Cell
biology/Biologie
cellulaire
First
evidence
of
DAAM1
localization
in
mouse
seminal
vesicles
and
its
possible
involvement
during
regulated
exocytosis
Massimo
Venditti
a,
Chiara
Fasano
a,
Alessandra
Santillo
b,
Francesco
Aniello
c,
Sergio
Minucci
a,*
a
DipartimentodiMedicinaSperimentale,Sez,FisiologiaUmanaeFunzioniBiologicheIntegrate,Universita` deglistudidellaCampania ‘‘LuigiVanvitelli’’,ViaCostantinopoli,16,80138Napoli,Italy
b
DipartimentodiScienzeeTecnologieAmbientali,BiologicheeFarmaceutiche,Universita` degliStudidellaCampania‘‘LuigiVanvitelli’’, Caserta,Italy
cDipartimentodiBiologia,Universita` degliStudidiNapoli‘‘FedericoII’’,Napoli,Italy
1. Introduction
Forminsarealargegroupof120-to220-kDaproteins thatregulatethenucleationandassemblyofactinfibresin responsetoseveralsignals[1,2].Todate,15genescoding forforminshavebeenidentifiedinMammals[3–5].They
weredividedintosevensubclassesbasedonthesequence similarities:
diaphanous(DIAs); formins(FMNs);
FMN-likeforminsor formin-relatedproteins in leuko-cytes(FMNLs/FRLs);
Dishevelled-associated activators of morphogenesis (DAAMs);
forminhomologydomainproteins(FHODs); delphilinandinvertedformins(INFs)[6]. ARTICLE INFO
Articlehistory:
Availableonline21March2018
Keywords: DAAM1 Actin Exocytosis Seminalvesicles Smoothmuscle ABSTRACT
Dishevelled-associatedactivatorofmorphogenesis1(DAAM1)isaproteinbelongingto theforminfamily,whichregulates,togetherwiththesmallGTPaseRhoA,thenucleation andtheassemblyofactinfibresthroughWnt-DishevelledPCPpathway.Itsrolehasbeen investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expressionofDAAM1mRNAandproteinbyPCRandWesternblotanalysesandits co-localizationwithactininadultmouseseminalvesiclesbyimmunofluorescence.Weshow thatbothproteinsarecytoplasmic:actinisevidentatcell–celljunctionsandatcellcortex; DAAM1hadamorediffusedlocalization,butisalsoprominentattheapicalplasmatic membraneofepithelialcells.ThesefindingssupportourhypothesisofaroleofDAAM1in cytoskeletalrearrangementthatoccursduringtheexocytosisofsecretoryvesicles,andin particularconcerningactinfilaments.We werealsoableto detectDAAM1and actin associationinthesmoothmusclecellsthatsurroundtheepitheliumtoo.Inthiscase,we couldonlyspeculatethepossibleinvolvementofthisformininmuscularcellsinthe maintenanceandtheregulationofthecontractilestructures.Thepresentresultsstrongly suggestthatDAAM1couldhaveapivotalroleinvesicleexocytosisandinthephysiologyof mouseseminalvesicles.
C 2018Acade´miedessciences.PublishedbyElsevierMassonSAS.Allrightsreserved.
* Correspondingauthor.
E-mailaddress:sergio.minucci@unicampania.it(S.Minucci).
ContentslistsavailableatScienceDirect
Comptes
Rendus
Biologies
ww w . sci e nc e di r e ct . com
https://doi.org/10.1016/j.crvi.2018.03.001
DAAM1showsatypicalforminstructure,includingthe ForminHomology1(FH1)domain,whichcaninteractwith profilin,andtheFH2domain,whichbindsactin,allowing the nucleation of new filaments [1,2]. This protein is stabilizedinanautoinhibitedstateduetotheinteraction between thediaphanous inhibitory domain(DID) at C-terminal and the GTPase-binding domain (GBD) at N-terminal[7].DAAM1switchestoanactiveconformation upon bindingattheC-terminalwiththePDZdomainof Dishevelled2,aphosphoproteininvolvedintheplanarcell polarity(PCP)non-canonicalWntpathway[8].Thisfrees theGBDdomainand allowsforitsinteractionwithRho GTPase[8,9],whileactinisrecruitedattheFH2;RhoA,in turn,can activateRho-associated protein kinase(ROCK)
