British journal of pharmacology 2015 volume 172 part 7

Both models implicate the binding of S100A10 and AnxA2 in the regulation of the surface proteases and the fact that genetic deletion of either protein shows roles for both proteins in ma

Themed Section: Annexins VII Programme

EDITORIAL

‘Annexins’ themed section

R J Flower and M Perretti

William Harvey Research Institute

Correspondence

R J Flower, Centre forBiochemical Pharmacology, TheWilliam Harvey ResearchInstitute, Barts and The LondonSchool of Medicine, Queen MaryUniversity of London,

Charterhouse Square, London,EC1M 6BQ, UK E-mail:

r.j.flower@qmul.ac.uk

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The ‘annexins’ are an evolutionarily ancient family of

mono-meric proteins which are widely distributed throughout

eukaryotic phyla – specifically the animal, plant and fungal

kingdoms – but which are largely absent from prokaryotes

and yeasts A characteristic feature of the family is the

pres-ence of an ‘annexin core domain’ which generally comprises

four (occasionally more) repeating subunits of approximately

70 amino acids (the ‘annexin’ repeat) These subunits usually

contain characteristic ‘type 2′ calcium binding sites although

in some members of the family, these have been replaced

with other motifs

More than 150 annexins have been identified over 50

species Twelve proteins have been identified in humans;

these are conventionally referred to as annexin (Anx) A1-13

(the Anx-A12 gene is unassigned) The descriptor ‘A’ denotes

their vertebrate origin as opposed to insect, fungal, plant or

protist annexins, which are denoted by ‘B’, ‘C’, ‘D’ or ‘E’

respectively Most human annexins are thought to be derived

from a single ancestral gene (Anx-A13)

In addition to the characteristic core domain, individual

vertebrate annexins have a unique N-terminal domain of

variable length This harbours motifs that can recognize and

bind to other intracellular protein partners, such as those of

the S100 family, and often contains residues that can be

modified by post-translational processing, including

phos-phorylation The N-terminus is a rapidly-evolving

compo-nent of these molecules and is probably responsible for the

diversity of functions found within the family

Structurally, in the right intracellular milieu, free annexins

fold into a concaveα-helical disk Calcium binding sites on

the convex side facilitate the attachment of this conformer to

plasma membranes or other phospholipid containing

struc-tures The N-terminal domain lies buried in the concave

surface, but may ‘flip’ out in the presence of calcium making

it available for binding to other partners

Why are the annexins of interest to pharmacologists?

Whilst we are only now beginning to understand theirbiology, it is clear that these proteins are involved in a greatnumber of intracellular processes such as membrane traffick-ing and organization as well as, surprisingly, functioning asextracellular local hormones as well The observation (forexample) that extracellular Anx-A1 has striking anti-inflammatory properties, and that the pharmacophoreresides within a sequence in the N-terminal domain, wascertainly not an intuitive finding It seems, once again, thathaving developed a useful motif, evolutionary pressure hasadapted it again and again to fulfill other jobs There is agrowing list of pathologies – ‘annexinopathies’ – associatedwith defects in annexin structure or function

A further discovery of interest to the pharmacologicalcommunity was the demonstration that several drugs related

to the benzodiazepine or phenothiazine structure can bind tothe core domain of these molecules modifying their behavior.Whether this is relevant to the mechanism of action of any ofthese drugs remains to be seen

Progress in the annexin field is reviewed by periodic ings of the annexin community The 7thInternational Con-ference on Annexins was the most recent in this series It washeld on the Charterhouse Square Campus of St Barts and TheLondon School of Medicine, Queen Mary University ofLondon in September 2013 The event was organised andhosted by the William Harvey Research Institute and sup-ported in part by a grant from the British PharmacologicalSociety Some of the papers presented at this conference arecollected together here in this ‘virtual’ themed issue andspeak to the many roles for annexins

meet-In the first paper in this issue, Jones and his and leagues highlight the role of annexins as prominent mem-brane proteins in the trematode integument and discuss theidea that these could be used to develop an immunotherapy

col-for Schistsomiasis (Leow et al 2015) Turning to mammalian

systems, Rentero’s group investigate the role of Anx-A6 – an

unusual annexin with 8 as opposed to the more usual 4

‘annexin repeats’ – as a membrane scaffolding protein and

the implications of this for regulation of signal transduction

(Alvarez-Guaita et al 2015) Anx-A2 is one of those annexins

with confirmed extracellular as well as intracellular actions

(it can act as a cell surface receptor for tissue plasminogen

activator) and in the final paper, Dekker’s laboratory has used

a ‘toolbox’ of peptides to study the interaction of Anx-A2

with its principal binding partner (S100A10) and to

deter-mine how this modifies and regulates its properties (Liu et al.

2015)

We hope that the papers published in this themed section

will serve to stimulate interest in this protein family, which

we commend as a fertile area for future pharmacological

investigation

References

Alvarez-Guaita A, Vilà de Muga S, Owen DM, Williamson D,

Magenau A, García-Melero A et al (2015) Evidence for annexin

A6-dependent plasma membrane remodelling of lipid domains Br J

Pharmacol 172: 1677–1690

Leow CY, Willis C, Hofmann A, Jones MK (2015)

Structure–function analysis of apical membrane-associated

molecules of the tegument of schistosome parasites of humans:

prospects for identification of novel targets for parasite control Br J

D’Acquisto F, Perretti M, Flower RJ (2008) Annexin-A1: a pivotalregulator of the innate and adaptive immune systems Br JPharmacol 155 (2): 152–169

Gerke V, Creutz CE, Moss SE (2005) Annexins: linking Ca2+signalling to membrane dynamics Nature reviews Molecular cellbiology 6 (6): 449–461

Gerke V, Moss SE (1997) Annexins and membrane dynamics.Biochimica et biophysica acta 1357 (2): 129–154

Iglesias JM, Morgan RO, Jenkins NA, Copeland NG, Gilbert DJ,Fernandez MP (2002) Comparative genetics and evolution ofannexin A13 as the founder gene of vertebrate annexins Molecularbiology and evolution 19 (5): 608–618

Perretti M, D’Acquisto F (2009) Annexin A1 and glucocorticoids aseffectors of the resolution of inflammation Nature reviews.Immunology 9 (1): 62–70

Themed Section: Annexins VII Programme

targets for parasite control

Chiuan Yee Leow1,2,3, Charlene Willis2,4, Andreas Hofmann4,5and

Malcolm K Jones1

1School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia,

2Infectious Diseases, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia,

3Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia,

4Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland,

Australia, and5Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria,

Australia

Correspondence

Malcolm K Jones, School ofVeterinary Sciences, TheUniversity of Queensland,Gatton, Qld 4343, Australia

E-mail: m.jones@uq.edu.au; orAndreas Hofmann, StructuralChemistry Program, EskitisInstitute, Griffith University, N75Don Young Road, Nathan, Qld

4111, Australia E-mail:

a.hofmann@griffith.edu.au -

infection with one of five species of blood fluke belonging to the trematode genus Schistosoma Although there is one drug

available for treatment of affected individuals in clinics, or for mass administration in endemic regions, there is a need for newtherapies A prominent target organ of schistosomes, either for drug or vaccine development, is the peculiar epithelial

syncytium that forms the body wall (tegument) of this parasite This dynamic layer is maintained and organized by concertedactivity of a range of proteins, among which are the abundant tegumentary annexins In this review, we will outline advances

in structure–function analyses of these annexins, as a means to understanding tegument cell biology in host–parasite

interaction and their potential exploitation as targets for anti-schistosomiasis therapies

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Abbreviations

Anx, annexin; NTDs, neglected tropical diseases; RA, radiation attenuated; Sm, Schistosoma mansoni; TEMs,

tetraspanin-enriched microdomains; TSP, tetraspanin

Neglected tropical diseases (NTDs)

NTDs include some 17 lesser known chronic infections that

affect poor and disenfranchised people, primarily, but not

exclusively, in developing nations (Hotez et al., 2007; Hotez

and Fenwick, 2009) Chronic infections caused by NTDs lead

to many adverse outcomes in affected populations and

con-tribute substantially to human morbidity In addition to

microbial and protozoan diseases, NTDs include a number of

helminth infections, such as diseases caused by flatworm

parasites, notably schistosomiasis, echinococcosis and liver

fluke diseases, as well as roundworm parasites, such as the

major soil transmitted helminth infections (ascariasis,

trichu-riasis and hookworm diseases) Although no individual NTD

rivals the major infectious threats of HIV, malaria or

tubercu-losis in terms of global impact of disease, collectively, the

NTDs contribute substantially to morbidity throughout the

world (Engels and Savioli, 2006)

A number of factors present major challenges for the

development of new treatments for NTDs Firstly, NTDs are

chronic diseases, which may reside in affected people as

life-long infections Secondly, NTDs are not always associated

with human mortality and the burden of these diseases can

be subtle, hidden among such other measures of disease

burden as hindered development, poor cognitive function

and chronic ailments Thirdly, as stated, NTDs often affect the

poorest of the poor, people often unable to pay for medical

treatments, especially for chronic illnesses Hence, these

dis-eases receive less attention than other, immediately

life-threatening infectious diseases Lastly, poor development of

infrastructure systems in impoverished countries also

irre-versibly impacts on efficient drug distribution for the

treat-ment of these NTDs (Chimbari et al., 2004).

Among the NTDs of major interest is the suite of diseases

known as human schistosomiasis These diseases are caused

by infection with any of a number of species of the genus

Schistosoma, a taxon of platyhelminth trematodes,

com-monly known as blood flukes (and historically, known as the

agents of bilharzia) (Ross et al., 2002) Five species are the

main contributors to human schistosomiasis, Schistosoma

mansoni (Sm), S japonicum, S mekongi, S intercalatum and

S haematobium Transmission of the parasites to humans

takes place in freshwater, typically in regions of poor

sanita-tion where human excreta contaminate water bodies The egg

hatches in freshwater to liberate a larva, which searches for

and infects a species of snail Schistosomes, like other

trema-todes, display high host specificity for their snail host Thedistribution of a schistosome species is largely dependent onthe geographic distribution of its snail host

Schistosomes infect over 200 million people in mately 74 nations, the majority of which are in Sub-Saharan

approxi-Africa and the Middle East (Steinmann et al., 2006) Distinct

foci of schistosomiasis also occur in Asia (China, the pines, along the Mekong River and Indonesia), as well asSouth America (notably Brazil and some Caribbean Islands).Disability-adjusted life years lost to human schistosomiasis in

Philip-2010 were measured at 48/100 000, an increase of 20% on

estimates made in 1990 (Murray et al., 2013).

There is a distinct dichotomy in schistosomiasis in tion to the host responsiveness to various life stages On theone hand, the invasive larvae and adult parasites are largelyable to avoid immunosurveillance of the hosts To that effect,these parasites employ a series of strategies including rapiddevelopment, stealth-like host interfaces and immunosup-pression (Wilson, 2009) On the other hand, the active secre-tion of immunogenic molecules by eggs provokes an intense

rela-immune response (Burke et al., 2009) This phenomenon is

characterized by a strong granulocytic response around theegg in affected tissues that may lead to fibrosis, particularly inthe liver The intense response enables the escape of the eggsfrom the host The cellular infiltrate forces a schistosome eggacross the vascular endothelium and into tissues of luminalorgans, such as the intestinal lining, the bladder wall orgenital organs, driving the egg ultimately into the lumen,from which the egg is voided into the environment The bulk

of chronic disease in schistosomiasis is related to hostresponses against parasite eggs deposited in the blood vessels

surrounding the gut (Sm and S japonicum) or bladder and genital organs (S haematobium) (Ross et al., 2002) However,

it has proven more effective to direct control towards killingadult worms or the invasive larvae that establish infection sothat the deposition of eggs is stopped

Schistosomes belong to the Clade Lophotrochozoa of theKingdom Animalia The multicellular animals are monophy-

letic (Walker et al., 2011) and there are substantial similarities

among the many cellular, biochemical and molecular tations in different animal clades The search for effectivetreatments against schistosomiasis thus needs to exploit keymolecular and conformational differences between targetmolecules of these parasites and their hosts This reviewexplores work focused on the search for novel moleculartargets of therapeutics and prophylactics, and examines new

adap-Table of Links

TARGETS

This Table lists key protein targets in this document, which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org,

the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the

Concise Guide to PHARMACOLOGY 2013/14 (a,b,c Alexander et al., 2013a,b,c).

insights from studies of a primary site of host interaction, the

schistosome tegument Some annexins of schistosomes are

abundant molecules in the proteome of the schistosomes

These proteins are found in close association with the apical

membrane of the tegument of the parasites In view of their

abundance, distribution and distinctive structure, these

pro-teins are of interest, both as targets and as vehicles to

under-stand the dynamic nature of the apical membrane complex of

these parasites, a complex that is crucial for survival of the

parasites in their hosts

Treatments for schistosomiasis – drugs

There currently exist few drugs for treatment of

schistosomia-sis: praziquantel, metrifonate, oxamniquine and artemether

(Cioli et al., 1995; Ross et al., 2002; Bartley et al., 2008) All of

these drugs have proven useful for therapeutic treatment of

individuals in the clinic or of communities in mass drug

administration Of the four drugs, oxamniquine is only

effec-tive against schistosomiasis mansoni; resistance to this drug

by the parasite is known and the mechanism of resistance

elucidated (Valentim et al., 2013) Metrifonate is only

effec-tive against urinary schistosomiasis, caused by S

haemato-bium, and its use is hampered by a complex administration

schedule with multiple doses required over a 2 week period

Frequently, this therapy is met with a low rate of compliance

among patients Furthermore, the drug is labile in warm

climates and is thus less useful in field settings Combination

therapy using praziquantel and metrifonate has been

effec-tive for urinary schistosomiasis (Danso-Appiah et al., 2009).

Artemether is aβ-methyl ether derivative of artemisinin, a

compound derived from the sweet wormwood Artemesia

annua Artemisinin and its derivatives are highly effective

against haematophagous parasites, notably malaria, but they

have also proven effective against schistosome infection (Liu

et al., 2012) One recent report suggests that artemisinin is

acted upon by elemental iron in the iron-rich environment of

haematophagous parasites and the complex, in turn, inhibits

calcium transport (Shandilya et al., 2013) Concerns about

resistance to artemisinin and its derivatives by the more

insidious human disease of falciparum malaria has precluded

the use of artemether against schistosomiasis where the two

diseases are co-endemic (Bergquist et al., 2005; Utzinger et al.,

2007)

The current drug of choice for treatment of

schistosomia-sis is praziquantel This drug has been used in mass treatment

campaigns in many countries and remains a primary tool in

the war against the disease (Knopp et al., 2013) The mode of

action of praziquantel remains unknown, although recent

developments strongly suggest a role for the drug in calcium

homeostasis in the parasites and notably in calcium transport

complexes (Greenberg, 2005; You et al., 2013) The drug

remains highly effective for a wide range of flatworm diseases

of humans and domestic animals Praziquantel has been

deployed for mass drug administration in endemic regions

and has been successful in pushing the disease from high to

low endemicity (Geary, 2012) This major achievement has

been facilitated in part by reductions in costs associated with

manufacture of the drug, and the development of public–

private partnerships that have led to the distribution of the

drug to many impoverished communities where miasis is endemic

schistoso-Despite its high efficacy, praziquantel has limitations(Geary, 2012) The drug is only effective against adult orpre-adult forms (Greenberg, 2005) Furthermore, praziquan-tel confers no protection against subsequent infection andpeople may become reinfected within days of treatment (Ross

et al., 2002) Treatment failures for S mansoni and S tobium infections have been observed, and the presence of

haema-resistant strains has been demonstrated experimentally(Greenberg, 2013) Although widespread resistance to prazi-quantel has not been observed clinically, the application ofthe drug in mass treatment campaigns may result in newresistant forms emerging and new replacement drugs andformulations are needed (Geary, 2012)

Prevention of schistosomiasis – vaccines

Many experts within the schistosomiasis community arguethat continued application of a single drug, praziquantel, forsingle treatments and as a mass control strategy is problem-atic and not likely to lead to effective control of the disease.The alternative, a subunit vaccine, has thus been promoted as

an important alternative strategy for the control and

elimi-nation of schistosomiasis (Bergquist et al., 2008; McManus and Loukas, 2008; Loukas et al., 2011; Kupferschmidt, 2013).

