the syndapin protein family: linking membrane trafficking ... · the interface between membrane...

IntroductionThe formation and movement of vesicles, as well as theorganization of different pools of vesicles within distinctcompartments of cells, are thought to involve cytoskeletalelements; however, how the different molecular machineriesinvolved are interconnected is mostly unclear (Qualmann et al.,2000b; Qualmann and Kessels, 2002; Engqvist-Goldstein andDrubin, 2003; Gundelfinger et al., 2003; Orth and McNiven,2003). Recently, a handful of candidates for proteins that canact at this interface have been identified. Among these aremembers of the syndapin family, which are Src-homology 3(SH3)-domain-containing proteins that exhibit severalisoforms and splice variants. SH3 domains recognize proline-rich motifs of the PXXP type and their specificity relies mainlyon the residues flanking such motifs. Syndapins belong to agrowing class of accessory proteins functioning in membranetrafficking that interact with the proline-rich domain of theGTPase dynamin. Because the name ‘syndapins’ (for synapticdynamin-associated proteins) currently seems to reflect bestwhat is known about the functions of these proteins in vivo,we refer to them as such here – avoiding use of other names(e.g. FAP52, SH3p14 and PACSIN) to keep the nomenclatureas simple as possible.

Dynamin is an important player in endocytosis, a processthat comprises several distinct steps. First, cell-surfacereceptors are bound by intracellular adaptors. A clathrin coatis then assembled on the underside of the membrane, whichthen invaginates. The developing clathrin-coated pits constrictat the neck, and finally they pinch off from the membrane. Thenewly formed vesicle is transported into the cytosol, where itis uncoated and can undergo further sorting. Dynamin is crucial

for the fission step, in liberating newly formed vesicles fromthe donor membrane (Hinshaw, 2000; Sever et al., 2000).

All syndapins cloned and/or identified as DNA sequencesshow remarkably high conservation of both domain structureand amino acid sequence in species as diverse as worms,insects, fish, birds and mammals. Each is composed of an N-terminal region predicted to be almost exclusively α-helicaland to engage in coiled-coil interactions, a flexible stretch thatmay contain up to three NPF motifs, and a C-terminal SH3domain. The SH3 domain is responsible for interactions withdynamin and N-WASP, a potent activator of the Arp2/3complex F-actin-nucleation machine. Syndapin complexesmay thus link membrane trafficking and the actin cytoskeleton.Recent evanescent field-microscopy studies have demonstratedthat actin, the Arp2/3 complex and N-WASP transientlyaccumulate at sites of endocytosis and that this is coordinatedwith dynamin-mediated vesicle fission (Merrifield et al., 2002;Merrifield et al., 2004). Here, we focus mainly on the roles ofthe actin cytoskeleton in vesicle formation, discuss howsyndapins might work at the interface of actin and membranetrafficking, and highlight the molecular requirements andmechanisms involved. We also analyse the phylogeneticrelationship between the different syndapin orthologs, isoformsand splice variants, which allows them to be organized intodistinct subgroups.

Syndapin interactions and functionsConnecting the cytoskeleton with vesicle formationSyndapins interact with N-WASP (a protein important for actinfilament formation) as well as several molecules implicated in

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Syndapins – also called PACSINs – are highly conservedSrc-homology 3 (SH3)-domain-containing proteins thatseem to exist in all multicellular eukaryotes. They interactwith the large GTPase dynamin and several other proteinsimplicated in vesicle trafficking. Syndapin-dynamincomplexes appear to play an important role in vesiclefission at different donor membranes, including theplasma membrane (endocytosis) and Golgi membranes. Inaddition, syndapins are implicated in later steps of vesiclecycling in neuronal and non-neuronal cells. Syndapins alsointeract with N-WASP, a potent activator of the Arp2/3complex that forms a critical part of the actinpolymerization machinery. Syndapin oligomers can

thereby couple bursts of actin polymerization with thevesicle fission step involving dynamins. This allows newlyformed vesicles to move away from the donor membranedriven by actin polymerization. Syndapins also engage inadditional interactions with molecules involved in severalsignal transduction pathways, producing crosstalk atthe interface between membrane trafficking and thecytoskeleton. Given the distinct expression patterns of thedifferent syndapins and their splice forms, these proteinscould have isoform-specific functions.

Key words: Syndapin, Actin polymerization, Vesicle trafficking

Summary

The syndapin protein family: linking membranetrafficking with the cytoskeletonMichael M. Kessels and Britta Qualmann*Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany*Author for correspondence (e-mail: [email protected])

Journal of Cell Science 117, 3077-3086 Published by The Company of Biologists 2004doi:10.1242/jcs.01290

Commentary

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membrane trafficking: the GTPase dynamin (which controlsendocytic vesicle formation) (Hinshaw, 2000; Sever et al.,2000), the phosphatidylinositol 5-phosphatase synaptojanin (aprotein that plays a crucial role in the uncoating of clathrin-coated vesicles; Cremona et al., 1999) and synapsin I (a proteinassociated with the reserve pool of synaptic vesicles) (Hilfikeret al., 1999) (Fig. 1). These interactions first raised thepossibility that syndapins have roles in both membranetrafficking and organization of the actin cytoskeleton(Qualmann et al., 1999), a hypothesis that was followed up bymore-detailed studies of the interactions with dynamin and N-WASP.

