in REFS 2–4). some CI pathways are dynamin-independent, at least mechanism. It is mediated by cup-like membrane extensions Recently, however, several endocytic pathways that when they are assessed by monitoring cargo internal- that are used to internalize do not use clathrin have also emerged (FIG. 1; TABLE 1). ization in the context of exogenous expression of mutant large particles such as bacteria. Electron micrographs of some of the early intermediates dynamin isoforms7 or from analysis of organisms that in clathrin-dependent and -independent pathways are are homozygous for mutant dynamin8. In addition, some shown in FIG. 2. Some of these pathways are constitutive, members of the ADP-ribosylation factor (Arf) and Rho *National Centre for Biological Sciences, whereas others are triggered by specific signals or are subfamilies of small GTPases have recently been sug- 5 UAS-GKVK Campus, even hijacked by pathogens . In addition, they differ in gested to have key roles in regulating different pathways Bangalore 560065, India. their mechanisms and kinetics of formation, associated of CI endocytosis9–13. ‡Mayo Clinic College of molecular machinery and cargo destination. We propose a classification scheme for CI pathways, Medicine, Departments of Here, we discuss the characteristics of clathrin- primarily for the purpose of arriving at a mechanistic Medicine, Biochemistry and Molecular Biology, independent (CI) endocytic pathways, the mechanisms understanding of the essential steps that are required to 200 First Street Southwest, of cargo selection and vesicle budding, the itineraries of generate an endocytic vesicle in these pathways (FIG. 3). Rochester, Minnesota 55905, internalized cargo, and the regulation of CI endocytosis. This is based on lessons that have been learned from the USA. The CI internalization pathways of macropinocytosis clathrin-mediated pathways, which show the irreduc- e-mails: [email protected]; phagocytosis [email protected] and are not discussed, primarily because ible requirements for a pinching mechanism (dynamin doi:10.1038/nrm2216 they belong to a separate class of endocytic processes GTPase) and for cargo concentration (the clathrin coat Published online 4 July 2007 that involve internalization of relatively large (>1 μm) and various adaptor proteins). The primary division

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Phagocytosis Macropinocytosis

Clathrin- Caveolin- Particle dependent dependent Clathrin- and caveolin- endocytosis endocytosis independent pathways Dynamin Actin filaments Clathrin Caveolin

CLIC

Uncoating Caveosome GEEC

Lysosome Early endosome

Figure 1 | Pathways of entry into cells. Large particles can be taken up by phagocytosis, whereas fluid uptake occurs by macropinocytosis. Both processes appear to be triggered by and are dependent on actin-mediated remodelling of the plasma membrane at a large scale. Compared with the other endocytic pathways, the size of the vesicles formed by phagocytosis and macropinocytosis is much larger. Numerous cargoes (TABLE 1) can be endocytosed by mechanisms that are independent of the coat protein clathrin and the fission GTPase, dynamin. This Review focuses on the clathrin-independent pathways, some of which are also dynamin independent (FIGS 2,3). Most internalized cargoes are delivered to the early endosome via vesicular (clathrin- or caveolin-coated vesicles) or tubular intermediates (known as clathrin- and dynamin- independent carriers (CLICs)) that are derived from the plasma membrane. Some pathways may first traffic to intermediate compartments, such as the caveosome or glycosyl phosphatidylinositol-anchored protein enriched early endosomal compartments (GEEC), en route to the early endosome.

in our classification scheme is the requirement for Dynamin-dependent CI pathways. Caveolae-mediated dynamin, thus, CI pathways are neatly divided in terms endocytosis is perhaps the best-characterized dynamin- of those that use a dynamin-mediated scission mech- dependent CI pathway. Caveolae are 50–80 nm flask- anism (dynamin-dependent), and those that require shaped plasma membrane invaginations that are marked other processes (dynamin-independent). A second by the presence of a member of the caveolin (Cav) Dynamin feature apparent from the literature is the involvement protein family and are present in many cell types19–21. A large GTPase of ~100 kDa. It mediates many, but not all, of small GTPases in several CI pathways. We use this fea- Biochemical and proteomic analyses of caveolar mem- forms of endocytosis as well as ture to classify the CI pathways further; the terminology brane fractions have shown that they are enriched in vesicle formation from various CDC42-, RhoA- or ARF6-regulated endocytic pathway sphingolipids and cholesterol22, signalling proteins and intracellular organelles through indicates that interfering with or modifying the function clustered glycosyl phosphatidylinositol-anchored proteins its ability to tubulate and sever 23–25 membranes. of these different GTPases affects the internalization (or (GPI-APs) . Indeed, caveolar cargoes are diverse, trafficking) of one set of CI endocytic markers, but not ranging from lipids, proteins and lipid-anchored proteins Caveolae another (see below and REFS 10,14,15). However, this to pathogens (TABLE 1). However, the study of caveolar 50–80 nm flask-shaped pits terminology is not meant to imply that a given GTPase endocytosis is complicated by the possibility that the that form in the plasma is directly involved only in a particular internalization same endocytic cargo may be internalized by different membrane and are enriched in caveolins, sphingolipids and mechanism — these pathways have been primarily mechanisms in different cell types or may switch path- cholesterol. defined by mutants of these GTPases, which are likely ways in a single cell type under different conditions. For to affect many cellular processes16–18. example, albumin is internalized by caveolae in some cell Caveosome By dividing the CI pathways into the potential sub- types (human skin fibroblasts and endothelial cells)26–28, An intracellular compartment that is involved in the classes outlined above, we aim to categorize the molecular whereas in Chinese hamster ovary cells it is internal- 29 intracellular transport of simian machinery associated with these pathways in a straight- ized by a RhoA-dependent mechanism (see below and virus-40 from caveolae to the forward manner that is also consistent with the existing REFS 28,30–34 for other examples). This suggests the endoplasmic reticulum. literature; however, alternative classification schemes presence of redundant CI endocytic mechanisms that have also been proposed (BOX 1). Although no system is can be used by some cargo molecules. Glycosyl phosphatidylinositol-anchored protein likely to be completely satisfactory at this time, we believe A second, dynamin-dependent CI mechanism is a 35 A protein that is anchored to that the scheme shown in FIG. 3 is helpful in providing a pathway that is dependent on the small GTPase RhoA . the extracellular membrane framework to organize and discuss the current literature This pathway was uncovered while studying the path- leaflet through the glycosyl in this rapidly growing field and to fuel systematic experi- way that is responsible for internalizing the β-chain phosphatidylinositol lipid modification rather than mentation. A major task ahead is to identify the mole- of the interleukin-2 receptor (IL-2R-β), although it is through a transmembrane cules that regulate particular CI endocytic mechanisms also used to internalize other proteins in both immune protein domain. and to learn whether additional CI pathways exist. cells and fibroblasts (TABLE 1). Following ligand binding,

