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CELL SCIENCE AT A GLANCE

Tetraspanins at a glance

Stephanie Charrin1,2, Stephanie Jouannet1,2, Claude Boucheix1,2 and Eric Rubinstein1,2,*

ABSTRACT

Tetraspanins are a family of proteins with four transmembrane

domains that play a role in many aspects of cell biology and

physiology; they are also used by several pathogens for infection

and regulate cancer progression. Many tetraspanins associate

specifically and directly with a limited number of proteins, and also

with other tetraspanins, thereby generating a hierarchical network of

interactions. Through these interactions, tetraspanins are believed

to have a role in cell and membrane compartmentalization. In

this Cell Science at a Glance article and the accompanying

poster, we describe the basic principles underlying tetraspanin-

based assemblies and highlight examples of how tetraspanins

regulate the trafficking and function of their partner proteins that

are required for the normal development and function of several

organs, including, in humans, the eye, the kidney and the immune

system.

KEY WORDS: Tetraspanin, Integrin, Membrane

compartmentalization

IntroductionEfforts in cloning membrane antigens in the early 1990s led to the

cloning of tetraspanin 8 (TSPAN8) and of several clusters of

differentiation (CD) proteins, including CD63, CD53, CD37,

CD81 and CD9, a then new family of molecules of unknown

function (Boucheix and Rubinstein, 2001; Charrin et al., 2009a;

Hemler, 2003; Maecker et al., 1997; Yanez-Mo et al., 2009).

These proteins are expressed by all metazoans, with 33

members in mammals, 37 in Drosophila melanogaster and 20

in Caenorhabditis elegans (Huang et al., 2005). Plants also

express tetraspanin-like proteins. Tetraspanins have been shown

to play a role in many biological processes. Some, such as CD81

and CD37, have an important function in the immune system

1Inserm, U1004, F-94807, Villejuif, France. 2Universite Paris-Sud, Institut AndreLwoff, F-94807 Villejuif, France.

*Author for correspondence (eric.rubinstein@inserm.fr)

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(Jones et al., 2011; Levy, 2014; van Spriel, 2011). Otherscontribute to the infection by several pathogens (Box 1). Several

tetraspanins gained special attention because they regulate theprogression of various types of cancers or the formation ofmetastasis, perhaps as a consequence of their ability to regulatethe migration of cancer cells and their ability to invade the

surrounding matrix (Boucheix and Rubinstein, 2001; Hemler,2014; Zoller, 2009). In particular, several experimental models,including genetic models of cancers (Copeland et al., 2013a;

Copeland et al., 2013b; Deng et al., 2012), as well as clinical data,suggest that CD9 and CD82 are suppressors of metastasis,whereas CD151 and TSPAN8 promote metastasis (Hemler, 2014;

Romanska and Berditchevski, 2011; Zoller, 2009).Despite considerable advances in the understanding of their

physiological importance and their implications in several

pathologies, the molecular function of tetraspanin proteinsremains hypothetical. An increasing number of data, nowsupported by genetic evidence, show the key role oftetraspanins in regulating the trafficking and function of other

membrane proteins and they might do so by regulating themembrane compartmentalization of the molecules they associatewith.

Structure of tetraspaninsTetraspanins are small integral proteins that protrude 3–5 nm

from the membrane; they contain four transmembrane domainsthat delineate two extracellular regions of unequal size and three

short intracellular regions (see Poster) (Boucheix and Rubinstein,2001; Charrin et al., 2009a; Hemler, 2003; Yanez-Mo et al.,

2009). Structural analysis of the large extracellular domain (LED)of CD81 (Kitadokoro et al., 2001; Rajesh et al., 2012) andmolecular modelling (Seigneuret et al., 2001) have providedessential insights into the structure of this domain, which

appears to comprise a structurally variable domain inserted intoa structurally conserved domain comprising three a helices, oneof which immediately follows transmembrane region (TM)3 and

another of which precedes TM4. This domain is stabilized bydisulfide bonds involving four, six or eight cysteine residues.Modelling of the entire molecule (Seigneuret, 2006) suggests that

the small extracellular domain fits into a groove of the LED andthat the transmembrane regions form a coil-coiled structure,which is stabilized by hydrogen bonds involving the polar

residues. Tetraspanins are glycosylated to variable extents and arepost-translationally modified through the addition of palmitatemoieties on their intracellular cysteine residues (Berditchevskiet al., 2002; Charrin et al., 2002; Yang et al., 2002).