The relevance of the interaction with dynamin isstrongly supported by coimmunoprecipitation studies of theendogenous proteins (Qualmann et al., 1999; Qualmann andKelly, 2000) and by the fact that a surplus of dynamin-bindingsyndapin SH3 domains inhibits receptor-mediated endocytosisin both permeabilized cell assays (Simpson et al., 1999) andintact cells (Qualmann and Kelly, 2000). This block ofendocytosis occurs at the transition from invaginated clathrin-coated pits to closed endocytic membrane compartments (i.e.at the step at which dynamin is crucial) (Simpson et al., 1999).In common with other SH3-domain-containing dynamin-binding proteins, syndapins might influence the subcellularlocalization, GTP-binding and/or GTP hydrolysis rate ofdynamin.

All syndapins also interact with the Arp2/3 complex

activator N-WASP (Qualmann et al., 1999; Qualmann andKelly, 2000; Modregger et al., 2000); they might thus connectthe actin cytoskeleton with dynamin-mediated vesicle fission(Fig. 1). The in vivo relevance of this interaction is supportedby studies showing coimmunoprecipitation of endogenoussyndapin I and N-WASP (Qualmann et al., 1999) and by thefact that overexpression of syndapin I or syndapin II inducesformation of numerous actin-rich filopodia. This requiresactivation of the Arp2/3 complex at the cell cortex (Qualmannand Kelly, 2000), and the phenotype is similar to that causedby activation of overexpressed N-WASP (Miki et al., 1998).

N-WASP exists in an autoinhibited state and needs to beopened up by effector molecules such as phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] and Cdc42 to associate withand activate the Arp2/3 complex (Kim et al., 2000; Higgs andPollard, 2001; Welch and Mullins, 2002). As syndapins canrecruit N-WASP to membranes and trigger local actinpolymerization in vivo in an SH3-domain- and Arp2/3-complex-dependent manner (Kessels and Qualmann, 2002),syndapins seem to belong to the diverse set of N-WASPeffectors that can trigger activation of the Arp2/3 complex andthereby actin polymerization. The cytoskeletal role ofsyndapins is also reflected by the fact that syndapins areenriched at sites of high actin turnover, such as lamellipodia(Qualmann and Kelly, 2000) and neuronal growth cones(Kessels and Qualmann, 2002).

Studies of the syndapin II isoform in chicken (FAP52)

Journal of Cell Science 117 (15)

ADAM

Ectodomain shedding

N-WASP

SH3

NP

F

NP

F

SH3

Dynamin

Vesicle formation

F-Actin nucleation

Clathrin-coated vesicle uncoating

Vesicle recycling and endocytosis

EHD proteins

Synaptojanin

Syndapin

Anchoring of synaptic vesicles to F-Actin

Synapsin

mSos

Huntingtin CD95L

GTP

GDP

GDP/GTP exchange (GEF)

Filamin

F-actin crosslinking

ApoptosisHuntington's disease

Oligomerization

PtdIns(4,5)P2

Fig. 1. Interactions of the syndapin proteinfamily. Depicted are all syndapininteraction partners described thus far,irrespective of species, syndapin isoform orsplice variant. Note that the depictedantiparallel dimers are merely ahypothetical model for syndapinoligomerization, which is not yet supportedby a crystal structure. The thickness of thearrows indicates whether the interactionsare based on in vitro data, supported by invivo interaction studies or confirmed byfunctional analyses of the respectivecellular functions and corresponding rescueexperiments.

3079Syndapin interactions and functions

reinforce this connection with the actin cytoskeleton. FAP52binds to the actin-crosslinking protein filamin/ABP-280 (Nikkiet al., 2002a) (Fig. 1) and localizes to focal adhesions(Meriläinen et al., 1997). Note, however, that this has not beenfound in other mammalian systems or in Xenopus laevis(Ritteret al., 1999; Cousin et al., 2000) (M.M.K. and B.Q.,unpublished). Moreover, filamin/ABP-280 is not a focaladhesion protein, and the distribution of the two proteins onlypartially overlaps at sites of contact between stress fibres andfocal adhesions (Nikki et al., 2002a).

A link between the cytoskeletal and endocytic functionsof syndapins was suggested by the observation thatoverexpression of N-WASP interferes with receptor-mediatedendocytosis, and this depends solely on the syndapin-binding,central proline-rich domain of N-WASP. The phenotype canbe rescued by syndapin co-overexpression (Kessels andQualmann, 2002). The involvement of N-WASP interactions in

endocytic vesicle formation is strongly supported by theobservation that endocytosis is inhibited in cells in whichendogenous N-WASP is confined to mitochondria or targetedby anti-N-WASP antibodies. One can rescue endocytosis byresupplying the cells with N-WASP (Kessels and Qualmann,2002). Analysis of lymphocytes from mice lacking WASP alsoimplicates WASP family members in endocytosis. These cellsexhibit defects in T-cell receptor endocytosis in addition todefects in actin polymerization (Zhang et al., 1999).

