[International Review of Cytology] Volume 220 || Endocytosis and the cytoskeleton

Endocytosis and the Cytoskeleton

Britta Qualmann and Michael M. Kessels Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-391 18 Magdeburg, Germany

In this review we describe the potential roles of the actin cytoskeleton in receptor-mediated endocytosis in mammalian cells and summarize the efforts of recent years in establishing a relationship between these two cellular functions. With molecules such as dynamin, syndapin, HIP1 R, Abpl, synaptojanin, N-WASP, intersectin, and cortactin a set of molecular links is now available and it is likely that their further characterization will reveal the basic principles of a functional interconnection between the membrane cytoskeleton and the vesicle-budding machinery. We will therefore discuss proteins involved in endocytic clathrin coat formation and accessory factors to control and regulate coated vesicle formation but we will also focus on actin cytoskeletal components such as the Arp2/3 complex, spectrin, profilin, and motor proteins involved in actin dynamics and organization. Additionally, we will discuss how phosphoinositides, such as Pl(4,5)P2, small GTPases thought to control the actin cytoskeleton, such as Rho, Rat, and Cdc42, or membrane trafficking, such as Rab GTPases and ARF proteins, and different kinases may participate in the functional connection of actin and endocytosis. We will compare the concepts and different molecular mechanisms involved in mammalian cells with yeast as well as with specialized cells, such as epithelial cells and neurons, because different model organisms often offer complementary advantages for further studies in this thriving field of current cell biological research.

KEY WORDS: Actin, Endocytosis, Dynamin, Syndapin, Abpl, HIP1 R, Myosin IV, N-WASP. 0 2002, Elsevier Science (USA).

Intematiomi Review of Cytology, Vol. 220 0074-7696/02 $35.00

93 Copyright 2002, Ekvier Science (USA). All rights resewed.

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I. Introduction

QUALMANNANDKESSELS

The last decades were marked by significant advances in our understanding of cellular uptake mechanisms, especially receptor-mediated endocytosis dependent on clathrin. More than 35 years ago, electron microscopic studies first suggested that invaginations of the plasma membrane, which are distinguished by electron-dense coats, are precursers of nascent vesicles used to take up material into cells. These vesicles were observed to carry characteristic bristle coats and it took more than a decade before the major component of these coats, clathrin, was discovered. Since the mid-1970s, however, the soccer ball-like structure of clathrin-coated vesicles has fascinated many researchers around the world. Extensive morphological, structural, and biochemical examinations of clathrin-coated pits and vesicles and their single components have generated many insights into the architecture of the endocytic machinery. In particular during the past 10 years, we have gained a better understanding of the mechanisms underlying the assembly of this huge protein machinery. We are also exploring how it is regulated, how it functions, and how it is finally disassembled after a new vesicle has been generated. Little, however, is known about how the complicated and dynamic array of proteins involved in receptor-mediated endocytosis crosstalks to the cortical cytoskeleton underlying and attached to the plasma membrane serving as a donor membrane for vesicle generation.

A few years ago, a handful of researchers started to search for molecular links between the actin cytoskeleton and the endocytosis machinery in mammalian cells. The first description and characterization of such molecules have spurred an ever- growing interest and many laboratories in the world are now following this research avenue, which will ultimately lead to a better understanding of how membrane budding and fission using a donor membrane intimately connected to supporting cytoskeletal structures is accomplished and how these cytoskeletal structures participate in this process. We will try to provide an overview of the theoretical concepts by which the actin cytoskeleton may be involved in receptor-mediated endocytosis, as briefly outlined before (Qualmann et al., 2000), and summarize the efforts to reveal connections of the two cellular functions and to address functions the actin cytoskeleton has been hypothesized to have within each step of receptor-mediated endocytosis.

II. Cortical Cytoskeleton and Endocytosis

A. Receptor-Mediated Endocytosis

Endocytosis is critical for a variety of functions in eukaryotic cells including receptor internalization, nutrient uptake, antigen presentation, and synaptic transmission. In this review, we will focus on the well-defined internalization pathway

ENDOCYTOSISANDTHECYTOSKELETON 95

mediated by clathrin-coated vesicles, also referred to as receptor-mediated endocytosis. Vesicle formation during receptor-mediated endocytosis involves complex structural and regulatory machinery and depends on two classes of proteins. First, structural components of the clathrin coat and second, a growing array of accessory proteins (Brodin et al., 2000; S&mid, 1997; Slepnev and De Camilli, 2000). According to current models, coated vesicle formation starts with the recruitment of the tetrameric adapter complex AP2 to the plasma membrane (Schmid, 1997) and is thought to involve AP2 interactions with the plasma membrane protein synaptotagmin (Zhang et al., 1994), tyrosine- and dileucine-based sorting signals in the cytoplasmic tails of cargo molecules (Jarousse and Kelly, 2000), and phosphoinositides (Gaidarov and Keen, 1999). AP2 complexes subsequently recruit clathrin to the plasma membrane.

Clathrin is composed of light and heavy chains, three of which form the basic building blocks of clathrin coats, the so-called triskelia. These three-legged structures can assemble into a lattice of pentagons and hexagons due to the relative flexibility of the angle formed by the legs of the triskelion (Owen and Luzio, 2000; Schmid, 1997). Genetic analyses demonstrated that clathrin-coat formation at the plasma membrane is furthermore modulated by the monomeric adaptor protein AP180, which binds to both clathrin and AP2 (Lindner and Ungewickell, 1992; Owen et al., 1999). It has been proposed that AP180 regulates vesicle size, because Drosophila and Caenorhabditis elegans mutants lacking AP180 proteins exhibited larger synaptic vesicles, which also displayed an increased size variability (Nonet et al., 1999; Zhang et al., 1998). Whereas in in vitro systems, clathrin and APs alone can form cages on liposomes and are thus sufficient to drive membrane de- formation (Takei et al., 1998), formation of constricted coated pits and the budding and detachment of vesicles in vivo require a variety of additional factors as has been demonstrated over the past years in a plethora of studies applying genetic methods or dominant-negative interference by overexpression or microinjection (Slepnev and De Camilli, 2000).

The next step in receptor-mediated endocytosis is the constriction of deeply invaginated coated pits and their pinching off from the membrane. This process involves the large GTPase dynamin (Hinshaw, 2000; Sever et al, 2000; also see below). The thus formed clathrin-coated vesicles are then detached from the plasma membrane, moved into the cytosol, and uncoated. Subsequently, they undergo further cellular sorting. In this review, we will not cover the different endosomal sorting pathways but will focus on the earlier steps of the endocytosis process and discuss the potential involvement of me actin cytoskeleton in these steps and the molecular players mediating these cytoskeletal functions in clathrin-dependent endocytosis.

B. Organization and Dynamics of Cortical Cytoskeleton

The actin molecule is one of the most abundant proteins in eukaryotic cells and shows an extremely high conservation throughout evolution. Actin consists of 375

96 QUALMANNANDKESSELS

amino acids and is a polypeptide chain folded into two large domains, each of which is again comprised of two subdomains numbering from 1 to 4 according to Kabsch et al. (1990). The two large domains create a hinged molecule with a deep cleft, which carries actin’s essential cofactors, an adenine nucleotide and a divalent metal ion-usually Mg*+--exhibiting interactions with either side of the cleft. Some lower eukaryotes, such as yeast, have only one actin gene encoding a single protein. All higher eukaryotes have several isoforms encoded by a family of actin genes; however, the basic properties of the at least six major types of actin in mammalian cells seem to be very similar.

Actin belongs to the very limited group of proteins, which can form extremely large homooligomeric structures; in the case of actin, these are about 7-nm-thick fibers. Negative staining electron microscopic experiments have shown that these structures can be viewed as two linear chains of actin monomers wound into a compact double helix (Fig. 1). The pitch of the double helix is very long: compared to the diameter of about 7 nm the helix makes a complete turn only once in every 71 nm, i.e., the chains appear to cross each other every 35.5 nm, at every half a turn (Fig. 1B). Like microtubles, actin filaments are polar structures, with a slow- growing minus or pointed end and a fast-growing plus or barbed end. The initial polymerization of monomers into filaments is not a kinetically favored process. The polymer needs to be stabilized by multiple contacts between adjacent subunits; actin dimers exhibit a relatively weak binding affinity to each other and fall apart easily. If a third monomer is bound by the dimer, the additional interactions make the entire group more stable so that now a core for rapid filament formation is presented to the monomers in solution and polymerization can proceed rapidly. This process is called nucleation (Fig. 1A).

Shortly after polymerization into filaments, actin molecules hydrolyze their bound ATP. This results in a rather closed, compact conformation of the actin monomer. Because this conformation is additionally supported by the manifold contacts to the other actin monomers within the polymer, adenosine diphosphate (ADP) cannot be exchanged for adenosine triphosphate (ATP) as long as the filament is not taken apart. The fact that interactions between ADP-containing monomers are weaker than between those carrying ATP adds to the polar properties of actin fibers. The critical concentration, i.e., the concentration of free actin molecules, which represents the threshold for further addition to the polymer, of the minus end is higher than that of the plus end still marked by ATP-actin. Thus, if both ends are exposed, polymerization proceeds until the concentration of free monomeric actin reaches a value minimally above the critical concentration of the plus end; this value, however, will be below the critical concentration of the minus end. This situation is referred to as steady state. At steady state, subunits will undergo net polymerization at the plus and net depolymerization at the minus end at an identical rate. Although there is a net flow of monomers from one end to the other, the length of the filament will remain unchanged. This biophysical concept is called treadmilling (Fig, 1B). It is a nonequilibrium phenomenon requiring a

ENDOCYTOSIS AND THE CYTOSKELETON 97

A. Nucleation

monomer dimer %

trimer

B. Treadmilling

minus plus

(pointed) (barbed)

end end

It 35.5 nm 3

a

--I) l f- 71 nm T-z

FIG. 1 The basic principles of actin dynamics. (A) The kinetically unfavored dimer and trimer formation creates a seed for subsequent rapid polymerization of monomers into filaments (nucleation). (B) Structure and dynamics of F-actin. Actin filaments are not static but undergo treadmilling at steady state, a nonequilibrium phenomenon requiring a constant supply of energy. ATP-bound actin monomers are in dark gray, ADP-bound actin molecules are in lighter gray. Three timepoints during treadmilling are depicted, note that the group of three monomers marked by asterisks “moves” from the plus to the minus end of the filament undergoing treadmilling.

constant supply of energy and serves as the basis for actin cytoskeleton dynamics (Theriot, 2000).

In cells, each step of the cycle including actin nucleation, polymerization, depolymerization, and nucleotide exchange for a new round of polymerization is regulated by a wealth of actin-binding proteins, which themselves are regulated


by many means (Pollard et al., 2000). One crucial machinery to ensure efficient nucleation of new filaments is the Arp2/3 complex. Two actin-related molecules embedded into this complex serve as seeds for rapid polymerization when the complex is activated (May, 2001; Welch, 1999). Currently, the list of proteins activating the Arp2/3 complex in in vitro experiments is growing rapidly and it will be extremely important to evaluate the relevance of such interactions in vivo and to

determine the cellular processes and cytoskeletal structures for which the different means of activation are used and how they are controlled. The best understood Arp2/3 complex activators are the multidomain proteins of the Wiskott-Aldrich Syndrome Protein (WASP) family, which are controlled by small GTPases of the Rho family. These GTPases associate with WAS family proteins only in their GTP state and can therefore act as molecular switches (Olazabal and Machesky, 2001; Takenawa and Miki, 2001).

Single actin filaments may also combine and form complex distinct F-actin superstructures within cells. Some of these superstructures are unique for certain cell types and others are abundant. All of these huge superstructures are not static but undergo dynamic changes in response to outer and inner clues, which change in time and space. Specialized actin-binding proteins help to organize these large superstructures. One of these specialized cytoskeletal arrays is the cortical actin cytoskeleton underlying the plasma membrane. It creates the environment to which all membrane-associated processes are subjected and gives rise to special cytoskeletal structures at the cell periphery, such as finger-like protrusions containing parallel bundles of F-actin, filopodia, and flat protrusions supported by highly crosslinked actin networks, in which fibers are oriented with their barbed ends toward the direction of cell movement or extension, lamellipodia.