Optimism for a vaccine rests on observations from the1970s on host responses to radiation-attenuated (RA) cer-

cariae in experimental infections (Bickle et al., 1979a,b) A

cercaria is the larval stage that penetrates human skin toinitiate infection This stage transforms rapidly in humanskin to become a host-adapted larva, the schistosomulum.This larva then follows a set pattern of migration and devel-opment over the following days and weeks, passing alongvasculature through the lung and liver In the liver, a maleparasite will mate with a female and carry her to mesenteric

or pelvic circulation, the final destination being parasitespecies specific (Wilson, 2009) It was shown that infection ofhumans with live, RA parasites led to strong protectionagainst subsequent challenge infections with normal cer-

cariae (Correa-Oliveira et al., 2000; Ribeiro de Jesus et al.,

2000) Vaccination of animal models with RA cercariae hasthus led to an adult worm burden reduction in experimental

schistosomiasis of 60–70% (Bickle et al., 1979a,b; Benedetti et al., 1991; Coulson et al., 1998; McManus, 1999; Dillon et al., 2008) The molecular mechanism of protection

Caulada-with RA is unclear; however, the immune response appears toresult from transcriptional suppression in the attenuated

parasites during the early stage of development (Dillon et al.,

2008) Transcriptional suppression in RA was observed for avariety of genes including those encoding tegument proteins,members of signalling pathways associated with GPCRs, neu-rotransmitters and cytoskeletal components The majorlessons learned from these studies are that parasite killing islargely dependent on host–parasite interaction during thehost establishment phase of the parasites, that is, within thefirst week after infection During this time, the cercaria under-goes an extensive remodelling of its surface body wall, the

tegument and becomes transcriptionally active for a series of

molecules associated with surface dynamics and nutrient

absorption (Gobert et al., 2009b), compared with the cercaria.

Indeed, some of the promising vaccine candidates come from

this tissue, and it seems that vaccine targeting of this layer is

crucial for parasite killing

Despite the high level of protection available with

radiation-attenuated vaccines, the unstable lifespan, delivery

problems and safety problems of these modified cercariae

makes them unsuitable for further development as a vaccine

(Bergquist et al., 2008) Therefore, efforts have been directed

to discover and identify suitable protective antigens from

schistosomes, leading to the development of recombinant

vaccines, DNA vaccines, peptide–epitope-based vaccines,

multivalent vaccines and chimeric vaccines (McManus and

Loukas, 2008)

Of the vaccines trialled, a number have been promoted

for human trials, including the Bilvax vaccine based on a

28 kDa S haematobium glutathione-S-transferase, which has

entered phase 3 clinical trials, and a S mansoni tetraspanin

(Sm-TSP-2) (Tran et al., 2006), which has entered phase 1 trials

(Loukas et al., 2011; Kupferschmidt, 2013) Other vaccines

presented at a recent vaccine discovery workshop sponsored

by the Bill and Melinda Gates Foundation in the United

States (Kupferschmidt, 2013) identified additional vaccines

still in experimental development, including Sm14, a fatty

acid binding protein, a calpain (Smp80) from S mansoni, and

Sj23, a TSP, a triose-phosphate isomerase, an insulin receptor,

and paramyosin from S japonicum (Zhu et al., 2004; 2006;

Siddiqui et al., 2005; Tendler and Simpson, 2008; You et al.,

2012) An advantage of vaccination strategies against the

zoonotic S japonicum is that the parasite is found in a variety

of domesticated animals, including water buffalo and goats in

China Researchers involved in controlling this species in

China and the Philippines have developed vaccines for use in

animals as transmission-blocking vaccines, based on

model-ling of transmission dynamics in endemic regions (McManus

et al., 2009) Antigen discovery studies are still progressing

using a variety of immunomics and proteomic approaches It

is now widely appreciated that targeted approaches are

required for antigen discovery, and there is continuing

inter-est in considering fundamental cell biological and

develop-mental understanding with molecular advances

The tegument of schistosomes

The tegument, or body wall, of schistosomes is a dynamic

host-adapted interface between the parasite and its vascular

environment The tegument is a highly polarized syncytium

and possesses functional analogy with transporting epithelia,

including the gut lining or the syncytiotrophoblasts of the

human placenta The tegument plays significant roles in

nutrient uptake, immune evasion and modulation, excretion,

osmoregulation, sensory reception, and signal transduction

(Jones et al., 2004; Kusel et al., 2007; Castro-Borges et al.,

2011) Given the importance of the schistosome tegument in

nutrition and immune evasion, proteins of this surface layer

are recognized as prime candidates to target for vaccine and

therapeutic drug development (Loukas et al., 2007).

Ultrastructure of schistosome tegument

The tegument is formed as a single syncytium that covers theentire body and is continuous with other epithelia (Figures 1–

2), notably the foregut lining (Silk et al., 1969) This surface

cytoplasmic layer is a highly ordered structure with distincttransporting regions, secretory components and absorptiveadaptations A peculiarity of the layer is the presence of a dualmembrane complex that forms the apical extremity of thetegument cytoplasm (Hockley, 1973; Hockley and McLaren,

1973; Castro-Borges et al., 2011).

The developmental activity of cercarial transformationreferred to above appears first and foremost to involve altera-tion of the apical membrane of these parasites soon afterinvasion (Hockley and McLaren, 1973; Skelly and Shoemaker,

1996; 2001; Keating et al., 2006) The single-unit membrane

of the cercaria, with its highly immunogenic glycocalyx,becomes replaced by a host-adapted dual membrane system,consisting of the membrane proper, overlain by an additionalunit membrane, the membranocalyx Although the mem-branocalyx is depauperate of parasite-derived proteins, theunderlying membrane is decorated with abundant mem-brane proteins (Braschi and Wilson, 2006) Membrane repairand maintenance is an ongoing process, as evidenced byabundant cytoplasmic inclusions and molecule associatedwith the apical membranes

The advantage to schistosomes in possessing a syncytialtegument is poorly understood, but appears to be an impor-tant strategy that ensures survival of parasites in the vascularenvironment Invaginations of the surface membranecomplex, as well as in the basal membrane of the cytoplasm

(Hockley, 1973; Gobert et al., 2003; Skelly and Wilson, 2006),

are structural evidence of high turnover of these membranes

(Brouwers et al., 1999), a process that is related to nutrient

uptake and a way of avoiding the host immune response byinternalizing antibodies and removing possible antigenicmolecules from the surface (Skelly and Wilson, 2006) Mem-brane internalization and translocation events are driven by acomplex interplay of multiple membrane proteins including

the TSP-enriched microdomains (TEMs) (Tran et al., 2010; Jia

et al., 2014) The TEMs are protein complexes formed about a

membrane-resident TSPs, which act as scaffold proteins forthe multiple fusion and scission activities of plasma mem-

brane (Hemler, 2008) For S.mansoni, TEM residents include a

variety of proteins strongly linked to the apical plasma brane, including schistosome annexins B30, Sm29, a dysfer-lin, calpain, fructose-biphosphate aldolase, heat shock

mem-protein 70 and actin (Jia et al., 2014).

The tegument is supported by cell bodies that lie

embed-ded in the parasite parenchyma (Hockley, 1973; Gobert et al.,

2003) The apical cytoplasm of the tegument and the cellbodies are linked by cytoplasmic bridges, which traverse themuscle bundles lying beneath the parasite tegument

(Figure 1B) (Hockley, 1973; Gobert et al., 2003) Tegumentary

cell bodies contain the synthetic machinery of the ium, including endoplasmic reticulum and Golgi apparatus,and produce abundant vesicular products that are trafficked

syncyt-to the tegument along cysyncyt-toplasmic bridges (Figure 3).The molecular interactions driving membrane formationduring transformation and in repair and renewal are far fromunderstood, but the abundance of adaptor and chaperone

proteins associated with the apical membrane complex(Table 1) and abundance of membrane vesicles (Figures 1–2)suggest a continuous cycle of renewal and repair throughoutadult life of the parasite.

Tegument proteins as vaccine targets

In mice immunized with tegument extract of newly

trans-formed S mansoni schistosomula, an induced Th1-type

pro-tection has been observed, which damages the adult wormtegument layer and reduces egg number and parasite burden

in challenge infections (Smithers et al., 1990) Therefore, it is

currently believed that tegument proteins of schistosomes are

a priority in antigen discovery Proteins potentially exposed

at its surface during intra-mammalian stages are possibly themost susceptible targets for vaccine development (Loukas

et al., 2007) A challenge in studies of schistosome biology is

the elucidation of when and how during the infection targetsare exposed to the host immune recognition According to

models of the S mansoni tegument, the primary vaccine target Sm-TSP-2 (tetraspanin-2) occurs in the plasma mem-

brane, that is, it lies hidden from the host under the branocalyx (Wilson, 2012) Recent immunolocalization datasuggest the molecule is even more hidden from the host, inadult parasites at least, occurring predominantly in associa-tion with surface invaginations of the hosts and in subsidiary

mem-membranes (Schulte et al., 2013).

Figure 1

Tegument of Schistosoma mansoni by transmission electron microscopy (A) Low magnification image from a cross-section of an adult The image

shows the apical cytoplasm of the tegument (Teg), which is the interface with the host vasculature The syncytial cytoplasm rests on bands ofmusculature and is supported and maintained by tegumentary cell bodies That depicted is rich in vesicles that are transported to the apicalcytoplasm along cytoplasmic bridges (arrows) The parasite digestive system, lined by a syncytial epidermis called a gastrodermis, lies deep withinthe body (B) High magnification view of the teguments of paired male and female adults The apical membrane complex (AP) consists of a plasmamembrane overlain by a subsidiary membrane-like structure, the membranocalyx, evident only after special fixation/staining of TEM tissues usinguranyl acetate The apical cytoplasm infolds frequently as surface invaginations, sometimes with secondary caveola-like outpocketings appearing.Other bodies decorate the tegument, including discoid bodies (DB) Tegumentary spines (SP), used for adhesion, are observed Original figures

Figure 2

Immuno-electron microscopy of Sm-Anx-B22, transmission electron

microscopy, using indirect immunocytochemistry incorporating

10 nm protein-A gold particles Sm-Anx-B22 was localized to surface

invaginations (SI) and other membrane compartments associated

with the apical plasma membrane complex (AP) After Leow et al.

(2014)

Analysis of the schistosome proteome has vastly increased

the speed of identification of tegument proteins (Braschi and

Wilson, 2006; Mulvenna et al., 2010; Castro-Borges et al.,

2011; Jia et al., 2014) (Table 1) A series of experiments have

allowed different proteins to be assigned to the distinct

mem-brane fractions of the apical memmem-brane complex (Wilson,

2012), although these assignments are likely to be crude and

require further analysis by refined localization tools A range

of molecules has been identified including glucose

transport-ers, proteases and other enzymes, receptors, chaperones and

structural proteins (Mulvenna et al., 2010; Castro-Borges

et al., 2011; Wilson, 2012) Further confirmation of the

co-location of many of these molecules has come from

inter-action studies of Sm-TSP-2 (Jia et al., 2014), which show

strong interactions, as stated, with a range of surface-linked

molecules including annexin B30, alkaline phosphatase,

actin, an aldolase, calpain, HSP70, dysferlin and Sm29, a

schistosome-specific molecule These interacting partners

widen the pool of available molecules for vaccination studies

Among the dominant surface-related proteins is

S mansoni annexin B30 (hereafter Sm-Anx-B30)

(Castro-Borges et al., 2011; Cantacessi et al., 2013; Jia et al., 2014).

This molecule is strongly associated with the tegument

(Tararam et al., 2010) and the Sm-TSP-2 TEMs, although how

it binds to other proteins is undetermined Sm-Anx-B30 lies in

direct association with the apical plasma membrane (C Leow,

unpubl obs.) Three other S mansoni annexins, namely,

Sm-Anx B7a, B22 and B5a, have been shown in various

studies of different schistosomes to be located to the

tegu-ment The abundance, as well as the peculiar features of

annexins of some parasite groups, makes them potential

targets for therapies

Schistosome annexins

Annexins are a family of proteins that are able to bind to

acidic phospholipid membranes Their membrane-binding

mode includes formation of a ternary complex involving the

protein, the calcium ions and the membrane The survey of

group B annexins from different invertebrate taxa revealed

that the proteins occur in the vast majority of species studied

so far (Cantacessi et al., 2013) The abundant annexin

pro-teins are conspicuously evident in many parasite groups,

including a series of arthropod vectors of disease, as well asbasal metazoans, but are apparently absent from others,notably the Mollusca Using structure-based amino acidsequence alignments and phylogenetic analyses, the recentanalysis provided a robust classification for this proteingroup, enabling information on structure–functional rela-tionships of these proteins, as well as to assign names tosequences with ambiguous annotations in public databases

(Cantacessi et al., 2013) It was immediately apparent in

phy-logenetic analyses that gene duplication in divergent cladeswas the major evolutionary event in annexins’ genesis, par-ticularly in schistosomes The highest representation of

annexin was found in S mansoni with 13 annexins, many

distributed on two chromosomes, suggesting linkage

(Cantacessi et al., 2013).

Evidence gained from tissue-specific transcriptional andproteomic profiling of adult parasites suggests that the differ-ent schistosome annexins are expressed differentially

throughout the body of the parasites (Gobert et al., 2009a) As stated, Sm-Anx-B7a, B22 and B30 are distinctly associated

with the syncytial tegument (Braschi and Wilson, 2006;

Mulvenna et al., 2010) Our tissue-specific transcriptomic survey of female S japonicum indicated that different annex-

ins were expressed preferentially by different cell types, withthe gut lining expressing annexin B7 and B22, while the

vitelline gland expressed annexin B5 (Gobert et al., 2009a) Although S japonicum is a distinctive parasite, as it diverged early from other species of Schistosoma, similar patterns of

annexin expression might reasonably be expected to be served within the genus The abundance of annexin B7 andB22 in gut and tegument allows the postulate that thesemolecules may be epithelial annexin in these parasites, andthus associated with syncytial epithelia Importantly, boththe gastrodermis and the tegument are predicted to have high

con-membrane turnover and reshaping (Nawaratna et al., 2011).