What part might actin play in vesicle formation? The actincytoskeleton might spatially organize the endocytic machinery.It might represent a barrier through which newly formedvesicles must be transported that needs to be removed by alocal increase in actin turnover. It might also provide structuralsupport for membrane topologies that facilitate vesicleformation, such as invaginated tubules, and/or promote vesicleformation by generating force through motor proteins and/or

Fig. 2. Interconnection of dynamin-mediatedvesicle fission with Arp2/3-complex-dependentF-actin nucleation triggered by N-WASP andsyndapins. (A) Early in vesicle formation, themembrane is deeply invaginated and dynaminstarts to concentrate at the vesicle neck, whichis still wide. Syndapin oligomers associatedwith dynamin may help recruit and activate theArp2/3 complex activator N-WASP (1). In thisway, actin nucleation by the Arp2/3 complexcan be linked to dynamin-mediated fissioncontrol (2). Actin filaments can be generated denovo (2) and as new branches from alreadyexisting actin fibres that may be part of thecortical cytoskeleton (3). It remains to beinvestigated whether syndapin-dynamincomplexes form first in the cytosol (4), afterdynamin has been recruited to the plasmamembrane (5) or both. (B) Late in vesicleformation, the vesicle neck is constricted andthe vesicle is subsequently pinched off anddetached from the plasma membrane. Dynaminoligomers surrounding the neck could be aspatial and temporal cue for Arp2/3-complex-mediated F-actin nucleation. Syndapins and N-WASP serve as connecting elements thatensure that actin polymerization is restricted tothe neck region. Such a restriction of actinbuild-up and a polarization of actin fibres in amanner that orientates the fast-growing plusends towards the forming/moving vesicleprovides force and ensures the directionality ofvesicle movement away from the donormembrane. Growing plus ends of actinfilaments are marked by ATP-loaded actinmonomers, which are depicted in darker blue.PIP2, phosphatidylinositol (4,5)-bisphosphate.

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actin polymerization (Qualmann et al., 2000b). The latter couldbe achieved by activation of the Arp2/3 complex by N-WASPand syndapin, if actin polymerization is spatially restricted andoccurs mainly at vesicle membrane areas facing the plasmamembrane (Fig. 2). By contrast, F-actin formation at vesiclemembrane areas facing the cytoplasm or within wide areas ofthe cortical cytoskeleton would create a barrier and thus insteadbe inhibitory.

The timing of local actin polymerization would need to betightly controlled to correlate with the fission reaction (Fig. 2).Short-lived actin structures at sites of endocytosis whoseappearance coincides with dynamin-mediated vesicle releasecan be observed by evanescent field microscopy (Merrifieldet al., 2002). Additionally, both N-WASP and the Arp2/3complex transiently appear at sites of endocytosis (Merrifieldet al., 2004). The kinetics of Arp2/3 complex recruitmentmirror those of formation of the actin structures – this isexpected because the complex becomes incorporated intoforming F-actin structures. By contrast, the catalytic Arp2/3complex activator N-WASP appears transiently, being presentat its highest levels during the initial phase of F-actin formationupon vesicle departure (Merrifield et al., 2004).

An attractive hypothesis is that the coincidence of actin

nucleation and dynamin-mediated fission reflects the use of acommon binding partner for both machineries, such assyndapin (Fig. 2). Dynamin forms a collar at the neck regionof plasma membrane invaginations in synaptosomes incubatedwith GTPγS and in nerve terminals of shibire flies (Hinshaw,2000). The interaction with the dynamin-associated syndapincould allow specific recruitment of N-WASP to the neck ofcoated pits and thereby produce polarity in the actinpolymerization and directed movement of newly formedvesicles away from the plasma membrane (Fig. 2). Indeed,recent studies have revealed that syndapins can recruit N-WASP to intracellular membranes (Kessels and Qualmann,2002). Such mechanisms might not only facilitate thedeparture of the vesicle from the donor membrane but mightalso create actin structures that have the appropriatelocalization, timing and polarity for moving detached vesiclesaway from the plasma membrane. Several studies showingactin tails attached to moving vesicles support this theory(Taunton, 2001). Interestingly, both dynamin and N-WASPhave been detected in such actin tails, mainly at the actin-membrane interface (Taunton, 2001; Orth et al., 2002; Lee andDe Camilli, 2002).