Whereas in epithelial cells, the cortical network of the apical side is elaborate and relatively dense (also called the terminal web), in most other resting cells, the cytoskeletal cortex is composed of a gel-like, loose network of actin filaments and associated cytoskeletal components, which is thus relatively difficult to observe with F-actin-staining fluorophors. However, electron microscopic methods, for example those based on freeze-etching procedures, have revealed many aspects of the architecture of the cortical network underlying the membrane. Much information derives from research on mammalian erythrocyte “ghosts” obtained by hypotonic disruption of the cells (Terada et al., 1996). The studies revealed a loose network, a major component of which are spectrin molecules. Spectrin exists as about a lOO-nm-long, highly elastic tetramer, which contains binding sites for other major components of the cortical cytoskeleton, actin, ankyrin, and band 4.1 protein. Both of the latter proteins anchor the spectrin network to the membrane via associations with integral membrane proteins. An important aspect in band 4.1 protein function is that it stabilizes the association of spectrins to the short actin filaments integrated in the cortical net by increasing the actin-spectrin binding affinity by several orders of magnitude (Hitt and Luna, 1994). Today we know that this organization of the cortical cytoskeleton first revealed for erytbrocytes reflects

ENDOCYlOSlSANDTHECYTOSKELETON 99

a basic cellular principle and that the proteins first described are just members of larger superfamilies of related proteins.

Little is known about the connections of cortical structures to the endocytosis machinery. Early examinations suggested that membrane cytoskeletal proteins are excluded from sites of receptor-mediated endocytosis (Marshall et al., 1984). In line with the hypothesis that spectrins might represent barriers for endocytic uptake and that a reorganization of the spectrin cytoskeleton might be necessary for endocytic vesicle formation, Kamal et aZ. (1998) found that annexin VI-dependent coated pit budding was accompanied by a reduction of spectrin at the membrane. Because the inhibition was overcome after an hour of incubation, cells, however, seem not to rely on such an annexin VI-mediated mechanism. One spectrin- associated molecule, which could mediate the link to the endocytic machinery, may be ankyrin. The membrane-binding domain D4 of ankyrin has recently been shown to interact strongly with the clathrin heavy chain and excess amounts of peptides including parts of this region of ankyrin consistently blocked endocytosis in vivo (Michaely et al., 1999). From these examinations it seemed likely that there is some interplay of the classical spectrin network with the endocytic machinery. Flexibility and the dissolution of cortical barriers are likely to be important for membrane trafficking. In part, the elasticity of different spectrin-related proteins involved may be sufficient to provide this flexibility. The degree of crosslinking within the membrane cytoskeleton is furthermore regulated by phosphorylation of the major components reducing the binding affinities of these components, and thereby reducing the extent and rigidity of the cortical network (Hitt and Luna, 1994). In the following sections we will focus on functions of microfilament components within the cortical cytoskeleton in endocytosis, which may exceed the restrictive role proposed for spectrins.

C. Possible Roles of Actin Cytoskeleton

As outlined above, the endocytic process can be broken down into an ordered array of morphologically defined steps, invagination of the plasma membrane, formation of coated pits, sequestration of the coated pits formed, and finally detachment of the newly formed vesicle and movement of this new endocytic compartment away from the plasma membrane into the cytosol (Fig. 2). Involvement of the actin cytoskeleton in each of these steps is possible. Over the past few years research in this field has been extremely productive, so that now the fact that the actin cytoskeleton is somehow functionally involved in endocytosis in mammalian cells seems most likely. In the following we present theoretical concepts of how the actin cytoskeleton could be involved in the different steps of endocytosis (Qualmann et al., 2000) and experimental hints for such functions.

First, the cortical cytoskeleton underlying the plasma membrane may localize the endocytic machinery to certain domains of the membrane (Fig. 2). It may do

QUALMANNANDKESSELS

Initiation of Coat Assembly

Clathrin

B Api? 0 Other Coat Proteins

l Dynrmln

0 Acc@%oyPloteins

-Actin Filaments

Coat Propagation

Vesicle Budding

Vesicle Detachment, Movement and Uncoating

l Cyto8ksktalProteino

FIG. 2 The different steps of endocytic vesicle formation and the roles the actin cytoskeleton may play in these (see text). Depicted are spatial restriction and organization of the endocytic machinery at initiation sites of endocytosis and during coat formation, barrier effect during coated pit generation and budding, which may need to be overcome, force generation during vesicle formation and/or detachment from the donor membrane, and finally actin polymerization-driven movement of vesicles, which may still be coated or undergo uncoating.

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so by providing a physical barrier to free diffusion of the endocytic machinery or components thereof (trapping) or by directly anchoring parts of this complex machinery, which may then function as cornerstones for the build-up of functional endocytic complexes (anchoring). A higher organization of endocytic sites within the plasma membrane has been observed at the Drosophila neuromuscular junction, Roos and Kelly (1999) described a highly ordered array of endocytic and exocytic areas. Furthermore, sites of clustered integral membrane proteins to be taken up and sites of clathrin-coated pits were described as ordered arrays corresponding to the pattern of underlying stress fibers of cultured cells and these arrays were shown to depend on an intact actin cytoskeleton (Ash et al., 1977; Puszkin et al., 1982). In a recent study, Bennett et al., (2001) demonstrated that overexpression of a central clathrin domain, the so-called Hub domain, caused the loss of the former linear order of clathrin-coated pits and noted that in clathrin Hub-overexpressing cells, the protein HIPIR (see Section III), an actin-binding coat component, was dissociated from clathrin-coated pits. The concept that coated pit formation is initiated at specific and restricted membrane sites was also supported by studies determining the occurrence and lateral mobility of GFP-labeled clathrin-coated pits (Gaidarov et al., 1999). Coated pits formed and disappeared many times at the same site. Their limited lateral movement was found to increase upon treatment with the actin monomer sequestering drug latrunculin B suggesting the removal of some kind of cytoskeletal constraint (Gaidarov et al., 1999). It is thus attractive to speculate that sites of endocytosis are spatially defined by linkages to the actin cytoskeleton via multifunctional scaffolding proteins.

Second, the cortical actin cytoskeleton also influences membrane topology. It could thus theoretically be used to create deformations and invaginations of the plasma membrane. However, there is currently no evidence that actin functions are required for the formation of deep invaginations giving rise to vesicles.

The third obvious possibility is that the subcortical cytoskeleton may simply be a barrier for endocytic vesicle formation and movement. Such a rigid cortical net would need to be removed to allow internalization processes. As already men- tioned, the spectrin network may need to be dissolved to allow for endocytosis (see Section 1I.B). In the case of F-actin structures the removal of such a barrier could easily be achieved by a local increase in actin dynamics (Fig. 2). A spatial and temporal coupling of increased actin turnover to ligand-activated receptor signaling, which triggers endocytic uptake, would be an attractive molecular mechanism to restrict a barrier dissolution to sites of endocytosis. In support of a barrier effect it has been reported that a rigid cortical actin cytoskeleton has an inhibitory effect on membrane traffic (Trifaro and Vitale, 1993). Fujimoto et al. (2000) showed by beautiful quick-freeze deep-etch electron microscopic experiments that the imme- diate vicinity of clathrin-coated pits seems almost devoid of cortical actin fibers. However, although rather static F-actin fibers are preserved by this method, more dynamic actin structures are likely not observable. Several studies suggest that actin cannot play an exclusively negative role. At the apical surface of polarized


epithelial cells, endocytosis was inhibited upon actin depolymerization (Gottlieb et al., 1993), whereas stabilizing actin filaments with jasplakinolide had no effect. Jasplakinolide, a potent stabilizer of F-actin fibers, neither inhibited nor stimulated endocytosis at the apical surface, but it stimulated basolateral uptake (Shurety et al., 1998).

The fourth potential function of the actin cytoskeleton is based on the idea that newly created dynamic actin structures may be actively involved in the membrane fission event that liberates vesicles from the plasma membrane because both force and directionality can be generated by actin polymerization (Fig. 1). It is likely that both are prerequisites for membrane fission. This concept would theoretically be implemented best if it were possible to ignite a burst of actin polymerization at the neck of an invaginated clathrin-coated pit (Fig. 2). Actin polymerization would thus need to be spatially and temporally coordinated with vesicle formation. Premature actin polymerization would in contrast lead to an increased F-actin barrier beneath the plasma membrane and would thus be counterproductive. When actin polymerization is inhibited in A43 1 cells by the drug latrunculin A, receptor- mediated endocytosis is arrested at the stage of invaginated coated pits (Lamaze et al., 1997). Thus, it is indeed possible that actin polymerization at the neck provides the force to drive membrane fission or vesicle detachment. Recent actin- depletion studies (Fujimoto et al., 2000) argue against an essential role of the cortical actin cytoskeleton in the sealing of invaginated vesicles. Detachment of clathrin-coated vesicles from the plasma membrane, however, was not measurable in the permeabilized cell assay used. Also, until recently it remained unclear how the required delicate regulation and coordination of actin dynamics and endocytosis could be achieved. It seems attractive to hypothesize that the GTPase dynamin and its interaction partners may be involved in such coordination (see Section IV).

The fifth possible role of the actin cytoskeleton in endocytosis involves steps subsequent to the vesicle formation. The cytoskeleton and associated components may help drive vesicle detachment from the membrane and move detached vesicles through the viscous cytoplasm (Fig. 2). These later stages of the endocytic pathway could either again involve forces generated by actin polymerization, i.e., a propulsion mechanism, or actin-based motor proteins (Wu et al., 2000). Assuming an F-actin orientation similar to lamellipodia and filopodia, with the fast-growing barbed ends directed toward the plasma membrane, for the latter mechanisms pointed end-directed motor proteins would be required (see Section V). The unconventional myosin VI moves toward the pointed end of actin filaments (Wells et al., 1999). In support of the propulsion concept, endosomes, pinosomes, and clathrin-coated and secretory vesicles have recently been described as associated with actin comet tails in the cytoplasm (Frischknecht et al., 1999; Merrifield et al., 1999; Rozelle et al., 2000) as have endosomes and lysosomes in in vitro systems (Taunton et al., 2000). Such an actin-based propulsion mechanism has first been described for certain pathogens. For example, Listeriu monocytogenes propels itself through the cytoplasm of infected host cells by triggering asymmetric Arp2/3


complex actin polymerization. The machinery is kept going by a steady presentation of a bacterial Arp2/3 complex activator at the outer bacterial membrane and by a massive recruitment of host cell actin cytoskeletal components (Cossart, 2000).

It should be stressed that the functions of the actin cytoskeleton in the different steps of endocytosis introduced above are not mutually exclusive, nor need they be found in all cell types and in all forms of plasma membrane vesiculation. Further- more, it seems possible that participation of the actin cytoskeleton is not restricted to the plasma membrane but may also be a basic principle used in other clathrin- dependent membrane budding processes, such as Golgi transport processes. Also the membranes of the Golgi apparatus are tightly interconnected with surrounding cytoskeletal structures, which may, to some extent, resemble the composition of the actin cytoskeleton underlying the plasma membrane. In support of this hypothesis, we were recently able to show that one of the functional links between actin and endocytosis we identified also served as a part of the cytoskeleton associated with the Golgi and played a role in Golgi trafficking (Fucini et al., 2002).

III. Coat Components

One potential role of the cortical cytoskeleton in endocytosis might be the organization of the protein machinery at specific sites at the plasma membrane (see Sec- tion 1I.C). In line with this hypothesis, recent live imaging studies using GFP- tagged clathrin light chain have beautifully demonstrated the reoccurrence of clathrin-coated pits at defined sites of the cell cortex over time (Gaidarov et al., 1999). The reappearance and disappearance of these clathrin-positive puncta very likely correspond to the formation of clathrin-coated pits and pinching off followed by vesicle uncoating, respectively. So far, no direct association of either clathrin or adaptor proteins to cytoskeletal components have been observed. Recent identification and characterization of Huntingtin interacting protein l-related (HIPlR) provide an attractive candidate molecular for a physical link between cortical F-actin and clathrin-coated pits and vesicles. HIPlR (Seki et al., 1998) is a member of an evolutionary well-conserved protein family including the yeast Sla2/End4 protein (see Section IX) and HIPl, which is expressed predominantly in the brain and has been identified as an interaction partner for huntingtin (Kalchman et al., 1997; Wanker et al., 1997). The ubiquitously expressed HIPlR appears not to be able to bind to huntingtin (Chopra et aZ., 2000). HIP1 and HIPlR share about 50% sequence identity. At the N-terminus, both proteins contain a region very similar to the ENTH domain, a PI(4,5)P,-binding domain. Consistently, HIP1 can be recruited to PI(4,5)Pz-containing liposomes in vitro (Mishra et al., 2001). A stable association with clathrin-coated pits and vesicles has been extensively demonstrated for both HIPlR (Engqvist-Goldstein et al., 1999, 2001) and HIP1 (Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). Real-time analyses


performed by Engqvist-Goldstein et al. (2001) have revealed that the dynamic behavior of fluorescently labeled clathrin and HIPlR in clathrin-coated pits at the cell cortex appeared to be almost identical.