Of webs and membrane domainsTetraspanins interact with one another and with a largely common

repertoire of other transmembrane molecules, including integrinsand other adhesion receptors, immunoglobulin (Ig)-domain-containing factors, growth factor and cytokine receptors and

ectoenzymes (Boucheix and Rubinstein, 2001; Charrin et al.,2009a; Hemler, 2003; Yanez-Mo et al., 2009). Most of theseinteractions are indirect and have been mostly identified by using

co-immunoprecipitation with mild detergents that preservetetraspanin–tetraspanin interactions. The use of some detergentshas allowed the identification of directly interacting smalltetraspanin–partner complexes (Serru et al., 1999; Yauch et al.,

1998). This suggests that various primary complexes assemblethrough tetraspanin–tetraspanin interactions to form a dynamicnetwork of secondary interactions, we have referred previously to

this as the tetraspanin ‘web’ (Boucheix and Rubinstein, 2001) (seePoster). Importantly, urothelial plaques assemble on a tetraspanin–partner pair basis (Box 2). Tetraspanin–partner interactions have

been shown to involve the LED or transmembrane domains of thetetraspanin (Charrin et al., 2009a; Hemler, 2003; Yanez-Mo et al.,2009). Tetraspanin–tetraspanin interactions are regulated by lipids,including the palmitate moieties that are attached to tetraspanins,

membrane cholesterol and gangliosides (Berditchevski et al., 2002;Charrin et al., 2002; Charrin et al., 2003c; Odintsova et al., 2006;Yang et al., 2002). In addition, similar to components of lipid raft

microdomains, tetraspanins float to some extent in sucrosegradients after cell lysis (Berditchevski et al., 2002; Charrinet al., 2002; Claas et al., 2001), indicating that they associate

with detergent-resistant membranes. These findings have ledto the idea that tetraspanins assemble tetraspanin-enrichedmicrodomains (Berditchevski et al., 2002; Hemler, 2003).

Importantly, many data suggest that tetraspanins and rafts aredifferent entities (Charrin et al., 2009a; Hemler, 2003; Yanez-Moet al., 2009), which does not exclude the possibility that thesetwo types of membrane ‘structures’ can cooperate in particular

circumstances (Hogue et al., 2011).Several predictions can be made from the tetraspanin–partner

pair model. First, a tetraspanin connects its partner proteins to

other tetraspanins and their partners, a prediction that has beenvalidated for interactions between CD151 and integrins, andbetween CD9 and CD9 partner 1 (CD9P-1, also known as EWI-F

and PTGFRN) (Berditchevski et al., 2002; Charrin et al., 2003b;

Box 1. Tetraspanins in infectious diseases

Tetraspanins participate in the infection by several viruses, bacteriaor protozoa (Monk and Partridge, 2012). Many studies haveunravelled a role of tetraspanins in the entry of the pathogen. Forexample, CD81 and TSPAN9 have been recently identified ingenome-wide small interfering (si)RNA screens for host factors thatregulate the early steps of influenza virus and Alphavirus infection,respectively (Karlas et al., 2010; Konig et al., 2010; Ooi et al.,2013); it has also been suggested that CD81 and TSPAN9 regulateviral fusion in endosomes (He et al., 2013; Ooi et al., 2013). CD151has been shown to contribute to the endocytosis of thehuman papillomavirus (HPV)16, an etiologic agent for cervicalcancers (Scheffer et al., 2013). This function of CD151 requires aninteraction with integrins (integrin a6 has previously been identifiedas a receptor for HPV) and its palmitoylation (and so presumablyinteraction with other tetraspanins). Of special interest is CD81,which plays a specific role in two major human diseases. It isessential for the entry of the malaria parasite into hepatocytes, thefirst stage of the Plasmodium life cycle in mammals and is anessential receptor for the envelope protein E2 of the hepatitis Cvirus (HCV) (Dubuisson et al., 2008; Silvie et al., 2003). Theinteraction of CD81 with claudin-1, another entry factor, might playa key role in the infection by HCV (Harris et al., 2008). Interestingly,the ability of CD81 to support Plasmodium and HCV infections isnegatively regulated by CD9P-1 and a cleavage fragment of EWI-2, respectively (Charrin et al., 2009b; Rocha-Perugini et al., 2008).The involvement of tetraspanins in HIV infection has been