Structure–function observations of schistosome annexins

Observations made by us and others in the recent past pointtowards a potential use of parasite annexins as therapeutic

Figure 3

Cartoon representations of (A) human annexin A5, (B)α1-giardin from Giardia intestinalis and (C) the dimerized Schistosoma mansoni annexin Sm-Anx-B22 For annexin A5 andα1-giardin, the annexin repeats are shown in different colours For all annexins shown the N-terminus is coloured

blue and the II/III linker in magenta Note the distinctly longer linker in Sm-Anx-B22, which packs against the N-terminal domain The disulphide

bond between Cys173 (molecule 1) and Cys173 (molecule 2) is rendered in green Protein structures were rendered with PyMOL (DeLano, 2002)

targets These findings include (i) immunoreactivity of some

parasite annexins (Hongli et al., 2002; Palm et al., 2003;

Weiland et al., 2003; Gao et al., 2007; Weeratunga et al., 2012;

Leow et al., 2014); (ii) localization of certain parasite

annex-ins to areas of potential exposure and/or structural integrity

(Braschi and Wilson, 2006; Jia et al., 2014); and (iii) the

existence of a unique structural feature, including the

extended helical linker between repeats II and III (Figure 3),

in parasite annexins that differentiates them from host

annexins (Hofmann et al., 2010; Weeratunga et al., 2012;

Leow et al., 2014).

The extended linker region is a primary source of

varia-tion between some group B and group A annexins Many

group B annexins, including those from the cestode Taenia

solium; annexin B36 (nex-4) from the model nematode norhabditis elegans; and some Group E annexins, including

Cae-α-12- and α-19 giardin, possess an unusually long linkersegment between repeats II and III on the concave side of the

protein (Hofmann et al., 2010) (Figure 3) Whereas the typical

length of this linker in annexin ranges from 10 to 15 aminoacids, the linker peptide of these groups B and E range from

25 to 38 amino acids (Hofmann et al., 2010) Secondary

struc-ture predictions consistently indicate that this elongatedlinker region adopts an α-helical structure, and the recent

crystal structure of Sm-Anx-B22 provided the anticipated experimental proof (Leow et al., 2014) We hypothesize that

Table 1

Apical membrane complex-associated proteins of schistosomes

Membranocalyx

Tetraspanin-2 Mediators of tetraspanin-enriched microdomain in surface

membrane complex; membrane remodelling

8 kDa low-molecular weight protein? Secreted protein

Sm29 Uncertain role, but potential ligand of tetraspanin

Bound host proteins-CD48, 90, immunoglobulins,complement factors

Host moleculesInter-membrane space 8 kDa low-molecular weight protein? Secreted protein

8 kDa low-molecular weight protein? Secreted protein

Aquaporins Water and small solute transport

Anion channelPlasmolipins Tetraspanin myelin proteins, membrane raftsCytosolic Dysferlin Calcium-dependent membrane fusion events; component

of tetraspanin-enriched microdomains in tegument

Dynein light chains Dynein-like motor function

Evidence or proposed location in the tegument is derived from the review of proteomic analyses of the tegument of Schistosoma mansoni by

Wilson (2012) In many cases, the link between the protein and unit membrane is inferred and further experimental evidence is required Themodel is based on a static two-dimensional structure only and ignores the dynamic nature of the schistosome tegument The membranecomplex is a dual membrane system, consisting of a unit membrane overlaid by an additional membrane structure, the so-calledmembranocalyx Only one annexin is listed here, although it is known that multiple annexins are present in the tegument of schistosomes

this additionalα-helical element on the concave side of the

molecule may provide a target for immunological

therapeu-tics (Hofmann et al., 2010) It is tempting to speculate that

other parasite annexins with an extended II/III linker peptide

may adopt a very similar conformation A comparison of the

extent of the N-terminal domains for annexins with the

unique linker shows that such a fold may be possible for most

of them

The crystal structure of Sm-Anx-B22 confirms the

pres-ence of the predictedα-helical segment in the II/III linker and

also reveals a covalently linked head-to-head dimer (Leow

et al., 2014) Sm-Anx-B22 and its homologues from S

japoni-cum (Cantacessi et al., 2013) and S bovis (de la Torre-Escudero

et al., 2012) are the only B annexins known to date that

possess an exposed cysteine residue in the IIDE loop

(Cys173), a position where most other annexins possess a

serine residue In Sm-Anx-B22, the involvement of Cys173in

an inter-molecular disulphide bond as well as several

inti-mate electrostatic side chain interactions add to the

stabili-zation of the unique head-to-head dimer topology where the

dimer interface is exclusively located in module II/III

Struc-turally, this is significantly different to other annexin

head-to-head dimers (Hofmann et al., 2010), where the dimer

interface comprises the entire convex surface of both

molecules

In addition, from the calcium-bound crystal structure of

Sm-Anx-B22, canonical as well as novel calcium binding sites

can been identified, which seems to be a recurring motif in

parasite annexins Intriguingly, the dimer arrangement

observed in the annexin B22 crystal structure revealed the

presence of two non-anticipated prominent features: a

poten-tial non-canonical membrane-binding site and a potenpoten-tial

binding groove opposite of the former (Figure 4)

Annexins in schistosomes

A variety of roles have been proposed for annexins In

verte-brates, annexins are known to display a broad range of

biological activities including response to inflammation,membrane traffic and adhesion, anticoagulation, signal trans-duction, developmental processes and membrane repair

(Bouter et al., 2011; Draeger et al., 2011) In parasites,

annex-ins are suggested to be involved in maintenance of

mem-brane structure (Peattie et al., 1989; Tararam et al., 2010), anti-inflammatory activity (Zhang et al., 2007) and fibrino- lytic activity (de la Torre-Escudero et al., 2012) Annexins may

thus have distinct roles in enabling survival of parasiteswhen they are within the hosts Some annexins are specu-lated to be involved in redox reactions and the regulation of

reactive oxygen molecules in plants (Hofmann et al., 2003) (Konopka-Postupolska et al., 2011) and in mammals (Tanaka

et al., 2004; Madureira et al., 2011; Madureira and Waisman,

2013)

Localization of Sm-Anx-B22 by fluorescence and electron

microscopy in different species of schistosomes (Tararam

et al., 2010; de la Torre-Escudero et al., 2012; Leow et al.,

2014) demonstrates that the molecule is strongly associatedwith the tegument and the plasma membrane structures ofthe apical regions of the tegument of adult parasites(Figure 2) The molecule is expressed in human-parasiticphases of the parasite life cycle, suggesting a major role insurface membrane dynamics during life in the human host

Although Sm-Anx-B22 shares many structural similarities

with other annexins, its dimeric nature as well as the uniqueextended linker region suggests that this molecule isco-adapted to function in the peculiar syncytial environment

of the tegument of these parasites (Leow et al., 2014) Among

the peculiarities, there is a prominent groove that occurswithin the dimeric species (Figure 4) This groove is postu-

lated to enable the Sm-Anx-B22 dimer to assume an adaptor

function, linking the apical membrane complex withproteins

Sm-Anx-B22 possesses another unique feature, namely,

the external arrangement of the II/III linker that is reflexedover the N-terminal region of the molecule There is nowsubstantial evidence that annexins, and indeed other mol-ecules of the schistosome tegument, adopt unique conforma-tions that might be exploited for therapeutics or prophylaxis

as they distinguish the parasite proteins from homologousproteins in the host

The question remains as to how these unique regionsmight be targeted if we are to develop anti-schistosomiasistherapies directed against these molecules Undoubtedly, aswith all areas of investigation concerning annexins from awide variety of organisms, more structure–function analyses

of the proteins in cells is required For schistosomes the liarities of the annexins, including the extended II/III linkerregions, non-canonical calcium binding sites and othermolecular anomalies are of interest, not only for enhancingfundamental understanding of membrane dynamics, but alsofor designing anti-parasite targets

pecu-Being highly adapted to life within hosts, helminth sites present considerable difficulties in functional genomicsanalyses They are not easily cultivated outside of the hostand require molecular signalling from their host to developfully Furthermore, transgenesis studies for schistosomesremain in their infancy, although these parasites are amena-ble to RNA-interfering technologies Thus, studies of annex-ins of schistosomes present some challenges Two important

para-Figure 4

Surface-rendered model of Schistosoma mansoni annexin

Sm-Anx-B22 A distinctive groove, lying at the junction of the two partners of

the dimer (coloured light blue and tan) may give the protein an

adaptor function The N-terminus (dark blue) and II/II linker region

(magenta) of one partner are shown Figure prepared with PyMOL

(DeLano, 2002)

outcomes of the recent comparative analysis of invertebrate

annexins (Cantacessi et al., 2013) is the occurrence of

common structural motifs in some group B (invertebrate) and

group E (Giardia) annexins and the growing diversity of

annexins among the single celled protists The encouraging

result suggests that there is substantial information to be

gained from comparative studies among parasites that are less

tractable in laboratory models and readily culturable

inverte-brate model species and parasites These comparative

structure–function investigations as models for

understand-ing annexins’ function at the host–parasite interface, as

het-erologous expression systems of parasite annexins and as

targets of inhibitor and drugs assays, will prove invaluable as

we move towards developing targeted therapies for parasites

of socio-economic importance

Acknowledgements

We gratefully acknowledge funding of our laboratories by the

National Health and Medical Research Council (A H., M K

J.) C Y L was supported by a scholarship from the Malaysian

Government and Universiti Sains Malaysia ASTS scholarship

C W was supported by a NHMRC Australian Biomedical

Research Fellowship

Conflict of interest

None

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Themed Section: Annexins VII Programme

Yidong Liu, Helene K Myrvang and Lodewijk V Dekker

School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham,

lodewijk.dekker@nottingham.ac.uk -

differentially expressed between normal and malignant tissue and potentially involved in tumour progression An importantaspect of AnxA2 function relates to its interaction with small Ca2+-dependent adaptor proteins called S100 proteins, which isthe topic of this review The interaction between AnxA2 and S100A10 has been very well characterized historically; morerecently, other S100 proteins have been shown to interact with AnxA2 as well The biochemical evidence for the occurrence

of these protein interactions will be discussed, as well as their function Recent studies aiming to generate inhibitors of S100protein interactions will be described and the potential of these inhibitors to further our understanding of AnxA2 S100 proteininteractions will be discussed

LINKED ARTICLES

This article is part of a themed section on Annexins VII Programme To view the other articles in this section visit

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Abbreviations

AnxA2, annexin A2; Plgn, plasminogen; tPA, tissue plasminogen activator

1664 British Journal of Pharmacology (2015) 172 1664–1676 © 2014 The Authors British Journal of Pharmacology published by John Wiley &

Sons Ltd on behalf of The British Pharmacological Society.

AnxA2 structure

Like all annexins, AnxA2 is a slightly curve-shaped protein

with a convex and a concave side It consists of a highly

conserved core domain of four homologous repeats of 70–80

amino acids called the annexin repeats and a unique

30-amino-acid long N-terminal ‘head domain’ (which is also

referred to as tail domain or N-terminal interaction domain

in some literature) (Waisman, 1995; Gerke et al., 2005) The

core domain region, encompassing residues 31–338, has

binding sites for calcium, phospholipids, heparin and F-actin

Although the core domain is conserved among the annexins,

subtle differences between them have been noticed For

example, the AnxA2 core domain is subtly different in its

Ca2+ sensitivity in Ca2+-dependent membrane interactions

(Drucker et al., 2013) Thus, different annexins may have

different functions in the cell

The head domain of AnxA2 contains a number of features

relatively unique to this particular annexin The first 12

resi-dues of the head domain constitute the binding site for

S100A10, a member of the S100 protein family (Johnsson

et al., 1986; 1988) This region also encompasses a binding

site for tissue plasminogen activator (tPA) mapped to residues

7–12 (Johnsson et al., 1988; Hajjar et al., 1998) and a nuclear

export signal (NES) mapped to residues 3–13 (Eberhard et al.,

2001) Residues Tyr23, Ser11and Ser25of AnxA2 can be

phos-phorylated by Src family tyrosine kinases and serine kinases

respectively (Gould et al., 1986; Khanna et al., 1986; Powell

and Glenney, 1987; Jost and Gerke, 1996)

The interaction of AnxA2 with

S100 proteins

Perhaps one of the most clearly defined features that

charac-terize AnxA2 is its capacity to interact with members of the

S100 protein family to yield so-called heterotetrameric

com-plexes, consisting of an S100 protein dimer and two AnxA2

proteins ((S100AXX-AnxA2)2) S100 proteins are a group

of small Ca2 +-binding proteins with molecular weight of

10–12 kDa (Donato, 1999; 2003) With the exception of

S100A10, they contain Ca2+-binding EF-hand motifs, and areregarded as the largest family grouping within the EF-handprotein superfamily Rather uniquely, compared with otherEF-hand proteins, S100 monomers contain two different EFhands with distinct affinities for calcium: a canonical

C-terminal EF hand (KD≈ 10–50 μM) and a pseudo-canonical

N-terminal EF hand (KD≈200–500 μM) S100 proteins alwaysfunction as dimers, mostly homodimers, but sometimes het-erodimers: S100A1/B, S100A8/A9, S100A1/A4 and S100A1/P

(Odink et al., 1987; Duda et al., 1996; Tarabykina et al., 2000; Wang et al., 2004) Ca2 + binding induces a conformationalchange in the S100 proteins due to repositioning of helix IIIfrom a near antiparallel position to helix IV to a nearlyperpendicular position Thus, a compact and closed confor-mation opens up and exposes a large hydrophobic area which

is capable of recognizing and binding potential targets

(Malashkevich et al., 2008) S100A10 is unique among S100

proteins in that it is locked in a permanently open mation, comparable to the Ca2+-bound configuration of theother S100 proteins Many S100 proteins play a role in cancer

confor-prognosis or progression (Schlagenhauff et al., 2000; Davies

et al., 2002; Cross et al., 2005; Vimalachandran et al., 2005;

De Petris et al., 2009) and some of them are suggested as biomarkers to certain types of cancer (Hamberg et al., 2003; Nedjadi et al., 2009; Tsuna et al., 2009).

The classic AnxA2-binding S100 protein is S100A10,which was identified as a binding partner almost 30 years ago

(Erikson et al., 1984; Gerke and Weber, 1985a,b) Binding to

S100A10 occurs at the helical AnxA2 N-terminus (Glenney

et al., 1986; Johnsson et al., 1988; Kube et al., 1992; Rety et al.,

1999) The AnxA2 N-terminus is accommodated in the freehydrophobic space between helix III and helix IV of the

S100A10 dimer (Rety et al., 1999) (Figure 1) Interaction with

the AnxA2 N-terminus appears sufficient for binding sinceproteolytic removal of this N-terminus from purified AnxA2

(cleaved at Gly14) (Johnsson et al., 1988) or deletion of dues 1–14 from recombinant AnxA2 (e.g Semov et al., 2005)

resi-results in a complete loss of the interaction with S100A10.Removal of the first methionine of the primary AnxA2 trans-lation product as well as acetylation of the serine at position

2 is necessary for AnxA2 binding to S100A10 (Johnsson et al., 1988; Becker et al., 1990; Konig et al., 1998; Nazmi et al.,

Src tyrosine kinaseTissue plasminogen activator (tPA)

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b Alexander et al., 2013a,b).

2012) In addition to the acetyl group, specific hydrophobic

residues are crucial for binding with S100A10 (Becker et al.,

1990; Rety et al., 1999).

