Microridges are apical projections formed of branched F-actin networks that organize the glycan layer

20 Pages·2017·7.63 MB·English

bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Microridges are apical projections formed of
branched F-actin networks that organize the
glycan layer
ClydeSavioPinto1,AmeyaKhandekar1,RajasekaranBhavna1,PetraKiesel2,GaiaPigino2,andMahendraSonawane1,(cid:0)
1DepartmentofBiologicalSciences,TataInstituteofFundamentalResearch,Colaba,Mumbai,India
2MaxPlanckInstituteofMolecularCellBiologyandGenetics,Dresden,Germany
Apical projections are integral functional units of epithelial of various shapes and sizes. Columnar epithelia possess
cells. Microvilli and stereocilia are cylindrical apical projec- actin-basedcylindricalmicrovilliortheirderivativessuchas
tions that are formed of bundled actin. Microridges on the stereocilia(ApodacaandGallo,2013;Sauvanetetal.,2015;
otherhand, growlaterallylong, forminglabyrinthinepatterns Tilney et al., 1980). On the contrary, the apical domain of
onsurfacesofvariouskindsofsquamousepithelialcells.Sofar,
severalofthenon-cornifiedsquamousepitheliaisthrowninto
the structural organization and functions of microridges have
laterally long protrusions called microplicae or microridges
remained elusive. We have analyzed microridges on zebrafish
(ApodacaandGallo,2013).Whilepreviousstudieshavesig-
epidermal cells using confocal and electron microscopy meth-
nificantly enhanced our understanding of actin organization
ods including electron tomography, to show that microridges
in microvilli (Crawley et al., 2014; Sauvanet et al., 2015),
are formed of a network of F-actin and require the function
oftheArp2/3complexfortheirmaintenance. Duringdevelop- how actin is organized to build microridges remains contro-
ment,microridgesbeginasF-actinpunctaeshowingsignatures versial.
of branching and requiring an active Arp2/3 complex. Using Maturemicroridgesarerelativelystablewalllikeprotrusions,
inhibitorsofactinpolymerizationandtheArp2/3complex,we arranged in a labyrinthine fashion (Bereiter-Hahn et al.,
show that microridges organize the surface glycan layer. Our 1979; Collin and Collin, 2000; Fishelson, 1984; Hawkes,
analyseshaveunraveledtheF-actinorganizationsupportingthe 1974; Lam et al., 2015; Schliwa, 1975). They are shown
mostabundantandevolutionarilyconservedapicalprojection,
to be present on several non-cornified epithelia in various
whichfunctionsinglycanorganization.
speciesacrossvertebratephyla,makingthemoneofthemore
widespread actin protrusions (Bereiter-Hahn et al., 1979;
microridges|branchedF-actinnetwork|Arp2/3complex|glycanorganization|
electrontomography|zebrafishepidermis Breipohletal.,1977;CollinandCollin,2000;Eroschenko
Correspondence:[email protected] and Osman, 1986; Fenger and Knoth, 1981; Hafez et al.,
1977;Johnstonetal.,1996;Makabeetal.,1998;Saitoand
Itoh,1993;Ueharaetal.,1991;Worawongvasu,2007). Mi-
Introduction
croridgesarethoughttohaveimportantrolesinmucusreten-
Epithelial tissues cover the outer surface of metazoans as tion, abrasion resistance and increasing the tensile strength
well as the outer and inner surfaces of organs, thus sep- oftheapicaldomain(Fishelson,1984;Sharmaetal.,2005;
arating two physiologically distinct compartments. Over SperryandWassersug,1976).Theyhavealsobeenproposed
the course of evolution, epithelia have undergone diversi- tofunctionasarapidlydeployablestoreofF-actinandmem-
fication and specialization allowing them to adapt to vari- braneduringwoundhealing(Sharmaetal.,2005).