[10].Theseeventsresultin actinstatemodificationand cytoskeletal rearrangement. Several studieshave shown that DAAM1 participates in many essential biological processes,suchascytoskeletalorganizationincellpolarity, movement and adhesion during morphogenesis and organogenesis[11–16].AlthoughDAAM1hasbeen impli-cated in a variety of developmental processes, most frequently associated with central nervous system and heart morphogenesis[17–22],thereisjustlittle infor-mation about its role in adult tissues. We previously reportedthepossibleinvolvementofthisforminduring thepost-nataldevelopment ofrattestisandinratand humanspermatozoa[23].WeshowedthatDAAM1and actinsharedtheirlocalizationinthecytoplasmofgerm cells, suggesting its role in the formation of complex structures, marked by a significant cytoskeletal remo-delling,which enablesgerm cellseparation protection and maintenance. Moreover, we found that DAAM1 localized intheflagellum ofratspermatozoa,while in human sperm it was retained inside the residual cytoplasmicdroplets.Inafurtherpaper,weinvestigated theinvolvementofthetesticularDAAM1expression in the possible protective role of zinc against cadmium-induced male reproductive toxicity at adult age after maternal Cd and/or Zn exposureduring gestation and lactation [24]. As of now, no reports have directly associated DAAM1 with theepithelial cells of seminal vesicles.Theseareexocrineglandsthatare partofthe malesexaccessoryorgans.Theseglandsarepresent in most familiesofplacentalmammals[25]andproduce, aftertestosteronestimulation[26]afructose,citricacid, proteinsandprostaglandinrichsecretionthatcanreach more than 60–70% of the seminal plasma in humans
[27].Duringtheexocytosisofthesecomponents,agreat remodelling ofcytoskeletalelementsoccurs,especially regarding actin and it is known that factors that participateinthesamemolecularpathwaysare expres-sed in other exocrine tissues: these include other formins,suchasmDia[28,29],aswellasseveralGTPases andRho-relatedproteins[30,31].Thus,toimprovethe understandingofthepotentialroleoftheforminDAAM1 in the cellular system, and in particular during the exocytosisevents,inthiswork,weassessedthepossible involvementofthisproteininthesecretoryepitheliumof mouse seminal vesicles, comparing its expression and localizationwith afundamental componentof cellular cytoskeleton,theactinfilaments.
2. Materialsandmethods
2.1. Animalcareandtissueextraction
Male C 57 black mice (Mus musculus) were housed underdefiniteconditions(12D:12L)andwerefedstandard foodand providedwithwateradlibitum.Animalswere perfusedwithphosphatebuffersaline,PBS(13.6mMNaCl; 2.68mM KCl; 8.08mM Na2HPO4; 18.4mM KH2PO4;
0.9mMCaCl2;0.5mMMgCl2;pH7.4)andthensacrificed
by decapitation under ketamine anaesthesia (100mg/kg i.p.) in accordance with national and local guidelines coveringexperimental animals.Foreach animal,seminal vesiclesweredissected;onehalfwasfixedin10%formalin andembeddedinparaffinforhistologicalanalysis,theother halfwasquicklyfrozenbyimmersioninliquidnitrogenand storedat–808CuntilRNAandproteinextraction. Addition-ally, male Sprague–Dawley rats (Rattus norvegicus)were housedinthesameconditionsandtestesweredissected, frozenandstoredat–808Ctobeusedasapositivecontrol. 2.2. Reversetranscriptionpolymerasechainreaction (RT-PCR)
TotalRNAwasextractedfromthesamplesusingTrizol reagent(Sigma-Aldrich, St.Louis,MO,USA). First-strand cDNA was synthesizedusing 5
m
g of totalpooled RNA, 250ng/m
Loligo(dT)18,0.8mM primer(Promega, Heidel-berg, Germany) and 100 U Super-script II RT enzyme (Invitrogen, Paisley, UK) in a total volume of 25m
L, accordingtothemanufacturer’sinstructions.Asanegative control,the reaction wascarried out on the same RNA without using the reverse transcriptase enzyme (RT). Twotothreem
LoftheobtainedcDNAtemplatewerethen usedforthePCRreactionasdescribedbyChemeketal.[24]. Based on thepublishedsequenceof Mus musculus Daam1 mRNA (GenBank accession No. NM_026102.3), specificoligonucleotideprimersweredesignedasfollows: Daam1 for: 50-GCCATGTTTGCACTTCCAGC-30 Daam1
rev:50-TCAGAATGAGCCAGGACATG-30.
AnappropriateregionofMusmusculusActinmRNA(Act; NCBIGenBankaccessionNo.NM_007393.5)wasamplified with specific oligonucleotide primers (Act for=50
-CTTTTCCAGCCTTCCTTCTT-30,Actrev=50
-CTGCTTGCTGATC-CACATC-30)andused ascontrol.Theamplifications were
carriedoutfor35cycles,withdenaturationat948Cfor30s, annealingat568Cfor45sandextensionat728Cfor45s.The expected PCR productsizes were 392bp for Daam1 and 300bp for Act, 391. The amplification products were electrophoresedon1.5%agarosegel.