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Table 1 | Mechanisms of clathrin-independent endocytosis* Characteristics Caveolar‡ RhoA-regulated CDC42-regulated ARF6-regulated Cargo§ Modified albumins IL-2R-β GPI-AP¶ MHC I GSL analogues IL-2R-αβ Fluid-phase markers β1 integrin SV40; EV1 γc-cytokine receptor|| CtxB¶ E-cadherin Some integrins (for example, β1) IgE receptor|| VacA toxin GPI-AP CtxB¶ Aerolysin CPE Ab-clustered GPI-AP# IL-2R-α (Tac) Structural components CAV1 RhoA CDC42 ARF6 and machinery PV1 Dynamin Flotillin-1** Flotillin-1** associated with pathway Src RAB5 RAB11 PKC-α PI3K RAB22 Dynamin EEA1 PI3K RAB5 Primary carriers 50–80 nm diameter caveolin- Uncoated vesicles Clathrin- and dynamin- Unknown coated vesicles and ‘grape-like’ independent carriers (CLICs) aggregates Identity of earliest Caveosome or early endosome Early endosome GPI-AP enriched early endosomal Early tubular recycling acceptor compartment compartments (GEECs) compartment Ab, antibody; ARF6, ADP-ribosylation factor-6; CAV1, caveolin-1; CPE, carboxypeptidase E; CtxB, cholera toxin B; EEA1, early endosomal antigen-1; EV1, Echo virus; GPI-AP, glycosyl phosphatidylinositol-anchored protein; GSL, glycosphingolipid; IgE, immunoglobulin E; IL-2R, interleukin-2 receptor; MHC I, major histocompatibility complex I; PI3K, phosphatidylinositol 3-kinase; PKC-α, protein kinase C-α; PV1, Polio virus; SV40, simian virus-40; VacA, Helicobacter pylori vacuolating toxin. *See Supplementary information S1 (table) for inhibitors and dominant negative proteins that are used to distinguish among the various clathrin- independent mechanisms. ‡The best caveolar markers vary with cell type (see text). §See text for representative references for different cargo molecules. ||Although the specific requirements for endocytosis of molecules via a given pathway have not been rigorously established, available evidence suggests this classification. ¶Originally described as a caveolar marker, but recent data suggests uptake by the CDC42-regulated pathway31,33,104. #Crosslinking of GPI-APs shifts internalization from the CDC42-dependent pathway to the caveolar pathway37,105,106. **Currently, it is not clear if flotillin-1 is part of one or both of these pathways (see text).