Interestingly, the association of the two related proteins HIP1 and HIPlR with other clathrin-coat components is mediated in a quite different manner. HIP1 displays canonical AP2 (DPF motifs)- and clathrin-binding (clathrin box) motifs within the central region of the molecule before the predicted coiled coil segment. Although HIP1 and HIPlR share about 50% sequence identity, this particular region of HIP1 is quite divergent from HIPlR. Several recent studies showed an in vitro association of HIP1 with both AP2 and clathrin applying pull-down assays (Metzler et al., 2001; Mishra et al., 2001; Waelter et al., 2001). The authors furthermore provide good evidence that the association of HIP1 with both AP2 and the terminal domain of the clathrin heavy chain is direct. In line with this interaction, expression of HIP1 fragments encompassing the clathrin- and AP2-binding region but not the coiled coil region or overexpressed full-length HIP1 interfered with transferrin endocytosis in nonneuronal cells (Metzler et al., 2001; Mishra et al., 2001). The endocytosis phenotype, however, appeared rather mild, because complete inhibition was observed in only less than a third of the cells (Mishra et al., 2001).

In contrast, HIPlR did not interact with the terminal domain of the clathrin heavy chain in vitro (Engqvist-Goldstein et aE., 2001). HIPlR binds directly to clathrin cages, but not to truncated cages lacking clathrin light chain. The interaction with clathrin, also shown by immunoprecipitation, is mediated by the predicted central coiled coil region of HIPlR. Furthermore, HIPlR induced clathrin cage assembly in vitro similar to AP2 and AP180. High levels of overexpression of full-length HIPlR on Cos-7 cells caused the redistribution of the clathrin light chain, but had no detectable consequence on clathrin heavy chain localization and transferrin internalization (Engqvist-Goldstein et aE., 2001). In complementary experiments, overexpression of the Hub fragment of clathrin, which comprises the C-terminal third of the clathrin heavy chain, caused the cytosolic redistribution of not only clathrin light chain but also HIPlR (Bennett et al., 2001). Additionally, clathrin Hub overexpression disrupted the spatial relationship between actin stress fibers and coated pits detected with anti-AP2 antibodies, which was observed in nontransfected cells. Actin filament assembly, however, seemed not to be altered (Bennett et al., 2001).

Pelleting experiments with pure clathrin cages, HIPlR, and F-actin further suggest that HIPlR can physically link F-actin and clathrin in vitro (Engqvist- Goldstein et al., 2001). This interaction with filamentous actin is most likely mediated via a talin-like domain at the C-terminus of HIPlR, which had been shown to bind to F-actin in vitro (Engqvist-Goldstein et al., 1999). Interestingly, according to initial data reported by Legendre-Guillemin et al. (2001) the talin-like domain in HIP1 appears to fail to bind to F-actin in vitro. Thus, a potential link of HIP1 to the cytoskeleton might be indirect and require heterodimer formation

ENDOCYTOSlSANDTHECYTOSKELETON 105

with HIPlR (Chopra et al., 2000). HIPlR might represent an important adaptor protein at the interface of phospholipids, clathrin, and the actin cytoskeleton (Fig. 3).

IV. Dynamin and Interacting Proteins

A. Dynamin

The large GTPase dynamin is critically required for receptor-mediated endocytosis, as first demonstrated in DrosophiZu melunogaster. The temperature-sensitive shibire fly harboring a mutation in the dynamin gene exhibits a rapid and reversible paralysis at nonpermissive temperatures. This phenotype was attributed to an inhibition of endocytic function because the nerve terminals of the shibire flies exhibited a depletion of the synaptic vesicle pool and an accumulation of coated pits at the plasma membrane at elevated temperatures (Koenig and Ikeda, 1983; Koenig et al., 1989). Subsequent examinations in particular studying the effects of dynamin mutants have established a generalized essential role for dynamin in clathrin-mediated endocytosis (Hinshaw, 2000). More recently, dominant-negative forms of dynamin have also been demonstrated to interfere with internalization processes from the plasma membrane other than receptor-mediated endocytosis including caveolae budding and phagocytosis (Gold et uZ., 1999; Henley et al., 1998; Oh et al., 1998).

The precise mechanism of action of the large GTPase in the budding and separation of clathrin-coated vesicles from the plasma membrane is still unclear, and numerous studies have promoted the suggestion of several models of dynamin function (Kirchbausen, 1999; McNiven, 1998; Sever et al., 2000; Yang and Cerione, 1999). Dynamin has the ability to self-assemble into helical structures either spontaneously (Hinshaw and Schmid, 1995) or around synaptosomal membranes or lipid vesicles forming membrane tubules (Sweitzer and Hinshaw, 1998; Takei et uZ., 1998). The addition of GTP to dynamin-decorated tubules caused their fragmentation into numerous small vesicles (Sweitzer and Hinshaw, 1998). These results led to the view that dynamin might be directly involved in the separation of endocytic vesicles from the plasma membrane acting as a mechanochemical enzyme. Alternatively, dynamin may act rather as regulatory GTPase-similar to small GTPases (see Section VII)-in endocytosis because overexpression of dynamin mutants that slowed GTP hydrolysis increased the rate of receptor-mediated endocytosis (Sever et al., 1999) suggesting that dynamin promotes endocytosis in its GTP form, for example, by recruiting and/or activating other factors that mediate fission.

Several recent reports furthermore support an association of dynamin with the actin cytoskeleton, which has been suggested by earlier studies describing the


thrin

\ I \ ‘i

Profilin II G-Actin

c 1


effects of dynamin mutants (Dan&e ef al., 1994) and dynamin depletion by applying antisense oligonucleotides (Torre et aZ., 1994) on cell morphology and cytoskeletal organizations. In both examinations, however, it could not be excluded that the observed defects might be secondary to a block of receptor-mediated endocytosis. Dynamin 2 colocalized with filamentous actin at podosome rosettes, adhesion sites between cells and the substratum, in cells transformed by Rous sar- coma virus (Ochoa et al., 2000), and dynamin mutants exhibited different effects on podosomes in vivo. Overexpression of a GFP-dynamin 2aa mutant corresponding to the Drosophila shibire mutant abolished podosomes. This effect is unlikely due to the endocytosis block caused by this construct, as another mutant, GFP-dynamin 2aa K44A, known to inhibit endocytosis, did not perturb podosome structures in the same system but instead only delayed actin turnover at podosomes (Ochoa et al., 2000). These results may suggest a direct or indirect functional link of dynamin to the actin cytoskeleton. Podosome rosettes were furthermore almost completely disrupted upon overexpression of a proline-rich domain (APRD) dynamin mutant (Lee and De Camilli, 2002). A localization of dynamin 2 to cellular sites characterized by F-actin accumulation, such as podosomes but also membrane ruffles and lamellipodia (Cao et aZ., 1998), might correlate with dynamin functions in processes other than its well-established role in receptor-mediated endocytosis.

In macrophages, dynamin 2 was detected at actin-rich phagocytic cups and the K44A mutant of dynamin 2 interfered with phagocytosis (Gold et al., 1999). Fur- ther support for a link between dynamin and the actin cytoskeleton is provided in recent studies by Lee and De Camilli (2002). The authors report the presence of dynamin in actin comet tail generated by Listeria monocytogenes infection or by overexpression of type I PIP kinase. Whether a significant proportion of the vesicles exhibiting actin tails in type I PIP kinase-transfected cells corresponds to organelles originating from clathrin-mediated endocytosis or rather corresponds to vesicles generated by fluid-phase endocytosis or Golgi budding (Lee and De Camilli, 2002; Rozelle et al., 2000) needs to be determined. The PRD of dynamin was sufficient and necessary for a targeting of dynamin to actin tails suggesting that the localization of dynamin to these actin-rich structures is mediated by dynamin-interacting partners such as profilin, cortactin, and Abpl (Fig. 3). These cytoskeletal components, which can bind to both actin and the dynamin PRD (see below), have been observed in actin comet tails (Kaksonen et al., 2000; Theriot et al., 1994; M. M. Kessels, unpublished observations). Overexpression of either K44A dynamin or APRD dynamin greatly reduced the number of tails induced

FIG. 3 Currently known protein interactions between components of the endocytosis machinery and those of the actin cytoskeleton (dark gray arrows) and their functional implications (dashed black arrows). Molecules, which seem to act at the functional interface of actin and endocytosis, are gathered in the central shaded area. For further possible crosstalk mechanisms between the cytoskeleton and endocytosis see the text. (See also color insert.)


in type I PIP kinase-transfected cells (Lee and De Camilli, 2002) indicating that an interplay between the PRD and further domains of the dynamin molecule is important for a role of the GTPase dynamin in the cytoskeletal context.

Dynamin interacts with a variety of proteins containing polyproline-binding sites or SH3 domains via its C-terminal proline-rich domain (Fig. 3). Interestingly, several of these dynamin-interacting proteins exhibit functional connections to the actin cytoskeleton, as described in the following. Dynamin-PRD protein interactions appear to be essential for the function of GTPase. First, the GTPase activity of dynamin in regulated by the binding of SH3 domains in vitro (Gout et al., 1993; Her- skovits et al., 1993). Second, the involvement of SH3 domain-containing proteins in the targeting of dynamin to clathrin-coated pits was suggested by mutational analyses (Shpetner et al., 1996). Furthermore, interfering with SH3/PRD interactions by overexpression or microinjection studies resulted in severe defects in clathrin-mediated endocytosis (Kessels et al., 2001; Owen et al., 1998; Qualmann and Kelly, 2000; Sengar et al., 1999; Shupliakov et al., 1997; Simpson et al., 1999; Wigge et al., 1997b) suggesting that dynamin interactions with proteins such as amphiphysin I (David et al., 1996) and II (Leprince et al., 1997; Ramjaun et al., 1997; Wigge et al., 1997a), endophilins (Micheva et al., 1997; Ringstad et al., 1997) DAP160/intersectin (Roos and Kelly, 1998; Yamabhai et al., 1998) syndapin I (Qualmann et al., 1999) and II (Qualmann and Kelly, 2000) as well as Abpl (Kessels et aE., 2001) and cortactin (McNiven et al., 2000) are essential for endocytic vesicle formation.

B. Interacting Proteins

1. Amphiphysin

As depicted in Fig. 3, the amphiphysin protein family has been shown to associate not only with dynamin (David et al., 1996) but also with the coat components AP2 (David et al., 1996; Wang et al., 1995) and clathrin (McMahon et al., 1997; Ramjaun et uE., 1997). The N-terminal region of amphiphysins furthermore mediates dimerization and an association with lipids (Ramjaun et al., 1999; Takei et al., 1999). Purified amphiphysin was shown to transform spherical liposomes into narrow tubules either alone or together with dynamin and to enhance the liposome-fragmenting activity of dynamin 1 in the presence of GTP (Takei et al., 1999). Amphiphysins might thus function as scaffolding molecules linking coat components, endocytic machinery, and lipid bilayers via regulated interactions (Slepnev et al., 1998). Interfering with amphiphysin’s protein interactions was shown to have a dominant-negative effect on clathrin-mediated endocytosis. Injection of peptides blocking the dynamin-amphiphysin interaction into the presynaptic compartment of giant lamprey axons inhibited synaptic vesicle recycling at the stage of deeply invaginated coated pits (Shupliakov et al., 1997).

ENDOCYlOSlSANDTHECYlOSKELETON 109

Overexpression of both amphiphysin I and II SH3 domains or full-length proteins domains in fibroblasts reduced receptor-mediated endocytosis of transferrin (Owen et al., 1998; Wigge et al., 1997a,b). Amphiphysin I antisense treatment of primary neuronal cultures, however, did not affect receptor-mediated and fluid- phase endocytosis (Mundigl et al., 1998). This result could reflect functional redun- dancy between the two amphiphysin isoforms I and II, which both occur in brain tissue.

The reduction in the level of amphiphysin I in primary hippocampal cultures, however, inhibited neurite outgrowth and caused the collapse of growth cones (Mundigl et al, 1998) implicating amphiphysin I in cytoskeletal regulation by unknown mechanisms. No direct interaction of amphiphysins with any cytoskeletal component has been unraveled so far. Amphiphysin I has been identified as a substrate for the cyclin-dependent kinase (cdk) 5 (Floyd et al., 2001; Rosales et al., 2000) and interacts with the regulatory subunit of the kinase, ~35, via its N-terminus (Floyd et al., 2001). In neuronal growth cones, amphiphysin I and p35 colocalize. Cdk5 and its activators p35 and p39 are essential for neuronal migration and neurite outgrowth (Humbert et al., 2000; Nikolic et al., 1996, 1998). Their effects on neuronal morphology might by mediated by Rho-family GTPases and their effecters. The neuronal p35/cdk5 kinase has been shown to associate with the Pakl kinase in an RacGTP-dependent manner resulting in hyperphos- phorylation of Pakl and down-regulation of Pakl activity (Nikolic et al., 1998; Rashid et al., 2001). Modulation of Pakl is in turn likely to influence the organization and dynamic of the actin cytoskeleton in neuronal growth cones and processes.