2.3. ProteinextractionandWesternblotanalysis
Mouseseminalvesiclesandrattesteswerelysedina specific buffer (1% NP-40, 0.1% SDS, 100mM sodium ortovanadate, 0.5% sodiumdeoxycholate in PBS) in the presence of protease inhibitors (4mg/mL of leupeptin, aprotinin,pepstatinA,chymostatin,PMSFand5mg/mLof TPCK).The homogenatesweresonicatedtwice by three strokes (20Hz for 20s each); after centrifugation for 30min at 10,000g, the supernatants were stored at
808C. Proteins (40
m
g) wereseparated by9% SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF)membranes(AmershamPharmacia Biotech, Buc-kinghamshire,UK)at280mAfor2.5hat48C.Thefilters weretreatedfor3hwithblockingsolution[5%skimmilk inTBS(10mMTris–HclpH7.6,150mMNaCl)]containing 0.25%Tween-20(Sigma–AldrichCorp.,Milan,Italy)before theadditionofanti-DAAM1(Sigma-Aldrich,Milan,Italy), oranti-ACTIN(ElabscienceBiotechnology,Wuhan,China) antibodiesdilutedrespectively1:3000and1:5000, incu-batedovernightat48C.AfterthreewashesinTBST(TBS including0.25%Tween20),thefilterswereincubatedwith horseradish peroxidase-conjugated anti-rabbit IgG (Sig-ma-Aldrich, Milan, Italy) for the rabbit anti-DAAM1 antibody diluted 1:10,000 in the blocking solution, or anti-mouseIgG(ImmunoReagents,Inc.,Raleigh,NC,USA) for the mouse anti-actin diluted 1:10,000 in the same blocking solution. Then, the filters were washed again three times in TBST and the immunocomplexes were revealedusingtheECL-Westernblottingdetectionsystem (AmershamPharmaciaBiotech,Buckinghamshire,UK). 2.4. HistologicalanalysisInordertoassessthequalityandtocharacterizethe histology of the tissue samples, 7-
m
m-thick mouse seminal vesicles sections were obtained, stained with hematoxylinandeosin(H&E),andexaminedunderalight microscope(LeicaDM2500) for histologicalevaluation. PhotographsweretakenusingtheLeicaDFC320R2digital camera.2.5. Immunofluorescenceanalysis
For theco-localization experimentsfor DAAM1+ AC-TIN, 7
m
m-seminal vesicles sections were dewaxed, rehydratedandprocessedasdescribedbyParianteetal.[23].Briefly,antigenretrievalwasperformedby pressure-cookingslidesfor3minin0.01Mcitratebuffer(pH6.0). Nonspecificbindingsiteswereblockedwithan appropri-atenormalserumdiluted1:5inPBScontaining5%(w/v) BSA before the addition of anti-DAAM1, or anti-ACTIN antibodies diluted 1:100, for overnight incubation at 48C.After washinginPBS,theslideswereincubatedfor 1hwiththeappropriatesecondaryantibody(AlexaFluor 488,Invitrogen;FITC-Jackson,ImmunoResearch,PeroMI, Italy; Anti-Mouse IgG 568, Sigma-Aldrich, Milan, Italy) diluted 1:500 in the blocking mixture.The slides were mounted with Vectashield+DAPI (Vector Laboratories, Peterborought,UK)fornuclearstaining,andthenobserved under the optical microscope (Leica DM 5000 B+CTR 5000)andimageswereviewedandsavedwithIM1000. 3. Results
3.1. DAAM1transcriptandproteinsinadultmouseseminal vesicles
Thepresenceof Daam1mRNAwasfirstinvestigated in adult mouse seminal vesicles by semiquantitative relative RT-PCR analysis. Agarose gel electrophoresisof
amplification products showed a single band of the expectedsize(392bpforDaam1)incDNAfromseminal vesicles(Fig.1A).TheamplifiedcDNAbandcorresponded to the Damm1 transcript, as demonstrated by the successive cloning and sequencing (not shown). The quality control of cDNA was performed using specific primers for Actin mRNA(Fig. 1A). The PCR analysis on negativecontrol(RT)samplesdidnotshowanysignal. TheexpressionofDAAM1wasalsoevidencedbyWestern blot analysis using polyclonal anti-DAAM1 antibody: a 123-kDaband(correspondingtoDAAM1)waspresentin totalextractsfromadultmouseseminalvesiclesandrat testis,usedasacontrol.Another,lowerbandwasdetected, probablyduetopost-translationalmodifications(Fig.1B). The same samples were employed for the analysis of ACTIN.Asexpected,itsbandwasdetectedinbothsamples, asaconfirmationoftheexpressionoftheproteininmouse seminalvesiclesandrattestis(Fig.1B).