Recent studies showed that in addition to S100A10, three

other S100 proteins, S100A6, S100A4 and S100A11, are also

able to bind to AnxA2 (Table 1) Various techniques have

been employed to investigate the binding between these S100

proteins and AnxA2 Isothermal titration calorimetry of 16

different S100 proteins with the AnxA2 N-terminus identified

S100A10 and S100A11 as binding partners (Streicher et al.,

2009) The interaction between S100A11 and AnxA2 was

also demonstrated by nuclear magnetic resonance (NMR),

isothermal titration calorimetry and immunoprecipitation

(Rintala-Dempsey et al., 2006) Although not observed using

isothermal titration calorimetry, S100A4 can accommodate

the AnxA2 N-terminus based upon nuclear NMR and

immu-noprecipitation evidence (Semov et al., 2005) The NMR

studies indicated that Glu6, Asp10, Leu42, Phe45, Ile82, Phe89andPro94in S100A4 could be involved in the binding with AnxA2and these amino acids are in similar positions to key aminoacids in S100A10 involved in the binding to AnxA2 (Glu5,Glu9, Phe38, Phe41, Leu78, Tyr85and Met90in S100A10) (Semov

et al., 2005) Finally, an AnxA2 complex with S100A6 was

identified using affinity chromatography and

immunopre-cipitation methods (Zeng et al., 1993) and further confirmed

by biochemistry and spectrometry methods (Filipek et al., 1995; Nedjadi et al., 2009) Given the similarity of the

calcium-bound conformations of S100A4, S100A6 andS100A11 to the S100A10 conformation, it may be argued thatAnxA2 is accommodated in a similar fashion in each of theseS100 proteins

Several studies have investigated the affinity of the AnxA2N-terminus for S100 proteins Using nuclear magnetic reso-nance techniques, a dissociation constant of 3.3± 0.6 μM wasderived for the binding of AnxA2 to S100A11 by titrating anAnxA2 N-terminus peptide into calcium-bound S100A11

(Rintala-Dempsey et al., 2006) Isothermal titration

calorim-etry experiments have also been used to determine the

dis-sociation constant of this interaction (Streicher et al., 2009).

It was found that the binding isotherm could not be fitted tothe simplest binding model, but fitted into a sequentialbinding model suggesting that the interaction involves non-symmetric binding to the two AnxA2 peptides: one bindingsite on the dimer needs to be occupied before the secondbinding event can take place Thus, two dissociation con-stants were determined for the binding of the AnxA2N-terminus to S100A11: 1.7± 1.2 μM and 9.2 ± 1.9 μM A verysimilar scenario applied to the binding of the AnxA2N-terminus to S100A10 albeit that the two sequential bindingevents appeared to have identical dissociation constants of0.5± 0.4 μM (Streicher et al., 2009) A comparable dissocia-

tion constant of 1.3± 0.3 μM was measured independentlyfor this interaction using equilibrium dialysis and fluores-

cence resonance transfer techniques (Li et al., 2010) and for the binding of full-length AnxA2 to S100A10 (Nazmi et al.,

2012)

Figure 1

Structure of S100A10 binding with annexin A2 peptide displayed as

ribbon diagram S100 proteins are coloured blue green yellow while

the AnxA2 N-terminus is coloured magenta (PDB: 1BT6) (Rety et al.,

Spectrometry or chromatography method

S100A10 Erikson et al., 1984

Gerke et al., 1985a

Gerke et al., 1985b

Rety et al., 1999 Streicher et al., 2009 Johnsson et al., 1988

Li et al., 2010 Streicher et al., 2009

–

S100A6 Zeng et al., 1993

Filipek et al., 1995

Nedjadi et al., 2009

Filipek et al., 1995 Nedjadi et al., 2009

The structure of the full

AnxA2-S100 complex

In appreciating the well-established binding mode of the

AnxA2 N-terminus to S100A10, the structure of the full

complex is more speculative Modelling the complex between

the S100A10 dimer and the AnxA2 N-terminus with the

solved AnxA2 core domain suggests two plausible

configura-tions In one model, the S100A10 dimer bridges two AnxA2

molecules arranged in opposite orientation whereas the

second model predicts the S100A10 dimer to sit on top of two

AnxA2 molecules arranged side by side (Sopkova-de Oliveira

Santos et al., 2000) It is extremely difficult to ascertain which

of these models would prevail in vivo; however, individually

they can explain various functions of the AnxA2 protein and

therefore both conformations may exist In terms of how

these various conformations are regulated, it is of interest

that the interaction of AnxA2 with the S100A10 dimer takes

place at the very end of the protein, leaving a loop region

between this S100A10 recognition region and the AnxA2 core

domain Flexibility in this loop may allow the one or the

other conformation Electron microscopy data of membrane

bridges at pH 7.4 in the presence of Ca2 + suggest that the

dimer of S100A10 be located in the centre of the protein

density, with one molecule of AnxA2 facing the bilayer on

each side (Lambert et al., 1997) However, AnxA2 can also

move around S100A10 as a hinge to acquire a more open

conformation where the S100A10 subunit is held away from

the phospholipid bilayer (Lambert et al., 2004; Menke et al.,

2004; Illien et al., 2010), compatible with the suggested

models (Sopkova-de Oliveira Santos et al., 2000) A third

more stretched conformation has been observed

experimen-tally at mild acidic pH in the absence of Ca2+ (Illien et al.,

2010) Cellular studies indicated that acidic pH can support

the membrane binding of the (S100A10-AnxA2)2

heterote-tramer complex and it is perhaps this stretched conformation

that is responsible for this (Monastyrskaya et al., 2008)

Inter-estingly, the putative hinge region of the annexin head

domain is subject to phosphorylation and phosphorylation

events may also regulate the specific loop architecture and

conformation of the tetramer (Grindheim et al., 2014).

The cellular complex of AnxA2 and

S100 proteins

The ratio of monomeric AnxA2 to (S100A10-AnxA2)2 can

vary widely, and the differences in ratio are due to

coordi-nated expression of AnxA2 and S100A10 (Munz et al., 1997)

as well as post-translational control (Puisieux et al., 1996).

Interfering with the expression of one of the partners in the

(S100A10-AnxA2)2 complex affects the expression of the

other partner, indicating their intimate relationship in vivo.

In most reports, knockdown of AnxA2 affects the levels of

S100A10; however, the reverse has also been observed in

some cases and thus the ‘direction’ of the regulatory effect

seems to differ between cell types For example, in

endothe-lial cells, knockdown of AnxA2 not only results in the

disap-pearance of AnxA2 but also in that of S100A10, while

knockdown of S100A10 does not affect expression of AnxA2

(Brandherm et al., 2013) In complex with S100A10, AnxA2

may protect S100A10 from being rapidly polyubiquitinated

and degraded (He et al., 2008) Interestingly, the residues

86–95 subject to ubiquitination of S100A10 are the residuesresponsible for binding with the AnxA2 N-terminal, suggest-ing that once ubiquitinated, S100A10 may not bind AnxA2anymore The amount of S100A10 in the cell would thusdictate the amount of the (S100A10-AnxA2)2complex in thecell (Figure 2) This may be important to determine the intra-cellular fate of AnxA2 Although AnxA2 itself can associate

with cellular membranes (Zobiack et al., 2001), S100A10

binding increases the Ca2+ sensitivity of AnxA2 and itscapacity to bind membranes and (submembranous) F-actin(Ikebuchi and Waisman, 1990; Harder and Gerke, 1994;

Filipenko and Waisman, 2001; Monastyrskaya et al., 2007).

Depletion of S100A10 by RNA silencing or phosphorylation

on Ser11on AnxA2 (which inhibits interaction with S100A10)

disrupts the membrane association of AnxA2 (Regnouf et al., 1995; Deora et al., 2004) Similar observations have been

made for S100A6 which has also been proposed to interactwith AnxA2 Depletion of S100A6 from pancreatic cancercells was accompanied by diminished levels of membraneAnxA2 associated with a pronounced reduction in the motil-

ity of pancreatic cancer cells (Nedjadi et al., 2009) Under

certain conditions of stress, AnxA2 can become expressed onthe cell surface, in a mechanism that requires the interactionwith S100A10 as well as phosphorylation of AnxA2 on tyros-

ine at position 23 (Deora et al., 2004).

Sequestration of AnxA2 by S100A10 in the cytosol also

prevents its nuclear localization (Eberhard et al., 2001) In

prostate cancer cells, monomeric AnxA2 can localize to thenucleus where it acts as negative regulator of cell proliferation

(Liu et al., 2003) Phosphorylation of AnxA2 may be tant for its nuclear localization (Chiang et al., 1996; Eberhard

impor-et al., 2001) AnxA2 contains a functional NES sequence at

the N-terminal which allows export via the

Ran/exportin-mediated export pathway (Eberhard et al., 2001) This

Figure 2

Simplified diagram to illustrate some aspects of the cellular tion of AnxA2 by S100 protein interactions

regula-sequence overlaps with the binding site of AnxA2 with

S100A10

As mentioned above, loss of S100A10 has also been

observed to affect the levels of AnxA2, in particular in studies

in which S100A10 was removed by genetic deletion In

S100A10 knockout mice, the AnxA2 level decreased in spleen,

kidneys, lungs and liver, but was not affected in intestine

(Madureira et al., 2012), suggesting that S100A10 could

sta-bilize and regulate the level of AnxA2 However, studies in

nociceptor neurons indicated that genetic deletion of

S100A10 did not affect AnxA2 levels (Foulkes et al., 2006).

Functions associated with the

AnxA2-S100 complex

Biochemical reconstitution experiments, mouse genetic

deletion models and RNA interference studies have yielded

much information on the effector functions of individual

annexins and S100 proteins, including AnxA2 and S100A10

It is beyond the scope of this review to discuss all the

evi-dence and the reader is referred to recent excellent reviews

in this area (Gerke and Moss, 2002; Rescher and Gerke,

2004; Gerke et al., 2005; Kwon et al., 2005; Flood and Hajjar,

2011; Madureira et al., 2011; Bharadwaj et al., 2013; Luo and

Hajjar, 2013) However, a few recent relevant examples are

cited here to provide an indication of the effector function

of (S100A10-AnxA2)2and related complexes in the cell It is

perhaps useful to consider these in relation to the two

con-formational models cited above While it is very difficult to

ascertain precisely which model applies to a particular

cel-lular context, in a broad simplification one could say that

in one conformation, the ‘opposite conformation’, the

tetramer can bring together different cell membranes, while

in the other conformation, the ‘lateral conformation’, the

tetramer can act as a platform for association with other

proteins This classification should, however, not be taken as

absolute, since the former conformation could

accommo-date additional proteins and the latter can serve to juxtapose

membranes

The former conformation points to a role in membrane

trafficking, secretory or endocytic processes A recently

pre-sented example shows that the (S100A10-AnxA2)2complex is

involved in the secretion of von Willebrand factor (vWF),

which is stored in the Weibel–Palade bodies (secretory

gran-ules) of endothelial cells (Knop et al., 2004; Brandherm et al.,

2013) It is normally released by agonists that raise

intracel-lular Ca2+or cAMP levels and a functional (S100A10-AnxA2)2

complex is required for the forskolin-induced,

cAMP-dependent release of vWF (Knop et al., 2004; Brandherm

et al., 2013) Forskolin triggers dephosphorylation of AnxA2

(Borthwick et al., 2007), mediated by a calcineurin-like

phos-phatase This stabilizes the (S100A10-AnxA2)2complex and

promotes vWF release (Brandherm et al., 2013) When the

(S100A10-AnxA2)2 complex cannot form, cAMP-dependent

vWF secretion is compromised (Brandherm et al., 2013) At

present, it is not clear whether additional protein interactions

contribute to the secretion of vWF such as observed in

secre-tory processes in stimulated chromaffin cells In these cells,

AnxA2 directly interacts with S100A10 to form a tetramer

at the plasma membrane (Chasserot-Golaz et al., 2005; Umbrecht-Jenck et al., 2010) S100A10 can interact with

vesicle-associated membrane protein 2 (VAMP2) which mayact as docking factor for S100A10 Prevention of S100A10binding to VAMP2 inhibits the translocation of annexin-A2

to the plasma membrane In bronchial epithelial cells, AnxA2associates with collagen VI and the SNARE proteins SNAP-23and VAMP2 at secretory vesicle membranes, and as such hasbeen implicated in the collagen VI secretion pathway (Dassah

et al., 2014) It is not clear whether this also involves the

S100A10 protein interaction

Localized at the cell surface, AnxA2 has been implicated

in cell-cell interactions and cell adhesion AnxA2 provides asignal for interaction with and phagocytosis of apoptoticcells, most likely via interactions with phosphatidyl serine on

the juxtaposed apoptotic cell surface (Fan et al., 2004; Law

et al., 2009; Fang et al., 2012) AnxA2 expressed on apoptotic

cells themselves binds complement factors as signal for

cell-cell interaction and phagocytosis (Leffler et al., 2010; Martin

has been implicated in tight junction maintenance in lial MDCK cell monolayers in a model in which AnxA2 isassociated with the lipid membrane with the S100A10 dimer

epithe-bridging two AnxA2 molecules (Lee et al., 2004; 2008) The

binding of surface AnxA2 to surface S100A10 also contributes

to heterotypic cell-cell interactions between breast tumourcells and microvascular endothelial cells An AnxA2 moleculepresent on an opposing cell, such as a breast cancer cell, canbridge to the endothelial cell by interacting with surface-

localized S100A10 located on the latter (Myrvang et al.,

2013)

A wide range of platform functions of the AnxA2)2 complex have been suggested Early researchrevealed that as well as bridging phospholipid vesiclesand binding biological membranes, the (S100A10-AnxA2)2

(S100A10-complex displays binding and bundling of F-actin (Gerke andWeber, 1985a) This occurs at physiological Ca2 +concentra-tions in theμM range (Ikebuchi and Waisman, 1990; Regnouf

et al., 1991) This activity can be specifically inhibited by

pre-incubation of F-actin with a nonapeptide to the

actin-binding site of AnxA2 at residues 286–294 (Jones et al., 1992).

The (S100A10-AnxA2)2complex is important for the zation of F-actin at lipid rafts and for the dynamic regulation

organi-and remodelling of the actin cytoskeleton (Hayes et al., 2004;

2006) As such, AnxA2 has been implicated in various cellularprocesses that involve the actin cytoskeleton

One of the other AnxA2 partners, S100A11, is requiredfor efficient plasma membrane repair which may support

the survival of invasive cancer cells (Jaiswal et al., 2014).

During cell migration and invasion, cells are exposed tophysical stress Injury to the cell membrane occurringduring this process results in entry of calcium into the cellwhich in turn can trigger recruitment of S100A11 and

AnxA2 to the site of injury (Jaiswal et al., 2014) The

complex of S100A11 with AnxA2 directs polymerization ofcortical F-actin and excision of the damaged part of theplasma membrane thereby resealing the plasma membrane

(Jaiswal et al., 2014).

On endothelial cells, the (S100A10-AnxA2)2complex hasbeen proposed as endothelial surface platform for tPA andplasminogen (Plgn), aiding the conversion to plasmin

Several somewhat conflicting models exist to explain the

exact contributions of these proteins individually (or as a

complex) to the plasmin activation process with either

AnxA2 (Cesarman et al., 1994; Hajjar et al., 1994; 1998; Flood

and Hajjar, 2011; Luo and Hajjar, 2013) or S100A10 (Kassam

et al., 1998; MacLeod et al., 2003; Madureira et al., 2011;

Bharadwaj et al., 2013) proposed as the main receptor of tPA

and Plgn Both models implicate the binding of S100A10 and

AnxA2 in the regulation of the surface proteases and the fact

that genetic deletion of either protein shows roles for both

proteins in maintenance of vascular patency, fibrin

resolu-tion, cell migration and neoangiogenesis also suggests a very

close relationship between them, most likely because they act

as a complex in these processes (Ling et al., 2004; Huang

et al., 2011; Madureira et al., 2011; Phipps et al., 2011; Surette

et al., 2011).