ousconditionsandperformnotonlythebarrierfunctionbut The nature of the protrusion is determined by the organiza-
also additional functions such as absorption, secretion and tion and dynamics of F-actin within it. Filaments in actin
mechanosensation(LeBivic,2013;Urdyetal.,2016). These protrusions take on one of two conformations - either as a
functions require special adaptions of their apical surfaces branched network of filaments, seen in lamellipodia (Svitk-
(ApodacaandGallo,2013;Rodriguez-BoulanandNelson, inaandBorisy,1999)orasparallelbundlesformingthecore
1989), which include diverse apical membrane protrusions. of projections like microvilli and stereocilia (Revenu et al.,
Additionally, the apical surface is kept hydrated and pro- 2004). Although the importance of the Arp2/3 complex, an
tected from pathogens and physical injury by the overlying importantregulatorofbranchedactinnetworks,inthemain-
glycocalyx and mucosal layers (Apodaca and Gallo, 2013; tenanceofmicroridgesinzebrafishepidermalcellsisknown
Cone, 2009). Aside from the glycocalyx, mucosal epithelia (Lametal.,2015),itisnotclearhowactinwithinthemisor-
havemucus-aviscoelasticfluid-ontheirsurface. Thus,the ganized. Whileactinwithinmicroridgesofguppyepidermal
apicalzoneofepithelialtissuesiscomprisedofthreecompo- cellsisreportedasbundled(Bereiter-Hahnetal.,1979),mi-
nents, namely, the apical plasma membrane with its projec- croridgesoncarporalmucosalcells,catepithelialcells,and
tions, the cytoskeleton supporting these projections and the guppycellsincultureareshowntocontainanetworkofactin
outermucus-glycocalyxlayer(ApodacaandGallo,2013). (Andrews,1976;Bereiter-Hahnetal.,1979;Ueharaetal.,
Invertebrates, epithelialcellsexhibitmembraneprotrusions 1991). Furthermore,basedontheoreticalanalysis,ithas
Pintoetal. | bioRχiv | October4,2018 | 1–20
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure1.EMtomographyofuntreatedsamplesandSEMondetergentextractedsamplesrevealstheultrastructuralorganizationoftheactinmicroridge,
sub-protrusivezoneandcortex.TEMthroughaheadperidermalcellrevealedthatperidermalcellscompriseofamicroridge/glycanzone,sub-protrusive
zoneandorganellezone(A).SEMofanAlcianblueandlysinefixedheadperidermsample(B)revealsathickglycanlayer,whichischippedoffonthe
leftrevealingtheunderlyingmicroridges.TomogramsofGluteraldehyde+OsO fixedsamplesthroughthelengthofthemicroridge(C)andperpendicular
4
tothecell-boundarymicroridge(I).ZoomedinareasofCandIareshownin(D-H)and(J,K),respectively.EachimageinC-HandI-Krepresentsa0.7
and0.55nmthicksliceofthetomogramintheZaxisrespectively. (L)SEMofadetergentextractedsample. Magentaandgreenarrowheadsindicate
actinandkeratinfilaments,respectively. Intheinsetin(D),thearrowheadspointtoafilamentwithbranchpoints. In(E)thearrowheadspointtoa
keratinfilamententeringthemicroridgefromthesub-protrusivezonewhereasthewhitedashedlinedemarcatestheboundarybetweenthemicroridge
abovefromthesub-protrusivezonebelow. Smallfilamentsthatappeartobeparalleltoeachother(F)areshownbythearrowheadsandinsetinF.
Thearrowheadsin(G)pointtobranchpointsinkeratinfilamentsinthesub-protrusivezone. In(H)thearrowheadspointtoafilamentthatisparallel
totheplasmamembrane. Thecytoskeletonfromthetopofthemicroridgetoitsbase(indicatedbywhitedashedlineinI)andthesub-protrusivezone
tillthebaseofthetightjunction(indicatedbytheblackdashedline)isdenserandmadeuplargelyofactin. In(J)smalleractinmembranecrosslinks
(whitearrowhead)andlongeractinmembranecrosslinks(blackarrowhead)areshown.Thecortexbetweenridges(K)ismadeuplargelyofactin,which
arethinnerfilaments(magentaarrowhead),distinctlydifferentfromthethickerkeratinfilamentbelow(greenarrowhead). Colouredarrowheadsin(L)
indicatefilamentsorfilamentbundlesextendingfromthecortexintothemicroridge.ThescalebarsinA,Bare2µm,inC,F,I,Lareequalto200nmand
thoseinD,E,G,H,J,Kare20nm.
2 | bioRχiv Pintoetal. | F-actinorganizationinmicroridges
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 2. Segmentation of an electron tomogram reveals the arrangement of actin to be a network within the microridge. An EM image section
tomogramwastakenwith2090×1740×249voxelsatanequalspacingof0.707nmineachdirection, hereshownatadepthof85.5nm(A).Higher
Eigenvalue2Dmatrixoutlinesoftheactinstructureswithinthemicroridgesshownin(B).Acubicsectionof283.5×318.8×43.1nm3(dottedcubein
B)iscroppedforillustrationofthesegmentationanalysis. Imagebinarizationoutlines(inblue)oftheactinstructures(C),shownat3differentdepths.