extensively studied. Several tetraspanins are recruited to thebudding site of HIV (Krementsov et al., 2010) and are incorporatedinto the viral membrane (Monk and Partridge, 2012). Furthermore,the presence of tetraspanins at exit sites and in viral particlesinhibits, to some extent, virus infection at a post-attachment step,as well as the formation of syncytium induced by the virus (Gordon-Alonso et al., 2006; Krementsov et al., 2009; Sato et al., 2008).

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Takeda et al., 2007). Second, tetraspanins can be expected to have

a specific role in the regulation of their partners and a non-specific role in contributing to the stoichiometry of all themolecular interactions inside of the tetraspanin web.

Dynamics inside of the tetraspanin webThe dynamic behaviour of tetraspanins has been addressed inseveral recent studies. For instance, analysis of CD9 at the single

molecule level using total internal reflection fluorescence (TIRF)microscopy revealed that CD9, at the cell basal surface, cyclesbetween tetraspanin-enriched areas (TEAs) and the rest of the

membrane (Espenel et al., 2008). In these enriched areas, it has aconfined motion and displays a lower diffusion rate, suggestingthat these areas correspond to regions of the membrane to which

several molecules are confined at the same time. Elsewhere, CD9molecules have a Brownian motion. Importantly, TEAs are notequivalent to tetraspanin-enriched microdomains becausetetraspanin–tetraspanin interactions occur inside and outside of

these structures.The function of TEAs, which also contain several tetraspanin-

associated molecules, is unknown, but TEAs are reminiscent of

the endothelial adhesive platforms that form upon the interactionof leucocytes with endothelial cells into which intercellular

adhesion molecule 1 (ICAM1) and vascular cell adhesionmolecule 1 (VCAM1) are recruited; several tetraspanins,

including CD9 and CD151, are also recruited and display alower diffusion rate in these platforms (Barreiro et al., 2008).Similarly, the human immunodeficiency virus (HIV) gag proteinrecruits several tetraspanins at the virus budding site, leading to a

confinement of tetraspanins at these sites (Krementsov et al.,2010). Although tetraspanins and their partners can mutuallyaffect the diffusion of each other (Mattila et al., 2013; Potel et al.,

2013; Yang et al., 2012), it remains to be determined whethertetraspanins actually recruit their partner proteins into TEAs.

The intracellular tails of tetraspaninsSeveral intracellular proteins have been shown to co-immunoprecipitate with tetraspanins. However, in many cases,

for example for phosphatidylinositol 4-kinase (PtdIns4) kinase,activated conventional protein kinase C (PKC) or heterotrimericG proteins (Berditchevski et al., 1997; Le Naour et al., 2006;Little et al., 2004; Zhang et al., 2001), it is unknown whether

these proteins associate directly with a particular tetraspanin.Consistent with the tetraspanin–partner pair model, the PKC-dependent phosphorylation of integrin b4 on serine residues is

regulated by CD151 (Deng et al., 2012; Li et al., 2013). Bycontrast, the C-terminus of CD63 contains a tyrosine-basedinternalization motif that confers a fast rate of endocytosis, as

well as a prominent localization in late endosomes, which ismediated through an interaction with adaptor protein complexes(Berditchevski and Odintsova, 2007; Pols and Klumperman,

2009). This motif overlaps with a PDZ-binding motif that isinvolved in the binding of CD63 to syntenin, suggesting that,because syntenin has two PDZ domains, some interactions withinthe tetraspanin web might occur through this (or other) PDZ

proteins (Latysheva et al., 2006). Other tetraspanins also havePDZ-binding or potential internalization motifs, but their roles arenot as clear as those in CD63 (Berditchevski and Odintsova,

2007). Furthermore, other proteins, such as ezrin and the smallGTPase Rac1, have been suggested to interact with the C-terminal domain of CD81 (Sala-Valdes et al., 2006; Tejera et al.,

2013), providing a potential link between tetraspanins and thecytoskeleton.