IL-2R-β partitions into detergent-resistant membranes or of a DN construct of CDC42 on the internalization (DRMs; BOX 2), a characteristic of membranes that is of GPI-APs8,31,36,37. This dynamin-independent pathway usually associated with CI endocytic processes. Neither seems to be the main route for the non-clathrin, non- dominant negative (DN) inhibitors of clathrin polymeriza- caveolar uptake of cholera toxin B (CtxB) as well as for tion, nor epidermal growth factor pathway substrate-15 the plant protein ricin38,39 and the Helicobacter pylori (EPS15; a protein involved in clathrin assembly), nor vacuolating toxin (VacA)40 (TABLE 1). These carriers also adaptor protein-180 (AP180, which is required for adaptor represent the main mechanism of fluid-phase internaliza- recruitment) affected endocytosis of IL-2R-β. However, tion in many cell types31,37, and are mainly devoid of cargo uptake was strongly inhibited by DN dynamin and RhoA. from the clathrin-dependent pathway36,37,41. However, it is Whether a RhoA-mediated process is required for the likely that GPI-APs may take other routes into the cell in correct sorting of IL-2R-β into the endocytic pathway or addition to the CDC42 pathway, especially if they laterally during the endocytic process itself is currently unknown. associate with proximal transmembrane proteins that are Because RhoA is a key player in the regulation of actin targeted to other pathways of internalization (discussed cytoskeleton dynamics, RhoA could possibly be required in REF. 42). For example, the GPI-anchored cellular prion for recruiting the actin machinery to regulate endocytosis protein (PrPC) is internalized by both clathrin- and via this pathway. caveolae-mediated pathways43–45. This could be due to the binding of specific molecules that might target PrPC Dynamin-independent CI pathways. A CI and dynamin- to different pathways in different cell types46. independent endocytic pathway was initially identified in The primary carriers that bud from the cell surface by HeLa cells, in which the expression of a mutant dynamin-1 CDC42-regulated endocytosis have long and relatively blocked receptor-mediated endocytosis but apparently wide surface invaginations, in contrast to the small increased fluid-phase uptake via a CI pathway7. Dynamin- spherical carriers that are characteristic of the clathrin independent fluid-phase uptake has also been observed at and caveolar pathways31,37. Therefore, a large volume of non-permissive temperatures in Drosophila melanogaster fluid phase is co-internalized in a single budding event haemocytes carrying a temperature-sensitive mutation compared with these other mechanisms of internaliza- in the GTPase domain of the protein that is encoded tion. This does not imply, however, that CDC42 is only by shibire, the sole dynamin gene in D. melanogaster8. involved in regulating this type of pathway: in dendritic Dominant negative protein A mutant version of a protein A common feature of constitutive dynamin-independent, cells, modulation of CDC42 function affects both macro- 47 that, when expressed, results CI endocytosis appears to be the use of small GTPases, pinocytic uptake and phagocytosis. A recent study in an inhibitory phenotype, either the Rho family member CDC42 or the Arf family suggests that cholesterol-sensitive CDC42 activation is usually by competing with member ARF6 (TABLE 1). coupled to the recruitment of an actin-polymerization interaction partners and thereby suppressing the The role of CDC42 in dynamin-independent endo- machinery, thus rendering this pathway sensitive to cho- function of the endogenous cytosis was first identified by studying the effects of a Rho lesterol levels in the plasma membrane and to inhibitors wild-type protein. GTPase inhibitor, Clostridium difficile toxin B (toxin B), of actin polymerization48.

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a b However, it should be noted that a direct role for ARF6 in CI internalization has not been explicitly established. Indeed, ARF6 has a potent role in actin remodelling, stimulates the formation of phosphatidylinositol 4,5- bisphosphate by activating phosphatidylinositol 4- phosphate-5-kinase, and has also been implicated in regulating endosome dynamics by influencing the recycling rates of various membrane components14,49. Furthermore, the effects of ARF6, at least in some instances, c d may be cell-type specific. For example, ectopic expression of activated ARF6 stimulated the internalization of multiple membrane cargo (including GPI-APs) in HeLa cells49, whereas perturbation of ARF6 function did not affect the endocytosis of GPI-APs in Chinese hamster ovary cells41. As mentioned above, GPI-APs have been demonstrated to be internalized by a CDC42-dependent pathway. Thus, whether the CDC42- and ARF6-regulated pathways efare related or whether they are distinct mechanisms of CI endocytosis still needs to be determined. It seems likely that these two pathways will share common features, and as the same cargoes are studied more comprehensively in 15 s many different cell types, we may find that CDC42- and ARF6-regulated mechanisms of CI endocytosis represent variations on a similar theme. Alternatively, CDC42 may have distinct roles in the formation of specific endocytic carriers, whereas ARF6 may be important for membrane trafficking and recycling.