(D)Stepsshowingsubsequent3Dmorphologicaloperationsonthereconstructed3Dbinarystackfollowedbycomputationofsegmentedstructures
properties.3Dreconstructionofactin(E)andcorresponding3Dskeletonimage(F)revealthearrangementofactinmeshwork.Depthisindicatedbya
colorbar.Examplesofsinglerenderedactinstructuresinmagenta(G1-2)andcorrespondingskeletons(H1-2)withinthemicroridgesindicatebranching
offilaments.Branchpoints(green)andendpoints(black)arehighlightedonthe3Dskeletonimages.Allthebranchpointsandendpointsarenumbered
andindicatedbyarrowmarks.TheEMtomogramanalysedherewasapartofthetomogramshowninFigure1C-HandVideo1.
Pintoetal. | F-actinorganizationinmicroridges bioRχiv | 3
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
beenproposedthatmicrovillicanmergetoformridges(Gov, (Fig. S1D),weusedElectronTomography(ET)tounravel
2006), suggestingthatmicroridgesformfrombundledactin the structural details of actin organization (Figure 1 C-K).
containingmicrovilli. We produced tomograms (Videos 1-3), with different ori-
The zebrafish periderm offers a genetically and microscop- entations of microridges. Samples fixed using osmium and
ically tractable model to study the formation, structure and GA(Figure1C-K;Video1;Video2)showedthatfilaments
function of microridges. It has been recently shown that withinamicroridgewerelargelyarrangedatdifferentangles
thecellpolarityregulatoratypicalProteinKinaseC(aPKC) toeachother(Figure1C-J),suggestiveofanetworkofactin
plays a role in restricting the elongation of microridges by alongwithafewparallelfilaments(Figure1F).Weusedan
controlling levels of Lethal giant larvae (Lgl) and nonmus- algorithm(seemethods)tosegmentfilamentsinatomogram
cle MyosinII at the apical domain of peridermal cells (Ra- of microridges (Figure 2). This allowed a reconstruction of
man et al., 2016). However, the ultrastructure and function thefilamentswithinthemicroridgeasshown,withinasmall
oftheseprotrusionshavenotbeencharacterizedwell. Here, regionofthetomogram(Figure2B-F,Video4). Thesecor-
wehaveperformedmolecularaswellasstructuralanalysisof roborated the fact that filaments are organized in a network
microridgesinthedevelopingzebrafishepidermisandprobed likefashion(Figure2E,F;Video4).Wewerealsoabletoob-
for their functional importance in the context of the mucus servebranchingoffilaments(Figure1D;Figure2G,H).ET
and glycocalyx, which we collectively call the glycan layer. ofsamplesfixedusingtannicacidanduranylacetate,avoid-
We show that the molecular composition of microridges is ingOsO ,corroboratedthefactthatactinwithinthemicror-
4
similar to lamellipodia and that Arp2/3 function is essential idgeisorganizedasanetwork(Video3). Asidefromafew
for their formation and maintenance. Electron tomography filamentsparalleltothemicroridgemembrane(Figure1H),
(ET)followedbysegmentationanalysisrevealsthattheactin we observed a number of actin-membrane crosslinks at the
isorganizedintheformofanetwork.Wefurtherdisprovethe interphasebetweenthemembraneandtheactinnetwork(Fig-
notionthatmicroridgesareformedfrommicrovillibyshow- ure 1 J). Interestingly, there were many instances in which
ingthatsmallactinspotsthatgeneratemicroridgesconsistof vesicleswerefoundinthemicroridge,particularlyatitsbase,
the Arp2/3 complex. Lastly, our analyses also indicate that indicatingthatitisnotanisolatedsystemlikeacilium(Fig-
microridgesareimportantfortheorganizationoftheglycan ure1I;Fig. S1E).