2. Syndapin and N-WASP

Syndapin I (synaptic, dynamin-associated protein I) has been identified in a screen for proteins interacting with the proline-rich domain of the large GTPase dynamin. The highly brain-enriched protein was named according to its localization in neurons and its first prominent interaction partner bound via its C-terminal SH3 domain (Qualmann et al., 1999). The protein shows considerable homology to the chicken protein focal adhesion protein FAP52 (Merilainen et al., 1997). The mouse ortholog of syndapin, termed PACSIN, has been reported as a hippocampal substrate for two protein kinases in vitro and displays decreased RNA levels after entorhinal-cortex lesion (Plomann et al., 1998). Syndapin I has also been shown to be a substrate for a yet to be identified kinase in rat brain activated by inositol hexakisphosphate, an abundant inositol metabolite of unknown function (Hilton et al., 2001). Today it is clear that three closely related isoforms of syndapins exist in higher vertebrates, while lower eukaryotes such as yeast seem to lack syndapins entirely.

The syndapin functions characterized best are those mediated by its SH3 domain. Biochemical analyses revealed that this highly conserved domain mediates


associations with three brain-specific proteins implicated in synaptic vesicle trafficking: dynamin I, synaptojanin, and synapsin I (Qualmann et al., 1999; Fig. 3). Coimmunoprecipitations of dynamin I and syndapin I as well as colocalization of the two proteins at vesicular structures in primary neurons indicated an association in vivo and a role for syndapin in synaptic vesicle endocytosis. Fur- thermore, the dynamin-associated protein syndapin I interacts with the neural Wiskott-Aldrich syndrome protein (N-WASP) (Qualmann et al., 1999). As outlined in Section II.B, N-WASP is a potent stimulator of the Arp2/3 complex-based actin polymerization machinery (Fig. 3). Taken together, syndapin interacts with three proteins implicated in cytoskeletal reorganization: N-WASP, the synaptic vesicle protein synapsin I, which can bundle actin filaments, and synaptojanin, which is thought to regulate actin dynamics via phosphatidylinositols (Fig. 3). Also the recently discovered association with the Ras guanine nucleotide exchange factor (GEF) mSos could represent an indirect functional connection to the actin cytoskeleton. However, this interaction may likely be restricted to a not yet understood syndapin function in MAP kinase signaling (Wasiak et al., 2001).

The identification and characterization of the ubiquitously expressed syndapin II isoform (Qualmann and Kelly, 2000) suggested that a molecular linkage between endocytosis and actin organization by syndapins is not restricted to the recycling of synaptic vesicles in the brain but represents a more general mechanism in a variety of mammalian cells. Functional analyses in vivo have substantiated this view (Qualmann et al., 2000). Dominant-negative experiments have demonstrated the physiological relevance of syndapin-protein interactions on receptor-mediated endocytosis: A surplus of the SH3 domain of both syndapin I and II inhibited clathrin- mediated endocytosis both in heterologous permeabilized cell assays (Simpson et al., 1999) and in vivo (Qualmann and Kelly, 2000). With the use of permeabilized cell assays it was also possible to assign this block of membrane transport to late endocytic steps corresponding to the transition from clathrin-coated invaginated pits to closed endocytic vesicles, a process controlled by dynamin (Simpson et al., 1999). These data are in good agreement with the syndapin/dynamin association demonstrated in vitro and in vivo and the colocalization of both proteins in neuronal cells (Qualmann et al., 1999).

The functional connection of syndapins to the actin cytoskeleton was also veri- fied by in vivo studies. Syndapin overexpression in both HeLa and 3T3 fibroblast cells resulted in the induction of numerous filopodia, finger-like protrusions containing bundled actin filaments, all over the cell surface (Qualmann and Kelly, 2000). The observed localization of syndapin to areas of high actin turnover such as lamellipodia and the very tips of filopodia is consistent with a role of syndapins in actin dynamics. The use of an N-WASP-derived protein tool permitted the further dissection of the cytoskeletal role of syndapins; cooverexpression of a C- terminal cytosolic N-WASP fragment, which mislocalizes the Arp2/3 complex, completely suppressed the syndapin-triggered filopodia induction (Qualmann and


Kelly, 2000). The syndapin-induced cortical actin reorganization therefore appeared to be mediated by the Arp2/3 complex at the cell periphery. This is in agreement with a colocalization of overexpressed syndapins and the Arp2/3 complex at the cell cortex (Qualmann and Kelly, 2000).

Although the biochemical and functional characterization of syndapin proteins strongly suggests that they have both endocytic and cytoskeletal functions and thus represent functional links at the interface of endocytosis and actin dynamics in mammalian cells, the cooperativity of these two functions and their importance for receptor-mediated endocytosis still have to be shown. As a first step, we recently examined whether the syndapin/N-WASP interaction plays an important role not only in the organization of the cortical actin cytoskeleton but also in endocytosis by analyzing the different domains of N-WASP for overexpression phenotypes in receptor-mediated endocytosis. Overexpression of all N-WASP constructs encompassing the proline-rich domain were found to block transferrin endocytosis whereas those containing the N-terminus, the actin-binding domain, and the Arp2/3 complex-binding and -activating domain caused no inhibition of ligand uptake (Kessels and Qualmann, unpublished). Mapping the syndapin-binding interface on the N-WASP molecule showed a complete overlap of fragments binding to syndapin and inducing an endocytosis block. In line with this, syndapin cooverexpression fully rescued the N-WASP proline-rich domain-dependent phenotype (Kessels and Qualmann, unpublished). These dominant-negative experiments also establish an essential role for syndapins in the internalization process. An involvement of N-WASP in receptor-mediated endocytosis is supported by the analysis of mice deficient for the blood cell-specific WAS protein. Lymphocytes from these mice exhibited defects both in actin polymerization and in T cell receptor endocytosis (Zhang et al., 1999). Thus, WAS family proteins may not exclusively act as components of actin cytoskeletal structures but have cellular functions that go beyond this.

The exact molecular mechanism of syndapin/N-WASP functions in endocytosis is still not fully resolved, but it is attractive to hypothesize that syndapins function to link endocytic vesicle formation with actin cytoskeletal functions supporting this process. Syndapins would be ideal candidates for such a role as they interact with the GTPase dynamin controlling the fission reaction. Since dynamins have been shown to form collars at the neck of constricted coated pits in synap- tosomes incubated with GTPT/S, associated syndapin molecules might also be at the place where force generation will be required for vesicle fission and/or detachment (Fig. 2). Actin polymerization is a very powerful source of spatially restricted force generation (Theriot, 2000). N-WASP would be an ideal candidate to ignite a burst of actin polymerization because it requires no preexisting F-actin structures to start actin nucleation but recruits monomeric actin molecules. Once actin polymerization is ignited, other proteins such as cortactin and perhaps also Abpl (see below) may create the more elaborately crosslinked F-actin structures optimal for transmission of the forces generated by polymerization.

112

3. Intersectin

QUALMANNANDKESSELS

Intersectins are large multidomain proteins capable of undergoing a variety of protein-protein interactions and were thus suggested to act as scaffolding or adapter molecules (Fig. 3). Intersectins, except for the first identified Drosophila ortholog DAP160 (Roos and Kelly, 1998), which contains only four SH3 domains, contain five consecutive SH3 domains in their C-terminal part mediating interactions with dynamin, synaptojanin, and mSos, a guanine nucleotide exchange factor for the small GTPase Ras (Roos and Kelly, 1998; Sengar et al., 1999; Tong et al., 2000). Further interactions with proteins of both the endocytic and exocytic machinery including SNAP-25 and the coat components Eps 15 and epsins are mediated via the N-terminal EH domains and the central predicted coiled coil domain of intersectins (Hussain et al., 1999; Okamoto et aE., 1999; Sengar et al., 1999). Several of the intersectin SH3 domains interfered with transferrin internalization in a permeabilized cell system, interestingly at different stages. Earlier stages in the endocytic process leading to the formation of constricted clathrin-coated pits were selectively blocked by the SH3A domain (Simpson et al., 1999) potentially reflecting the interaction with the Ras-GEF mSos (Tong et al., 2000). Overexpression of the full-length protein in whole cells also had a dominant-negative effect on transferrin endocytosis (Sengar et al., 1999); this could also reflect an excess of the inhibitory SH3 domains.

The long, brain-specific splice variant of intersectin furthermore contains a DH domain that catalyzes guanine-nucleotide exchange on the Rho-type GTPase Cdc42. Microinjection of DH domain-containing intersectin-l constructs in 3T3 fibroblast cells resulted in a cortical actin phenotype similar to that obtained by microinjection of dominant-active Cdc42 (Hussain et al., 2001). Similar to other SH3 domain-containing proteins (Miki et al., 1996; Qualmann et al., 1999), intersectin binds directly to N-WASP via its SH3 domains. The intersectin interaction with the N-WASP PRD domain seems sufficient to increase the guanine nucleotide exchange activity of full-length intersectin-l toward Cdc42 in vitro. In heterologous immunoprecipitations the intersectin DH domain was shown to bind both wild- type Cdc42 and the dominant-negative Cdc42N17 but not the dominant-active Cdc42L61 (Hussain et aZ., 2001).

4. Profilin

Dynamin furthermore interacts with proteins that directly associate with G-actin and F-actin, respectively, profilin, as well as Abpl and cortactin (see below). Profilins are small proteins of 12-14 kDa that bind to monomeric actin and stimulate the ATP exchange on actin (Mockrin and Kom, 1980) and thus act as actin polymerization promoters in vivo (Pantaloni and Carlier, 1993). Be- cause actin is a rather slow ATPase, the catalysis of the ADP/ATP exchange on actin represents an important mechanism of regulating actin dynamics. Profilin’s


nucleotide-exchange activity is regulated by phosphoinositides. Binding of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] releases G-actin from profilin (Lassing and Lindberg, 1985). Besides actin, profilin binds polyproline stretches of proteins of the VASP/MENA/diaphanous family (Gertler et al., 1996; Reinhard et al., 1995; Watanabe et al., 1997) and the Arp2/3 complex (Machesky et al., 1994), all important mediator proteins of F-actin filament polymerization. In mammals, two profilin isoforms have been described, the ubiquitously expressed profilin I and the brain-enriched isoform profilin II, which is also detectable in muscle and weakly in kidney and uterus (Honore et al., 1993; Kwiatkowski and Bruns, 1988). Profilin II was recently shown to be able to interact with dynamin and synapsins by affinity chromatography of high-speed brain supematants, suggesting a role for profilin II in membrane trafficking events in the brain (Witke et al., 1998). Schmidt and Huttner (1998) reported that the addition of recombinant profilins I and II to diluted cytosol stimulated the biogenesis of synaptic-like microvesicles in a perforated PC12 cell system (Schmidt et al., 1999), but it has to be determined whether profilins represent a necessary component for the formation of synaptic-like microvesicles. A potential role for profilins might be to promote the assembly of F-actin at sites of endocytosis by increasing the local concentration of ATP-bound G-actin.

5. Abpl

Interestingly, the GTPase dynamin has been recently shown to be both physically and functionally interconnected with an F-actin-binding protein (Kessels et al., 2001) mAbp1 (Fig. 3). The mammalian homologue of yeast actin-binding protein 1, mAbp1 (Kessels et al., 2000; Lappalainen et al., 1998), which carries the screen name SH3P7 (Sparks et uZ., 1996), was also identified as an Src substrate (Larbolette et al., 1999; Lock et al., 1998). It binds specifically to F-actin in vitro and in vivo using two independent F-actin-binding domains (Kessels et al., 2000) an N-terminal actin depolymerizing factor-homology (ADF-H) region (Lappalainen et al., 1998) and a central helical domain. In vivo, mammalian Abpl is specifically recruited to dynamic actin structures. This manifests in a strong accumulation at the leading edge of moving and of spreading cells, whereas in resting cells, Abpl shows a more uniformly distributed, punctate immunostain- ing pattern. The shift to the cell periphery is dependent on actin polymerization, as demonstrated by latrunculin A treatment, and coincides with an accumulation of the Arp2/3 complex. This relocation is controlled by several signal transduction pathways, such as activation of growth factor receptors or PKC activation by phorbol esters. It can also be induced by expression of a dominant-active mutant version of Racl (Kessels et al., 2000). A role for Abpl in actin dynamics is also supported by biochemical data recently generated in the S. cerevisiue model system. Yeast Abpl was copurified with the Arp2/3 complex and demonstrated to be able to activate it in vitro (Goode et al., 2001). The two acidic sequence


motifs in yeast Abpl shown to be required for Arp2/3 complex activation by mutational analysis (Goode et&., 2001) are, however, not conservedinthe mammalian protein.