5 min Mechanisms of cargo selection Figure 2 | Electron micrographs of early intermediates in clathrin-dependent and Little is known about how cargo is selected for the different independent pathways of endocytosis. a | Thin-section views of reticulocytes CI endocytic pathways. In contrast to clathrin-dependent incubated with gold-conjugated transferrin (AuTf) for 5 min at 37°C, which shows AuTf endocytosis, in which specific adaptor molecules have clustering into clathrin-coated pits and subsequently being endocytosed via coated been identified that recruit cargo to coated pits, no such vesicles. b | Rapid-freeze, deep-etch views of clathrin lattices on the inner surface of a well-defined adaptors for CI endocytosis have been normal chick fibroblast. c | Thin-section (left panel) and rapid-freeze, deep-etch images identified. One potential mechanism that has been con- (right panel) of caveolae in endothelial cells. The arrow in the left panel points to the sidered is sorting based on the association of cargo with endoplasmic reticulum near deeply invaginated caveolae. d | Thin-section surface view microdomains at the plasma membrane. However, it of horseradish peroxidase (HRP)-conjugated cholera toxin in the process of should be noted that the association of cargo with DRMs internalization via grape-like caveolae in mouse embryonic fibroblasts. e | Thin-section (BOX 2) does not assure subsequent internalization by a CI images of green fluorescent protein (GFP) with a glycosyl phosphatidylinositol (GPI) 50–52 anchor expressed in Chinese hamster ovary cells and incubated with gold-conjugated mechanism . The potential mechanisms for CI cargo antibodies against GFP. The antibodies show putative sites for clathrin- and dynamin- selection have been divided into lipid- and protein-based independent endocytosis. These represent surface-connected tubular invaginations mechanisms, although the various options within each where the gold probe is concentrated with respect to the rest of the plasma membrane. type of mechanism are only beginning to be determined f | Thin-section micrographs of internalized HRP-conjugated cholera toxin incubated at and their relative importance is unknown. 37°C in mouse embryonic fibroblasts, showing early intermediates in clathrin- and caveolin-independent endocytosis. Clathrin- and dynamin-independent carriers (CLICs) Protein-based mechanisms. Recently, it has been sug- are observed after 15 s (top panel) and GPI-anchored protein enriched early endosomal gested that ubiquitylation could have a role in determining compartments (GEECs) are observed after 5 min (bottom panel). The scale bar in a and b whether a protein is internalized via a clathrin-dependent is 100 nm, in c is 200 nm (left panel) and 100 nm (right panel), and in d–f is 200 nm. or -independent mechanism. Ubiquitylation of some Part a reproduced with permission from REF. 101 © (1983) Rockefeller University Press. receptor tyrosine kinases such as the epidermal growth Part b reproduced with permission from REF. 102 © (1989) Rockefeller University Press. E3 ubiquitin Part c reproduced with permission from REF. 103 © (1998) Annual Reviews. Parts d factor (EGF) receptor is mediated by c-Cbl, an and f reproduced with permission from REF. 31 © (2005) Rockefeller University Press. ligase. Although EGF receptors have been found to be Part e reproduced with permission from REF. 37 © (2002) Elsevier. internalized through clathrin-mediated and CI pathways, recently it has been suggested that ubiquitylation might be used to sequester EGF receptors into CI routes, A role for the Arf family GTPase ARF6 has been despite the fact that they also have clathrin-pit localiza- suggested in the CI and dynamin-independent endo- tion signals. For example, high concentrations of EGF cytosis of several proteins such as class I major histo- promote ubiquitylation of EGF receptors, association compatibility complex molecules (MHC I), β1 integrin, with DRMs, and endocytosis by a cholesterol-sensitive carboxy peptidase E (CPE), E-cadherin and GPI-APs. CI mechanism, possibly by caveolae51.

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Endocytic cargo

Clathrin dependent Clathrin independent

Dynamin dependent Dynamin dependent Dynamin independent

ConstitutiveLigand-induced Other? CaveolarRhoA-regulated Other? CDC42-regulated ARF6-regulated Other? Figure 3 | Proposed classification system for endocytic mechanisms. A cargo protein can be endocytosed by either clathrin-dependent or clathrin-independent (CI) mechanisms. We propose that the CI pathways can be further categorized first by their dependence on the large GTPase dynamin, and then by other mechanistic components of the internalization pathway. This system is based on the current literature, which shows that interfering with or modifying the function of a particular GTPase affects the internalization or trafficking of one set of CI endocytic markers but not another. See TABLE 1 for endocytic markers that have been studied. The aim of this classification system for CI pathways is to categorize the molecular machinery that is associated with these pathways in the most parsimonious fashion consistent with the current literature. It is not meant to imply that a given GTPase is directly involved in regulating a particular internalization pathway because many other important cellular functions may also be regulated by that GTPase. There may be other, as-yet-unidentified CI pathways.