Proteins such as Abp1 and cortactin could have functions


0.10

Confidence level > 90%





Rattus norvegicus Syndapin II

Mus muscu lus PACSIN 2Homo sapiens PACSIN 2

Gallus ga llus FAP52Xenopus laevis X-PACSIN II

Fugu ru bripes Syndapin II

Homo sapiens PACSIN 3Mus muscu lus PACSIN 3

Rattus norvegicus Syndapin III

Gallus ga llus Syndapin III

Danio rerio Syndapin III

Fugu ru bripes Syndapin V

Rattus norvegicus Syndapin IMus muscu lus PACSIN 1 Homo sapiens PACSIN 1

Fugu ru bripes Syndapin I

Echinococcus granu losus

EG13Echinococcus multi locu larisEM13

Caenor habditis elegans Syndapin

Worms

Anopheles gambiae Syndapin

Droso phila melanogaster Syndapin

Pro

tosto

mia

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Fugu ru bripes Syndapin IV

Deuterostomia Vertebrates

Sus scro fa Syndapin IIBos taurus Syndapin II

Icta lurus punctatus Syndapin IIDanio rerio Syndapin II

Xenopus laevis Syndapin I

Danio rerio Syndapin I

Danio rerio Syndapin IV

Sus scro fa Syndapin III

Xenopus laevis Syndapin III

Fugu ru bripes Syndapin III

Icta lurus punctatus Syndapin III

Danio rerio Syndapin V

Cyprinus car pio Syndapin V Oncor hynchus

myk iss Syndapin V

III

II

I

V


similar to those of syndapins in connecting vesicle fission withthe cytoskeleton (Kessels and Qualmann, 2002; Orth andMcNiven, 2003). Both bind to actin and dynamin throughindependent domains (Kessels et al., 2001; McNiven et al.,2000). By contrast, syndapins use their single SH3 domain forassociations with both N-WASP and dynamin. They musttherefore either switch between interacting with N-WASP anddynamin, or use bridging molecules or oligomerize to interactwith the two simultaneously (see below).

In common with the plasma membrane, Golgi membranesare associated with a specialized actin-spectrin cytoskeleton(Beck and Nelson, 1998; De Matteis and Morrow, 2000;Stamnes, 2002) that seems to support membrane topology andorganelle organization (Valderrama et al., 1998; di Campli etal., 1999) and might also be involved in membrane trafficking(Müsch et al., 2001; Valderrama et al., 2001; Fucini et al.,2002). Both dynamin and N-WASP localize to the trans-Golginetwork (TGN) and play a role in vesicle budding at Golgimembranes (Jones et al., 1998; Luna et al., 2002), and the F-actin-binding Abp1 (Kessels and Qualmann, 2002) has alsobeen reported to play a role in Golgi trafficking (Fucini et al.,2002). Recent data suggest that syndapins also associate withGolgi membranes. Interference with complexes of syndapin IIand dynamin II by antibodies or dominant-negative constructsstrongly inhibits budding from Golgi membranes (Kessels et

al., 2003) (M.M.K. and B.Q., unpublished). The involvementof actin polymerization in vesicle formation might thus not bea speciality of the plasma membrane but a more generalmechanism within the cell.

Syndapin oligomerization might physically link differentsyndapin interaction partnersAll syndapin isoforms contain stretches of amino acids thatare predicted to engage in coiled-coil interactions (Qualmannet al., 1999). Indeed, both homo-oligomers and hetero-oligomers composed of different syndapin isoforms can beobserved, and the coiled-coil-domain-containing N-terminusis sufficient for syndapin-syndapin interactions in vitro andin vivo (Qualmann et al., 2000a) (M.M.K. and B.Q.,unpublished). The hypothesis that syndapins oligomerize isfurthermore supported by yeast two-hybrid studies showingthat all isoforms can interact with each other (Modregger etal., 2000) and by in vitro work including gel filtration andsurface plasmon resonance analyses of the syndapin-relatedchicken focal adhesion protein FAP52 (Nikki et al., 2002b).As the SH3 domain of syndapins is not involved, sucholigomerization could create a multivalent platform to whichdifferent interaction partners of the syndapin SH3 domain areconnected (Figs 1 and 2).

Fig. 3.Unrooted phylogenetic tree of syndapins produced from aClustalW alignment of 36 syndapin sequences by the TreeTopphylogenetic tree reconstruction software (http://www.genebee.msu.su/services/phtree_reduced.html). Published syndapin sequences orconsensus sequences from as many expressed sequence tag (EST)clones as could be identified in the NCBI databases were used. Morethan 150 syndapin-related DNA sequences were analysed. Few ofthose have been described at the protein level (only some vertebratesyndapins and the antigens EG13 and EM13 from band worms). Thetree is based on an alignment of the first 120 residues of rat syndapinI with corresponding regions of all syndapin proteins and predictedproteins from DNA sequences in the databases. Parallel phylogenetictree constructions were performed with the first 210 residues (32sequences) and 305 residues (28 sequences), respectively. These gavevery similar results. The same is true for alignments with blunted N-termini. Note that the confidence levels of the branch points that havescores of 63-73% in the above analysis are enhanced to 82-99% inanalyses using longer sequences. Protostomia: parasitic band worms,Echinococcus granulosus(EG13, GI:158845) and Echinococcusmultilocularis(EM13, GI:158849); roundworms, Caenorhabditiselegans(GI:17567724, gene XI608); identified but not included (dueto degenerated DNA sequence or lack of N-terminus) were,Caenorhabditis briggsae(genome contig FPC4044) and a sequencefrom the most primitive plathelminthes, the turbellaria (Schmidteamediterranea; GI:21308965). Insects: Drosophila melanogaster,GI:28571784; Anopheles gambiae, overlapping ESTs (GI:31224233and GI:31224240) and new entry for assembled gene GI:21300122;Bombyx mori(domestic silk worm), GI:37662803, not included.Deuterostomia: there are extremely few sequence data for allorganisms originating from the basis of this line (hemichordata andechinodermata, such as starfish) and for the most primitive chordata(the tunicata, the copelata and the acrania). Fish and highervertebrates, however, were analysed. Fish: Fugu rubripes(fugu fish):syndapin I (SINFRUP00000064571 and FuguGenscan_5227),syndapin II (SINFRUP00000062952 and FuguGenscan_1173),syndapin III (SINFRUP00000059173), syndapin IV(FuguGenscan_14767) and syndapin V (FuguGenscan_30629);Danio rerio (zebra fish), syndapin I (GI:156355 and GI:17239474),