The interaction of the C-terminal SH3 domain of mammalian Abpl with dynamin, synapsin 1, and synaptojanin 1 in vitro suggested that Abpl may play a role in membrane trafficking. Coimmunoprecipitations from brain extracts demonstrated that Abpl and dynamin associate in vivo (Kessels et al., 2001). In line with this, overexpression of the SH3 domain led to a potent block in receptor- mediated endocytosis of transferrin. In contrast, neither overexpression of the F-actin-binding N-terminal half nor of full-length Abpl interfered with transferrin uptake. Interestingly, the endocytosis block caused by the SH3 domain could be rescued by combining this domain with one--or better both-actin-binding mod- ules of the Abpl protein (Kessels et al., 2001) suggesting that Abpl may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions (Fig. 3). This dual role for Abpl is furthermore supported by the fact that dynamin and Abpl colocalize at cortical spots after growth factor stimulation, which are also immunopositive for other proteins of the endocytic machinery such as AP2, eps15, and HIPlR. In contrast, other cellular sites with a high F-actin concentration, which have been described as containing dynamin such as podosomes, the phagocytic cup, and the lamellipodium (see Section IV.A), are devoid of clathrin coat components. Considering these properties it can be hypothesized that Abpl plays a role in the organization of the endocytic machinery at the cell cortex and/or coordinates endocytic and cytoskeletal function in a timely or spatial manner. Be- cause the actin-binding protein interacts with dynamin, which is asymmetrically distributed during the fission reaction of the newly formed vesicle, it could participate in the proposed formation of actin tails on newly budded vesicles (Fig. 2). The observation that Abpl is recruited to and enriched in the actin comet tails of List&a monocytogenes (M. M. Kessels, unpublished) supports this hypothesis. Assuming a molecular mechanism of Arp2/3 complex activation similar to the yeast protein, which has been shown to require the ADF-H domain and thus most likely F-actin binding for this purpose (Goode et al., 2001) a potential role for Abpl in tail formation is unlikely to include the ignition of actin tails corresponding to de novo actin polymerization but may rather be the formation of branched actin structures that may be required for optimal force transmission. More attractive candidates for the first stages of actin tail ignition are WASP proteins, which bind to actin monomers (see above) and have been localized to the interface of vesicles and actin tails in Xenopus extracts (Taunton et al., 2000).

6. Cortactin

Most of the dynamin interaction partners described above including amphiphysin, syndapin, intersectin, and profilin, exist in several isoforms in higher organisms.

ENDOCYTOSISANDTHECYTOSKELETON

In contrast, Abpl seems to be encoded by a single gene in all species analyzed thus far; however, it shares some intriguing biochemical and functional properties with another F-actin-binding protein, cortactin (Olazabal and Machesky, 2001). Cortactin is a prominent substrate of nomeceptor protein tyrosine kinases such as src (Wu et al., 1991) and is overexpressed in several types of cancer (Schuuring et al., 1993; Wu et al., 1991). The direct binding to actin filaments is mediated through a series of six and a half 37-amino acid tandem repeats (Wu and Parsons, 1993) and, like Abpl, cortactin localizes to lamellipodia upon Rat activation (Wu et al., 1991). Furthermore, cortactin has been shown to bind directly to the Arp2/3 complex (Uruno et al., 2001; Weed et al., 2000) and to modestly activate Arp2/3 complex-induced actin filament formation (Uruno etal., 2001; Weaver etaE., 2001). As for yeast Abpl, this ability of cortactin to stimulate the Arp2/3 complex was dependent on F-actin binding (Uruno et al., 2001; Weaver et al., 2001). Consis- tently, cortactin was demonstrated to inhibit the debranching of filament networks (Weaver et al., 2001).

Cortactin also appears to be involved in endocytosis. First, it was shown to colocalize with actin and Arp2/3 on endosomal vesicles in the cytoplasm (Kaksonen et al, 2000). This localization might be mediated through a direct interaction of the cortactin SH3 domain with dynamin (McNiven et al, 2000; Fig. 3). Second, microinjection of antibodies against specific domains of cortactin resulted in both actin reorganization and a reduction of transferrin uptake but not an inhibition of fluid-phase marker endocytosis (M. A. McNiven, personal communication). Considering that cortactin requires F-actin binding for Arp2/3 activation and thus preferentially induces branched actin filament networks, the molecular details of an involvement in membrane traft%cking events may be related to those of Abpl and will have to be examined further.

V. Motor Proteins

Motor proteins perform directional movement along polarized tracks such as polymerized actin filaments. The only known type of actin-based motor so far is the myosins, a large family of proteins comprising 15 or more classes. As outlined in Section ILB, actin filaments have an inherent polarity with a fast-growing plus end and a minus end. Individual myosin molecules convert energy from ATP hydrolysis into unidirectional movements toward only one specific end of actin filaments. Near the cell cortex, actin filaments are generally oriented in a way that their plus ends face the plasma membrane, in particular in cortical specializations such as filopodia, microvilli, and stereocilia. Myosin-based cargo transport in cells occurs preferentially at the cell cortex where actin filament arrays with uniform polarity are located. For a long time, only plus end-directed myosin motility was

116 QUALMANN AND KESSELS

known. Such myosins can mediate the transport of attached cargo toward the cell surface, such as secretory vesicles in the exocytic pathway (Wu et al., 2000).

In contrast, myosin-based vesicle transport in the endocytic pathway would require a myosin molecule moving in the opposite direction. Only a minus-end- directed myosin would allow the movement of organelles and particles toward the center of the cell. Interestingly, the abundant myosin VI has recently been demonstrated to move in a direction opposite to all other myosins analyzed so far, toward the minus end of actin filaments (Wells et al., 1999). Myosin VI is involved in cell motility and shape change events in a variety of organisms and cell types and plays an important role in hearing processes in mice and fertility in flies (Cramer, 2000; Rodriguez and Cheney, 2000). Recently, a myosin VI splice variant with a large insert in the tail domain has been implicated in clathrin- mediated endocytosis (Buss et al., 2001). This specific myosin VI isoform, which is preferentially expressed in polarized cells (see Section VIII.A), colocalizes with clathrin-coated vesicles at the apical domain and can be coimmunoprecipitated from cytosolic extracts with antibodies against clathrin and AP2. A GFP construct of the whole myosin VI tail containing the large insert, which localizes to clathrin-coated pits and vesicles, had a dominant negative effect on transferrin internalization in nonpolarized cells (Buss et al., 2001) suggesting a role for this motor protein in clathrin-mediated endocytosis. Involvement of myosin VI in moving organelles toward the minus end of actin filaments still needs to be shown. To transport organelles, myosin VI would need to be a processive mechanoenzyme as has been demonstrated for myosin V (Mehta et al., 1999). As a prerequisite for long-range organelle movement by a single molecule, myosin V was shown to move in a processive manner taking large steps approximately corresponding to the pseudorepeat within the actin filament structure (Mehta et al., 1999). Al- ternatively, such a minus-end myosin-if attached to a stationary support-might mediate outward movement of actin filaments. Myosin oligomers when attached to neighboring actin filament bundles also function in sliding of actin filaments. Whereas a plus-end directed myosin dimer would pull a second filament toward a filament associated with the membrane with its plus end until the second filament is in contact with the membrane, a minus end-directed myosin would push the second filament back into the cytosol resulting in a thinning of local actin filament networks. Although this might facilitate vesicle budding and transport within the cortical actin network underlying the plasma membrane, it has to be stressed that such filament sliding can occur only within actin structures with antiparallel orientation of filaments.

Genetic evidence argues against a fundamental role of myosin VI in the endocytic trafficking process. The only phenotypic abnormalities in mice with a null mutation in myosin VI (Snell’s Waltzer) observed were defects in hearing and balancing (Avraham et al., 1995). Given the fact that no other minus end-directed myosins have been identified, functional compensation by other myosins seems rather unlikely.

ENDOCYTOSlSANOTHECYlOSKELETON

VI. Phospholipids and Their Metabolizing Enzymes

117

Signaling molecules might play a pivotal role in the spatial and temporal regulation of the different stages of the endocytic process and also in their coordination with accompanying actin cytoskeletal reorganizations. Thus, proteins involved in the generation or metabolism of regulatory components, such as phosphoinositides, which affect signaling processes, membrane trafficking events, and the actin cytoskeleton (Martin, 1998; Martin, 2001; Simonsen et al., 2001), represent further possible candidate molecules that might act at the interface of endocytosis and actin dynamics. The cellular functions of phosphatidylinositol 4,5-bisphosphate [PI(4,5)Pz] are mediated through proteins required for membrane trafficking and cytoskeletal organization that contain PI(4,5)PT-binding domains, including the well-characterized pleckstrin homology (PH) domain. Recent studies demonstrated that PI(4,5)Pz is nonuniformly distributed on membranes. Localization studies using antibodies and GFP-labeled PI(4,5)Pz-binding domains revealed an organization in raft-like structures (Martin, 2001; Simonsen et al., 2001) suggesting that PI(4,5)Pz establishes discrete, defined sites for vesicular trafficking, membrane movement, and actin cytoskeletal assembly. PI(4,5)Pz was reported to be necessary for endocytosis in permeabilized cells (Jost et al., 1998) and several proteins implicated in different stages of clathrin-mediated endocytosis (coat assembly, membrane invagination, fission and vesicle uncoating; Fig. 2) have been shown to bind PI(4,5)Pz indicating an essential role for PI(4,5)P* in the sequential recruitment of clathrin coat components and accessory proteins to endocytic sites (Gaidarov and Keen, 1999).

The initial stages of clathrin-mediated endocytosis are critically dependent on a PI(4,5)Pz-mediated recruitment of major clathrin coat and coat-associated proteins including AP2 (Gaidarov and Keen, 1999), AP180, the ubiquitously expressed AP180 homologue CALM, and epsin. The latter three proteins bind PI(4,5)Pz via epsin amino-terminal homology (ENTH) domains (Ford et al., 2001; Itoh et al., 2001). The important role of PI(4,5)Pz in coat recruitment and clathrin- coated pit formation was supported by two recent studies. Overexpression of an epsin mutant unable to bind PI(4,5)P2 inhibited epidermal growth factor intemal- ization (Itoh et al., 2001). The formation of a clathrin lattice was reconstituted on PI(4,5)P2-containing lipid bilayers in the presence of clathrin and wild-type AP180, but not AP180 harboring mutations in the ENTH domain (Ford et al., 2001).

Subsequent to the formation of clathrin-coated membrane invaginations, fission at the neck of the clathrin-coated pits liberates vesicles from the plasma membrane (Fig. 2). These endocytic stages require the activity of the large GTPase dynamin (see Section IVA). Membrane recruitment and function of dynamin require PI(4,5)Pz binding via its PH domain. The dynamin PH domain has been shown to be crucial for membrane localization (Salim et al., 1996) and receptor-mediated


endocytosis (Achiriloaie et al., 1999; Lee et al., 1999; Vallis et al., 1999). PI(4,5)P2 binding stimulates dynamin’s GTPase activity (Salim et al., 1996).

After the formation and sequestration of a clathrin-coated vesicle, the hydrolysis of PI(4,5)Pz might be involved in the uncoating of the newly formed vesicle by lowering the membrane association of AP2, AP180, and additional PI(4,5)P2- binding coat and accessory proteins. Several lines of evidence suggest a role for the polyphosphoinositide phosphatase synaptojanin 1 (McPherson et al., 1996) in the uncoating of endocytic-coated vesicles (Fig. 3). Synaptojanin 1 contains SAC1 homology and 5’-phosphatase catalytic regions and catabolizes PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate (PIPs) (Woscholski et al., 1997). The interaction of synaptojanin with several accessory proteins implicated in clathrin-mediated endocytosis via its proline-rich domain including amphiphysin (David et al., 1996) endophilin (de Heuvel et al., 1997; Ringstad et al., 1997), DAP160/intersectin (Roos and Kelly, 1998), syndapin (Qualmann etal., 1999) and Epsl5 (Haffner et al., 1997) has been reported (Fig. 3). In neurons of synaptojanin l-deficient mice, clathrin-coated vesicles accumulate in nerve endings (Cremona et al., 1999). In hippocampal slices of these mutant animals, enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery was observed. These electrophysiological examinations thus also suggest a reduced efficiency of synaptic vesicle recycling. In C. elegans, mutations in synaptojanin led to multiple defects in synaptic vesicle formation and uncoating (Harris et al., 2000). The mutant animals displayed defects in the budding of synaptic vesicles from the plasma membrane, the uncoating of the vesicles, recovery of the vesicles from endosomes, and additionally in the tethering of the vesicles to the cytoskeleton. The latter was suggested by an abnormal organization of the vesicles in nerve terminals. Most vesicles in mutant animals were distant from the active zone and organized in a linear string-of-pearl configuration (Harris et al., 2000). An accumulation of free clathrin-coated vesicles was furthermore observed in lamprey nerve terminals after injection of a peptide, which interferes with the interaction of synaptojanin and endophilin (Gad et al., 2000).