Carboxypeptidase E An endopeptidase that is found in secretory vesicles and can activate neuropeptides. Mechanistically, the sequestration of cargo in caveo- clustered when it is located in clathrin-coated pits, It may also function as a lae has also been difficult to elucidate. EGF receptors whereas considerable clustering is observed in other sorting receptor that sorts are guided into the caveolar pathway by three ubiquitin parts of the membrane56. It has been shown that mono- cargoes into the regulated interacting motif-containing proteins: EPS15, epsin and sialoganglioside GM1, the receptor for CtxB, is not secretory pathway. EPS15R (EPS15-related). However, all three proteins are distributed uniformly over the plasma membrane, but Ubiquitylation somewhat redundant in their function because a strong is concentrated approximately four-fold in non-coated A post-translational inhibitory phenotype is only observed in triple small invaginations57. Considering this, it will be important modification that is added to interfering RNA (siRNA) knock-down studies51. to examine the nanoscale organization of CtxB as it is some receptor tyrosine kinases following ligand binding. Protein-based mechanisms of cargo sorting have internalized by different CI and dynamin-independent It mediates internalization and also been implicated in the RhoA- and ARF6-regulated pathways. Nanoscale domains might come together to subsequent sorting at the level pathways of CI endocytosis. Specific internalization form active endocytic domains58 that are regulated by of endosomes. sequences are thought to be present in the cytoplasmic a specific pathway, or different CI mechanisms may E3 ubiquitin ligase tail of the γc-cytokine receptor, which is internalized in show a preference for different sizes of microdomains. An enzyme that functions a RhoA-dependent manner53. Therefore, a protein-based Alternatively, if there are multiple types of micro domain downstream of or in adaptor machinery that targets molecules specifically to (as suggested by several studies42,59), these may serve combination with a ubiquitin- this pathway could exist, although these adaptors remain to sort membrane components associated with these conjugating enzyme (E2) to attach ubiquitin molecules to to be discovered. In the ARF6-regulated pathway, CPE has domains into different endocytic pathways. The challenge a target protein, marking the a cytoplasmic tail sequence that confers binding to ARF6. is to identify how lipid-based microdomains of different protein for subsequent Deletion of this tail region results in the inhibition of types may be assembled and recognized by cytoplasmic recognition by ubiquitin- endocytosis, as does expression of DN ARF654. However, constituents. binding domains. many cargoes that are internalized by the ARF6-regulated Ubiquitin interacting motif pathway and other CI pathways lack any known cyto- Combined lipid–protein mechanisms. Integral mem- A protein motif of ~24 amino plasmic signal; thus, other unknown mechanisms must brane proteins with unusual topology, such as caveolin, acids that interacts with regulate cargo sequestration for CI internalization. may serve to link mechanisms of cargo selection that are ubiquitin and, in some instances, is also necessary for based on lipid organization with a specific cytoplasmic the ubiquitylation of proteins Lipid-based mechanisms. In the absence of any protein machinery for subsequent internalization. Fluorescent that contain this motif. adaptors and without the extension of a cytoplasmic analogues of glycosphingolipids (GSLs; a subclass of tail for membrane-anchored proteins, other mechan- sphingolipids) are internalized primarily by caveolae Fluorescence resonance 28,60,61 energy transfer isms for CI cargo selection must be invoked. One such in various cell types ; however, the basis for this (FRET). A fluorescence-based mechanism is the nanoscale clustering of lipid-tethered selective internalization is not known. A recent study method for detecting proteins, which is emerging as an intrinsic sorting signal by Singh et al.62 indicated that the stereochemistry interactions between proteins for the CDC42-dependent pathway for internalization of of a GSL, as well as its ability to cluster in membrane that are <10 nm apart in a native GPI-APs55. Artificial crosslinking of GPI-APs into microdomains, have important roles in determining the cell. This method is dependent on the spectral overlap larger-scale structures instead directs these proteins pathway of internalization. GSL analogues of different between donor and acceptor into caveolar or other CI pathways. stereochemistries are internalized by different endocytic chromophores and uses the CtxB can be internalized by clathrin-, caveolar- and pathways and, furthermore, a non-natural GSL analogue transfer of (non-radiative) CDC42-dependent pathways, suggesting it is organized that does not cluster in membrane microdomains is not energy from an initially excited fluorescence donor molecule to in the membrane in different ways. Using a taken up in caveolae. Thus, the proper organization of subsequently excite an resonance energy transfer (FRET)-based assay, Nichols GSLs into plasma-membrane domains appears to be acceptor molecule. and colleagues have shown that CtxB is not significantly required for targeting to caveolae.

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56 Box 1 | Classification criteria for endocytic mechanisms in the membrane , which defines a further location that could recruit or trap specific membrane components. Fission mechanism Finally, siRNA-mediated depletion of endogenous flo- Dynamin CtBP3/BARS tillin-1 in cells caused a marked decrease in dynamin- Lipid-based independent endocytosis of fluid-phase markers, GPI-APs and CtxB. This implicates flotillin-1 as one of the cytoplasmic components of CI endocytosis56. However, it remains to be determined whether flotillin-1 has a direct role in cargo selection and vesicle budding, Lipid composition Cholesterol or whether it is a cargo molecule that is recognized by Raft components the CI endocytic machinery. GTPases Sphingolipids Cargo CDC42 Ligand or receptor ARF6 Mechanisms for membrane budding Fluid RhoA The generation of endocytic carriers at the cell surface GPI-AP involves membrane deformation, its growth into a Adhesion molecules Virus spherical bud or tubule and, finally, membrane scission. Toxin In the clathrin-mediated pathway, many membrane- Bacteria deforming proteins such as clathrin, epsin and dynamin Coat components 65 Clathrin have been reported to be involved . However, even in Caveolin this well-studied pathway, the physico-chemical mecha- Flotillin nism of membrane deformation and pinching are poorly Adaptors Actin cytoskeleton Actin understood. Recently, two theoretical models have been Regulation Actin-binding proteins proposed that consider the material characteristics Kinases Actin-regulatory proteins Phosphatases of the membrane and the machinery that is necessary for Ubiquitylation the generation of membrane buds (BOX 3). These theo- GEFs, GAPs retical approaches are important because they provide Many different factors affect clathrin-independent (CI) endocytosis (see figure) and new ways of thinking about the budding process, and could potentially be used to enumerate and classify these uptake mechanisms. may rationalize the classification of these endocytic Alternatives to the endocytic mechanism classification system that is used in this pathways based on the actin regulatory GTPases. Review (FIG. 3) might include cholesterol dependence, actin sensitivity or association with lipid rafts (which are often synonymous with detergent-resistant membranes Itineraries of CI endocytic cargo (DRMs); see the list below). Another scheme is based on the pathways that different Extensive studies that detail the itineraries of various viruses can use when they enter cells. cargo molecules have been carried out. For example, • Cholesterol dependence. Most endocytic pathways are sensitive to cholesterol endocytosed GPI-APs can be detected in tubular invagin- perturbation, and clathrin-dependent92,93 and CI pathways29,48,49 are inhibited by the ations that subsequently undergo fusion to generate removal of cholesterol. distinct early endosomal compartments, termed GPI-AP- • Lipid rafts. DRM-associated proteins can be accommodated in almost all endocytic enriched early endosomal compartments (GEECs)31,37,41; pathways42. CtxB is endocytosed via small tubular or ring-like carriers • Actin sensitivity. Many clathrin-dependent and -independent endocytic pathways known as clathrin- and dynamin-independent carriers show a differential sensitivity towards actin perturbation, depending on the cell (CLICs) that bud directly from tubular invaginations at type29,94. the plasma membrane31; and some cargo molecules of the • Viral entry. This system reflects the range of endocytic machinery that may be ARF6-mediated CI pathway also localize to a distinct recruited for the endocytic uptake of different viruses5. However, it is not clear how type of early endosome. Although not a focus of this much of the mechanism that is associated with viral entry is assembled by induction through the interaction of virus and the cell-membrane receptors. In addition, there Review, an important finding is that virtually all routes of are little data that compare virus uptake with the uptake of the many endocytic entry eventually merge at the RAB5 and early endosomal markers that have been used to analyse CI endocytosis, which makes it difficult to use antigen-1 (EEA1)-marked early endosome. Following this system as a tool to organize and discuss the literature on CI pathways. internalization by distinct endocytic mechanisms and subsequent merging at the early endosome, cargo mole- ARF6, ADP-ribosylation factor-6; CtBP3/BARS, C-terminal binding protein 3/brefeldin cules are then sorted for delivery to various intracellular A-ribosylated substrate; GAPs, GTPase-activating proteins; GEFs, guanine nucleotide exchange destinations or for recycling back to the plasma mem- factors; GPI-AP, glycosyl phosphatidylinositol-anchored protein. brane. It is interesting to note that there can be differences in intracellular destinations even when two cargo molecules are internalized by the same mechanism. Various The integral membrane proteins flotillin-1 and flotil- mechanisms have been suggested for sorting at the early lin-2 (also called reggie-2 and -1, respectively63) may have endosome and are supported to varying degrees. These a role in selecting lipid cargo for dynamin-independent include sorting based on the unique tubulovesicular endocytosis. First, crosslinked or ‘activated’ GPI-APs and geometry of the early endosome66, selective retention67 CtxB are found to be co-localized with flotillin-1 and -2 at or recycling of membrane components, lipid physical the surface of cells that lack caveolin64. Second, flotillin-1 properties68,69 and the presence of a mosaic of proteins is assembled in dynamic punctate structures at the cell (for example, Rab GTPases) that are required for vesicle surface that are distinct from clathrin and caveolin sites tethering and subsequent fusion steps70.