3.2. Co-localizationofDAAM1+ACTINinmouseseminal vesicles
First,inordertochecktissuequalityandtodistinguish the morphology of the tissue, a haematoxylin-eosin staining was performed on seminal vesicles sections (Fig.2).Theseminalvesicleislinedbyapseudostratified epithelium with columnar secretory cells (arrows) and smallerbasal cells. Theepitheliumis highly folded and deepnarrowcryptslinedwithepitheliumextendfromthe main sac-like lumen (asterisk). A lamina propria of connective tissue and thick coat of smooth muscle surround theepithelial lining(arrowhead). The nucleus ofeachcolumnarcellislocatedinthebasalportionandis elongated in the direction of the long axis of the cell
[32].Then,toobtaindetaileddataonDAAM1distribution insidetheseminalvesicles,aco-localizationexperimentof DAAM1+ACTIN was studied by immunofluorescence analysis(Figs.3–4).Weshowedthatapositivesignalfor thetwoproteinswasfoundinthecellsofsmoothmuscles thatsurroundtheepithelium(arrowheads,Fig.3). More-over, both proteins showed a cytoplasmic localization inside theepithelial cells, but withslightdifferences in their expression pattern: ACTIN is evident at cell-cell junctions(arrowheads,Fig.4A,D;betterhighlightedinthe inset) and at cell cortex (arrows, Fig. 4A, D; better highlighted in the inset); DAAM1 had a more diffused Fig.1.ExpressionofDAAM1mRNAandproteininadultmouseseminal vesicles.A.AgarosegelelectrophoresisofRT-PCRproductsperformedon totalRNAfromadultmouseseminalvesicles.Theamplificationproducts wereobtainedusingspecificprimersformouseDaam1andActinand mRNA. M:molecular weight markerXIV (Roche Diagnostics);RT: negative control. B. Western blot analysis for DAAM1(123kDa, top section)andACTIN(42kDa,bottomsection)performedontotalprotein extractsfromadultmouseseminalvesicles(mSV)andrattestis(rTest), usedasapositivecontrol(Parianteetal.,2016).
Fig.2.Haematoxylin-eosinstainingofatissuesectioninwhichthenormalhistologyoftheadultmouseseminalvesiclesishighlighted(A).Thescalebars represent20mmin(A)and5mmintheinset.Thepointerlegendisprovidedinthebottom-righttable.
Fig.3. ImmunolocalizationofDAAM1inadultmouseseminalvesicles.GeneralappearanceofDAAM1(green,BandC)co-localizationbydoubleIFwith ACTIN(red,AandC)onmouseseminalvesiclessections.ThedatashowthatDAAM1co-localizeswithACTINinthemuscularcells(arrowheads).The nucleusismarkedinblue(A–D).D.Negativecontrol,obtainedbyomittingtheprimaryantibodies.Thescalebarsrepresent40mm.
localization,butisalsoprominentattheapicalmembrane (Fig.4B, Ebetterhighlightedintheinset).Fig.4CandF show that DAAM1 signal noticeably overlaps with the fluorescenceproducedbyACTIN(seearrowsand arrow-heads),confirmingtheirassociationattheapicalplasmatic membrane,wherethetwoco-localizationsignalsproduce theintermediateyellow–orangetint.
4. Discussion
Theseminalvesicles(SV),togetherwiththeprostate and Cowper’s gland, constitute the male sex accessory glands. These glands are present in most families of placentalmammals[25]wheretheirsecretionscanreach more than 60–70% of the seminal plasma [27]. SV secretions are mixed with spermatozoa and secretions from other accessory reproductive glands before being ejaculated intothe vagina. The contributionof seminal vesiclestotheseminalplasmaincludesions(e.g.,Na+,K+,
Zn+), sugars (e.g., fructose, glucose), peptides (e.g.,
glutathione)and,aboveall,highlyconcentratedproteins. TherelevanceofSVforfertilityisrelatedtotheinfluenceof theirsecretionsonspermmotility,metabolismandsurface components[33–36]andtotheirroleintheimmunology ofreproductivefunctions[36–39].Oncesynthesized,these componentsarereleasedinthelumenofthegland,where they contribute to form the ejaculate. The secretion is regulatedbytestosterone[26],andduringexocytosis,the secretory vesicles are delivered to the cell periphery, wheretheyresideuntilthehormonalstimulusstartstheir fusionwiththeplasmamembrane.Themolecularpathway
controlling regulated exocytosis has been extensively investigated.In fact,several factorshavebeenidentified askeyregulatorsintheprocess,suchasintracellularCa2+