Phosphoinosites fulfill a pleiotropic role in cells. PI(4,5)P2 represents an important signaling molecule not only in the regulation of intracellular membrane trafficking events, but also in the regulation of actin cytoskeletal rearrangements via binding to a variety of actin-regulatory proteins, influencing the degree of actin filament polymerization (see below and Fig. 3). Thus, synaptojanin’s cellular function might not be limited to a role in endocytosis. In line with this, synaptojanin has been shown to be able to hydrolyze PI(4,5)Pz bound to actin-regulatory proteins such as profilin, cofilin, and czl-actinin in vitro (Sakisaka et al., 1997). Furthermore, a rearrangement of actin stress fibers was observed upon overexpression of wild-type synaptojanin, but not of a phosphatase-negative mutant in COS-7 cells (Sakisaka et al., 1997). Synaptojanin might thus coordinate both the uncoating of newly formed endocytic vesicles and local actin turnover, e.g., re- moving physical barriers for the inward movement of now uncoated endocytic vesicles.


An additional mechanism by which synaptojanin function might be linked to the actin cytoskeleton was suggested when the ubiquitously expressed synaptojanin isoform 2 was identified as a binding partner for the small GTPase Racl (Malecz et al., 2000). The translocation of synaptojanin 2 to the plasma membrane either by expressing constitutively active Racl or an engineered construct comprising a membrane targeting sequence and the central phosphatase domain of synaptojanin 2 inhibited receptor-mediated uptake of both transferrin and epider- ma1 growth factor receptors.

A potential countetplayer for synaptojanin 1 has recently been described at presynaptic nerve endings: Phosphatidylinositol phosphate kinase type Iy has been reported to be the major PI(4,5)P2-synthesizing enzyme in the synapse and to antagonize the effects of synaptojanin 1 in the recruitment of clathrin coats to membranes in a cell-free system (Wenk et al., 2001).

Actin cytoskeletal rearrangements depend on the tight temporal and spatial regulation of actin filament polymerization, organization, stability, and degrada- tion. In cells, this is accomplished via the regulation of the activity of a large array of actin-associated proteins, which nucleate, polymerize, cap, cross-link, sever, and/or depolymerize actin filaments or monomers, respectively. Phospho- inosites, in particular PI(4,5)Pz, seem to be an important signal originating from membranes, which regulates the cortical actin cytoskeleton. PI(4,5)Pz has been shown to influence the function of a variety of actin-associated proteins in vitro including profilin (see Section IV.B.4) and WAS proteins (see below), thereby favoring a “polymerized state” (Sechi and Wehland, 2000). A variety of recent studies point to an intimate association between PI(4,5)Pz levels at the plasma membrane and the coordination of actin organization and membrane trafficking. A change in intracellular PI(4,5)Pz levels, for example by overexpression of synaptojanin (Sakisaka et al., 1997; see above), alters the organization of the cortical actin cytoskeleton and actin stress fibers. An interconnection between the plasma membrane and the cortical cytoskeleton was furthermore strengthened by recent studies using optical tweezers tether force measurements (Raucher et al., 2000) showing that plasma membrane PI(4,5)Pz acts as a second messenger that regulates cytoskeleton-plasma membrane adhesion. A reduction in PI(4,5)P2 levels either by sequestration or hydrolysis of PI(4,5)Pz reduced the energy required to displace the plasmalemma from the underlying cytoskeleton (Raucher et al., 2000) and might thus facilitate cortical membrane trafficking. The data suggest that plasma membrane PI(4,5)Pz concentration controls dynamic membrane functions by regulating the adhesion force between the actin-based cytoskeleton and the plasma membrane. This might be achieved either by directly altering interactions between PI(4,5)Pz and cytoskeletal anchoring proteins in membrane rafts or by regulating the degree of actin polymerization via proteins such as gelsolin, profilin, and cofilin.

PI(4,5)Pz-enriched microdomains both on the plasma membrane and intracellular vesicles may spatially regulate a coordinated action of the machinery for membrane fission on one hand and cytoskeletal components on the other hand


(Fig. 3). Studies using Xenopus egg extracts show a corequirement for phosphoinositides and Cdc42 to promote actin assembly in this cell-free system capable of inducing actin comet tails around endogenous membrane vesicles or exogenous lipid vesicles prepared from purified phospholipids containing PI(4,5)P2 or PIP3 (Ma et al., 1998). Recent studies have shown an important role for PI(4,5)P2 in regulating actin polymerization-driven vesicle movement. Overexpression ofphos- phatidylinositol4-phosphate [PI(4)P] 5-kinase markedly enhanced actin comet tail formation on a variety of intracellular vesicles and resulted in the propulsion of par- ticularly Golgi-derived vesicles, which were enriched for sphingolipid-cholesterol rafts and contained PI(4)P 5-kinase, through the cytosol (Rozelle et aZ., 2000). These experiments suggest that local synthesis of PIPS in membrane raft domains might trigger an actin-mediated mechanism of vesicle motility and that the lipid microenvironment might provide a scaffold to recruit and activate proteins involved in both membrane trafficking and cytoskeletal regulation. The involvement of the Arp2/3 complex in the observed actin tail formation was established using dominant-negative WASP constructs (Rozelle et al., 2000).

PI(4,5)Pz acts synergistically with GTP-bound Cdc42 in WAS protein activation promoting a conformational change, which leads to an exposure of the Arp2/3- activating C-terminus (Higgs and Pollard, 2000; Prehoda et al., 2000; Rohatgi et al., 2000). A second protein family, which potentially integrates PI(4,5)P2- and Rho-family-protein-mediated actin remodeling, is represented by ezrin/radixin/ moesin (ERM) (Bretscher et al., 2000) proteins that bind to PI(4,5)Pz, plasma membrane proteins, Rho-GDI, F-actin, and EBP-50 (Sechi and Wehland, 2000) and might thus be good candidates for linking interactions between the plasma membrane and the actin cytoskeleton. ERM-binding phosphoprotein 50 (EBPSO) binds to both the cortical cytoskeleton via an ERM-binding domain and to the cytoplasmic tail of the /?z-adrenergic receptor through a PDZ domain (Cao et al., 1999).

In cells, where these protein interactions were disrupted, a missorting of en- docytosed &adrenergic receptor, but not of transferrin receptor, was observed: Mutations in the cytosolic tail of the &adrenergic receptor that abolish EBPSO binding did not affect retention on the plasma membrane in unstimulated cells, but sorting in agonist-stimulated HEK 293 cells. A similar phenotype was observed in cells harboring a wild-type receptor but overexpressing a mutant form of EBPSO lacking the ERM-binding domain. Similar effects on Bz-adrenergic but not transferrin receptor sorting were observed upon application of the actin depolymerizing drug latrunculin B (Cao et al., 1999). The trafficking of /3z-adrenergic receptor thus appears to require an interaction of EBPSO and ERM proteins as well as an intact actin cytoskeleton. How an EBPSO/ERM/F-actin linkage participates in receptor recycling and whether it is regulated by PI(4,5)Pz remain elusive.

PI(3,4,5)Ps may synergize with PI(4,5,)P2 in some of the effects described above. Recently, a class II PI3kinase was reported to bind directly to clathrin and localize to clathrin-coated vesicles. Overexpression of PI3K-C2a! inhibited clathrin- dependent endocytosis, suggesting that the production of 3-phosphorylated phosphoinositides is important for receptor-mediated endocytosis (Gaidarov et al.,

ENDDCYTOSISANDTHECYTOSKELETON 121

2001). Furthermore, recent work has revealed an intimate crosstalk between phosphoinositide metabolism and small GTPases (Se&i and Wehland, 2000). The involvement of small GTPases in regulating the actin cytoskeleton and membrane trafficking is discussed in the next section.

VII. Small GTPases

A. Rab Family GTPases

Rab GTPases and their Ypt homologues in yeast constitute the largest group within the Ras GTPase superfamily with a total of 60 human Rab proteins predicted from genome analyses. Rab proteins represent key regulators of vesicular trafficking in eukaryotic cells including both constitutive and regulated exocytosis, transcyto- sis, and endocytosis (Rodman and Wandinger-Ness, 2000; Segev, 2001). Initially, control of targeting, docking, and fusion of vesicles with acceptor membranes was suggested to be the principal function of all Rab proteins, however, recently, their involvement in multiple and various aspects of membrane trafficking has become evident. Evidence for an involvement of Rab proteins in clathrin-coated vesicle formation was first suggested by overexpression studies with wild-type Rab5 and dominant-negative Rab5 mutants influencing the rates of receptor-mediated endocytosis of transfer-tin (Bucci et al., 1992). A role for Rab5 in vesicle formation at the plasma membrane in addition to its essential function in the homotypic fusion of early endosomes (Gorvel et al., 1991) was furthermore supported by in vitro studies identifying a complex of Rab5 and guanine-nucleotide dissociation inhibitor (GDI) required for the sequestration of receptor-bound ligands into clathrin-coated pits (McLauchlan et al., 1998). In addition to this well-established role in membrane trafficking, Rab5 might also play a role in controlling the organization of the actin cytoskeleton as suggested by recent overexpression studies. Both Rab GDI and a dominant-negative Rab5 mutant inhibited phorbol ester-induced reassembly of stress fibers and focal adhesions in Madin-Darby canine kidney (MDCK) cells (Imamura et al., 1998). Actin filament reorganization resulting in lamellipodia formation was observed upon introduction of active Rab5 into fibroblasts (Spaar- garen and Bos, 1999). The molecular basis for this effect remained unclear; it did not require the activation of PI3kinase or the GTPases Ras, Rat, Cdc42, or Rho (Spaargaren and Box, 1999).

6. Rho Family GTPases

The general role of Rho GTPases seems to be the regulation of the actin cytoskeleton (Hall, 1998; Kaibuchi et al., 1999; Schmidt and Hall, 1998). Recent evidence suggests that members of this subfamily may additionally be involved in various


aspects of membrane trafficking events (Ellis and Mellor, 2000). Some of these processes, such as phagocytosis, require massive actin remodeling and thus an involvement of Rho family GTPases is not surprising. Here, we will focus on evidence for a role of Rho family GTPases in the formation of clathrin-coated vesicles from the plasma membrane. Activated forms of Rat and Rho have been shown to inhibit receptor-mediated internalization of transferrin and EGF when expressed in intact HeLa cells (Lamaze et al., 1996).

Consistently, addition of Rho GDI and recombinant C3 transferase (a toxin that specifically inactivates Rho), respectively, stimulated transferrin endocytosis in a cell-free system, suggesting that endogenous Rho and Rat might be negative regulators of endocytosis under the experimental conditions applied (Lamaze et al., 1996). Mutant forms of the GTPases Cdc42, RhoA, and Rat 1 have also been shown to affect the rate and extent of both apical and basolateral endocytosis in polarized epithelial cells (Jou et al., 2000; Leung et al., 1999; Rojas et al., 2001). Recently, the clathrin heavy chain has been identified as a binding partner for the activated Cdc42-associated kinase 2 (ACK2), a specific target/effecter for the GTPase Cdc42 (Yang et al., 2001). Overexpression of the nonreceptor tyrosine kinase ACK2 interfered with transferrin receptor endocytosis most likely due to competition by ACK2 and AP2 for the same clathrin binding site (Yang et al., 2001). Activated Cdc42 was demonstrated to weaken the interaction between clathrin and exogenous ACK2. Consequently, it restored endocytic function in ACKZtransfected cells (Yang et al., 2001).