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Box 2 | Detergent-resistant membranes, rafts and lipid organization However, other endocytic mechanisms are also cholesterol- sensitive and, depending on the cell type and the extent of depletion, might also be disrupted (Supplementary information S1 (table)). A general depletion of sphingolipids (that is, GSLs and sphingomyelin) also blocks multiple CI pathways, including caveolae-mediated endocytosis Cholesterol GPI-AP as well as the RhoA- and CDC42-regulated pathways. Raft-localized lipid In contrast, depletion of GSLs alone selectively blocks 29 Caveolae Caveolin caveolar endocytosis . Furthermore, the effects of GSL Non-raft lipid depletion can be restored by incubation with exogenous Transient confinement zones GSLs, whereas disruption of RhoA- and CDC42-regulated endocytosis by sphingolipid depletion is restored by exogenous sphingomyelin (but not GSLs). These results suggest the importance of cellular sphingolipids in the regulation of different CI endocytic mechanisms. Cargo molecules that are associated with clathrin-independent (CI) endocytic Caveolar endocytosis is an inducible process and can 57,73–75 pathways have often been thought to be endocytosed by lipid-raft-based mechanisms. be stimulated by the presence of cargo . Indeed, Rafts have been proposed to be lipid-based sorting and signalling platforms that are acute treatment of human skin fibroblasts with non- enriched in cholesterol and sphingolipids22, mainly because of the observed fluorescent GSLs or elevation of cellular cholesterol dependence of sorting and signalling of lipid-tethered membrane components on levels stimulated the internalization of caveolar markers cholesterol and sphingolipid levels in cell membranes95. Sphingolipids and without affecting other clathrin-dependent and CI 61,76 phosphatidylcholine segregate into liquid-ordered (lo) and liquid-disordered (ld) phases pathways . One plausible model for the stimulation above a certain cholesterol concentration in artificial membranes. Cholesterol-rich l o of caveolar endo cytosis by exogenous lipids is that they 96 domains are relatively insoluble in cold non-ionic detergents , whereas extraction of alter the domain organization of the plasma membrane, cell membranes by cold non-ionic detergents gives rise to a cholesterol-rich membrane thereby inducing the clustering and activation of signal- fraction that is enriched in specific membrane proteins and lipids97. These observations have converged on a popular notion that rafts in cell membranes resemble the ling proteins at the plasma membrane that are needed to β 72,76 cholesterol-based phase segregation that is observed in artificial membranes and that initiate caveolar uptake (for example, 1 integrins ). rafts are ‘captured’ as a detergent-resistant membrane (DRM) fraction. Unfortunately, The effects of exogenous lipids on caveolar endocytosis the picture is not so simple (see REF. 58). The challenge ahead is to find new ways to are likely to be physiologically important. For example, probe lipid-dependent assemblies in the membranes of living cells. DRM association gangliosides are shed from tumour cells, and thereby may be one, albeit imperfect, way to probe the potential association of membrane contribute to the tumour microenvironment53,77. Acute, components in such assemblies. The figure shows two models of membrane rafts. local changes in cellular cholesterol might also occur In one model (top), the plasma membrane appears as a mosaic of domains, including as a result of many processes in vivo (for example, cell caveolae, nanodomains that are enriched in glycosyl phosphatidylinositol-anchored injury), which, in turn, could affect endocytosis as proteins (GPI-APs), and transient confinement zones98. In a second model (bottom), noted above. pre-existing lipid assemblies are small and dynamic and coexist with lipid monomers (left); however, they can be actively induced to form large-scale stable rafts (right)58. Top panel modified with permission from REF. 98 © (2002) Elsevier. Bottom panel Regulation of distinct pathways through GTPases. In modified with permission from REF. 58 © (2004) Blackwell Publishing. the absence of acute stress, it seems reasonable that the surface area of the cell will be relatively constant over time. Therefore, a change in the activity of a particular endocytic mechanism might be compensated for by Regulation of CI endocytosis alterations in other mechanisms of endocytosis. One The wide variety of cargo molecules that are able to of the first important clues supporting the concept of stimulate and be internalized by CI pathways, including cross-regulation came from the work of Schmid and pathogens, lipids and growth factors, suggests that these colleagues, who showed that the inhibition of recep- pathways must be regulated. Indeed, various kinases and tor-mediated endocytosis following the expression of signalling molecules have been associated with membrane a temperature-sensitive dynamin mutant resulted in microdomains and caveolae, and a recent screen of the an upregulation of fluid-phase endocytosis7,78. Another human kinome identified a total of 80 different kinases that example of the co-regulation of different CI endocytic Transient confinement zone A region of the cell membrane are somehow involved in endocytosis of the caveolar cargo mechanisms comes from studies of cells that were 71 that is defined in single-particle simian virus-40 (SV40) . Furthermore, lipids may have a treated with tetrafluoroaluminate (AlF4), an agent that diffusion studies of mobile role in regulating CI pathways of endocytosis through their locks G-proteins in an activated state. This caused an molecules as an area where effects on the formation of distinct membrane domains upregulation of ARF6-mediated endocytosis in cells that mobile species are likely to and the clustering of proteins into these domains72. Finally, were transfected with ARF6, whereas the CDC42-regu- spend more time than expected from the analysis of the dependence of many CI pathways on distinct small lated pathway was inhibited, which implies that there the trajectory of diffusing GTPases also provides a likely target for regulation. are different modes of regulation for these pathways, species. potentially via G-proteins41. Membrane composition and caveolar endocytosis. An important clue as to how cross-regulation between Kinome The entire repertoire of kinases Pharmacological depletion or sequestration of cho- the caveolar and CDC42-dependent (or RhoA-dependent) encoded by the genome of an lesterol disrupts the integrity of caveolae and inhibits pathways might occur comes from a recent study by organism. caveolar endocytosis of several cargo molecules (TABLE 1). Nevins and Thurmond79. They showed that CAV1