Tetraspanins that regulate the trafficking of theirpartner proteinsThere are a few examples of tetraspanins of which the primaryfunction is to control the trafficking of their partner proteins. For

instance, a key function of CD63, consistent with the presence ofa tyrosine-based internalization motif, might be to facilitate theinternalization of discrete partner proteins and/or their targeting

to late endocytic organelles (Pols and Klumperman, 2009) (seePoster). Such proteins include synaptotagmin 7, a protein thatregulates lysosome exocytosis and membrane repair (Flannery

et al., 2010); the stromal cell-derived factor 1 (SDF1) and HIVreceptor C-X-C chemokine receptor type 4 (CXCR4) (Yoshidaet al., 2008); H+/K+ ATPases (Codina et al., 2005; Duffield et al.,2003); and PMEL17 (also known as PMEL), which is involved in

melanogenesis (van Niel et al., 2011). The Drosophila tetraspaninSunglasses (Tsp42Ej) is another example; it is predominantlyexpressed in the eye and is enriched in lysosomes where it

associates with the G-protein-coupled light receptor rhodopsinand regulates its trafficking to lysosomes for degradation. Fliesthat lack Sunglasses have an impairment in this process that

results in light-dependent retinal degeneration (Xu et al., 2004).

Box 2. Specialized tetraspanins

The tetraspanin uroplakins UPIa and UPIb (also known as UPK1Aand UPK1B, respectively) together with two type I integralmembrane proteins, UPII and UPIII (also known as UPK2 andUPK3A, respectively), are the major constituents of ‘urothelialplaques’, hexagonally packed 16-nm uroplakin particles that coverthe urothelial apical surface. Consistent with the tetraspanin–partner pair model, UPIa and UPIb associate directly with UPII andUPIII, respectively, and are required for the ER exit of their partners(Wu et al., 2009). The 16-nm particles can be resolved into sixidentical dumbbell-shaped subunits, each comprising an inner andan outer subdomain, which are occupied by one UPIa–UPIIheterodimer and one UPIb–UPIII heterodimer, respectively (Minet al., 2006). Mice that lack the uroplakins UPII and/or UPIII show aloss of apical urothelial plaques and a compromised barrierfunction of the urothelium. Of note, UPIb serves as the receptorfor the uropathogenic type 1-fimbriated E. coli (Wu et al., 2009).The tetraspanins peripherin/RDS (also known as PRPH2) and

ROM-1 are two related tetraspanins of the photoreceptor outersegment, which comprises hundreds of stacked membranous disksand is involved in the transduction of light signals into a gradedmembrane potential. These tetraspanins are highly expressed at therim of the disks. Mutations in peripherin/RDS are responsible for abroad range of progressive human retinal diseases and, in themouse, yield dysmorphic outer segments in heterozygous animalsand a lack of outer segments in homozygous animals (Goldberg,2006). Peripherin/RDS forms homo-oligomers and hetero-oligomerswith ROM-1, which polymerize through intermolecular disulfidebonds involving a seventh cysteine residue that is uniquely present,among tetraspanins, in peripherin/RDS and ROM-1 (Loewen andMolday, 2000). With more than 50 residues, the C-terminalcytoplasmic domains of peripherin/RDS and ROM-1 are, amongtetraspanins, exceptionally long. This domain in peripherin/RDS hasbeen suggested to have a fusogenic activity (Boesze-Battaglia et al.,2003) and was recently shown to contain an amphiphatic helix that isable to generate membrane curvature (Khattree et al., 2013),suggesting that there is a direct role of peripherin/RDS in generatingthe high-curvature bend of the disk rim.