Like the complex of AnxA2 and S100A10, the complex of

AnxA2 and S100A4 may also regulate the tPA/Plgn cascade on

the cell surface Addition of S100A4 to umbilical vein

endothelial cells stimulated tPA/Plgn on these cells This

stimulation was reversible upon addition of a synthetic

peptide based upon the AnxA2 N-terminus, suggesting that a

complex between S100A4 and AnxA2 is involved in tPA/Plgn

regulation (Semov et al., 2005) This scenario may be relevant

when tumour cells produce S100A4 which, once bound to

endothelial cells and activating pericellular proteases, can aid

the growth of blood vessels into the tumour

While the F-actin-binding and protease-regulating

plat-form functions of the (S100A10-AnxA2)2 complex are not

clearly defined in structural terms, interactions with the

protein AHNAK and SMARCA3 have been solved by protein

crystallography AHNAK, a Hebrew word for ‘giant’, is a

629 kDa protein involved in membrane repair (Shtivelman

et al., 1992; Zhang et al., 2004) A multi-protein complex of

(S100A10-AnxA2)2and AHNAK is a target of dysferlin, a core

protein in wound repairing process of ‘injured’ epithelial cells

(Huang et al., 2007) The minimal binding site of AHNAK

protein, AHNAK5654–5673, with the (S100A10-AnxA2)2complex

has been mapped (De Seranno et al., 2006; Rezvanpour et al.,

2011) and was used to derive a crystal structure (Ozorowski

et al., 2013) The 20-amino-acid length AHNAK peptide

binds asymmetrically across the (S100A10-AnxA2)2complex

Hydrogen bonding between backbone atoms of ANNAK

pep-tides and (S100A10-AnxA2)2and the hydrophobic interaction

of ANNAK side chains with S100A10 are the main binding

forces responsible for the ternary complex (Ozorowski et al.,

2013)

It was recently found that SMARCA3, a protein involved

in chromatin remodelling in different nuclear processes in an

ATP-dependent manner (Debauve et al., 2008), is also a target

of the (S100A10-AnxA2)2 complex (Oh et al., 2013) The

co-crystal structure of SMARCA3 with this complex shows

that the binding site is very similar to that of AHNAK peptide:

two SMARCA3 peptides symmetrically bind to the

(S100A10-AnxA2)2 complex at the ‘back’ of S100A10 reaching into a

small hydrophobic cavity created by the ‘C-terminal’ of

AnxA2 peptide and helix IV(IV′) (Oh et al., 2013) It was

observed that the (S100A10-AnxA2)2 complex can increase

the DNA binding affinity of SMARCA3 and help SMARCA3

localize to the nuclear matrix; this would require S100A10 to

be present in the nucleus

A peptide toolbox to study AnxA2-S100 protein interactions

Given the detailed knowledge of the binding interactionsbetween the N-terminus of AnxA2 and S100A10, variousgroups have reported the use of peptides based upon thisN-terminus to perform competition experiments with theaim of disrupting the endogenous complex of the two pro-teins and understanding its functions An isolated acetylatedsynthetic peptide comprising residues 1–14 of AnxA2 candisrupt a preformed complex between S100A10 and a labelled

annexin 1–14 peptide (O’Connell et al., 2010) Furthermore,

the same peptide also disrupts a preformed full-length(S100A10-AnxA2)2 complex (Konig et al., 1998) Thus, it is

feasible to use synthetic peptides to disrupt endogenous

com-plexes between AnxA2 and S100A10 in vivo (Table 2).

Because of their nature, peptide interference studies havelargely been confined to scenarios where AnxA2 and S100A10are localized at the outer face of the plasma membrane, orwhere the peptide could somehow be introduced into thecells, for example, by microinjection or in patch clampexperiments (Table 2) Very elegant studies have been per-formed in which the action of an acetylated peptide wascompared with a non-acetylated peptide It is known that thenon-acetylated version of AnxA2 (or its N-terminus) bindsweakly to S100A10 dimers, therefore it is expected that such

a peptide cannot disrupt the endogenous complex (Becker

et al., 1990) By studying both peptides in parallel, a

convinc-ing argument can be made for or against the involvement ofthe (S100A10-AnxA2)2 complex in a cellular process understudy In this way, it was shown that an acetylated version ofthe annexin N-terminus peptide reduced the volume activa-tion of a chloride current in pulmonary artery endothelialcells, whereas a non-acetylated version of the same peptidedid not affect the current, implicating the S100A10 proteininteraction with AnxA2 in activation of these ion currents

(Nilius et al., 1996) The same strategy has revealed the

involvement of the (S100A10-AnxA2)2complex in histamine

induced secretion of vWF from endothelial cells (Knop et al.,

2004)

The AnxA2 N-terminus peptide is able to compete withcell-cell interactions between breast cancer cells and endothe-

lial cells while a scrambled peptide is not (Myrvang et al.,

2013) It was observed that AnxA2 is present on the surface ofbreast cancer cells, and S100A10 on the surface of endothelialcells Thus, these proteins may function as bridge betweenthese cell types in cell-cell interactions Similar studies usingcompeting N-terminus peptides indicate that (S100A10-AnxA2)2complexes are involved in tight junction assemblybetween kidney epithelial cells, suggesting a role in cell-cell

interactions (Lee et al., 2004).

The adhesion of prostate cancer cells to bone marrowendothelial cell is also inhibited by AnxA2 N-terminus pep-tides A putative AnxA2 receptor has been identified on pros-tate cancer cells, which may aid the cell-cell interaction

(Shiozawa et al., 2008).

A peptide based upon the AnxA2 N-terminus, but not ascrambled peptide, has been shown to inhibit neoangiogen-

esis into Matrigel plugs (Ling et al., 2004), suggesting that

protein interactions at the AnxA2 N-terminus participate in

this process The peptide may compete with an endogenous

(S100A10-AnxA2)2complex at the endothelial cell surface, or

alternatively it may inhibit directly the interaction between

AnxA2 and tPA

This last scenario illustrates the power of the use of

synthetic peptides in elucidating the involvement of the

(S100A10-AnxA2)2 complex in physiological processes but

also the problem, since additional interactions are possible (at

least in principle) to explain the observations

Chemical manipulation of the

AnxA2-S100 protein interaction

Protein interactions are generally considered not amenable to

blockade with small molecules This is largely because they

involve shallow and extensive interfaces with no features that

could support effective small molecule binding However,

there are cases of successful targeting of protein interactions

For example, the interaction between Mdm2 and p53, and

the interaction between Bcl2 and Bak have both been

explored pharmacologically using small molecule inhibitors,

which have subsequently shown promise as therapeutic

agents (Shangary and Wang, 2008; 2009; Gandhi et al., 2011).

It is of interest that both p53 and Bak contain a short helical

sequence that docks into a well-defined groove-like feature on

the surface of the respective binding partners, which in bothcases constitutes a small globular protein The protein inter-action between the AnxA2 N-terminus and S100A10 proteinssimilarly involves a well-defined and comparatively deepconcave binding pocket accommodating a small helicalpeptide The AnxA2 N-terminus conceals approximately

660 Å2 of solvent-accessible surface area in the lipophilic

pocket of S100A10 (Rety et al., 1999) Most of the binding

energy derives from hydrophobic interactions in the most portion of the pocket and from charge-enhancedH-bonds with the carboxyls of E5 and E9 of S100A10 (Becker

inner-et al., 1990; Rinner-ety inner-et al., 1999) Residues V3, I6, L7 and L10

alone displace approximately 430 Å2 of solvent-accessiblesurface area This is a size of binding pocket that is withinreach of what are commonly considered drug-like molecules(Lipinski, 2004)

A receptor-guided as well as a ligand-guided virtualscreening approach was recently used to identify a novel class

of small molecules that inhibit the interaction between

AnxA2 and S100A10 (Reddy et al., 2011; 2012; 2014)

(Figure 3) This virtual screening approach allowed the tification of candidate blockers that were able to dock intothe AnxA2-binding site on S100A10 or that mimicked thebinding pose of the AnxA2 N-terminus as defined in the

iden-complex crystal structure (Rety et al., 1999) Candidate

mol-ecules were then screened in a biochemical FRET assay thatmeasured the binding between the AnxA2 N-terminus and

Table 2

Peptide inhibitors used to elucidate the function of annexin A2 protein interactions

AA2 (1–14) Chloride channel activation measured by patch

AA2 (1–14) Formation of epithelial cell tight junctions in vitro Reduced by peptide Lee et al., 2004

AA2 (1–14) FGF- and VEGF-driven angiogenesis into Matrigel

plug in vivo

80% decrease in vascularization by thepeptide

Ling et al., 2004

AA2 (7–12) Pancreatic cancer cell migration Inhibited by peptide at high concentrations Diaz et al., 2004

AA2 (1–14) S100A4-induced, tPA-mediated plasminogen

activation on endothelial cells

Inhibited by the peptide Semov et al., 2005

AA2 (1–12) Adhesion of embryonic stem cells to annexin A2

in vitro

∼80% inhibition Jung et al., 2007

AA2 (1–14) AnxA2/S100A10 complex formation with CFTR in

Reduced by peptide Shiozawa et al., 2008

AA2 (1–12) Homing of prostate cancer cells to bone marrow

in vivo (metastasis)

Reduced by peptide Shiozawa et al., 2008

AA2 (1–14) Adhesion of breast cancer cells to endothelial cell

the S100A10 protein This identified two classes of

com-pounds: 3-hydroxy-1H-pyrrol-2(5H)-one analogues and

sub-stituted 1,2,4-triazoles as effective blockers of the binding of

S100A10 and AnxA2 (Reddy et al., 2011; 2012) The docking

suggested that both kinds of inhibitors could bind to three

pockets on S100A10 that are normally occupied by an acetyl,

valine and leucine moiety on the AnxA2 N-terminus (Reddy

et al., 2011; 2012) Selected blockers were also able to inhibit

the interaction of the native complex of AnxA2 and S100A10

and some were shown to inhibit the complex inside the cell

These compounds may be used to further elucidate the

func-tion of the (S100A10-AnxA2)2complex

The compound withaferin A has been shown to bind to

the N-terminus of AnxA2 via covalent bonding to the

cysteine residue at position 9 (Ozorowski et al., 2012) This

residue is solvent exposed in the (S100A10-AnxA2)2complex

and withaferin A did not inhibit the protein interaction

between the proteins However, it may inhibit functions of

the monomeric form of AnxA2

In addition to the S100A10 AnxA2 blockers described

above, a number of additional S100A4 protein blockers have

been described that could conceivably be useful in

under-standing interactions between S100 proteins and annexins

Merocyanine can covalently bind to Cys81of S100A4 and act

as an irreversible inhibitor of the binding of S100A4 to

myosin IIA (Garrett et al., 2008) Cys81is a part of the phobic area on S100A4 that could possibly be involved in the

hydro-binding with AnxA2 (Semov et al., 2005), indicating that

interactions with AnxA2 may also be inhibited by this pound A set of Food and Drug Administration-approveddrugs was tested for their ability to inhibit the Ca2+-inducedconformational change of S100A4 as determined by a fluo-rescence increase of the linked biosensor This identified anumber of phenothiazine compounds Phenothiazines werefound to defunctionalize S100A4 by polymerizing the protein

com-(Malashkevich et al., 2010) Other compounds affecting the

S100A4 conformation included ketoconazole, bepridil and

nicergoline (Garrett et al., 2008) Bepridil can inhibit the

myosin IIA filament depolarizing effect of S100A4 It is notknown whether compounds like these interfere with theAnxA2-binding properties of S100A4

Oxyclozanide has been shown to inhibit the interactionbetween S100A4 and receptors for advanced glycation end

products (RAGEs) or toll-like receptor 4 (TLR4) (Bjork et al.,

2013) It appears to bind to the homodimeric form or to anS100A4/A9 heterodimer to interfere with the binding with

RAGE and TLR4 (Bjork et al., 2013) These interactions are

implicated in inflammation and tumour growth (Foell and

Roth, 2004; Apetoh et al., 2007; Gebhardt et al., 2008; Bjork

et al., 2009) as well as cell matrix invasion (Yammani et al.,

Figure 3

Chemical structures of S100 protein interaction inhibitors

2006) Again, it remains to be established whether this

com-pound interferes with the AnxA2-binding properties of

S100A4

Conclusion

The interaction between S100 proteins and AnxA2 plays a

role in various processes in the cell The recent identification

of small molecule inhibitors of this interaction, combined

with known peptidic inhibitors, will allow further functional

elucidation of these complexes

Acknowledgements

We acknowledge the support of Cancer Research UK and the

Biotechnology and Biological Sciences Research Council UK

Conflict of interest

There is no conflict of interest to disclose

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Themed Section: Annexins VII Programme

Anna Alvarez-Guaita1*, Sandra Vilà de Muga1*, Dylan M Owen2†,

David Williamson2‡, Astrid Magenau2§, Ana García-Melero1,

Meritxell Reverter1, Monira Hoque3, Rose Cairns3, Rhea Cornely2,

Francesc Tebar1, Thomas Grewal3, Katharina Gaus2,

Jesús Ayala-Sanmartín4, Carlos Enrich1,5and Carles Rentero1

1Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina,

Universitat de Barcelona, Barcelona, Spain,2Center for Vascular Research, The University of New

South Wales, Sydney, NSW, Australia,3Faculty of Pharmacy, University of Sydney, Sydney,

NSW, Australia,4Centre National de la Recherche Scientifique (CNRS), École Normale Supérieure

(ENS) and Université Pierre et Marie Curie (UPMC), Paris, France, and5Centre de Recerca

Biomèdica CELLEX, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),

carles.rentero@ub.edu;

enrich@ub.edu -

*Both authors contributedequally

Present addresses:†Department ofPhysics and Randall Division ofCell and Molecular Biophysics,King’s College London, LondonWC2R 2LS, UK

-‡Faculty of Life Sciences,University of Manchester,Manchester M13 9PT, UK

-§Garvan Institute of MedicalResearch and Kinghorn CancerCentre, Cancer ResearchProgram, St Vincent’s ClinicalSchool, Faculty of Medicine,University of New South Wales,Sydney, NSW 2010, Australia. -

BACKGROUND AND PURPOSE

Annexin A6 (AnxA6) is a calcium-dependent phospholipid-binding protein that can be recruited to the plasma membrane tofunction as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution ofcellular cholesterol Here, we have investigated how AnxA6 influences the membrane order

EXPERIMENTAL APPROACH

We used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (ii) live cells to investigate membrane packing andordered lipid phases; and (iii) a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley’s Ksecond-order point pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and proteinclustering

KEY RESULTS

In artificial membranes, purified AnxA6 induced a global increase in membrane order However, confocal microscopy usingdi-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels andmodifies the actin cytoskeleton meshwork, displayed a decrease in membrane order (∼15 and 30% in A431 and MEF cellsrespectively) PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression inducedclustering of both raft and non-raft markers Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also

observed after cholesterol extraction with methyl-β-cyclodextrin or actin cytoskeleton disruption with latrunculin B

CONCLUSIONS AND IMPLICATIONS

AnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased membrane order throughthe regulation of cellular cholesterol homeostasis and the actin cytoskeleton This study provides the first evidence from livecells that support current models of annexins as membrane organizers

LINKED ARTICLES

This article is part of a themed section on Annexins VII Programme To view the other articles in this section visit

http://dx.doi.org/10.1111/bph.2015.172.issue-7

Abbreviations

A6ko, annexin A6 knockout; AnxA1, annexin A1; AnxA2, annexin A2; AnxA6, annexin A6; cPLA2, cytoplasmic

phospholipase A2; DRM, detergent-resistant membrane; EGFR, epidermal growth factor receptor; Lat34, transmembranedomain of linker for activation of T-cells; LatB, latrunculin B; Laurdan, 6-dodecanoyl-2-dimethylaminonaphthalene;Lck10, 10 first amino acids of Lck; Ld, liquid-disordered domain; Lo, liquid-ordered domain; LUV, large unilamellarvesicle; mβCD, methyl-β-cyclodextrin; MEF, mouse embryonic fibroblast; PALM, photoactivated localization

microscopy; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PFA, paraformaldehyde; PIP2,

phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine; r, radius; Src15, 15 first amino acids of Src; TIRF, total

internal reflection fluorescence; WT, wild type

Introduction

The cellular plasma membrane was initially described as a

two-dimensional homogeneous fluid cell compartment in

which lipids and embedded proteins are able to diffuse in the

lateral dimension (Singer and Nicolson, 1972; Nicolson,

2014) The plasma membrane sets a physical barrier and

selectively regulates the molecules that pass through,

delim-iting an intra- and an extracellular space This selective

per-meability is critical for drug diffusion into the cell (Sugano

et al., 2010).