syndapin II (GI:31063171, GI:39660160, GI:6949740 andGI:16098827), syndapin III (GI:28279267), syndapin IV(GI:38647966 and GI:13104055) and syndapin V (GI:38554082,GI:38540910 and GI:23193087); Ictalurus punctatus(channel catfish), syndapin II(GI:40583787) and III (GI:40581408, GI:18646500and GI:33607133); Cyprinus carpio(carp), syndapin V(GI:37560134, GI:37557575 and GI:27491180) and Oncorhynchusmykiss(rainbow trout), syndapin V (GI:39964270, GI:29590006,GI:24697026 and GI:24681637). Syndapins from other fish, such asOryzias latipes(Japanese rice fish) were identified (GI:17373342 and17368378) but not included in the above analysis. Birds and frogs:Gallus gallus(chicken), syndapin I (GI:25737679 and GI:15085432,not included), syndapin II/FAP52 (GI:2217963); syndapin III(GI:25904662 and GI:25953223) and Xenopus laevis(Africanclawed frog), syndapin I (GI:31090847), X-PACSIN2/syndapin II(GI:11558503) and syndapin III, GI:27469860). Mammalia: Susscrofa(pig), syndapin II (GI:37854627), syndapin III (GI:40437003,GI:11075716 and GI:40437003), Bos taurus(cow), syndapin II(GI:9747526, GI:24332175 and GI:9601216), Canis familiaris(dog),syndapin II (GI:23699945 and GI:23699935, not included), syndapinIII (GI:34413292 and 23707795, not included). The sequences of thethree isoforms from rat, mouse and human included in thephylogenetic analyses have mostly been published, and these have inpart been studied at the protein level: Rattus norvegicus(rat),syndapin I (GI:4324451), syndapin II consensus sequence ofsyndapin IIaa, IIbb, IIab and IIba (GI:6651162, GI:6651168,GI:6651164, GI:6651166), syndapin III (GI:27702145 and M.M.K.and B.Q., unpublished, respectively); Mus musculus(mouse),syndapin isoform I called h74 or PACSIN (GI:2632077), PACSIN2(GI:19483912) and PACSIN3 (GI:13539689); Homo sapiens(man),PACSIN1 (GI:25955520), a consensus of the long PACSIN2 versionand a shorter syndapin II splice variant (GI:6005825 andGI:12053194) and PACSIN3 (GI:11127645). Note that the databaseentries for so-called syndapin-II-related proteins in Dictyosteliumdiscoideumrather represent a homologue of PSTPIP (GI:28828180)and a formin-binding protein 17 homologue (GI:21240669),respectively.

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Crosstalk between syndapins and signalling pathwaysSyndapins interact with several signalling moleculesdownstream of activated membrane receptors. These includethe mammalian Sos (for ‘son-of-sevenless’) protein(Qualmann et al., 2000a; Wasiak et al., 2001), which acts as aguanine nucleotide exchange factor (GEF) for the smallGTPases Ras and Rac (Scita et al., 1999) (Fig. 1). Ras isinvolved in growth factor signalling, whereas the mostprominent role of Rac is the regulation of actin cytoskeletondynamics. The interaction between mSos and syndapinis direct and relies on an intact SH3 domain.Coimmunoprecipitation and colocalization studies indicatethat syndapin and Sos interact in vivo (Qualmann et al., 2000a;Wasiak et al., 2001).

Syndapins also bind to the cytoplasmic tails of members ofthe ADAM family of metalloprotease disintegrins in vitro(Cousin et al., 2000; Howard et al., 1999; Mori et al., 2003).ADAM proteins are transmembrane proteins involved in cell-cell communication and proteolytic ectodomain shedding,which is required for a variety of developmental andmaturation processes (Schlöndorff and Blobel, 1999;McFarlane, 2003) (Fig. 1). Developmental alterations inducedby overexpression of ADAM13 can be rescued by co-overexpression of the syndapin II isoform X-PACSIN2 inXenopus (Cousin et al., 2000). This suggests some form ofnegative regulation of ADAMs by syndapins. However, Moriet al. have shown that syndapin III overexpression increases theectodomain shedding of heparin-binding epidermal growthfactor-like growth factor (HB-EGF) and that knocking downsyndapin III by RNA interference partially attenuates thisproteolysis, which is thought to involve ADAM12 (Mori et al.,2003).

Much less is known about the interaction of the SH3 domainof syndapins with the proline-rich cytoplasmic portion of theCD95/Fas/Apo-1 ligand CD95L (Ghadimi et al., 2002).CD95L is a 40 kDa type II transmembrane receptor thatbelongs to the tumour necrosis factor (TNF) family of deathfactors and induces apoptosis through the cell death receptorCD95 but has also been described as costimulatory receptor forT-cell activation in mice in vivo (Janssen et al., 2003).