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Tetraspanins also sometimes (but not always) control thetrafficking of their partner proteins in the secretory pathway (see

Poster). For example, the metalloproteinase ADAM10 is thepartner protein of the tetraspanins that have eight cysteineresidues in their LED (TSPAN5, TSPAN10, TSPAN14,TSPAN15, TSPAN17 and TSPAN33), referred to collectively

as the TspanC8 subgroup. TspanC8 proteins regulate the exit ofADAM10 from the endoplasmic reticulum (ER) and differentiallycontrol its targeting to either late endosomes or to the plasma

membrane (Dornier et al., 2012; Haining et al., 2012; Prox et al.,2012). Consistent with the key role of ADAM10 in mediating thecleavage of Notch and thus the release of its cytoplasmic domain,

which is a transcription cofactor (Hori et al., 2013), certainTspanC8 proteins regulate Notch signalling. Importantly, theTspanC8 proteins are among the few mammalian tetraspanins

that have apparent orthologues in D. melanogaster and C.

elegans, which also regulate Notch signalling (Dornier et al.,2012; Dunn et al., 2010). These findings clearly show thatspecific tetraspanins can influence a signalling pathway by

regulating the cell surface expression of their partner protein.Another example is CD81, which regulates the trafficking of

CD19 along the secretory pathway (Shoham et al., 2003). A

mutation in CD81 has recently been shown to be the cause of theimmunodeficiency of a child displaying a defective humoralresponse (van Zelm et al., 2010). This mutation causes a strong

reduction in the surface levels of CD19, an essential co-stimulatory molecule of lymphoid B cells and a well-characterized CD81 partner, and consequently impaired

signalling upon B-cell receptor stimulation. In addition, CD81can contribute to the activation of B cells through theorganization of CD19 nanoclusters (Mattila et al., 2013). CD81in the mouse also facilitates cognate B-cell–T-cell interactions

with an impact on the polarization of helper T cells towards the Thelper type 2 profile, which play an essential role in the humoralresponse (Deng et al., 2002). In this regard, CD81 is concentrated

at the immune synapse during B-cell–T-cell cognate interactionsand regulates the timing of the maturation of the synapse andsignalling in T cells (Rocha-Perugini et al., 2013). Whether this is

directly linked to the ability of CD81 and other tetraspanins tointeract with CD3, CD4, CD8 or major histocompatibilitycomplex (MHC) class II molecules remains to be determined(Boucheix and Rubinstein, 2001; Levy, 2014).

Regulation of membrane-anchored enzymes and growthfactor signallingTetraspanins regulate the function of a diverse range ofmembrane proteins. Two recent studies combining biochemicaland genetic analyses have highlighted the ability of tetraspanins

to regulate the function of associated oligomeric complexeswithout apparent influence on the trafficking of their partner (seePoster). The first study showed that the C. elegans tetraspanin

TSP-15 associates with the transmembrane proteins of the dualoxidase (DUOX) pathway (BLI-3 and DOXA-1) and cruciallyregulates H2O2 production, which is required for collagen cross-linking through tyrosyl residues. As a consequence, worms that

lack TSP-15 have an abnormal cuticle, the exoskeleton of theworm (Moribe et al., 2012; Moribe et al., 2004). In the secondexample, it was found that TSPAN12, but not other tetraspanins,

enhances b-catenin signalling in response to norrin (encoded byNDP) by promoting the multimerization of its receptor complex,comprising Frizzled-4 and low-density lipoprotein-related protein

5 (Lrp5) co-receptor (Junge et al., 2009). The functional

relevance of signalling between TSPAN12 and norrin isstrengthened by the finding that alterations in TSPAN12, NDP,

FZD4 and LRP5 genes cause similar vascular retinal defectsleading to familial exudative vitreoretinopathy in humans (Yeet al., 2010).