Following many observations showing that proteins are

not homogeneously distributed in the plasma membrane,

Simons and Ikonen (1997) proposed the lipid raft hypothesis

In this model, lipids not only play a structural role but

mediate protein clustering and diffusion parameters within

the bilayer inducing lateral heterogeneity of proteins and

lipids It postulates the existence of cholesterol- and

sphingolipid-enriched, ordered-phase liquid (Lo) domains

surrounded by the bulk liquid-disordered (Ld) membrane

These domains create lateral heterogeneity and functionality

as highly efficient transient platforms for clustering of specific

proteins in cell membranes (Parton and Richards, 2003)

Many of these raft clustered proteins function in cell

signal-ling and endocytosis, as described for immune and other cell

types (Simons and Toomre, 2000; Jacobson et al., 2007).

Membrane cholesterol concentration has also been described

as a regulator of membrane permeability of both hydrophilicand hydrophobic solutes by changing the phospholipidpacking conformation of the lipid bilayer affecting drug

delivery (Zocher et al., 2013).

New findings have prompted researchers to revisit andupdate the lipid raft hypothesis in the last few years Kusumiand colleagues proposed that the cortical actin meshwork,together with lipid rafts and membrane proteins, regulateslateral diffusion of plasma membrane components (Ritchie

et al., 2003; Kusumi et al., 2004) A transient tethering of the

cortical actin filaments (fences) with transmembrane teins (pickets) would divide the plasma membrane incorrals, which would orchestrate protein and lipid lateraldiffusion and, among others, contribute to several cellularprocesses such as cell migration, mechanotransduction and

pro-immune cell activation (Head et al., 2014) Other studies

identified actin-associated ‘protein islands’ in the plasmamembrane surrounded by protein-free membrane character-ized by very low amounts of cholesterol In contrast, the

‘protein islands’ can have high (raft) or low (non-raft)

cholesterol content (Lillemeier et al., 2006) Targeting

fluo-rescent proteins to different domains of the plasma brane, for instance, Lck10 (first 10 amino acids of Lck) and

PE, phosphatidylethanolaminePIP2, phosphatidylinositol-4,5-bisphosphate

PS, phosphatidyl-L-serine

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b Alexander et al., 2013a,b).

transmembrane domain of linker for activation of T-cells

(Lat34) to lipid rafts, and Src15 (first 15 residues of Src) to

non-lipid rafts, allowed the definition of the role of the actin

cytoskeleton in inducing co-clustering of raft-associated

proteins by FRET microscopy (Chichili and Rodgers,

2007) Strikingly, actin promotes protein clustering and

regulates the protein phosphorylation of raft-associated

signalling proteins More recent findings suggest that the

actin meshwork can induce membrane partitioning with

Lo-enriched compartments surrounded by Ld-enriched

domains that correlate with the actin fibres (Honigmann

et al., 2014).

Adding further complexity, plasma membrane

cholesterol/membrane rafts can also regulate the cortical

actin cytoskeleton structure For instance, cholesterol

deple-tion induced stress fibre formadeple-tion through Rho activadeple-tion in

both mesenchymal and epithelial cell lines (Qi et al., 2009).

However, other studies suggested less stable actin stress fibres

after cholesterol depletion through the disorganization of

phosphatidylinositol-4,5-bisphosphate (PIP2) microdomains

at the plasma membrane in both fibroblasts and

lymph-oblasts (Kwik et al., 2003).

It is generally believed that annexins contribute to the

architecture, functioning and structural organization of

membranes through their binding to negatively charged

phospholipids in a calcium-dependent manner, but also

through protein-protein interactions with a variety of

membrane-associated proteins In addition, several annexins,

including AnxA1, AnxA2 and AnxA6, have shown some

affin-ity for cholesterol and cholesterol-enriched membranes

(Ayala-Sanmartin, 2001; Hulce et al., 2013) We and others

showed that AnxA2 and AnxA6 translocate to

detergent-resistant membranes (DRMs, membrane rafts) in a

calcium-dependent manner (Gokhale et al., 2005; Illien et al., 2012),

and were found enriched in caveolae (Schnitzer et al., 1995;

Calvo and Enrich, 2000) However, probably based on their

strong affinity to negatively charged phospholipids, these

annexins were also found in non-raft domains such as

clathrin-coated pits, which are required for receptor-mediated

endocytosis (Kamal et al., 1998; Zobiack et al., 2003).

In addition to its lipid-binding properties, we and others

identified that AnxA6 acts as a scaffolding protein for

signal-ling proteins such as PKCα and p120GAP (Grewal et al., 2005;

Rentero et al., 2006; Koese et al., 2013), but also interacts with

cytoskeleton proteins such as actin and spectrin (Kamal et al.,

1998; Monastyrskaya et al., 2009) This tethering ability of

AnxA6 may indicate an anchoring role of AnxA6 to link lipid

bilayers and cell cytoskeleton structure

Although annexins have been proposed as membrane

organizers since their discovery as calcium-dependent

membrane-binding proteins more than three decades ago,

experimental evidence for this hypothesis in living cells is

still lacking To address if AnxA6 alters membrane

organiza-tion, we compared membrane order and protein clustering

using the fluorescent dyes Laurdan and di-4-ANEPPDHQ, and

super-resolution photoactivated localization microscopy

(PALM), in live cells lacking or expressing AnxA6 Direct

visualization of membrane lipid structure of living cells

indi-cated that elevated AnxA6 expression significantly decreased

membrane order through the regulation of cholesterol

cellu-lar homeostasis and actin cytoskeleton meshwork These

findings are in agreement with our previous data identifyingreduced cholesterol levels at the plasma membrane upon

AnxA6 up-regulation (Cubells et al., 2007) Studies presented

here provide the first evidence from live cells that a member

of the annexins family, AnxA6, is capable of inducingchanges in the membrane architecture

by extrusion as previously described (Zibouche et al., 2008)

and Laurdan was added at a final concentration of 0.1%.Briefly, lipids were mixed together in chloroform The solventwas removed under a stream of nitrogen and the residualsolvent was removed under vacuum Lipids were then resus-pended in buffer A (40 mM HEPES pH 7, 30 mM KCl, 1 mMEGTA) at a final concentration of 1 mg·mL−1 by vortexingvigorously The liposomes were then extruded by passing thesuspension 21 times through a polycarbonate membranewith 0.1μm pores (Avestin, Mannheim, Germany)

Fluorescence measurements were performed as describedpreviously with a Cary fluorimeter (Varian, Agilent Technolo-gies, Santa Clara, CA, USA) in cuvettes thermostated at 37°C

(Maniti et al., 2010) In five independent experiments, three

spectra were recorded before and 7 min after addition of 5μgpurified AnxA6 to 5μg LUVs in the presence of 500 μM Ca2+.All fluorescence spectra were corrected for the baseline signal.Laurdan emission spectra were recorded from 400 to 600 nmusing a 365 nm excitation wavelength, and its generalized po-larization (GP) index was calculated according to the equation:

GP=(I( 440 )−I( 490 ))(I( 440 )+I( 490 ))

where intensities at 440 and 490 nm (I(440)and I(490), tively) represent the fluorescence intensities at the maximumemission wavelength in the ordered (440 nm) and disordered

respec-(490 nm) phases (Parasassi et al., 1990) The means of three replicates from each independent experiment (n = 5) wereused for the statistical analysis

Cell culture, transfection and treatments

A431 adenocarcinoma cells were supplied by ATCC 1555; Middlesex, UK) and mouse embryonic fibroblast (MEF)cells were isolated from wild-type (WT) and AnxA6 knockout

(CRL-mice (Hawkins et al., 1999) and immortalized by stable

trans-fection of the SV40 large T antigen mammalian expressionvector pBsSVD2005 Both cell types were grown in DMEM,10% fetal calf serum, L-glutamine (2 mM), penicillin(100 U·mL−1) and streptomycin (100μg·mL−1), and incubated

at 37°C, 5% CO2 The generation of stable AnxA6-expressing

A431 cells has been described in detail (Grewal et al., 2005).

For transient transfection, A431 or MEF cells were transfectedusing Lipofectamine LTX (Life Technologies, Thermo FisherScientific, Inc., Waltham, CA, USA) following the manufac-turer’s instructions

For di-4-ANEPPDHQ or PALM imaging, cells were treated

with 1μM (di-4-ANEPPDHQ) or 5 μM (PALM) LatB for

10 min, or with 10 mM mβCD for 30 min For the addition of

cholesterol, cells were incubated with 50μg·mL−1cholesterol

for 2 h

Fluorescence microscopy

For cholesterol imaging, cells were grown on glass coverslips,

fixed with 4% paraformaldehyde (PFA) at 37°C, stained with

200μg·mL−1Filipin, mounted in Mowiol (Calbiochem, Merck

Millipore, Darmstadt, Germany) and imaged with a Leica

DMI 6000B epifluorescence inverted microscope (Leica

Microsystems GmbH, Wetzlar, Germany) equipped with an

HCX PLAN APO 63× oil immersion objective lens For actin

staining, cells were grown on glass coverslips, fixed and

per-meabilized with methanol for 2 min at −20°C,

immunola-belled againstβ-actin and mounted in Mowiol (Calbiochem,

Merck Millipore) Images were acquired with a Leica TCS-SL

inverted spectral confocal microscope with a 63× oil

immer-sion objective lens

For di-4-ANEPPDHQ, live cells grown on glass coverslips

were stained for 30 min at 37°C with 1.5μM di-4-ANEPPDHQ

in DMEM, and in vivo imaged as described previously (Owen

et al., 2012b) in a Leica TCS-SL inverted spectral confocal

microscope with a 63× oil immersion objective lens

Di-4-ANEPPDHQ was excited at 488 nm and two simultaneous

images were acquired at 540–580 and 620–700 nm channels

Di-4-ANEPPDHQ intensity images were converted into GP

images (Gaus et al., 2006; Owen et al., 2012b), with each pixel

calculated in ImageJ (Schneider et al., 2012) from the two

di-4-ANEPPDHQ intensity images according to the equation:

GP=(I( 540 580 − )−I( 620 700 − ))(I( 540 580 − )+I( 620 700 − ))

GP distributions were obtained from the GP images

histo-gram values and non-linearly fitted to one Gauss

distribu-tions using a custom-built macro in ImageJ (Owen et al.,

2012b) GP images were pseudocoloured The induced

mem-brane changes were estimated by theΔGP (GPtreated− GPuntreated)

value

Cholesterol measurements

Total cellular cholesterol was determined using the Amplex™

Red Cholesterol Assay Kit (Molecular Probes, Thermo Fisher

Scientific, Inc.) according to the manufacturer’s instructions

Results were normalized to total cellular protein

Photoactivated localization

microscopy (PALM)

A431 and MEF cell lines were plated onto ozone-cleaned total

internal reflection fluorescence (TIRF)-suitable 18 mm

cover-slips and transfected with Lck10-PS-CFP2 or Src15-PS-CFP2

Twenty-four hours after transfection, cells were treated with

mβCD or LatB and fixed in 4% PFA at 37°C Seven to ten

PALM images were acquired on a TIRF microscope (ELYRA

PS-1; Carl Zeiss MicroImaging, GmbH, Jena, Germany) with a

100× NA 1.46 oil immersion objective Eight microwatts of

405 nm laser radiation was used for photo-conversion and

18 mW of 488 nm light was used for imaging of

green-converted PS-CFP2 Fifteen thousand images were acquired

per sample with a cooled, electron-multiplying

charge-coupled device camera (iXon DU-897D; Andor Tech., Ltd.,Belfast, UK) with an exposure time of 30 ms Images wereanalysed with Zeiss ZEN 2010D software (Carl Zeiss MicroIm-aging GmbH) Drifting of the sample during acquisition wascorrected relative to the position of surface-immobilized

100 nm colloidal gold beads (BB International, Cardiff, UK)placed on each sample

PALM image analysis

Events from raw fluorescence intensity images were Gaussianand Laplace filtered, and were judged to be originated from

single molecules when I − M > 6S, where I is event intensity,

M is mean image intensity and S the SD of image intensity.

The centre of each point-spread function was then calculated

by fitting intensity profiles to a two-dimensional Gaussiandistribution After correction for sample drift with immobilecolloidal gold bead markers, the x-y particle coordinates ofeach molecule were stored in a table Two-dimensionalmolecular coordinates were cropped into non-overlappingregions of 3μm × 3 μm in size Stringent parameters for singlemolecule detection were applied as previously described,excluding molecules with localization precisions smaller than

30 nm and re-excited fluorophores from further analysis

(Williamson et al., 2011; Rossy et al., 2013) Because

indi-vidual fluorophores can undergo several ‘blinking-cycles’, weaccounted for multiple blinks by selection of an appropriate

off-gap, as published previously (Annibale et al., 2011; Rossy

et al., 2013) Ripley’s K function analysis and quantitative

cluster maps were generated as described (Owen et al., 2010).