It is tempting to speculate not only that syndapins do interactwith proteins involved in different signal transductionprocesses but also that syndapin function is controlledby signalling cascades, because syndapins containphosphorylation sites for protein kinase C and casein kinase 2.Recombinant mouse syndapin I can be phosphorylated by thesekinases in vitro (Plomann et al., 1998). Furthermore, an as-yet-uncharacterized signalling pathway regulated by inositolhexakisphosphate (InsP6) leads to the phosphorylation ofsyndapin I – a modification that seems to increase theassociation of glutathione-S-transferase (GST)-syndapin I withdynamin by a factor of 2-3 in vitro (Hilton et al., 2001).

The above observations represent promising starting pointsfor studying the crosstalk of syndapins with differentsignalling pathways. It will be exciting to examine whetherand to what extent the interaction of syndapins with mSoscorrelates with the endocytic and cytoskeletal functions ofsyndapins and to unravel the molecular details and thephysiological relevance of their interactions with thesignalling molecules mentioned.

Syndapin isoforms and their evolutionDatabase analyses reveal that syndapins only exist inmulticellular animals. Plants do not seem to contain syndapins;neither do single-celled eukaryotes such as Dictyosteliumdiscoideumand the different yeasts. On the basis of allcurrently available sequence information, it seems that theappearance of syndapins correlates with the arrival ofcoelomata, which are characterized by the presence of a bodycavity (coelom) and a gut system that spans the body from onepole (mouth) to the other (anus). Other new structures thatappeared at this stage were blood vessels, nephridiae and thebrain. It remains unclear whether porifera, cnidaria and/orctenophora contain syndapins because sequence data for theseanimals are generally not available; however, lower worms areknown to possess a syndapin gene (Fig. 3). We have identified(partial) syndapin-related sequences in the most primitiveplathelminthes, the turbellaria (not included in Fig. 3), as wellas parasitic band worms (Echinococcus). We have alsoidentified syndapins in roundworms, such as the nematodeCaenorhabditis elegans(Fig. 3). Because the genomes of C.elegans and Drosophila melanogastercontain a singlesyndapin gene, this seems a general property of protostomia.

By contrast, deuterostomia, which ultimately gave rise to thevertebrates, possess several syndapin isoforms. The geneduplications must have occurred as much as 400-440 millionyears ago because lower vertebrates contain all three syndapingenes known in mammalia. Our phylogenetic analyses suggestthe following evolutionary sequence: first, a syndapin I/IIancestor duplicated to give rise to syndapin III; then the formerduplicated again (Fig. 3). In line with this, we have found as-yet-undescribed syndapin I, II and III sequences not only inmammals but also in the segregated branch of highervertebrates formed by birds and reptiles. We have identified allthree isoforms in Gallus gallusas well as in Xenopus laevis(Fig. 3).

Interestingly, in fugu and zebra fish, we have identified fivesyndapin genes. Sequences from other fish support theexistence of the two additional groups of syndapin isoforms.The syndapin V group is clearly at the basis of all vertebratesyndapins. The data from our phylogenetic analysis could beinterpreted as indicating loss of these most ancient syndapinsduring the evolution of higher chordata and of life on land (Fig.3). The group of syndapin IV genes currently contains only twosequences suitable for phylogenetic analysis. We have termedthe partial Danio reriosyndapin sequence put in isoform groupI (shown in grey in Fig. 3) syndapin IV because alignments andphylogenetic analyses with the entire sequence suggest it to bea member of this subgroup. Further sequence data will berequired for a firmer analysis of the nature and phylogeneticrelationships between syndapin IV isoforms.

The picture is further complicated by the fact that notonly do different syndapin isoforms exist but also they arealternatively spliced. As in the case of the rat syndapin IIisoforms (Qualmann and Kelly, 2000), alternative splicing isalso evident from database sequences of the other isoforms inall vertebrates. (Because this predominantly affects the flexibleregion preceding the SH3 domain, this region was excludedfrom the sequence alignments used to generate thephylogenetic analysis shown in Fig. 3.)

The evolution of different isoforms and splice variants might



reflect a need for differential regulation and/or differentialaffinities for interacting molecules, but none of this has beenstudied in detail yet. Our current knowledge is therefore largelyrestricted to the differential distribution of syndapins inmammalian tissues (Table 1).

The syndapin III isoform is the least characterized and seemsmainly to occur in skeletal muscle and heart (Table 1). Indifferentiated C2F3 myotubes, syndapin III has a cytosolicimmunolabelling pattern (Modregger et al., 2000) (Fig. 4).

The syndapin II isoforms are expressed more ubiquitously(Table 1). Interestingly, the short and long splice variantsdisplay different tissue distributions (Qualmann and Kelly,2000). Xenopussyndapin II proteins are observed as earlyas the two-cell stage of development. Immunostaining ofsectioned embryos reveals expression in all cells of the threegerm layers with varying intensities (Cousin et al., 2000).