Among the membrane proteins that are functionally regulated

by tetraspanins, there are several membrane-bound proteases,including ADAM10, ADAM17, MT-MMP1 (also known asMMP14) and the urokinase-type plasminogen activator

(Arduise et al., 2008; Bass et al., 2005; Gutierrez-Lopezet al., 2011; Lafleur et al., 2009; Shiomi et al., 2005; Xu et al.,2009; Yanez-Mo et al., 2008), and several membrane-anchored

growth factor precursors, such as transforming growth factor a(TGFa) or heparin-binding EGF-like growth factor (HBEGF)(Higashiyama et al., 1995; Imhof et al., 2008). In addition,

several tetraspanins, in particular CD9, CD82 and CD151,associate with and/or modulate downstream signalling oftyrosine kinase receptors, including epithelial growth factorreceptor (EGFR), ERBB2, c-MET and vascular endothelial

growth factor receptor (VEGFR)-3 (also known as FLT4)(Franco et al., 2010; Iwasaki et al., 2013; Murayama et al.,2008; Novitskaya et al., 2014; Odintsova et al., 2003; Sridhar

and Miranti, 2006; Takahashi et al., 2007). However, so far,there is no evidence that a particular tetraspanin associatesdirectly with these receptors. By contrast, several studies

suggest that the underlying mechanisms, at least for CD82,might be indirect (Odintsova et al., 2006) and that the effectof CD151 on the signalling of these receptors might be a

consequence of a functional or physical coupling withlaminin-binding integrins (Franco et al., 2010; Novitskayaet al., 2014).

The regulation of integrin function by CD151 and othertetraspanins is important in adhesion strengtheningTetraspanins have long been known to associate with various

integrins. Among these, the laminin-binding integrins a3b1, a6b1and a6b4 directly associate with CD151 (Serru et al., 1999;Yauch et al., 1998) (see Poster). In general, CD151 only

minimally affects adhesion or spreading on matrix proteins towhich these integrins bind. Instead, CD151 has been shownto regulate the activation of several signalling moleculesdownstream of these integrins, including Akt, Src, focal

adhesion kinase (FAK), p130CAS (also known as BCAR1),paxillin and Rho family GTPases (Hong et al., 2012; Takedaet al., 2007; Yamada et al., 2008; Yang et al., 2008). Importantly,

the influence of CD151 on the activation of these moleculesappears to depend on the cellular context, suggesting that CD151does not function as an adaptor molecule for a particular

signalling pathway. There is also no indication that CD151could regulate the binding of soluble laminins to cells. Bycontrast, several studies point to a role of CD151 in regulating

adhesion strengthening (Lammerding et al., 2003; Nishiuchiet al., 2005; Sachs et al., 2012), a dynamic process that followsintegrin-mediated adhesion and involves receptor clustering andinteractions with cytoskeletal, structural and signalling elements

(Puklin-Faucher and Sheetz, 2009).Loss of CD151 has a strong effect in vivo, both in mouse and

human. Particularly dramatic is kidney failure due to glomerular

alterations (Baleato et al., 2008; Karamatic Crew et al., 2004;Sachs et al., 2006). Importantly, specific deletion of integrin a3b1in podocytes causes a similar phenotype (Sachs et al., 2012). The

abnormalities that are observed in the absence of CD151 worsen

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with age and are influenced by blood pressure, consistent with adefect in adhesion strengthening in podocytes (Sachs et al., 2012).

CD151 can also regulate adhesion strengthening followingengagement of the platelet fibrinogen receptor, and integrinaiibb3 (Lau et al., 2004) and the tetraspanins CD81 and CD37regulate adhesion of lymphoid B cells to integrin a4b1 ligands

under flow (Feigelson et al., 2003; van Spriel et al., 2012).The impaired activity of integrin a4b1 that is observed in CD37-null B cells is associated with a change in the cell membrane

distribution of the integrin and could be the cause of theimpairment of Akt-dependent survival of long-lived antibody-secreting cells, which, in turn, leads to impaired IgG1 production

in response to T-cell-dependent antigens (van Spriel et al.,2012).

In contrast to many other tetraspanins, CD151 expression

has an impact on the growth of primary tumours or tumouronset (Deng et al., 2012; Hemler, 2014; Romanska andBerditchevski, 2011; Zoller, 2009), and this might be owing toits regulation of integrin function. For example, in a genetic

model of breast cancer, phosphorylation of FAK has beenshown to be diminished in the tumours of CD151-null animals,suggesting a potential defect in integrin signalling. Additionally,

data suggest that the reduced susceptibility to two-stage skincarcinogenesis of CD151-null mice is linked to the ability ofCD151 to regulate integrins a6b4 or a3b1 (Li et al., 2013; Sachs

et al., 2014).