The Getis and Franklin’s L function for local point pattern

analysis was calculated with a cluster threshold of 60 (L(r)>

60) with the radial scale r= 30 nm, corresponding to mately 30% of each region’s cluster-map maximum (Owen

approxi-et al., 2013) Confidence intervals were generated by

simulat-ing 100 spatially random distributions with the same averagemolecular density as the data regions

Isolation of DRMs

Isolation of DRMs was performed as described previously

(Reverter et al., 2011) Fractions were separated by SDS-PAGE

and transferred to Immobilon-P membrane (Merck Millipore)followed by incubation with primary antibodies and theappropriate peroxidase-conjugated secondary antibodies andECL detection (Amersham Biosciences, GE Healthcare LifeSciences, Pittsburgh, PA, USA)

Data analysis

Data were analysed for normality using the Pearson omnibus K2normality test (D’Agostino et al., 1990)

D’Agostiono-from Prism 5.02 software (GraphPad Software, Inc., La Jolla,

CA, USA) to ensure Gaussian distribution and its suitabilityfor further analysis Statistical significance was determined by

two-tailed unpaired Student’s t-test or Bonferroni post-tested

two-wayANOVAusing Prism 5.02 Differences were considered

statistically significant at P < 0.05 Graphs are given as barplots± SD or SEM as indicated in the figure legends

Materials

DMEM was from Biological Industries (Kibbutz Beit-Haemek,Israel) Methyl-β-cyclodextrin (mβCD), egg yolk L-α-phos-

phatidylcholine (PC), egg yolk

L-α-phosphatidylethanol-amine (PE), brain L-α-glycerophosphatidyl-L-serine (PS) and

cholesterol were purchased from Sigma-Aldrich (St Louis,

MO, USA) Latrunculin B (LatB) was purchased from

Calbio-chem (Merck Millipore) Brain

L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) was from Avanti Polar Lipids (Alabaster,

AL, USA) Laurdan, di-4-ANEPPDHPQ and Filipin were from

Molecular Probes (Thermo Fisher Scientific, Inc.) Annexin A6

from pig brain was a kind gift of L.A Pradel (Paris, France)

Mouse monoclonal β-actin and rabbit polyclonal

anti-body against GFP were from Abcam (Cambridge, UK) Rabbit

polyclonal antibody against RFP was from Genscript

(Piscata-way, NJ, USA) HRP-conjugated secondary antibodies were

from Bio-Rad Laboratories (Hercules, CA, USA) pBsSVD2005

(Addgene plasmid 21826) was a kind gift of D Ron

(Cam-bridge, UK)

For the generation of eGFP, monomeric Cherry (mCherry)

and photoswitchable cyan fluorescent protein 2 (PS-CFP2)

fusion proteins containing either the Lck10 or the Src15, sense

and antisense oligonucleotides for the respective human

sequences plus a spacer of four glycines were annealed and

subcloned into the BamHI and EcoRI sites of pEGFP-N1

(Tanaka Bio Europe/Clontech, Saint-Germain-en-Laye,

France), pmCherry-N1 (kindly provided by R.Y Tsien, La Jolla,

CA, USA) and pPS-CFP2-N1 (Evrogen, Moscow, Russia)

Results

AnxA6 changes membrane order in LUVs

The potential involvement of several annexin family

members in the compartmentalization of membrane lipids

and cortical actin cytoskeleton using in vitro vesicle and

cel-lular models has been described previously (Illien et al., 2012;

Drucker et al., 2013), supporting models of annexins as

mem-brane organizers (Gerke et al., 2005) This might involve the

ability of annexins to bind negatively charged phospholipids

as well as cholesterol However, there is still a lack of an

integrated model to explain how annexins may regulate the

formation and/or maintenance of lipid microdomains

Taking advantage of Laurdan, an environment-sensitive

membrane probe, to label LUVs, we investigated whether the

addition of AnxA6 could induce modification in membrane

order in the presence or absence of cholesterol Like other

annexins, AnxA6 preferentially binds PS and other negatively

charged phospholipids (Gerke et al., 2005) AnxA6 may also

show some affinity for PIP2in vitro (Enrich et al., 2011; Hoque

et al., 2014), which is often enriched in specialized,

cholesterol-rich domains (DRMs, lipid rafts) at the plasma

membrane (Hayes et al., 2004; Rescher and Gerke, 2004).

Therefore, membrane order of PC and PE vesicles with PS or

PIP2 ± AnxA6 together with calcium and with or without

cholesterol was compared

As shown in Figure 1, the addition of cholesterol to

PC/PS/PE and PC/PIP2/PE LUVs strongly increased membrane

order; Laurdan fluorescence intensity increased at 440 nm

and decreased at 490 nm (compare the spectral shape of

Figure 1B vs 1A and Figure 1D vs 1C), and the GP value

[ratiometric function to quantify membrane order (Owen

et al., 2012b)] dramatically increased in those LUVs In

cholesterol-free Ld membranes (Figure 1A and C), AnxA6induced a statistically significant increase in membrane order

in both PS and PIP2LUVs (Figure 1A and C) At the same time,

a statistically significant increase of membrane order (Lo)could be observed with the addition of purified AnxA6 tocholesterol-containing membranes (Figure 1B and D) Inter-estingly, addition of AnxA6 increased membrane orderregardless whether LUVs contained PS or PIP2 Takentogether, these data indicate that AnxA6 is able to induce

membrane lipid redistribution in vitro.

Annexin A6 regulates membrane order in living cells

To assess the possible role of AnxA6 in plasma membraneremodelling in living cells, we next examined the membraneorder of two cellular models using: (i) the AnxA6-deficientepithelial adenocarcinoma cell line A431-WT and a well-characterized AnxA6-overexpressing A431 cell line (A431-A6)

(Grewal et al., 2005); and (ii) wild-type mesenchymal mouse

embryonic fibroblasts (MEF-WT) and AnxA6 knockout MEFs(MEF-A6ko) derived from the AnxA6 knockout mice

(Hawkins et al., 1999).

Membrane order was assessed by labelling the plasmamembrane of these cells with the fluorescent probe

di-4-ANEPPDHQ (Figure 2A) (Owen et al., 2012b)

Di-4-ANEPPDHQ specifically labels the cell membranes (Figure 2A)and is associated with a spectral shift when membrane orderdecreases The membrane order can be quantified by the

ratiometric GP function (Figure 2B and C) (Owen et al.,

2012b), and the induced membrane changes can be mated by theΔGP (GPtreated− GPuntreated), where a positiveΔGPindicates an increase in membrane order and a negativeΔGPindicates a decrease in membrane order

esti-The comparison ofΔGP demonstrated that AnxA6 sion in A431 and MEF cells (A431-A6 and MEF-WT) induced

expres-a diminution in the plexpres-asmexpres-a membrexpres-ane order (Figure 2D expres-andE) In line with published data, 30 min 10 mM mβCD treat-ment decreased membrane order in A431 and MEFs, indepen-dently of AnxA6 expression (Figure 2D and E) However, bothup-regulation and loss of AnxA6 in A431-A6 and MEF-A6kocells were associated with a significantly increased sensitivitytowards mβCD compared with their respective controls Incontrast, the addition of exogenous cholesterol increasedmembrane order in both A431-WT and A431-A6 cells, restor-ing the membrane order perturbed by up-regulated AnxA6expression (Supporting Information Fig S1A) We have pre-viously shown that elevated AnxA6 levels cause intracellularcholesterol imbalance, characterized by a strong reduction of

the cholesterol levels at the plasma membrane (Cubells et al.,

2007) Filipin staining showed prominent cholesterol ing at the plasma membrane in AnxA6-deficient A431-WT

stain-(Cubells et al., 2007) and MEF-A6ko cells (Supporting

Infor-mation Fig S1B), suggesting a redistribution of cholesterol tothe plasma membrane in the absence of AnxA6 In line withprevious data, plasma membrane cholesterol levels in cellswith low/high AnxA6 levels did not correlate with total cho-lesterol levels in the two cell lines studied (Supporting Infor-mation Fig S1C)

Next, we studied the contribution of the cortical actincytoskeleton for plasma membrane organization in the pres-

ence or absence of AnxA6 Upon cortical actin network

depo-lymerization after 10 min of 1μM LatB treatment, we

observed changes in membrane order of A431-A6 cells

(Figure 2D) However, LatB induced a dramatic increment of

membrane order in both MEF-WT and MEF-A6ko cell lines

(Figure 2E) These findings correlated with AnxA6-dependent

changes in actin cytoskeleton organization: (i) AnxA6

expres-sion in A431 cells reduced cortical actin staining (arrowheads,

Supporting Information Fig S2) (ii) In contrast, AnxA6

expression in MEF cells increased stress fibres and reduced cell

surface area

Taken together, up-regulation of AnxA6 expression levels

is associated with plasma membrane lipid redistributions,

probably due to elevated AnxA6 levels causing intracellular

cholesterol imbalance, in particular reducing cholesterol

levels at the plasma membrane This may contribute to create

a microdomain environment that renders the plasma

mem-brane in AnxA6-expressing cells more sensitive towards

mβCD-induced lipid disorder Furthermore, increased

sensi-tivity towards actin-depolymerizing agents in our gain- and

loss-of-function AnxA6 models may be indicative of AnxA6

providing a bridging function for the actin cytoskeleton toattach to the plasma membrane, with potentially drasticconsequences for establishing membrane microdomainpartitioning

AnxA6-induced plasma membrane organization regulates clustering of raft and non-raft membrane proteins

Results presented above implied that AnxA6 contributed

to membrane remodelling To determine the effect of induced membrane order changes in protein domain parti-tioning at the plasma membrane in more detail, we nextanalysed the clustering of membrane-anchored fluorescentproteins expressed in the presence (A431-A6 and MEF-WT) orabsence (A431-WT and MEF-A6ko) of AnxA6 by PALM super-resolution microscopy Lck10 and Src15 membrane-targetingmotifs were fused to the photoconvertible fluorescent proteinPS-CFP2 Lck10 corresponds to the first 10 N-terminal aminoacids of Lck, which contain a myristoylation and two palmi-toylation groups and partitions into DRMs In contrast, Src15corresponds to the first 15 N-terminal residues of Src, which

AnxA6-Figure 1

AnxA6 modifies membrane order in LUVs Normalized fluorescence spectra of Laurdan stained (A) PC/PS/PE, (B) PC/PS/PE/Chol, (C) PC/PIP2/PEand (D) PC/PIP2/PE/Chol LUVs in the absence or presence of purified porcine AnxA6 (5μg) Mean and SD graphical representation of itscorresponding GP values See Methods for the preparation of LUV details The presented spectra are representative of five independentexperiments (three replicates per condition for each experiment), where Lo (440 nm) and Ld (490 nm) emission wavelengths are represented The

means of three replicates from five independent experiments were used for statistical analysis Unpaired Student’s t-test showed statistically significant differences *P < 0.05, **P < 0.01, ***P < 0.001 Chol, cholesterol.

contains a myristoylation group and several positively

charged amino acids and is not enriched in DRMs but in

Triton X-100 soluble fractions In line with total cell lysate

fractionation data, these motifs are targeted to raft (DRM) or

to non-raft (TX-100 soluble) fractions of the plasma

mem-brane respectively (Chichili and Rodgers, 2007) (Supporting

Information Fig S3)

The clustering level of Lck10 and Src15 membrane

domain markers imaged by PALM microscopy was calculated

using the Ripley’s K function (L(r)-r), which indicates the

degree of clustering (density of molecules) at a specific radius

r (Owen et al., 2010) Both these membrane domain markers

clustered in A431 and MEF cells, with a clustering radius of r

= 60 nm in all cases (Figures 3A,C and 4A,C) At this radius,

we also analysed the percentage of molecules per cluster, the

cluster density and the cluster radius (Figures 3B,D and 4B,D)

A431 cells with and without AnxA6 displayed no significant

changes in regard to raft marker clustering (Lck10, Figure 3A

and B) However, Lck10 clustering, proportion of molecules

in cluster and cluster density significantly increased in

MEF-WT cells (Figure 4A and B) On the other hand, the

expression of AnxA6 increased Src15 clustering, proportion

of molecules in cluster and cluster density in both A431-A6and MEF-WT cells, with slight increase in cluster radius(Figures 3C,D and 4C,D)

Hence, AnxA6 expression differentially affects the bution of raft and non-raft markers depending on the celltype analysed While AnxA6 increases membrane raft proteinclustering only in MEF-WT, AnxA6 expression in bothA431-A6 and MEF-WT increases non-lipid raft proteinclustering

distri-AnxA6 modulates the regulatory role of cholesterol and cortical actin cytoskeleton in plasma membrane protein partitioning

Since the lipid raft model was proposed in 1997 by Ikonenand Simons (Simons and Ikonen, 1997), the cholesterol func-tion on the protein partitioning at the plasma membrane has

been studied extensively (Owen et al., 2012a) As outlined

above, we and others identified a role for AnxA6 in the

regulation of cholesterol homeostasis (Enrich et al., 2011),

with elevated AnxA6 levels causing cholesterol accumulation

in late endosomes, thereby reducing cholesterol at the Golgi

complex and the plasma membrane (Cubells et al., 2007) As

Figure 2

Membrane order of AnxA6-overexpressing A431 and AnxA6 knockout MEF cells A431-WT, A431-A6, MEF-WT and MEF-A6ko cells were stainedwith 1.5μg·mL−1di-4-ANEPPDHQ for 30 min and imaged with a confocal microscope (A) Representative GP pseudocoloured images of A431 andMEF cells Bar, 10μm GP value histogram graphical representations of di-4-ANEPPDHQ stained (B) A431 and (C) MEF images from (A) (D) Meanand SD of A431-WT versus A431-A6 and (E) MEF-WT versus MEF-A6koΔGP representation under normal conditions, 30 min 10 mM mβCD and

10 min 5μM LatB treatments of di-4-ANEPPDHQ stained images The mean GP values of five images from five independent experiments wereused to generate theΔGP values for the statistical analysis Two-wayANOVAtests were conducted on (C) and (D), and statistically significant

interaction between AnxA6 levels and drug treatment (F(2, 84) = 8.907, P = 0.0003 in A431 cells; F(2, 66) = 11.20, P < 0.0001 in MEF cells) was determined Bonferroni post-test analysis showed significant differences for drug treatment (#P < 0.05, ##P < 0.01, ###P < 0.001) and AnxA6 expression (*P < 0.05, **P < 0.01, ***P < 0.001).

shown above, AnxA6 modified membrane order of PS- and

PIP2-containing membrane bilayers in a calcium-dependent

manner in vitro (Figure 1), but most strikingly, also in living

cells (Figure 2) Furthermore, these AnxA6-induced changes

in membrane order in live cells correlated with altered

membrane-anchored raft and non-raft marker proteins

parti-tioning at the plasma membrane (Figures 3 and 4)

To further assess the role of AnxA6 in

cholesterol-dependent plasma membrane protein partitioning,

Lck10-and Src15-PS-CFP2 transfected cells were treated with 30 min

10 mM mβCD, fixed and PALM images were acquired The

cluster analysis revealed that cholesterol depletion did not

significantly affect Lck10 clustering in MEF-A6ko cells, but

induced a broad Lck10 Ripley’s K function curve, indicating

higher heterogeneity of raft cluster size (Figure 5C,

blue-dotted vs red-blue-dotted line; Supporting Information Fig S4) In

both AnxA6-expressing MEF-WT and A431-A6 cells, mβCD

treatment significantly reduced Lck10 clustering and

increased cluster size heterogeneity (Figure 5A and C, blue

line; Supporting Information Fig S4) On the other hand,

mβCD treatment did not affect Src15 clustering of deficient A431-WT cells (Figure 5B, blue-dotted vs red-dottedline; Supporting Information Fig S4), but increased Src15clustering, proportion of molecules in cluster, cluster densityand cluster size in AnxA6-deficient MEF-A6ko cells(Figure 5D, blue-dotted line; Supporting Information Fig S4).When we treated AnxA6-expressing A431-A6 cells withmβCD, Src15 clustering was comparable to non-treatedA431-A6 cells (Figure 5B, blue vs red line; Supporting Infor-mation Fig S4) However, mβCD treatment of MEF-WT cellsdropped Src15 clustering to levels observed in non-treatedMEF-A6ko cells (Figure 5D, compare blue vs red-dotted lines;Supporting Information Fig S4)

AnxA6-Taken together, our PALM microscopy data furtheremphasize differential and cell-specific differences of AnxA6

on the cholesterol-sensitive microdomain distribution of raftand non-raft markers