Syndapin I is mainly restricted to the brain (Table 1). Incommon with other proteins involved in membrane trafficking,such as clathrin and dynamin, which are present at 10-50-foldhigher concentrations in neuronal cells compared with non-

neuronal cells (Morris and Schmid, 1995), syndapin I isdetectable at high levels in the (adult) brain and accumulatesin synaptic compartments (Plomann et al., 1998; Qualmann etal., 1999; Kessels and Qualmann, 2002; Modregger et al.,2002) (Fig. 4). This might reflect the need for high-capacityand high-speed recycling of synaptic vesicles in neurons,which might be facilitated by the coupling of membranetrafficking to the cytoskeleton by syndapins (Gundelfinger etal., 2003).

Such coupling might also be highly important in non-neuronal, regulated secretory cells. Lacrimal acini cells, forexample, are the principal source of tear proteins that arereleased into nascent tear fluid at the apical plasma membrane.These cells perform massive exocytosis and compensatoryendocytosis. Membrane trafficking processes in polarized cellslike these must be tightly controlled in order to generate andmaintain polarity. Endocytosis and exocytosis at the apicalsurface of many epithelial cells has to occur within an elaboratecortical actin network. Interference with syndapin interactionsby introduction of the syndapin I or syndapin II SH3 domain

Table 1. Differential expression of syndapin isoformsAmino

Isoform acids RNA expression Protein expression References

I Mouse PACSIN 1 441 4.1 kb transcript in total adult brain; absent 50 kDa signal in total brain; absent in thymus, Plomann et al., 1998in total brain P10, thymus, liver, spleen, liver, spleen, kidney, heart and lungkidney and heart

Rat syndapin I 441 n.d.* 52 kDa signal in brain and low levels in PC12 Qualmann et al., 1999cells. Not detectable in liver, kidney, spleen, lung, heart and skeletal muscle

Human PACSIN 1 444 4.4 kb transcript in brain; lower in heart n.d. Sumoy et al., 2001and pancreas; absent in placenta, lung, liver, skeletal muscle and kidney

II Chicken FAP52 448 Two transcripts of 3.7 and 7.2 kb in all 63 kDa signal in all tissues tested (cardiac muscle, Meriläinen et al., tissues tested (gizzard, liver, cardiac muscle, brain, lung, intestine and chicken embryonic 1997skeletal muscle, brain, lung, intestine, kidney, heart fibroblast cells)skin, eye, chicken embryonic heart fibroblast cells)

Mouse PACSIN 2 486 3.5 kb transcript; ubiquitous (brain, thymus, 65 kDa signal in brain, thymus, liver, spleen, Ritter et al., 1999liver, spleen, kidney, heart, lung, muscle, kidney, heart, lung, muscle, testis and uterustestis, ovaries); highest levels in brain, heart,skeletal muscle and ovaries

Human PACSIN 2 486 3.4 kb transcript, ubiquitous (brain, heart, n.d. Ritter et al., 1999pancreas, placenta, lung, liver, skeletal muscle, kidney)

Rat syndapin II n.d. Ubiquitously expressed with a different tissue Qualmann and Kelly, IIbb 445 distribution for the short and long splice variants. 2000IIab 447 65 kDa signal preferentially in PC12 cells and heart; IIba 486 52 kDa signal in most tissues examined (brain, liver, Iiaa 488 kidney, spleen, heart, testis and skeletal muscle)

Frog X-PACSIN 2 477 n.d. Doublet of 65/72 kDa as early as two-cell stage Cousin et al., 2000In all three germ layers (with varying intensities); high levels in neural crest cells, lens, pronephros tissue and neural tube

III Mouse PACSIN 3 424 2.0 kb transcript in skeletal muscle and heart 48 kDa signal in skeletal muscle, heart and lung Modregger et al., (weak in kidney, uterus and brain); no signal in 2000; Sumoy et al., liver, spleen, testis and thymus 2001

Human PACSIN 3 424 2.0 kb transcript, enhanced in heart and n.d. Modregger et al., skeletal muscle; low levels rather ubiquitous 2000; Sumoy et al., (brain, heart, pancreas, placenta, lung, liver 2001skeletal muscle and kidney)

*n.d., not determined.

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significantly increases the F-actin content of these cells (daCosta et al., 2003) and blocks endocytosis at the stage ofclathrin-coated pit formation. The result is a remarkableaccumulation of components of the endocytic machinery at theapical plasma membrane and an increase in the number ofclathrin-coated structures. Both phenotypes depend on theArp2/3 complex, which suggests that the endocytosis block iscaused by extensive actin polymerization elicited by the

syndapin SH3 domains (da Costa et al., 2003). The syndapinisoform expressed in these specialized secretory cells is mainlythe long version of syndapin II. Syndapin II but not syndapinI accumulates together with other endocytic components, suchas clathrin and AP2, at the apical plasma membrane when thesecells are stimulated by secretagogues, such as carbachol, orincubated with cytoskeletal toxins (da Costa et al., 2003) (Fig.4). This suggests that the syndapin II isoform preferentiallyparticipates in apical endocytosis.