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Box 3 | Mechanisms of budding: recent theoretical perspectives Coupling of secretion and endocytosis. To maintain a constant cell surface area, the delivery of new lipids and Actin-nucleating proteinsTubule coat proteins Plasma membrane proteins to the plasma membrane might be balanced by the internalization of components at the cell surface. In the regulated endocytosis that is observed at the synapse in nerve termini, there is compelling evidence to implicate clathrin-dependent endocytosis in main- taining membrane homeostasis80. However, there is little evidence that supports this concept in constitutive systems, except in yeast, where most secretory mutants were also found to be defective in endocytosis81. Ito and Komori demonstrated that the enzymatic cleavage of Interfacial cell-surface GSLs causes a rapid upregulation of GSL region synthesis and delivery to the plasma membrane82, which suggests the presence of a homeostatic mechanism for plasma membrane GSLs. A recent study by Choudhury et al. provided new evidence for the coupling of caveolar endocytosis and secretory transport83. They showed that inhibition of the SNARE protein syntaxin-6 blocked delivery of microdomain-associated lipids and proteins from the Golgi to the plasma membrane in human skin fibroblasts, and this resulted in an inhibition of caveolar internalization. In another study, it was shown that if Clathrin secretion by a dynamin-independent exocytic pathway is Actin filaments inhibited through siRNA-mediated knock-down of the membrane-fission protein C-terminal binding protein 3/ brefeldin A-ribosylated substrate (CtBP3/BARS), there is The generation of endocytic carriers at the cell surface involves membrane a concomitant block in dynamin-independent fluid- deformation, its growth into a spherical bud or tubule and, finally, membrane scission. phase uptake84. Although this result was interpreted as Recently, a general mechanism of membrane budding has been proposed in which a direct effect of the CtBP3/BARS protein on dynamin- the phase segregation of lipids could serve as a basis for initiating a membrane bud. independent endocytosis, it is equally likely that it could The mechanism of generating this phase segregation might include protein-based be due to a coupling of dynamin-independent endocytosis accretion at a particular site that allows for the accumulation of specific cargo and secretion. (for example, at clathrin-coated pits or at caveolae), which results in the spontaneous Finally, an important potential regulator of all of the CI curvature of these membrane domains and their subsequent pinching by dynamin. pathways is the actin cytoskeleton — endocytosis via all However, it is unlikely that lipid-based mechanisms of cargo selection operate on of these pathways is sensitive to agents that inhibit actin similar principles, especially if they are raft-like or liquid-ordered (BOX 2). The chiral composition of rafts could spontaneously initiate buds of the appropriate morphology polymerization. Space constraints do not permit a discus- and dimension commensurate with those observed in clathrin-independent endocytic sion of this topic here, but several key references discuss vesicles99. The figure shows a theoretical model for endocytosis in which actin the roles of the actin cytoskeleton and its regulatory filaments exert protrusive surface stresses on the bud and tubule100. The tubule coat machinery in various endocytic mechanisms15,85,86. proteins create a lipid-phase boundary between the bud and the tubule. The clathrin adaptor proteins (or alternative coats) may add to the bending modulus and Conclusions and perspectives spontaneous curvature of the bud region, and fission of membranes might be driven by The field of CI endocytosis has undergone enormous © a phase-separation mechanism. Figure modified with permission from REF. 100 growth in recent years with the realization that these (2006) National Academy of Sciences. pathways have important roles in the regulation of cell growth and development, as well as important implica- tions in the study of certain diseases and pathogens. functions as a novel CDC42 guanine nucleotide dissociation Intense investigation over the past 30 years has uncov- inhibitor (GDI) in pancreatic β-cells. Analysis of the ered different types of clathrin-mediated endocytic pits CAV1 amino-acid sequence revealed a motif that is and pathways87–89. A similar debate is now underway conserved in GDIs, and this finding was further sup- regarding the distinct CI endocytic mechanisms that ported by in vitro binding assays that showed a direct exist in eukaryotic cells, the best cargo molecules with interaction between CAV1 and GDP-bound CDC42. which to study a particular pathway, the underlying Importantly, CAV1 depletion resulted in an induction of protein machinery that regulates these pathways, and Guanine nucleotide activated CDC42 and an increase in CDC42-dependent whether certain CI mechanisms are found only in dissociation inhibitor insulin release from these cells. So, it is possible that specialized cell types. A protein that inhibits the factors affecting CAV1 levels or that alter its intracellular The classification system (FIG. 3) that we have used removal of guanosine distribution might also alter the activation state of here is based on the roles of dynamin and several small diphosphate from the nucleotide-binding pocket of a CDC42 (and possibly other Rho GTPases), resulting in a GTPases, and is likely to evolve as we learn more about GTP-binding protein, thus compensatory change in the levels of CDC42-dependent this complex field. For example, the classification into keeping it in the inactive state. (or RhoA-dependent) endocytosis. dynamin-dependent versus dynamin-independent