layer. ET revealed that the sub-protrusive zone contained cortical
actinandanetworkofthickkeratinfilamentswithdiscernible
branchingpoints(Figure1C,G,I,K).Somekeratinfilaments
Results
—discernable by their thickness as compared to actin (Fig-
Ultrastructural characterization of the apical zone of perid- ure1K)—clearlyenteredthemainbodyofthemicroridge,
ermalcells.Theultrastructuralcharacterizationoftheentire indicating that keratin and actin filaments arising from the
apicalzoneincludingthemicroridgeanditssurroundingshas cortexsupportitsarchitecture(Figure1E).Toconfirmsucha
notbeencarriedoutsofarinthedevelopingzebrafishperid- contributionfromthecortex,weuseddetergentextractionto
erm. Welookedattheorganizationoftheperidermalcellin removeapicalmembranesofperidermalcellsandperformed
transmissionelectronmicrographs(TEM)aswellasinscan- SEM.Inthesepreparations,actinfilamentsinthecortexwere
ningelectronmicrographs(SEM)at48hpf—atimepointat clearlyvisible(Figure1L).Thesefilamentswerearrangedin
whichembryosbegintohatchandpresumablyrequirefunc- a web like pattern. Interestingly, we found that actin fila-
tionalmicroridges. mentsandfilamentbundlesextendedaconsiderabledistance
TEM analysis using routine glutaraldehyde (GA) and os- within the cortex and into the microridge (Figure 1 L). Fur-
mium tetroxide fixation revealed three distinct zones in the thercorroboratingwiththeseETandSEMdata,weobserved
peridermal cell- the protrusive zone consisting of micror- thelocalizationofkeratin-usingtheAE1/AE3antibody-to
idges,thesub-protrusivezonecomprisedofactinandkeratin theapicalcortexandoccasionallyinthemicroridge(Fig. S2
filaments and a basal region containing various organelles A).Wedidnotobserveanydirectassociationofmicrotubules
(Figure1A).Microridgeshadameanheightof315±82nm withthemicroridgeeitherbyETorimmunostainingusingan
(n= 245, N=3; Fig. S 1 B), as measured from TEM. Using α-tubulinantibody(Fig. S2B).
SEM(Fig. S1A),wefoundtheirmeanwidthtobe162±36 To conclude, the apical zone of zebrafish peridermal cells
nm(n=645,N=3;Fig.S1B).Toobservetheorganizationof consists of a glycan layer, an apical domain consisting of
the glycan layer, we fixed samples in the presence of alcian shortprojectionscalledmicroridgesandthecytoskeletonthat
blueandlysine,whichhasbeenshowntopreservetheglycan supportsthem.Withinthemicroridge,F-actinisorganizedin
layerbetter(Fasseletal.,1997,1992;Fischeretal.,2012). the form of a network, supported by filaments arising from
SEM of heads of animals fixed in such a way showed that thesub-protrusivezoneorterminalwebandcortex. Thein-
the peridermal surface was covered by a thick glycan layer, terior of the microridge space is accessible to vesicles indi-
whichcompletelyobscurestheunderlyingmicroridgesonthe catingthatthemicroridgeisnotanisolatedsystem.
headepidermis(Figure1B).TEManalysisofsuchasample
revealedglycanenrichmentaroundthemicroridges(Fig. S1 Molecularsignatureofthemicroridgeinzebrafish.The mi-
C). croridge is strikingly different in both morphology and F-
SinceconventionalTEMdidnotrevealtheF-actinorganiza- actinorganizationascomparedtomicrovilli(Crawleyetal.,
tionwithinthemicroridgeduetothedensepackingofactin 2014;Sauvanetetal.,2015). Thispromptedustolookatthe
4 | bioRχiv Pintoetal. | F-actinorganizationinmicroridges
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure3. Localizationofactinbindingproteinsatthemicroridge.Confocalmicrographsshowingimmuno-localizationofArpC2(A,D),WASL(B,E),
Cofilin(C)at48hpf(A-C)andat50%epiboly(D,E).PlasmidencodedexpressionofEplinβ(F),andFilaminA(I)ingreenlocalizetomicroridgesmarked
withphalloidin(G,J)inmagentaandrespectivemerges(H,K).Plasmidswereinjectedatthe1cellstageandexpressedinaclonalfashion.Scalebars
inEforA,B,D,E,inCforC,inKforF-Kareequalto10µm.
Pintoetal. | F-actinorganizationinmicroridges bioRχiv | 5
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure4.Arp2/3complexactivityisrequiredformicroridgeformationandmaintenance.Confocalmicroscopyanalysesofphalloidinstainingofembryos
andlarvae(A-I)treatedwith1%DMSO(A,D,G),100µMCK689(inactivedrugcontrol;B,E,H)and100µMCK666(Arp2/3complexinhibitor;C,F,I)for1
hourfrom9-10hpf(A-C),18-19hpf(D-F)and47-48hpf(G-I).Inallcasesinhibitionresultedinabreakdownofmicroridgesoradecreaseinphalloidin
punctaerelativetocontrols(N=2sets,n=10).ScalebarinIforA-Iisequivalentto10µm.
molecularcompositionofmicroridges. Thusfar,onlyafew plex, localizes to microridges at 48 hpf (Figure 3 A). The
actin-binding proteins are known to localize to the micror- nucleation-promoting-factor WASp-like (WASL) (Figure 3
idge. These include α-actinin, ezrin, myosin II, VASP and B)andCofilin,anactinseveringanddepolymerizingprotein,
cortactin (Bereiter-Hahn et al., 1979; DePasquale, 2018; were also found to localize to the microridge (Figure 3 C).