A further isoform-specific function might exist for the SH3-domain-mediated interaction with the huntingtin protein. Thisprotein, which contains extended polyglutamine stretches inpatients with Huntington’s disease, binds directly to the brain-specific syndapin I isoform in vitro but not to the other twoisoforms (Modregger et al., 2002). If this occurs in vivo,huntingtin would be the first SH3-domain-binding partner ofsyndapins that is specific for one isoform. Interestingly, thepresence of an extended polyglutamine stretch in the huntingtinprotein seems to enhance the binding of syndapin I inyeast two-hybrid analyses. Biochemical fractionation andimmunocytochemical analysis has suggested that syndapin isrelocalized in tissue from one Huntington’s disease patient(Modregger et al., 2002). Huntingtin can associate with aplethora of factors involved in clathrin-mediated endocytosis,including α-adaptin, huntingtin-interacting protein 1 (HIP1),HIP1-related protein (HIP1R), endophilin and syndapin. Allof these interactions are modulated by the length of thepolyglutamine repeat (Harjes and Wanker, 2003). It thus seemspossible that defects in membrane trafficking triggered byextended polyglutamine stretches participate in the pathologyof Huntington’s disease.

Finally, syndapins have very recently been shown to interactwith EHD (eps15-homology domain) proteins (Braun et al.,2004), which are implicated in endocytic vesicle formationand/or recycling (Grant et al., 2001; Lin et al., 2001; Guilhermeet al., 2004). The interaction is mediated by the syndapin NPFmotifs and the highly conserved EH domain present in all fourEHD isoforms (Braun et al., 2004). All syndapin III proteinsknown thus far (Fig. 3) lack NPFs; the interaction is thereforespecific for the phylogenetically younger syndapin I and IIisoforms.

PerspectivesCoordination of the cytoskeleton with membrane traffickingprocesses at various sites within cells is a complex task that isprobably very important for cellular organization and for thefunction of individual cells within the context of complextissues and organs. Studying syndapins as molecularcomponents that can link the actin cytoskeleton with vesicleformation processes at the plasma membrane and at the Golgiapparatus will continue to provide us with a valuable researchavenue that leads to a deeper understanding of the individualprocesses, their interconnection and the physiologicalprocesses within multicellular organisms that rely on them.

Syndapin functions are probably similar to those of othermolecular links between actin and membrane trafficking, suchas the F-actin-binding proteins Abp1 and cortactin. In commonwith syndapins, Abp1 and cortactin associate with dynaminand thus appear to play a role in endocytosis (Kessels et al.,2001; Mise-Omata et al., 2003; Cao et al., 2003). A plausible


Fig. 4. Immunofluorescence microscopy images of syndapinisoforms I, II and III in different cell types. (A) Rat hippocampalneurons in culture immunostained for syndapin I (green) and for thesynaptic vesicle marker synaptophysin (red); merged confocal image,colocalization appears yellow. Reproduced with permission from TheAmerican Society for Cell Biology (Qualmann et al., 1999).(B,C) Isolated rabbit lacrimal acini (lumen marked by asterisks)treated with the cytoskeletal toxin cytochalasin D display anaccumulation of syndapin II (B) close to the actin-rich (C) apicalmembrane; confocal images. Reproduced with permission from TheAmerican Society for Cell Biology (da Costa et al., 2003).(D) Differentiated C2F3 myotubes immunostained for the syndapinIII isoform (image kindly provided by M. Plomann).


hypothesis is that the three proteins work together in sequentialsteps of a self-accelerating process of actin polymerization atsites of endocytosis. Syndapins seem to couple de novo actinnucleation at the vesicle membrane to the dynamin-mediatedvesicle fission step by activating the Arp2/3 complex activatorN-WASP, which has been localized to actin-membraneinterfaces. By contrast, cortactin has the ability to activate theArp2/3 complex directly (Olazabal and Machesky, 2001). Asa starting point for filament polymerization, cortactin might useexisting actin fibres, such as those created by syndapin, N-WASP and the Arp2/3 complex at the vesicle surface. Thiswould create branched dynamic actin structures that could becoupled to the vesicle fission machinery by both cortactin andAbp1 (Orth and McNiven, 2003). Indeed, the endocytic role ofAbp1 depends on its ability to bind to F-actin (Kessels et al.,2001). Furthermore, Abp1 and cortactin might help organizeactin tails to propel vesicles.

To identify how syndapins coordinate actin nucleation withthe fission event, it will be important to dissect syndapinregulation and to study the interactions with the signallingcomponents in more detail. Furthermore, studies comparingvarious syndapins in terms of (different) sets of interactingproteins, their means of regulation and their functions indifferent cell systems should shed light on the functions ofindividual syndapin isoforms and splice variants. This mightreveal how the cellular functions of syndapins can be fine-tuned and adapted to cope with the different needs of variouscell types. Comparing the specialized roles of the individualsyndapins in different cell types and studying gene-knockoutmodels, particularly in worms and insects, which posses onlya single syndapin gene, will also highlight common principlesin vesicle trafficking from different cellular membranes suchas the plasma membrane and Golgi membranes.

We thank Sarah Hamm-Alvarez and Markus Plomann for theirimmunofluorescence images, and we apologize to those whose workcould not be cited and covered in more detail because of spacelimitations. This work was supported by fellowships from theDeutsche Forschungsgemeinschaft (Qu116/2-3; Qu116/3-1) and theKultusministerium Land Sachsen-Anhalt (LSA 3451A/0502M).

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