The function of the cortical actin meshwork in membraneprotein partitioning and clustering has been examined

extensively (Owen et al., 2012a; Gomez-Llobregat et al.,

Figure 3

Cluster analysis of Lck10-PS-CFP2 and Src15-PS-CFP2 in A431-WT and A431-A6 cells A431-WT and A431-A6 cells were transfected with (A andB) Lck10-PS-CFP2 and (C and D) Src15-PS-CFP2 and fixed 20 min with 4% PFA PALM images were acquired and cluster analysis of 35–50non-overlapping 3× 3 μm regions at the plasma membrane from 7 to 10 PALM images was performed as explained in Materials and Methods.(A) Graphical representation of mean Ripley’s K functions of 35–50 non-overlapping regions of Lck10-PS-CFP2 in A431-WT and A431-A6 cells Itreports the degree of clustering relative to a random distribution (indicated by the 95% CI, grey dotted line) (B) Graphical representation of mean

± SEM of maximum L(r)-r at radius = 60 nm, molecules in cluster, cluster density and cluster radius of Lck10-PS-CFP2 in A431-WT and A431-A6

cells (C) Mean Ripley’s K functions of 35–50 non-overlapping regions of Src15-PS-CFP2 in both A431-WT and A431-A6 cells Grey dotted line,95% CI (D) Graphical representation of mean± SEM of maximum L(r)-r at radius = 60 nm, molecules in cluster, cluster density and cluster radius

of Src15-PS-CFP2 in A431-WT and A431-A6 cells Unpaired Student’s t-test showed statistically significant differences in (D) **P < 0.01, ***P <

0.001

2013) To analyse if AnxA6 may contribute to cortical

actin cytoskeleton-dependent compartmentalization of

membrane-anchored proteins such as Lck10 and Src15, A431

and MEF± AnxA6 cell lines transfected with fluorescent raft

(Lck10) and non-raft (Src15) markers were treated with 5μM

LatB for 10 min, fixed and imaged by PALM microscopy

Cluster analysis of these PALM images revealed that actin

cytoskeleton disruption did not significantly affect Lck10

clustering in AnxA6-deficient A431-WT cells, but increased

cluster heterogeneity in MEF-A6ko fibroblasts (Figure 5A and

C, green-dotted vs red-dotted line; Supporting Information

Fig S4) In contrast, AnxA6 expression in A431-A6 and

MEF-WT correlated with LatB treatment inducing Lck10

clus-tering (proportion of molecules in cluster, cluster density and

cluster size) (Figure 5A and C, green line; Supporting

Infor-mation Fig S4) On the other hand, LatB treatment induced

Src15 clustering in both A431-WT and MEF-A6ko cells

(Figure 5B and D, green-dotted vs red-dotted line;

Support-ing Information Fig S4) The expression of AnxA6 in

LatB-treated A431 cells reduced Src15 clustering to almost the

clustering levels of untreated A431-WT cells (Figure 5B, green

vs red-dotted line; Supporting Information Fig S4) In

MEF-WT, the presence of AnxA6 was associated with slightly more

Src15 clustering, proportion of molecules in cluster, clusterdensity and cluster size than in LatB-treated MEF-A6ko fibro-blasts (Figure 5D, green vs red line; Supporting InformationFig S4)

Altogether, these results highlight that differential sion levels of AnxA6 not only alter the cholesterol-sensitivedistribution of raft and non-raft proteins, but can also modu-late the actin-dependent microdomain environment in a cell-specific manner

expres-Discussion and conclusions

Utilizing two gain- and loss-of-function cellular models forAnxA6, this study provides the first evidence from live cellsthat members of the annexin family have the ability toremodel plasma membrane order Results presented herestrongly suggest that AnxA6 modulates plasma membraneorder through two different mechanisms: (i) directly affectingphospholipid bilayer organization and the actin corticalcytoskeleton; and (ii) indirectly through alterations in choles-terol homeostasis, thereby inducing plasma membrane cho-lesterol depletion and plasma membrane order diminution

Figure 4

Cluster analysis of Lck10-PS-CFP2 and Src15-PS-CFP2 in MEF-WT and MEF-A6ko cells MEF-WT and MEF-A6ko cells were transfected with (A andB) Lck10-PS-CFP2 and (C and D) Src15-PS-CFP2 and fixed 20 min with 4% PFA PALM images were acquired and cluster analysis of 35–50non-overlapping 3× 3 μm regions at the plasma membrane from 7 to 10 PALM images was performed as explained in Materials and Methods.(A) Graphical representation of mean Ripley’s K functions of 35–50 non-overlapping regions of Lck10-PS-CFP2 in both MEF-WT and MEF-A6kocells Grey dotted line, 95% CI (B) Graphical representation of mean± SEM of maximum L(r)-r at radius = 60 nm, molecules in cluster, cluster

density and cluster radius of Lck10-PS-CFP2 in MEF-WT and MEF-A6ko cells (C) Mean Ripley’s K functions of 35–50 non-overlapping regions ofSrc15-PS-CFP2 in both MEF-WT and MEF-A6ko cells Grey dotted line, 95% CI (D) Graphical representation of mean± SEM of maximum L(r)-r

at radius= 60 nm, molecules in cluster, cluster density and cluster radius of Src15-PS-CFP2 in MEF-WT and MEF-A6ko cells Unpaired Student’s

t-test showed statistically significant differences in (B) and (D) *P < 0.05, ***P < 0.001.

AnxA6 is well known to play a role in calcium

homeosta-sis, membrane traffic and membrane organization Negatively

charged phospholipids [phosphatidylserine (PS),

phosphati-dylinositol, phosphatidic acid] are the preferred binding

part-ners of annexins It has been suggested that, despite

calcium-dependent interaction with anionic phospholipids, AnxA6

displays calcium-independent cholesterol-binding properties

(de Diego et al., 2002) In addition, AnxA6 also shows affinity

for PE and arachidonic acid (Edwards and Crumpton, 1991),

which are enriched in membrane rafts and generated by

cytoplasmic phospholipase A2 (cPLA2) (Brown et al., 2003).

Apart from the role in phospholipid reorganization and

membrane aggregation, AnxA6 expression also regulates

intracellular cholesterol homeostasis (Grewal et al., 2010).

Cells with elevated AnxA6 levels are characterized by an

accumulation of cholesterol in late endosomes, with a

con-sequent cholesterol diminution at the plasma membrane and

Golgi apparatus, which inhibits caveolin-1 transport to the

plasma membrane (Cubells et al., 2007) Interestingly,

choles-terol depletion in the Golgi of AnxA6-expressing cells

interferes with the recruitment and activity of cPLA2 at the

trans-Golgi-network (TGN) (Cubells et al., 2008) Given that

cPLA2 is required to drive cholesterol-dependent formation

and transport of secretory vesicles from the TGN to the

plasma membrane, this might further contribute to changes

in membrane order at the cell surface

A role for cholesterol to stimulate the binding of AnxA6 to

liposomes in vitro (Ayala-Sanmartin, 2001) and changes in

cholesterol and/or pH stimulating Ca2+-independent tions of AnxA6 with endosomal and cell surface membranes

interac-had already been observed in earlier studies (de Diego et al., 2002; Monastyrskaya et al., 2009) AnxA6 has been postulated

as a bona fide cholesterol-binding protein; in vitro binding

studies identified tryptophan-343 (W343) within the linkerregion of AnxA6 as important for the proposed interaction

between recombinant AnxA6 and cholesterol (Domon et al.,

2010) More recently, a comprehensive proteome-widemapping of cholesterol-interacting proteins in mammaliancells recognized AnxA6 as a potential cholesterol-binding

protein (Hulce et al., 2013) Interestingly, while AnxA6 has

been linked to cholesterol homeostasis, similar modes ofaction have not been made for other annexins like AnxA2,indicating different mechanisms of AnxA2 and AnxA6 to

affect plasma membrane order (Illien et al., 2012).

In Figure 6, we propose a model that summarizes andlinks AnxA6 expression with cholesterol- and actin-mediatedstructural and functional changes at the plasma membrane.This model outlines two different plasma membrane domaindistributions depending on cellular cholesterol levels andcortical actin cytoskeleton meshwork features In MEF cells,the cortical actin cytoskeleton forms corrals where the Lo raftdomains are confined This hypothesis is in agreement with

Figure 5

Ripley’s K function of Lck10-PS-CFP2 and Src15-PS-CFP2 in mβCD and LatB-treated A431 and MEF cells Mean Ripley’s K function graphicalrepresentations of 35–50 non-overlapping 3× 3 μm regions at the plasma membrane from 7 to 10 PALM images of (A and B) A431-WT andA431-A6 and (C and D) MEF-WT and MEF-A6ko of (A and C) Lck10-PS-CFP2 and (B and D) Src15-PS-CFP2 transfected cells Graphs show the mean

of Ripley’s K functions under normal conditions, 10 mM mβCD and 5 μM LatB treatments Grey dotted line, 95% CIs

recent findings that describe, using fluorescent correlation

spectrometry and stimulated emission depletion

super-resolution microscopy, membrane partitioning into

Lo-enriched domains surrounded by Ld-enriched domains

that correlate with actin fibres (Honigmann et al., 2014) In

both models, the degree of the actin meshwork density will

determine the corral size, and the level of membrane

choles-terol and other raftophilic lipid moieties will determine the

different membrane phase proportions In A431 cells,

however, cortical actin cytoskeleton delimits Ld non-raft

domains into corrals, with the Lo membrane phase associated

with the actin filaments Experimental data supporting this

hypothesis, using a phasor approach to fluorescence lifetime

imaging microscopy data analysis and 7-ketocholesterol

treatment, allowed us to propose a model where, in reduced

plasma membrane order conditions, Ld phase is present in

the corral lumen while the Lo raft phase is associated with

actin filaments (Owen et al., 2012c) The proposed model

here suggests that the regulation of cholesterol levels, other

raftophilic lipid moieties and/or the actin cytoskeleton

mesh-work, by means of AnxA6 and other membrane regulators or

even specific drugs such as mβCD or LatB, might modulate

the plasma membrane structure and partitioning In

addi-tion, fluctuations in intracellular calcium levels are well

known to strongly affect the membrane-binding ability of

AnxA6 in vitro, but also in cellular models For instance, we

showed that calcium ionophores, but also activation of

epi-dermal growth factor receptor (EGFR), which triggers localintracellular calcium concentration increase, induces AnxA6

translocation to the plasma membrane (Vila de Muga et al., 2009; Grewal et al., 2010) Hence, a combination of choles-

terol, actin and calcium-driven events probably enablesAnxA6 to not only affect the membrane order locally, but also

to affect recruitment of signalling proteins to the plasmamembrane In line with this hypothesis, AnxA6 promotescalcium-dependent membrane recruitment of the GTPase

activating protein p120GAP (Grewal et al., 2005) as well as

PKCα (Koese et al., 2013) This is associated with AnxA6 acting with active H-Ras and EGFR, promoting EGF-inducibleRas and EGFR inactivation in a calcium-dependent manner

inter-(Vila de Muga et al., 2009; Koese et al., 2013) One can

envisage that the dual role of AnxA6 affecting membraneorder through cholesterol- and actin-dependent eventsidentified here, together with calcium-sensitive AnxA6 mem-brane association, modulates the recruitment of signallingproteins, and consequently strength and duration of cellularsignalling

We and others have shown different AnxA6 functionsregulating important cellular/physiological events such asendocytosis, exocytosis and cell migration, where membranepartitioning is considered essential Firstly, AnxA6 is located

at clathrin-coated pits and caveolae at the plasma membrane(Calvo and Enrich, 2000), specific membrane structures char-acterized by Ld and Lo phases respectively Anderson and

Figure 6

Proposed model for AnxA6-induced membrane organization The proposed model suggests that fibroblasts plasma membrane (A) has lipid raftconfined into actin fibre corrals due to its cholesterol content and its cortical actin cytoskeleton (B) When AnxA6 is knocked out, higher plasmamembrane cholesterol content and a prominent cortical actin cytoskeleton can be observed, which could drive domain partitioning of both Loand Ld phases On the other hand, epithelial cells (C and D) might have non-rafts confined into corrals where cortical actin cytoskeletondetermines corral size and cluster partitioning (D) In this setting, AnxA6 expression may induce diminution of cortical actin cytoskeleton andplasma membrane cholesterol, allowing larger non-raft Ld domains

co-workers, based on AnxA6 interacting with spectrin,

pro-posed a role for AnxA6 in receptor-mediated endocytosis

(Kamal et al., 1998) Upon AnxA6 binding to spectrin, calpain

I cleaves spectrin and ‘opens’ the actin cytoskeleton

facilitat-ing the endocytosis These AnxA6-dependent dynamic

changes in membrane–cytoskeleton interaction are likely to

involve changes in membrane order

Secondly, currently available data suggest that AnxA6

probably inhibits the secretory pathway (Creutz, 1992;

Donnelly and Moss, 1997; Podszywalow-Bartnicka et al.,

2010) Our recent findings are in line with this concept as we

identified a significant diminution of retrograde transport of

vesicular stomatitis virus G protein transport from the cell

surface to the TGN in cells with up-regulated AnxA6 levels

(Cubells et al., 2007) Furthermore, high levels of AnxA6

interfered with cholesterol-sensitive and t-SNARE (SNAP23

and syntaxin-4) dependent secretion of cargo (fibronectin,

TNF-α) (Reverter et al., 2011)

Finally, our most recent data provide novel molecular

insights into our understanding of constitutive protein

traf-ficking at the TGN/endosomal boundaries and identify the

delivery of late endosomal cholesterol to the Golgi as a new

pathway linking cholesterol with t-SNARE functioning and

integrin recycling (Reverter et al., 2014).

Future studies will have to determine whether AnxA6

expression levels and its influence in the remodelling of

membrane microdomains are a common determinant for

cholesterol regulation of t-SNARE localization, assembly and

functioning in various cellular processes and cell types

Acknowledgements

This study was supported by grants BFU2012-36272 and

CSD2009-00016 from Ministerio de Economía y

Competitivi-dad (MINECO, Spain) and PI042182 from Fundació Marató

TV3 (Spain) to C E K G would like to acknowledge funding

from the National Health and Medical Research Council of

Australia (NHMRC; 1059278, 1022182 and 1037320) and the

Australian Research Council (ARC; CE140100011) T G is

supported by the NHMRC of Australia (510294) and the

Uni-versity of Sydney (2010-02681) F T is supported by MINECO

(BFU2012-38259) We are thankful to M Calvo (Centres

Científics i Tecnològics, Universitat de Barcelona) for her help

in confocal microscopy, and M Molinos for technical

assis-tance A A G and M R are grateful to MEC for a short-term

fellowship at the Center for Vascular Research, University of

New South Wales in Sydney, Australia

Author contributions

A A-G.: PALM experiments and biochemistry S V d M.:

Di-4-ANEPPDHQ experiments D M O and D W.: PALM

quantification A M.: Lck10 and Src15 cloning A G-M.:

Immunocytochemistry M R.: Cholesterol quantification M

H and R C.: Biochemistry experiments R C.: Lck10 and

Src15 cloning F T.: Discussion of results T G.: Discussion

and proofreading K G.: PALM experiments design J A-S.:

LUV experiments and discussion C E.: Financial support,

discussion and proofreading C R.: Experiment design, cussion of results and writing the paper

dis-Conflict of interest

The authors disclose no conflicts

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Supporting information

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:http://dx.doi.org/10.1111/bph.13022

Figure S1AnxA6 alters cholesterol levels and distribution inA431 and MEF cells

Figure S2AnxA6 modulates actin cytoskeleton distribution

in A431 and MEF cells

Figure S3 Lck10-EGFP and Src15-mCherry distribution inDRMs and bulk membranes after subcellular fractionation ofA431 and MEF± AnxA6 cells

Figure S4 Cluster analysis of Lck10-PS-CFP2 and CFP2 in mβCD and LatB-treated A431 and MEF cells