Lam et al., 2015; Raman et al., 2016). Since actin-binding Our analysis using various tagged proteins revealed that the
proteins regulate and organize actin filaments into specific actinbundlingandcrosslinkingproteinsEplin/Lima-1,found
arrangements such as parallel or antiparallel bundles and instressfibersorattheadherensjunction(AbeandTakeichi,
branched networks, we analyzed the localization of addi- 2008; Maul et al., 2003), and Filamin, found in branched
tionalregulatorsoftheactincytoskeletontomicroridges. We structureslikelamellipodiaormembraneruffles(Smalletal.,
utilizedtwocandidate-basedstrategies—a)antibodystain- 2002;Vadlamudietal.,2002),alsolocalizedtomicroridges
ingstoidentifyproteinsendogenouslypresentatthemicror- (Figure3F-K).
idge and b) localization of plasmid encoded expression of SincetheactivityoftheArp2/3complexisessentialformain-
tagged proteins. Our immunolocalization analysis revealed tenanceofmicroridges(Lametal,2015),weaskedwhether
thatArpC2,acomponentoftheactinnucleatorArp2/3com- thereisatemporalcorrelationbetweenthelocalizationofthe
6 | bioRχiv Pintoetal. | F-actinorganizationinmicroridges
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Arp2/3 complex, WASL and microridge formation. As the tion of the glycan layer, we used chemical inhibitors to dis-
Arp2/3 complex and WASL are bona-fide components of rupt the actin cytoskeleton and consequently the microridge
branchedactin,wealsoreasonedthatsuchananalysiswould pattern. While in control embryos, the WGA fluorescence
allow us to test the notion whether microridges are formed followedthemicroridgepattern(Figure5A-C),intheCK666
frommicrovilli. Asaprerequisiteforthisanalysis,wechar- treated embryos the WGA fluorescence was comparatively
acterizedthedevelopmentofmicroridgesstartingfrom50% more uniform suggesting a mild effect on the organization
epiboly (Fig. S 3). We found that at 50% epiboly, F-actin oftheglycanlayer(Figure5E-G).SEManalysisonCK666
punctaewerepresentattheapicaldomainofEVL(Envelop- treated samples did not reveal a major change in glycan or-
inglayer)cells(Fig. S3A),whichlatergiverisetotheperi- ganization (Figure 5 D,H), again pointing to the mild effect
derm(Kimmeletal.,1990).Suchpunctaewerevisibletill12 ofCK666ontheglycanlayer(Figure5D,H).Weachieveda
hpf(Fig.S3B,C),afterwhichslightlylongF-actinstructures severe perturbation of microridgesusing theactin monomer
wereobservednearthecellperiphery. Theseseemtofurther bindingdrugLatrunculinA(LatA).LatAtreatmentresulted
elongateovertime(Fig. S3D-I).Tocheckifthesepunctae inthedecreaseddensityandlengthofmicroridges(Figure5
were associated with protrusions, we preformed SEM. We I-P).Weanalysedtheventralregionoftheflankwheretheef-
observedsmallprotrusionsattheperipherybutnotthecenter fectofLatAonmicroridgeswasmostsevere.Ascomparedto
of the cell in most parts of the EVL at 9 hpf (Fig. S 3 J). thecontrol(Figure5I-K),theLatAtreatedsamplesshowed
By 18 hpf, microridges at the cell boundary became promi- moreintenseWGAstainingaroundthemicroridgeremnants
nent and by 48 hpf most of the peridermal cell surface was indicating accumulation of glycans around them (Figure 5
coveredwithmicroridges(Fig. S3K,L). M-O).SEManalysisrevealedthattheglycanlayerpersisted
Both the Arp2/3 complex and WASL showed punctate lo- inthetroughsbetweenmicroridgesatalowerdensity. How-
calization at the apical cortex of EVL cells as early as 50% ever,itwasclearlyenrichedaroundthemicroridgeremnants
epiboly,similartoF-actinpunctae(Figure3D,E).Inhibition (Figure5L,P).