levels,ionchannels,Gproteins-coupledreceptors,small GTPasesandtheactincytoskeleton[40–45].Inparticular, actincytoskeletonremodellingplaysacrucialroleduring regulatedexocytosis,includingmembranebudding,fusion andfission[46–48].Moreover,ithasbeenproposedadual actionofactinfilamentsduringtheseprocesses.Actincan negatively regulateexocytosis,creatinga sort ofbarrier beneaththeplasmamembrane,preventingitsfusionwith secretoryvesicles;ontheotherhand,togetherwith actin-basedmotors,thisproteincanfacilitatethetransportof thevesiclestowardthefusionsite. Thisdoublebehavior suggeststhatregulatedexocytosisrequiresafinecontrolof assemblyanddisassemblyofactinfilaments[49,50]. To-day,itisknownthatpartofactinregulationisperformed by Wnt proteins [51]. Upon interactionwiththe mem-brane frizzled/co-receptor complex [52], Wnt recruits Dishevelled(Dvl),whichcanactivatedifferentpathways. In planar-cellpolarity(PCP)non-canonicalpathway,Dvl canactivatetheforminDAAM1,interactingwithitsDAD (DAAM autoinhibitory domain) region. The Dvl/DAAM1 complexis,then,abletorecruitandactivate,throughthe forminhomology(FH)1domain,theguaninenucleotide exchange factor WGEF, which, in turn, activates a Rho GTPase [8,9,53]. The subsequent activation of Rho-associatedkinase(ROCK)[10]resultsinactin polymeriza-tionatthedimerizedFH2domain[54]and,consequently, in cytoskeletalreorganization.Giventheinvolvementof bothactinandsmallGTPasesduringexocytosis,andbeing Fig.4.Co-localizationofDAAM1andACTINinadultmouseseminalvesicles.Twodifferentfieldswerecaptured[field1:(A/C);field2:(D/F)].A,D. DAPI-fluorescentnuclearstaining(blue)andACTINstaining(red).ACTINlocalizesbothatcell–celljunctions(arrowheads)andattheapicalplasmaticmembrane (arrows).B,E.DAPI-fluorescentnuclearstaining(blue)andDAAM1staining(green).DAAM1isprominentattheapicalmembrane(arrows).C,F.Merged fluorescentchannels(blue/red/green),whichenhancestheco-localizationofDAAM1andACTINattheapicalplasmaticmembrane(arrows).Thescalebars represent20mm,exceptfortheinsets,wheretheyrepresent10mm.
DAAM1itselfageneralregulatorofactinnucleation,the major aim of the present study was to investigatethe possible association of DAAM1 and actin in the SV epithelium.Thefirstsevidences comefromthePCRand Westernblotexpressiondata:DAAM1is,indeed, expres-sed in theadultSV.Thesuccessivelocalizationanalysis highlightedthattheproteinlocalizestothecytoplasmof epithelial cells. It is also possible to note congruence between DAAM1 profile and actin distribution, and in particularatthecellcortex.Thisisveryinteresting:ithas beenreportedthatanotherproteinbelongingtotheformin family,mDia,co-localizeswithactinattheapicalsurfaceof the exocrinepancreas cells, regulatingtheformation of actinbundles[29,30].Thisinformation,coupledwithour result, may suggest a possible involvement of DAAM1 duringexocytosis,astheproteinhasananalogueactivity asmDia.Infact,uponstimulation,sequentialeventstake place,includinganinitialactin depolymerization,which freesthesecretoryvesiclesandletthemreachtheplasma membrane, followed by a rapid polymerization [55,56]. Thisregulationrequiresthecoordinateactivityofthetwo GTPases:RhoA andcdc42[57,58].In particular,thefirst oneisactivatedtorestoretheactinbundlesbeneaththe plasma membrane. In this scenario,DAAM1, regulating RhoA activity, could participate in the polymerization eventsthatoccurafterthereleaseofthesecretoryvesicles. Finally,itisinterestingtonotethatsmoothmuscularcells areimmunopositiveforDAAM1andthattheyareassociated withactin.Smoothmusclecellsaremajorconstituentsof thewallsofbloodvesselsandvisceralorgans(apartfrom theheart)andareresponsiblefordynamicchangesinthe diameter and volume of these organs.Also in this case, forminsandsmallGTPasesoftheRhofamilyplayacrucial role in actin cytoskeleton remodelling and actomyosin-based contractility[59,60]. Being formin DAAM1itself a positive regulator of RhoA, it is possible to assert that DAAM1canbeinvolvedinthedynamicrearrangementof the actin cytoskeleton in the contractile machinery of smoothmuscle.Inconclusion,ourworkshows,forthefirst time,theexpressionandthelocalizationofDAAM1inadult mouseSV.Thoughfurtherinvestigationisrequiredtobetter understandthepossibleimplicationsofsuchvariedpattern, ourcurrentdataprovideaprofileofDAAM1co-localization withactin,suggestingitspossibleinvolvementasanactor inmorphofunctionalremodellingandorganizationofthe epitheliumandsmoothmuscleoftheseexocrineglands. Disclosureofinterest
Theauthorsdeclarethattheyhavenocompetinginterest.
Acknowledgements
This work is supported by University of Campania ‘‘LuigiVanvitelli’’.