C. ARFs

ADP-ribosylation factors represent a group of six small ubiquitous GTPases that regulates membrane traffic and organelle structure in eukaryotic cells (Chavrier and Goud, 1999; Donaldson and Jackson, 2000). Among these, ARF6 functions exclusively in the endosomal-plasma membrane system. ARF6 appears to cycle between the plasma membrane and recycling endosomes depending on its nucleotide status (D’Souza-Schorey et al., 1998) and overexpression of a dominant-active form of ARF6 or its guanine-nucleotide exchange factor EFA6 inhibited transferrin uptake (Franc0 et uZ., 1999; Radhakrishna and Donaldson, 1997). ARF6 was also demonstrated to regulate selectively apical clathrin-mediated endocytosis in polarized epithelial cells (see Section VIIIA). Among the Arf GTPases, ARF6 is unique in its ability to rearrange the cortical actin cytoskeleton (D’Souza-Schorey et al., 1997; Radhakrishna et al., 1996) and ARF6 activation has been shown to be required for cell spreading (Song et al., 1998). Similarly, overexpression of EFA6 resulted in the induction of actin-rich membrane ruffles, an effect that could be inhibited by coexpression of dominant-negative forms of ARF6 and Rat 1 (Franc0 et al., 1999). Interestingly, the effects of EFA6 on endocytosis and actin organization were separable: The effects of EFA6 on transferrin trafficking required a


functional Sec7 domain, which catalyzes the nucleotide exchange on ARF6 but does not affect actin remodeling. The latter function was instead dependent on the C-terminal part of the exchange factor (France et al., 1999). The demonstration that Arf GTPases can activate type I PI(4)P 5-kinase (Godi et al., 1999; Honda et al.,

1999; Jones et al., 2000), an enzyme that catalyzes the synthesis of PI(4,5)Pz, suggests that Arf6 functions through the regulation of PI(4,5)Pz synthesis and turnover in membrane trafficking (Brown et al., 2001).

VIII. Modifications of the Actin Cytoskeleton and Membrane Trafficking in Specialized Cells

Cytoskeletal organization and dynamics seem to be of general importance for membrane budding processes in many if not all cells. Such a coordination of these two cellular functions will be of special importance in cell types, which are characterized by either elaborate cortical cytomatrix structures, through which vesicles have to be transported, and/or in cells types, which rely on highly efficient membrane recycling processes.

A. Polarized Cells

Epithelial cells are polarized cells. They contain apical and basolateral surfaces with distinct compositions and functions. At the apical site, the actin cytoskeleton is divided into two specialized domains consisting of the microvilli and the underlying terminal web. Membrane trafficking processes in epithelial cells have to be highly regulated to achieve and maintain polarity. In particular, endocytosis and exocytosis at the apical surface of many epithelial cells are tightly controlled. The basal rate of clathrin-mediated endocytosis at the apical site of MDCK cells was reported to be only about one-fifth of the level at the basolateral plasma membrane or the surface of nonpolarized fibroblast cells (Naim et al., 1995) but it may be greatly stimulated through various signaling pathways. Internalization processes at the different plasma membrane domains of polarized epithelial cells differ in sensitivity to overexpression of different dynamin constructs (Altschuler et al.,

1998) and are differentially influenced by the small GTPase ARF6, whereas the small GTPases RhoA and Racl affected both apical and basolateral endocytosis in MDCK cells (Mostov et al., 2000). ARF6 was found to associate exclusively with the apical, but not basolateral plasma membrane and to regulate clathrin-mediated endocytosis at the apical surface (Altschuler et al., 1999). The role of ARF6 in the regulation of apical endocytosis is probably mediated through downstream effecters (Donaldson and Jackson, 2000) including enzymes involved in phos- pholipid generation, which in turn affect membrane trafficking either directly or


via modification of the actin cytoskeleton. In line with the latter, the use of drugs modulating the degree of actin polymerization has been reported to have different effects on the distinct sides of polarized cells plasma membranes. Depolymeriza- tion of filamentous actin with cytochalasin D inhibited endocytosis selectively in the apical membrane of polarized epithelial cells, which is marked by an elaborate actin cytoskeleton, although actin filament integrity was destroyed throughout the cell (Gottlieb et al., 1993). An accumulation of coated pits was observed by electron microscopy at the apical surface of the drug-treated MDCK cells suggesting defects in pinching off coated vesicles. Cytochalasin D furthermore interfered with the displacement of microvillar surface components to the intermicrovillar space suggesting that actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface toward intermicrovillar spaces, or provides force required for converting a membrane invagination or pit into an endocytic vesicle selectively at the apical surface (Gottlieb et al., 1993).

Treatment of MDCK cells with the actin filament-stabilizing drug jasplakinolide on the other hand did not affect the uptake and accumulation of fluid-phase endocytosis markers at the apical surface but at the basolateral surface. The authors did not observe impairment on the basolateral uptake and recycling of transferrin, indicating that jasplakinolide-induced actin stabilization may primarily affect clathrin-independent endocytosis (Shurety et al., 1998). A recent study by Buss et al. (2001) revealed that a specific isoform of myosin VI (see Section V) encompassing a large insert in the tail domain is predominantly expressed in tissues containing many polarized cells with apical microvilli. This isoform with a large insert exhibited a very good colocalization with clathrin-coated pits and vesicles exclusively at the apical surface, whereas the spatial distribution of myosin VI lacking this insert only partially overlapped with clathrin. Overexpression of the myosin VI tail domain with but not without the large insert furthermore interfered with transferrin endocytosis in nonpolarized cells (Buss et al., 2001). These results suggest a role for myosin VI in clathrin-mediated endocytosis at the apical surface of polarized cells.

B. Compensatory Endocytosis

Compensatory refers to a specialized form of lipid and protein internalization from plasma membranes used by regulated secretory cells, such as neurons, exocrine and endocrine cells, mast cells, and neutrophils. After massive stimulated vesicle exocytosis, the vesicle pool needs to be regenerated by endocytic retrieval of vesicle membrane and proteins in a fast and efficient way to maintain cellular structure and function. Interfering with compensatory endocytosis, as shown in the lamprey axonal terminal after injection of antibodies against a protein of the endocytic machinery, endophilin, had drastic effects on structure and function of the


presynaptic terminal after tonic stimulation. The synaptic vesicle pool was almost depleted and the plasma membrane of the presynapse was altered and extended (Ringstad et al., 1999). In general, the molecular machinery and fundamental features of compensatory endocytosis seem to differ little from those in constitutive or ligand-stimulated endocytosis. However, regulated secretory cells-studied best are neurons-exhibit some unique characteristics. Several components of the endocytic machinery are expressed at very high levels in these cells and/or exist as specific genes or splice variants, some of which exhibit distinct regulatory properties (Dresbach et al., 2001; Morris and Schmid, 1995; Slepnev and De Camilli, 2000). Presynaptic nerve terminals exhibit considerably higher levels of general endocytic proteins, such as clathrin, AP2, epsin, and epsl5, as well as large amounts of specific isoforms or splice variants of proteins, such as dynamin, AP180, amphiphysin, syndapin, intersection, and endophilin.

Elaborate cytomatrix structures exist both pre- and postsynaptically, which seem to play a pivotal role in synaptic transmission. The speed and efficiency of synaptic vesicle exocytosis and endocytosis in the presynapse may depend on arrays of adaptor and scaffold molecules predestinated to catalyze and regulate the different steps in the recycling process with high speed and efficiency (Dresbach et al., 2001). Cytoskeletal proteins are important components of these structures. Actin filaments localize to both pre- and postsynaptic elements and recent studies indicate that F-actin disassembles reversibly during synaptic vesicle recycling (Bernstein et al., 1998).

The cortical actin cytoskeleton in presynaptic nerve terminals might play a role in the structural organization of specialized areas that contain endocytic coat and accessory protein in a concentrated form. Several studies over the past few years reported the concentration of the machinery for the recovery of synaptic vesicles in close proximity to regions of neurotransmitter exocytosis at the Drosophila neuromuscular junction. Several proteins involved in clathrin-mediated compensatory endocytosis such as dynamin, a-adaptin, and DAP160 (Estes et al., 1996; Gonzalez-Gait&i and Jackie, 1997; Roos and Kelly, 1998) are highly enriched in so-called hot spots of endocytosis indicating that the endocytic machinery is not freely diffusible, but instead anchored in close vicinity to exocytic zones.

Intermediates, such as clathrin-coated pits, are rarely observed in resting synapses but can be found after massive exocytosis from neurotransmitter-containing synaptic vesicles, in particular under conditions interfering with compensatory endocytosis. In line with a spatial separation of the presynaptic plasma membranes into zones of endocytosis and exocytosis, these invaginated coated pits are observed rather at the edges of the active zones, where synaptic vesicles dock and fuse with the plasma membrane upon neurotransmitter release (Gad et d., 1998; Heuser and Reese, 1973; Ringstad et al., 1999; Shupliakov et al., 1997). Also in snake motor boutons, clathrin-mediated endocytosis was observed near active zones. Newly internalized coated vesicles and to a fewer extent pits appeared to be


clustered near, but not directly at active zones (Teng and Wilkinson, 2000). These hot spots of endocytosis have recently been reported to be enriched in F-actin at the frog neuromuscular junction. At target-deprived synaptic sites F-actin did not colocalize with the synaptic vesicle marker sv2, suggesting a concentration of actin fibers in the nonrelease domains. This may suggest that the actin-based network participates in restricting synaptic vesicles to release domains (“cage function”), in the recycling process and/or in stabilizing the nerve terminal at the neuromuscular junction (Dunaevsky and Connor, 2000). Interestingly, Morales et al. (2000) reported that the major GFP-actin pool in the presynaptic terminal of cultured hippocampal neurons did colocalize with a marker protein of the active zone, bassoon. It is, however, possible that this actin pool at exocytic sites corresponds rather to monomeric actin because it was not colabeled with phalloidin derivatives (Morales et al., 2000). Thus, the exocytic and the surrounding endocytic zones at the presynapse might be characterized by different pools of actin corresponding to distinct functional stages and different functions.

Organizing the machineries for endocytosis and exocytosis in distinct but adjacent domains, in which the proteins involved in these functions are thus highly concentrated and organized, might represent a powerful mechanism to achieve the high speed and efficiency of compensatory endocytosis. Cytoskeletal structures might help to maintain the highly ordered spatial organization of active zones, of opposing postsynaptic receptor clusters, and of endocytic hot spots by anchoring or trapping the machineries required. Surrounding exocytic zones by adjacent concentrated endocytic machinery would allow for a recycling quite near and rapidly after neurotransmitter secretion and would reduce vesicle sorting.

IX. Insights from Yeast

Several model systems exist, which are easily accessible for powerful genetic approaches, and thus offer attractive routes toward a better understanding of cellular processes and the molecules involved. In general, Saccharomyces cerevisiae may be the organism studied most extensively by genetic approaches. The isolation and characterization of Saccharomyces cerevisiae mutants have been especially productive during the last decade and led to the identification of many proteins involved in both organization of the actin cytoskeleton and the internalization step at the plasma membrane.

The yeast model system rapidly became a widely accepted system used to study the huge protein machinery building, rearranging, and controlling the actin cytoskeleton. The yeast actin cytoskeleton shows a very simple organization, e.g., actin cables running along the mother-daughter axis of budding cells, F-actin- rich patches specifically colocalized to the forming bud during the initial steps of cell division and the actin-rich cleavage site during cell division (Holtzman et al.,


1993; Welch et al., 1994). Both the simple architecture and the well-characterized changes of the actin cytoskeleton during the different steps of the yeast cell cycle provided an excellent read-out for any effects caused by mutations and/or deletions of single or several genes. Using these advantages rapid progress was made and often appeared consistent with data known from vertebrate systems. Furthermore, the Saccharomyces cerevisiae system permitted the functional connections of the actin cytoskeleton to cell polarity and division to be addressed and to be compared with those of higher eukaryotes (Drubin and Nelson, 1996).

In contrast, the use of yeast as a model to study endocytic uptake processes of higher eukaryotes was impaired by the lack of quantitative in vivo endocytosis assays, by the lack of in vitro reconstitution systems, and by the technical diffi- culties of ultrastructural studies in yeast. Although it was possible to overcome the first problem by use of internalization of radiolabeled a-factor pheromone bound to its receptor Ste2p (Dulic et al., 1991), the other limitations still persist. Only three morphological studies following the endocytic pathway have been reported thus far (Mulholland et al., 1999; Prescianotto-Baschong and Riezman, 1998; Prescianotto-Baschong and Riezman, 2002). Furthermore, the use of yeast as a model system for studying receptor-mediated endocytosis was questioned by results that suggested fundamental differences of endocytosis in yeast and other organisms (Trowbridge et al., 1993). For example, the endocytosis signal identified in Ste2p did not resemble the tyrosine-based signals known in mammals (Rohrer et al., 1993). Knocking out the single gene for the clathrin heavy chain (CHCI) or inactivating it via a temperature-sensitive allele led to only a 50% reduction of the a-factor uptake rate but did not abolish internalization, as commonly expected (Tan et al., 1993). The GTPase dynamin, which appears to play a central role in the vesicle fission reaction in mammals, seems not to exist in yeast; only three dynamin-like proteins have been identified, Mgmlp (Jones and Fangman, 1992), Vsplp (Vater et al., 1992), and Dnmlp (Gammie et al., 1995). These proteins do seem to play a role in membrane fission events, however, at mitochondria and not at the plasma membrane [Dnmlp (Otsuga et al., 1998); Mgmlp (Wong et al., 2000)]. Another surprising finding was that the disruptions of the two AP180 genes and several genes encoding for proteins of heterotetrameric adaptor complexes did not cause endocytosis defects (Huang et al., 1999). In contrast, y-adaptin-deficient mice and a-adaptin mutants in Drosophila melanogaster were not viable (Gonzalez-Gait&r and Jackie, 1997; Zizioli et al., 1999). On the other hand, many yeast proteins crucial for endocytic function have been identified through genetic screens. Although some of them were already known in mammals, many more were novel and/or were not thought to be functionally linked to endocytosis. Meanwhile many of those proteins have subsequently been identified in other eukaryotes and shown to play a role in endocytosis, as suggested by the yeast data (Geli and Riezman, 1998; Munn, 2001). These findings suggest that although it is necessary to be aware of the phylogenetic difference between animal and yeast cells, common principles used by all eukaryotes can be revealed.