oftheArp2/3complexbyCK666at9hpfresultedinastark During our analyses, we observed that microridge patterns
reductioninF-actinpunctae(Figure4A-C),inhibitionat18 differed on the flank (Figure 5 S,T) from those on the head
hpf prevented the formation of microridges (Figure 4 D-F) (Figure5Q,R),withahigherpackingdensityofmicroridges
andat48hpfresultedintheirbreakdown(Figure4G-I),indi- on the head. SEM analysis revealed that the glycan layer
catingthattheArp2/3complexisimportantforboththefor- coats microridges on the head in a sheet like fashion with-
mation and maintenance of microridges and punctae. Since out revealing their underlying pattern (Figure 5 R). In con-
Keratinscontributetotheterminalweb,wealsoinvestigated trast,theglycanlayerontheflankfollowsthepatternofun-
if a temporal link exists between the formation of the ker- derlying microridges, with glycans enriched on microridges
atin cytoskeleton and microridges. Keratin levels, analyzed withapparentgapsinbetween(Figure5T).Thisfurtherin-
byAE1/AE3antibodystaining,werelowat9hpf,increasing dicates thatmicroridges have the ability toretain glycans in
overtimeasmonitoredtill15hpf(Fig. S4). theircloseproximity.
Thus, bona fide markers of branched actin networks such Inconclusion,peridermalcellsonthezebrafishlarvalepider-
as the Arp2/3 complex, WASL and Filamin, as well as the misare coveredwitha thickglycanlayer. Theorganization
actin-bundling protein Eplin, localize to microridges. The ofthislayerreliesontheunderlyingmicroridges.
Arp2/3complexisessentialfortheformationormaintenance
ofthe actinpunctae andmicroridges. Atall time-pointsan-
Discussion
alyzed, actin structures contained the Arp2/3 complex, con-
sequentlymicrovilli-likestructureswerenotobservedduring Theapicalsurfaceisfunctionallythemostimportantpartof
thetimeresolvedexperiments. Therefore,microridgesform an epithelial tissue. It is decorated with actin based projec-
from actin punctae, presumably having branched configura- tions and a glycan layer. Laterally long protrusions such as
tion,andnotfrombundlebasedmicrovilli. microridges have been known to morphologists for over 50
yearsnowbuthavenotbeenwellstudied.Thezebrafishperi-
Physiologicalrelevanceofmicroridges.Thewidespreadoc- derm, which is completely covered with microridges, offers
currenceofmicroridgesonmucosalepithelialtissueshasled anidealsystemtostudysuchprotrusionsandtheirroleingly-
researchers to propose that microridges function in reten- canbiology(Lametal.,2015;Ramanetal.,2016).Here,we
tionandordistributionofmucus(Fishelson,1984;Schliwa, usedimmunofluorescence,EMtomographyandSEManaly-
1975; Sperry and Wassersug, 1976). The developing ze- sistounderstandhowactinmicroridgesform,aremaintained
brafish embryo is covered by a glycan layer as early as the andwhethertheyfunctioninglycanorganization.
epiboly stages (Baskin et al., 2010), making it an excellent Ourultra-structuralanalysespresentedhereindicatethatthe
modeltostudytheroleofmicroridgesinmaintainingtheor- microridge is largely made up of a network of actin. This
ganizationoftheglycanlayer. organization is in fact similar to the one in lamellipodia,
Inordertovisualizetheglycanlayerwithaconfocalmicro- whereinthelamellipodialnetworkmeetsthetransverseactin
scope,weusedafluorescentlytaggedwheatgermagglutinin arc (Burnette et al., 2011). Our results, in zebrafish larvae
(WGA) lectin. This approach revealed that the glycan layer at 48 hpf are consistent with Uehara et al, who have shown
wasarrangedaroundmicroridges(Figure5A-C,E-G,I-K,M- that microridges in carp oral mucosa exhibit actin filaments
O). To assess if microridges are important for the organiza- oriented randomly (Uehara et al., 1991). However, unlike
Pintoetal. | F-actinorganizationinmicroridges bioRχiv | 7
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure5.Microridgesplayaroleintheorganizationoftheglycanlayer.EffectoftheinhibitionoftheArp2/3complexontheglycanlayer(A-H).Confocal
micrographsofPhalloidin(A,E),WGAstainings(B,F),Merges(C,G)andSEM(D,H)ofanimalstreatedwith1%DMSOasvehiclecontrol(A-D)or
100µMCK666(E-H)andrespectiveorthogonalsectionsbelowtheimages(A’-C’,E’-G’).EffectofLatrunculinAtreatmentontheglycanlayer(I-P).