CD9 and CD81 in cell–cell fusion and other processes: anexample of cooperation between two tetraspaninsCD9 and CD81 are closely related tetraspanins, sharing 45%identity and two common partner proteins – the two related Ig-domain molecules EWI-F/CD9P-1 and EWI-2 (also known as

IGSF8) (Charrin et al., 2003a; Charrin et al., 2001; Stipp et al.,2001a; Stipp et al., 2001b). These two tetraspanins have beenshown to regulate several cell–cell fusion processes (see Poster).

Mouse eggs that lack CD9 are greatly deficient in their ability tofuse with sperm (Kaji et al., 2000; Le Naour et al., 2000; Miyadoet al., 2000), and this defect is attenuated by expression of CD81

(Kaji et al., 2002; Rubinstein et al., 2006). The shape of the eggmicrovilli are also altered in the absence of CD9 (Runge et al.,2007). Furthermore, CD9 and CD81 cooperate in macrophagefusion and muscle cell fusion, but as negative regulators of these

processes (Charrin et al., 2013; Takeda et al., 2003). Theincreased muscle cell fusion observed in their absence has beenrecapitulated by RNA interference (RNAi)-mediated depletion of

CD9P-1, pointing again to the importance of tetraspanin-associated molecules (Charrin et al., 2013). Finally, doubleCD9 CD81 knockout mice also display a number of pathologies

that are not, or only minimally, observed in single-mutant mice,such as pulmonary emphysema (a major pathological componentof chronic obstructive pulmonary disease) (Takeda et al., 2008),

osteopenia (Takeda et al., 2008) or defects of blood andlymphatic vessels (Iwasaki et al., 2013).

Conclusions and perspectivesRecent studies combining genetic and biochemical analyses havehighlighted the key roles of distinct tetraspanins in regulating thefunction and/or trafficking of their partner proteins at the cell

membrane. However, there are still a number of functions oftetraspanins for which molecular details are lacking. For example,how do CD9 and CD81 regulate cell–cell fusion? Why does

experimental modification of tetraspanin expression levels or the

use of antibodies against tetraspanin often induce membraneremodelling (Zhang and Huang, 2012)? How exactly do

tetraspanins regulate the immune response, besides the CD81-mediated regulation of CD19 expression? Why are tetraspaninsenriched in exosomes (Raposo and Stoorvogel, 2013)? It islikely that a more comprehensive knowledge of the molecular

organization of tetraspanins, and in particular the identification ofadditional tetraspanin–partner pairs, will help to resolve some ofthese questions. However, some tetraspanins might also play a

role by themselves, independently of any protein partner.The hypothesis that tetraspanins have a role in membrane

compartmentalization comes from their ability to interact with

many other membrane proteins and their ability to partition intothe light fractions of sucrose gradients. Although supported bymost of the recent data, this hypothesis still requires a formal

demonstration. Super-resolution microscopy techniques andanalysis of the dynamics of tetraspanins and of their partnerproteins under various conditions are likely to provide essentialnew insights into the function of these molecules, as these

techniques did for the field of lipid microdomains.

AcknowledgementsA short review as this one, concerning molecules with so diverse functionaleffects is inevitably incomplete. We apologize to all those colleagues whosestudies could not be covered due to space limitations. The original references canbe found in the different reviews we refer to. We would like to thank Olivier Silvie(Centre d’Immunologie et des Maladies Infectieuses, Paris, France) for criticalreading of the manuscript.

Competing interestsThe authors declare no competing interests.

FundingThe work of the authors has been supported by grants from Agence Nationalepour la Recherche; the Association pour la Recherche contre le Cancer;Nouvelles Recherches Biomedicales-Vaincre le Cancer; and Institut du Cancer etd’Immunogenetique.

Cell science at a glanceA high-resolution version of the poster is available for downloading in the onlineversion of this article at jcs.biologists.org. Individual poster panels are available asJPEG files athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154906/-/DC1.

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