References
[1]D.Pruyne,M.Evangelista,C.Yang,E.Bi,S.Zigmond,A.Bretscher,C. Boone,Roleofforminsinactinassembly:nucleationandbarbed-end association,Science297(2002)612–615.
[2]I.Sagot,S.K.Klee,D.Pellman,Yeastforminsregulatecellpolarityby controllingtheassemblyofactincables,Nat.CellBiol.4(2002)42–50.
[3]M.Evangelista,S.Zigmond,C.Boone,Formins:signalingeffectorsfor assemblyandpolarizationofactinfilaments,J.CellSci.116(2003) 2603–2611.
[4]H.N.Higgs,Forminproteins:adomain-basedapproach,Trends Bio-chem.Sci.30(2005)342–353.
[5]B.J.Wallar,A.S.Alberts,Theformins:activescaffoldsthatremodelthe cytoskeleton,TrendsCellBiol.13(2003)435–446.
[6]H.N.Higgs,K.L.Peterson,Phylogeneticanalysisoftheforminhomology 2domain,MolBiolCell.15(2004)1–13.
[7]B.L.Goode,M.J.Eck,Mechanismandfunctionofforminsinthecontrol ofactinassembly,Ann.Rev.Biochem.76(2007)593–627.
[8]R.Habas, Y.Kato, X.He, Wnt/frizzledactivationofRhoregulates vertebrategastrulationandrequiresanovelForminhomologyprotein Daam1,Cell107(2001)843–854.
[9]K.Tanegashima,H.Zhao,I.B.Dawid,WGEFactivatesRhointhe Wnt-PCPpathwayandcontrolsconvergentextensioninXenopus gastrula-tion,EMBOJ.27(2008)606–617.
[10]F.Marlow,J.Topczewski,D.Sepich,L.Solnica-Krezel,ZebrafishRho kinase2actsdownstreamofWnt11 tomediatecellpolarityand effective convergence and extension movements, Curr. Biol. 12 (2002)876–884.
[11]P.Aspenstro¨m,N. Richnau,A.S.Johansson,Thediaphanous-related forminDAAM1collaborateswiththeRhoGTPasesRhoAandCdc42, CIP4andSrcinregulatingcellmorphogenesisandactindynamics,Exp. Cell.Res.312(2006)2180–2194.
[12]T.Matusek,A.Djiane,F.Jankovics,D.Brunner,M.Mlodzik,J.Miha´ly, TheDrosophilaforminDAAMregulatesthetrachealcuticlepattern throughorganizingtheactincytoskeleton,Development133(2006) 957–966.
[13]A.Sato,D.K.Khadka,W.Liu,R.Bharti,L.W.Runnels,I.B.Dawid,R. Habas,ProfilinisaneffectorforDaam1innon-canonicalWntsignaling andisrequiredforvertebrategastrulation,Development133(2006) 4219–4231.
[14]J.Lu,W.Meng,F.Poy,S.Maiti,B.L.Goode,M.J.Eck,StructureoftheFH2 domainofDaam1:implicationsforforminregulationofactin assem-bly,J.Mol.Biol.369(2007)1258–1269.
[15]M.Yamashita,T.Higashi,S.Suetsugu,Y.Sato,T.Ikeda,R.Shirakawa,T. Kita,T.Takenawa,H.Horiuchi,S.Fukai,O.Nureki,Crystalstructureof humanDAAM1forminhomology2domain,GenesCells12(2007) 1255–1265.
[16]J.A.Zallen,Planarpolarityandtissuemorphogenesis,Cell129(2007) 1051–1063.
[17]Y.S.Kida,T.Sato,K.Y.Miyasaka,A.Suto,T.Ogura,Daam1regulatesthe endocytosisofEphBduringtheconvergentextensionofthezebrafish notochord,Proc.Natl.Acad.Sci.USA104(2007)6708–6713.
[18]R.Ju,P.Cirone,S.Lin,H.Griesbach,D.C.Slusarski,C.M.Crews, Activa-tionoftheplanarcellpolarityforminDAAM1leadstoinhibitionof endothelialcellproliferation,migrationandangiogenesis,Proc.Natl. Acad.Sci.USA107(2010)6906–6911.
[19]D.Li,M.A.Hallett,W.Zhu,M.Rubart,Y.Liu,Z.Yang,H.Chen,L.S. Haneline,R.J. Chan, R.J. Schartz,L.J. Field, S.J.Atkinson, W.Shou, Dishevelled-associatedactivatorofmorphogenesis1Daam1 is re-quiredforheartmorphogenesis,Development138(2011)303–315.
[20]R.K.Miller,S.G.Canny,C.W.Jang,K.Cho,H.Ji,D.S.Wagner,E.A.Jones,R. Habas,P.D.McCrea,PronephrictubulogenesisrequiresDaam1-mediated planarcellpolaritysignalling,J.Am.Soc.Nephrol.22(2011)1654–1664.