Phalloidin(I,M),WGAstainings(J,N)andMerges(K,O)ofanimalstreatedwith1%DMSOasvehiclecontrol(I-K)or2µMLatrunculinA(M-O)and
respectiveorthogonalsectionsbelowtheimages(M’-O’).InsetsinM-Oshowamicroridgeremnant(phalloidin,green)surroundedbytheglycanlayer
(WGAstaining,magenta).SEMusingAlcianblueandLysinefixationof1%DMSOasvehiclecontrol(L)or2µMLatrunculinA(P).TheinsetsinD,H,L,P
showhighermagnificationSEMimagesoftheirrespectivetreatments.Differencesintheglycanlayerorganizationindifferentpartsoftheanimal(Q-T).
Phalloidinstaining(Q,S)andSEM(R,T)ofthehead(Q,R)andflank(S,T)peridermalcells. Notethatmicroridgesontheheadarerelativelydensely
packedwithaflatglycanlayerascomparedtotheflank.ScalebarsinCforA-C,inGforE-G,inKforI-K,inOforM-O,inSforQ,S,inTforR,Tandin
D,H,L,Pare10µmandintheinsetsinD,H,L,Pare1µm.
8 | bioRχiv Pintoetal. | F-actinorganizationinmicroridges
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure6. AschematicrepresentationoftheactinmicroridgebasedonEMandimmunolocalizationanalyses. Theactinmicroridgeisalaterallylong
protrusion(A).Itconsistsofabranchednetworkofactinwithanunderlyingsub-protrusivezoneofactin(magenta)andkeratin(blue). Occasionally,
keratinfilamentsalsoentertheprotrusion(B).Theactinmicroridgeprotrusionitselfconsistsofvariousmolecules(C),manyofwhichassociatewith
branchednetworksThesub-protrusivezoneoftheactinmicroridgeresemblesthesub-protrusiveterminalwebofbrushbordermicrovilli(D),whichare
protrusionsmadeupofbundledactinwithaterminalwebofactinandkeratin. Ontheotherhand,thebranchedactinarchitectureofthemicroridge
protrusionhassimilaritieswiththelamellipodialprotrusion(E),thoughlamellipodiahaveanactinbasedsub-protrusivezone,withoutanoticeablekeratin
cytoskeleton.
Pintoetal. | F-actinorganizationinmicroridges bioRχiv | 9
bioRxiv preprint doi: https://doi.org/10.1101/442871; this version posted October 15, 2018. The copyright holder for this preprint (which was not
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
our ET analysis, this earlier analysis was done with deter- Although, Arp2/3complexinhibitionhadonlyamildeffect
gent extracted samples and hence did not reflect the com- ontheglycanlayer,ourTEM,SEMandWGAstainingdata
plexity of the network in its entirety. Our structural analy- asawholesuggeststhatglycansareenrichedaroundmicror-
sis is largely in agreement with the fact that all the major idges. WGA staining also follows the microridge pattern in
regulatorsofbranchedactinconformation-theArp2/3com- thecaseofkoiepidermalcells(DePasquale,2018). Itisthus
plex,WASL,CofilinandFilamin-localizetothemicroridges likely thatthe distancebetween microridges isan important
andthatArp2/3complexfunctionisrequiredinformingand factor in the organization of the glycan layer, which is cor-
maintaining microridges. In this sense, microridges can be roboratedbythefactthattheglycanlayeronthehead,which
thought of as several stable lamellipodium like protrusions hascloselypackedmicroridges,exhibitsadenseanduniform
organized in a labyrinthine manner on the apical surface of organizationascomparedtothatontheflank,wheremicror-
cells(Figure6A-C).BothSEMandtomographicdatashow idgesarespacedapart.
thatthemicroridgecytoskeletoniscontiguouswiththecortex Insummary,theactinmicroridgeisauniqueprotrusionthat
andkeratinnetwork,suggestingthattheorganizationofthis haspotentialevolutionaryoriginfromandstructuralhomol-
layer would influence the microridge pattern (Figure 6 B). ogy to the leading edge of migrating cells, with an under-
Curiously,inlamellipodiathesub-protrusivezoneisformed lying actin and keratin containing sub-protrusive zone and
of only actin, whereas in microridges it is formed of both functionsintheorganizationoftheoverlyingglycanlayer. It
actin and keratin, similar to microvilli (Figure 6 B,D,E). In willserveasanexcellentmodeltofurtherprobetheroleand
fact, keratin filaments from the sub-protrusive zone also en- regulationofactinnetworksinvivo.
ter microridges, possibly offering additional structural sup-
port(Figure6B).