One of these basic principles is the involvement of the actin cytoskeleton in endocytosis. In yeast, these two cellular functions appear to be more tightly interwoven than initial studies in mammalian cells may have suggested. In mammals, the involvement of the actin cytoskeleton was mainly studied with reagents destabilizing or stabilizing F-actin structures, such as cytochalasin D, latrunculin, phalloidin, and/or jasplakinolide. The results of these studies, however, did not show a clear picture. With the actin-destabilizing drug cytochalsin D, for example, it was shown that apical endocytosis in polarized cells was inhibited whereas basolateral endocytosis remained unaffected (Gottlieb etal., 1993; Jackman et al., 1994). In other cell types, there are conflicting results on the effect of various actin-depolymerizing drugs on receptor-mediated endocytosis (Lamaze et al., 1996, 1997; Salisbury et al., 1980; Sandvig and van Deurs, 1990; Wolkoff et al., 1984 and references therein). Many of these apparent inconsistencies may depend on differences of assays and cell types used as revealed in an extensive study by Fujimoto et al. (2000).

Yeast genetics and the characterizations of the resulting phenotypes in contrast clearly showed that interference with many proteins of the actin cytoskeleton and of the endocytosis machinery caused both endocytosis and actin cytoskeleton organization defects (Geli and Riezman, 1998; Wendland et al., 1998). Among the long list of such proteins are actin itself, cofilin, the Arp2/3 complex components Arp2 and Arp3, the yeast protein related to N-WASP (Lasl7p), MyoSp, the yeast proteins showing homologies to Eps15 (End3p and Panlp), the synaptojanin- like protein Sjllp, and finally the proteins physically or genetically connected to Abplp. These include Srv2p, Slalp, and Sla2p (a mammalian homologue of Sla2p is HIPlR; see Section III), and the proteins showing some homology to amphiphysins (Rvs167p andRvsl6lp) as well as the two kinases Arklp and Prklp. For many of these proteins it remains unclear whether and how they participate directly in the endocytosis process or whether their mutation or knock out only indirectly affected endocytosis.

Biochemical characterizations of protein functions and of mechanistic defects caused by certain mutations, however, were able to reveal more concretely the cytoskeletal functions required for endocytosis in yeast and thus suggested that the proteins listed above may indeed represent required cross-connections of actin cytoskeletal functions and endocytosis. Using mutants of the actin depolymerizing protein cofilin, Lappalainen and Drubin (1997) were able to demonstrate that rapid actin turnover is required for endocytosis. Rapid turnover requires both rapid actin depolymerization and polymerization. Because biochemical analysis revealed that actin polymerization depends on the Arp2/3 complex and its activators Lasl7p (Winter et al., 1999), Myo5p (Evangelista et al., 2000; Lechler et al., 2000),

Panlp (Duncan et al., 2001), and Abplp (Goode et al., 2001), the genetic data implicating all these proteins in endocytosis now appear in a new light.

The interface of actin and endocytosis as currently seen from the yeast analyses represents a complex network of physical and genetic protein interactions. It may, however, be possible to organize the overwhelming wealth of data into four


organization cores built around the Arp2/3 complex activators listed above. These complexes involved in endocytosis and actin organization, a concept introduced in the review of Wendland et al. (1998) may have in part similar and thus redundant functions, as suggested from examinations of the in vitro Arp2/3 complex activation. This hypothesis is strengthened by genetic analyses showing that many genetic interactions between components of the different “complexes” exist. Genetic lethalities of two genes are commonly interpreted as two genes having similar functions integrated in parallel pathways with redundant functions. In cases in which a knock out of a single gene is lethal, these connections cannot be established, but for genetic alterations with less drastic consequences such analyses can prove very informative. In the following we will describe the four complexes, which should, however, not be viewed as huge and static protein complexes but rather as dynamic functional organization cores at the interface between actin and endocytosis and we will also try to point out how these complexes in turn are interconnected.

The first complex would be the myosin complex (MyoSp/Myo3p). It includes the Myo5p interactions with calmodulin, a calcium sensor, which in turn interacts with the Arp2/3 complex component Arc35 in a Ca*+ -dependend manner (Schaerer- Brodbeck and Riezman, 2000). In mammals, an isoform of the unconventional myosin VI has been identified as playing a role in receptor-mediated endocytosis but it is still unknown whether a functional connection to Arp2/3 complex activation and calcium signaling exists (see Section V).

The second complex is centered on the Arp2/3 complex activator Abplp. It includes physical connections of Abplp to Rvs167p and Rvsl6lp (proteins showing homologies to amphiphysins) and the CAMP and Ras signaling component Srv2 mediated by the Abplp proline-rich and SH3 domain, respectively (Lila and Drubin, 1997). Interestingly it is the SH3 domain of Abplp, which becomes crucial for endocytosis, when central domains of Sla2p are deleted. SLA2 is a gene that like SLAl (see below) shows synthetic lethality to ABPI. In mammals, the Abpl homologue identified (mammalian Abpl) has been shown to interact with dynamin via its SH3 domain (see Section IV) and the SLA2 homologue identified (HIPlR) has been shown to interact with clathrin via its central domain (see Section III). Both the yeast and the mammalian Abpl were demonstrated to bind to F-actin via N-terminal parts of the proteins (Goode et al., 2001; Kessels et al., 2000). It remains, however, to be determined whether mammalian Abpl, which does not show the Arp interaction interfaces identified for yeast Abplp, can promote actin nucleation via the Arp2/3 complex or whether this is a yeast-specific function.

The third organization core at the interface of actin dynamics and endocytosis may be headed by Lasl7p (also named Beel), the yeast homologue of the mammalian Arp2/3 complex activating protein N-WASP (see Section IV). Interestingly, Lasl7p was shown to interact with Slalp (Li, 1997), another gene synthetic lethal withABP1, and thereby probably of related function to Abp lp and/or its interaction partners. It is currently unclear which protein in mammals may be a functional


homologue of Slalp. It is a multi-SH3 domain protein, which may serve as a cortical adapter protein (Ayscough et al., 1999), a function similar to that proposed for intersectins, which have been shown to play a role in endocytosis and to interact with N-WASP (see Section IV).

The fourth complex is centered on the Arp2/3 complex activator Panlp, a protein showing considerable homologies to the mammalian EH domain protein Epsl5. Whether Epsl5 can also promote actin nucleation via the Arp2/3 complex is currently unknown, but both the mammalian and the yeast protein interact with the clathrin-binding AP180 proteins and epsins (Slepnev and De Camilli, 2000; Wend- land and En-u, 1998). Mammalian Epsl5 and epsin proteins are dephosphins, i.e., these proteins are phosphorylated in resting nerve terminals and coordinately dephosphorylated upon stimulation (Chen et al., 1998). Interestingly, the yeast proteins also seem to be negatively regulated by phosphorylations. The phosphorylation of LxxQxTG repeats within the Panlp molecule, which are also present in the region that is responsible for End3p binding, has been shown to regulate the cellular function of Panlp (Zeng and Cai, 1999). This phosphorylation as well as that of the two yeast epsins Entlp and Ent2p is dependent on the kinase Prklp and at least Entlp has been shown to be a direct substrate of Prklp (Watson et al., 2001).

The kinase Prklp has been identified by its homology to the actin-regulating kinase Arklp, which was discovered as a protein interacting with Sla2p. Both yeast Sla2p and its mammalian homologue HIPlR bind to F-actin via their N-terminal domain (Engqvist-Goldstein et al., 1999; McCann and Craig, 1997). Arklp and Prklp show considerable homology to mammalian cyclin G-associated kinase (GAK) (Greener et al., 2000) an auxilin-like kinase, which is involved in clathrin coat dissociation (Umeda et al., 2000). Both yeast kinases can be viewed as important links between the four organization cores at the interface between actin and endocytosis introduced above and both appear to control functionally overlap- ping but distinct pathways (Cope et al., 1999). Arklp associates with Sla2p and with the Abplp SH3 domain and could thus represent a link between the Abplp complex and Sla2p. The localization of Arklp to cortical actin patches was shown to rely on the Abplp SH3 domain (Fazi et al., 2001). This interaction could be a molecular basis for the crucial role of the Abplp SH3 domain in SLA2 mutant backgrounds. The peculiar binding specificity of the yeast Abplp SH3 domain to extended class II ligands, as determined by Fazi et al. (2001), may not be a feature strictly conserved to mammals, as the critical glutamate identified is replaced by a leucine in mammalian Abpl. Consistent with the kinases Arklp and Prklp playing important roles in both cytoskeletal organization and endocytosis, ark1 A/prkl A cells showed a severely disrupted cytoskeletal organization (Cope et al., 1999), a reduced endocytic uptake, and an accumulation of endocytic intermediates (Watson et al., 2001). The ARKl-related gene PRKI shows genetic interactions with ABPl and SLA2, suggesting functions independent from but redundant to the Abplp complex. Prklp has been shown to be capable of phosphorylating Slalp, Panlp,

ENDOCYTOSlSANDTHECYlOSKELETON 131

and yeast epsins; it should therefore be considered to functionally belong to the Panlp complex. Furthermore, Slalp not only associates with Lasl7p but has recently been shown to bind to the Panlp/End3p complex (Zeng et al., 2001).

Given the importance of the kinases Arklp and Prklp for the regulation of actin cytoskeletal organization and endocytosis in yeast, considerable efforts are currently being made to identify functional mammalian homologues. Recently, the Schmid laboratory identified the mammalian kinase adaptor-associated kinase 1 (AAKl), which binds to and phosphorylates AP2 and shows about 40% similarity to the yeast kinases (S. Schmid, personal communication). It is, however, yet unknown whether AAKl also phosphorylates EpslS and/or has any functions in actin cytoskeleton regulation.

Another exciting recent development in yeast is the mounting evidence that sphingoid base synthesis is required for the internalization step of endocytosis and again for actin cytoskeletal organization (Zanolari et al., 2000). Because it is known that sphingosine inhibits some kinases and activates others, the functions of sphingoid bases may be to control protein phosphorylations. It will be extremely interesting to explore this new field further.

X. Concluding Remarks

The elucidation of functional connections of the actin cytoskeleton and receptor- mediated endocytosis is still in its infancy. The discovery of a set of molecular links between these two cellular functions provides an attractive research avenue currently followed by a growing number of laboratories in the world. Because it is reasonable to assume that we still have not discovered all the molecular players involved, efforts in this direction will continue to add to the ever mounting complex- ity of the dynamic protein arrays responsible for receptor-mediated endocytosis on one hand and the actin cytoskeleton on the other hand by the identification of further molecular links.

To obtain a better understanding of the interplay of the actin cytoskeleton and the vesicle formation machinery, we will have to thoroughly explore whether and how the functions of the individual molecules are employed during the process of vesicle formation and movement. Furthermore, considerable efforts need to be focused on unraveling the delicate spatial and temporal regulation of the functional interface of these two cell biological fields. Model systems with unique and often complementary advantages, such as yeast, other organisms accessible for genetic analysis, as well as specialized mammalian cell systems with their unique morphological and functional features, will certainly continue to be of great importance during this work.

Finally, we should then be able to answer the following currently entirely open questions. Which of the potential functions of the actin cytoskeleton proposed in


this review for the different steps of endocytosis are indeed used by cells? Are these functions crucial contributions or do they rather ensure high efficiencies or other requirements of specialized cells? How are these functions of the actin cytoskeleton set up and controlled on the molecular level?

Acknowledgments

We would like to thank Drs. Sandra Schmid, Howard Riezman, Pietro De Camilli, Francis Brodsky, and Mark McNiven for communicating additional unpublished data. This work was in part supported by grants from the Deutsche Forschungsgemeinschaft and from the Kultusministerium Land Sachsen- Anhalt to B.Q. and M.M.K.

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[International Review of Cytology] Volume 220 || Endocytosis and the cytoskeleton

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