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SNARE mechanisms has, in most cases, been assessed by over- Many exciting, yet challenging, areas of investigation Soluble N-ethylmaleimide expression of individual dynamin-1 or dynamin-2 lie ahead for those studying the CI pathways of internaliz- sensitive factor attachment mutants (for example, DYN2 Lys44Ala). However, other ation. For example, it will be important to obtain a better protein receptor. A family of methods are needed to assess the effects of the various understanding of the mechanisms for cargo selection in coiled-coil proteins that dynamin isoforms on CI internalization, such as siRNA- the absence of coats and adaptors as well as dissecting the operate in paired complexes (vesicle SNAREs and target mediated depletion and the use of more specific inhibi- mechanism(s) for vesicle fission that occur in the absence SNAREs) to mediate the fusion tors of dynamin90. Thus, the division of CI mechanisms of dynamin. Specifically regarding lipids, another major of donor and acceptor into dynamin-dependent and -independent mechanisms area that requires further study is the role(s) of choles- membranes, usually between a may need to be re-evaluated in the future. Use of the terol in each pathway and the molecular basis by which vesicle and an organelle or two vesicles. terminology CDC42-, RhoA- or ARF6-regulated may cholesterol influences the different internalization mecha- also change if more specific recruiting molecules and nisms. It is also not clear whether additional CI endocytic C-terminal binding protein effectors are uncovered along with the mechanisms for mechanisms remain to be discovered, nor is it known for 3/brefeldin A-ribosylated targeting them to different endocytic pathways. In addi- certain whether some bacteria or viruses induce their substrate tion to delineating the nature and number of CI mecha- own endocytic itinerary, rather than following an existing (CtBP3/BARS). A member of the CtBP transcription co-repressor nisms, it is important to resolve the confusion between trafficking pathway in the cell. Finally, a major challenge family of proteins. It is involved the definitions of existing CI pathways. Morphometric in the field will be to understand how all of the various in the dynamin-independent measurements similar to those undertaken by Griffiths91 endocytic mechanisms are regulated and integrated with fission of vesicles from the and, recently, by Parton and colleagues31 will be impor- each other and with other transport processes such as plasma membrane as well as from the Golgi apparatus. tant for determining the surface area of the membrane secretion. Undoubtedly, there will be a few surprises to that is endocytosed by each pathway. come as new studies of CI endocytosis are carried out.

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