[21]B.Bao,L.Zhang,H.Hu,S.Yin,Z.Liang,Deletionofasingle-copyDAAM1 geneincongenitalheartdefect:acasereport,BMCHum.Mol.Genet.13 (2012)13–63.,http://dx.doi.org/10.1186/1471-2350-13-63. [22]H.S.Seo,R.Habas,C.Chang,J.Wang,Bimodalregulationofdishevelled
functionbyVangl2duringmorphogenesis,Med.Genet.26(2017) 2053–2061.
[23]P.Pariante,R.Dotolo,M.Venditti,D.Ferrara,A.Donizetti,F.Aniello,S. Minucci,FirstevidenceofDAAM1localizationduringthepost-natal developmentofrattestisandinmammaliansperm,J.Cell.Physiol.231 (2016)2172–2184.
[24]M.Chemek,M.Venditti,S.Boughamoura,S.B.Mimouna,I.Messaoudi, S.Minucci,InvolvementoftesticularDAAM1expressioninzinc pro-tectionagainstcadmium-inducedmaleratreproductivetoxicity,J.Cell. Physiol.233(2018)630–640.
[25]Y.Rumpler,Seminalvesiclesinevolution,in:C.Bollack,A.Clavert (Eds.),Seminalvesiclesandfertility,biologyandpathologypp,Karger, Basel,Switzerland,1985,pp.1–3.
[26]G.Aumuller,J.Seitz,Proteinsecretionandsecretoryprocessesinmale accessorysexglands,Int.Rev.Cytol.121(1990)127–231.
[27]E.Spring-Mills,E.S.E.Hafez,Functionalanatomyandhistology,in: E.S.E.Hafez,E.Spring-Mills(Eds.),Accessoryglandsofmale reproduc-tiveTract.Ann.Arbor.Sci.,AnnArbor,MI,USA,1979,pp.29–57.
[28]E.Geron,E.D.Schejter,B.Z.Shilo,Targetingsecretiontotheapical surfacebymDia1-builtactintracks,Commun.Integr.Biol.6(2013) e25660,http://dx.doi.org/10.4161/cib.25660.
[29]E.Geron,E.D.Schejter,B.Z.Shilo,Directingexocrinesecretoryvesicles totheapicalmembranebyactincablesgeneratedbytheforminmDia1, Proc.Natl.Acad.Sci.USA110(2013)10652–10657.
[30]P.Croise´,C.Estay-Ahumada,S.Gasman,S.Ory,RhoGTPases, phos-phoinositidesandactin:atripartiteframeworkforefficientvesicular trafficking,SmallGTPases5(2014)e29469,http://dx.doi.org/10.4161/ sgtp.29469.
[31]Z.Wang,D.C. Thurmond, Mechanismsof biphasicinsulin-granule exocytosis–roles ofthecytoskeleton, smallGTPases and SNARE proteins,J.Cell.Sci.122(2009)893–903.
[32]C.J.Flickinger,Synthesis,intracellulartransportandreleaseof secreto-ryproteinintheseminalvesicleoftherat,asstudiedbyelectron microscoperadioautography,Anat.Rec.180(1974)407–427.
[33]A.Clavert,O.Gabriel,D.Montagnon,Physiologicalroleoftheseminal vesicles,in:C.Bollack,A.Clavert(Eds.),Seminalvesiclesandfertility. biologyandpathology,Karger,Basel,Switzerland,1985,pp.80–94.
[34]P.T.Curry,R.W.Atherton,Seminalvesicles:development,secretory productsandfertility,Arch.Androl.25(1990)107–113.
[35]X.D.Zhang,B.F.Jin,Theroleofseminalvesiclesinmalereproduction andsexualfunction,ZhonghuaNan.Ke.Xue.(2007)1113–1116.
[36]M.Emoto,E.Kita,F.Nishikawa,N.Katsui,A.Hamuro,D.Oku,S.Kashiba, Biologicalfunctionsofmouseseminalvesiclefluid.Roleof water-solublefractionofseminalvesiclefluidasanonspecific immunomod-ulator,Arch.Androl.25(1990)75–84.
[37]W.M.Maxwell,S.P.deGraaf,H.GhaouiRel,G.Evans,Seminalplasma effectsonspermhandlingandfemalefertility,Soc.Reprod.Fertil. Suppl.64(2007)13–38.
[38]R.Porta,S.Metafora,C.Esposito,L.Mariniello,P.Persico,F.Mancuso,G. Peluso,Biologicalactivitiesofamajorproteinsecretedfromtherat seminalvesiclesafterstructuralmodificationcatalyzedby transgluta-minaseinvitro,Immunopharrnacology25(1993)178–188.