Materials and Methods
Intriguingly, we also observed localization of Eplin, a com-
ponent of adherens junctions, as well as Fascin (Khandekar Fish strainsFor experiments, the zebrafish Tübingen (Tü)
and Sonawane, unpublished observations), a component of wild-typestrainorGFP-negativeindividualsfromtheback-
bundledactin,tomicroridges.ItispossiblethatthoughtheF- ground of the Tg(actb1:GFP-utrCH) line (Behrndt et al.,
actinnetworksupportingmicroridgesislargelycontrolledby 2012)wereused. Forzebrafishmaintenanceandexperimen-
regulatorsofbranchedactin, theremightbeasmallbutsig- tation,theguidelinesrecommendedbytheCommitteeforthe
nificant contribution from regulators of bundled actin. This PurposeofControlandSupervisionofExperimentsonAni-
isnotsurprisinggiventhefactthatstructuresformedofbun- mals(CPCSEA),Govt. ofIndia,werefollowed.
dledactin,suchasmicrospikesandfilopodia,areassociated
with lamellipodia. Alternatively, such components of bun- PlasmidinjectionsPlasmids,EGFP-EPLINbeta-agiftfrom
dledactinmaybepresenttoenablethetransformationofthe ElizabethLuna(Addgeneplasmid#40948)-andpcDNA3-
microridge actin cytoskeleton conformation from a largely myc-FLNaWT-agiftfromJohnBlenis(Addgeneplasmid
branchedtoacombinationofbranchedandbundledactin,un- #8982)(Wooetal.,2004)-werepurifiedusingInvitrogen’s
der certain physiological conditions. In fact, Bereiter-Hahn (USA) plasmid mini-prep kit and dissolved in autoclaved
andco-workershaveshownthatmicroridgesonthescaleepi- Milli-Q or nuclease free water. Plasmids were injected at a
dermis of adult guppy females exhibit a few actin bundles concentrationof40ng/µlinautoclavedMilli-Qwateratthe
spaced along the length of the microridges (Bereiter-Hahn 1cellstage.
etal.,1979).
During development, both the Arp2/3 complex as well as InhibitortreatmentsCK666(182515, Calbiochem), itsinac-
WASLlocalizetonascentmicroridges. Inaddition,thefunc- tivecontrolCK689(182517,Calbiochem)andLatrunculinA
tionoftheArp2/3complexisessentialforformationofactin (L5163,Sigma)weredissolvedinDMSO.Fortreatmentwith
punctae suggesting that they are formed of branched actin. inhibitors,10dechorionatedembryos/larvaewereaddedin1
Our data refutes the possibility that microridges are formed mlE3withoutmethylenebluetoawellofa12wellplateand
frommicrovilli(Gov,2006). IncontrasttotheArp2/3com- 1mlof2xinhibitororvehicleinE3withoutmethyleneblue
plex, whichisassociatedwithpunctaeevenat50%epiboly, wasaddedtoitandmixedgentlywithapipette. CK666and
keratin levels in the tissue are relatively low at early time CK689treatments(100µM)werecarriedoutfor1hwhereas
pointsandriseconcomitantwiththegrowthofmicroridges. Latrunculintreatment(2µM)wasperformedfor30min.
This increase in keratin levels might be linked to the speci-
fication of the periderm and could therefore be a trigger for
Immunohistology and imagingFor immunostainings, larvae
microridgeformation.
werefixedin4%PFAfor30minsandthenovernightat4°C.
Theubiquitouspresenceofmicroridgesduringevolutionsug- TheywerewashedinPBSandpermeablilizedwithPBScon-
gestsanimportantfunctionforthem. Microridgeshavebeen taining0.8%TritonX-100(PBSTx),blockedin10%NGSin
proposedtohavevariousimportantfunctions,themostcom- PBSTxfor3h,incubatedwithprimaryantibodyin1%NGS
mononebeingtheretentionofmucus. Indeed,disruptionof in PBSTx for 3 h to overnight (O/N), washed with PBSTx
microridgesbyLatA,visiblyaffectedtheglycanlayer. From 5timesfor30mineach,incubatedwithsecondaryantibody
bothconfocalandSEMdata,itisclearthatglycansarepref- in1%NGSinPBSfor3-4h., followedby5washesinPB-
erentiallyretainedaroundthespot-likemicroridgeremnants. STxeachfor10min. Sampleswerethenfixedfor30minor
10 | bioRχiv Pintoetal. | F-actinorganizationinmicroridges