[39]C.J.Thaler,P.M.Knapp,J.A.Mclntyre,C.B.Coulam,J.K.Critser,W.P. Faulk,Congenitalaplasiaofseminalvesicles:Absenceof trophoblast-lymphocytecross-reactiveantigensfromseminalplasma,Fertil.Steril. 53(1990)948–949.
[40]D.Communi,R.Janssens,N.Suarez-Huerta,B.Robaye,J.M.Boeynaems, Advancesinsignallingbyextracellularnucleotidestheroleand trans-ductionmechanismsofP2Yreceptors,CellSignal.12(2000)351–360.
[41]M.Fukuda,RegulationofsecretoryvesicletrafficbyRabsmallGTPases, Cell.Mol.LifeSci.65(2008)2801–2813.
[42]F.Momboisse,S.Houy,S.Ory,V.Calco,M.F.Bader,S.Gasman,How importantareRho GTPasesinneurosecretion?J. Neurochem.117 (2011)623–631.
[43]S.Ory,S.Gasman,RhoGTPasesandexocytosis:whatarethemolecular links?Semin.Cell.Dev.Biol.22(2011)27–32.
[44]A.B.Parekh,J.W.PutneyJr., Store-operatedcalciumchannels,Physiol. Rev.85(2005)757–810.
[45]C.D.Sladek,Z.Song,DiverserolesofG-proteincoupledreceptorsinthe regulationofneurohypophysealhormonesecretion,J. Neuroendocri-nol.24(2012)554–565.
[46]M.Anitei,B.Hoflack,Bridgingmembraneandcytoskeletondynamicsin thesecretoryandendocyticpathways,Nat.Cell.Biol.14(2011)11–19.
[47]G.Egea,F.Lazaro-Dieguez,M.Vilella,ActindynamicsattheGolgi complexinmammaliancells,Curr.Opin.Cell.Biol.18(2006)168–178.
[48]G.Eitzen,Actinremodellingtofacilitatemembranefusion,Biochim. BiophysActa1641(2003)175–181.
[49]L.M.Gutierrez,Newinsightsintotheroleofthecorticalcytoskeletonin exocytosisfromneuroendocrinecells,Int.Rev.Cell.Mol.Biol.295 (2012)109–137.
[50]N.Porat-Shliom,O.Milberg,A.Masedunskas,R.Weiger,Multipleroles fortheactincytoskeletonduringregulatedexocytosis,Cell.Mol.Life. Sci.70(2013)2099–2121.
[51]G.E.Kerr,J.C.Young,K.Horvay,H.E.Abud,K.L.Loveland,Regulated Wnt/beta-cateninsignalingsustainsadultspermatogenesisinmice, Biol. Reprod. 90 (2014) 3, http://dx.doi.org/10.1095/biolre-prod.112.105809.
[52]K. Marinou,C.Christodoulides,C.Antoniades,M.Koutsilieris,Wnt signalingincardiovascularphysiology,TrendsEndocrinol.Metab.23 (2012)628–636.
[53]W.Liu,A.Sato,D.Khadka,R.Bharti,H.Diaz,L.W.Runnels,R.Habas, MechanismofactivationoftheForminproteinDaam1,Proc.Natl.Acad. Sci.USA105(2008)210–215.
[54]S.Ku¨hn,M.Geyer,ForminsaseffectorproteinsofRhoGTPases,Small GTPases5(2014)e29513,http://dx.doi.org/10.4161/sgtp.295. [55]S.Muallem,K.Kwiatkowska,X.Xu,H.L.Yin,Actinfilamentdisassembly
isasufficientfinaltriggerforexocytosisinnonexcitablecells,J.Cell. Biol.128(1995)589–598.
[56]L.Orci,K.H.Gabbay,W.J.Malaisse,Pancreaticbeta-cellweb:its possi-bleroleininsulinsecretion,Science175(1972)1128–1130.
[57]M.F.Bader,F.Doussau,S.Chasserot-Golaz,N.Vitale,S.Gasman, Cou-plingactinandmembranedynamicsduringcalcium-regulated exocy-tosis:aroleforRhoandARFGTPases,Biochim.Biophys.Acta.1742 (2004)37–49.
[58]S.Gasman,S.Chasserot-Golaz,M.Malacombe,M.Way,M.F.Bader, Regulatedexocytosisinneuroendocrinecells:arolefor subplasma-lemmal Cdc42/N-WASP-inducedactinfilaments,Mol.Biol.Cell.15 (2004)520–531.
[59]S. Puetz,L.T. Lubomirov,G. Pfitzer,Regulation ofsmooth muscle contractionbysmallGTPases,Physiology24(2009)342–356.
[60]L.Weise-Cross,J.M.Taylor,C.P.Mack,Inhibitionofdiaphanousformin signaling in vivo impairs cardiovascular development and alters smoothmusclecellphenotype,Arterioscler.Thromb.Vasc.Biol.35 (2015)2374–2383.
