The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

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brazilian journal of microbiology47(2016)266–269

h tt p:/ / w w w . b j m i c r o b i o l . c o m . b r /

Microbial

Physiology

The

dynamic

structure

of

Spitzenkörpers

of

Trichosporon

asahii

examined

by

the

ﬂuorescent

probe

FM4-64

Yuntong

Lv

♦

,

Zhikuan

Xia

♦

,

Wenling

Wang

∗

DepartmentofDermatology,GeneralHospitalofBeijingMilitaryCommandofPLA,5Nanmencang,DongchengDistrict,Beijing100700, PRChina

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Articlehistory:

Received11April2013 Accepted17June2015 AssociateEditor:LuisHenrique SouzaGuimarães Keywords: Trichosporonasahii Spitzenkörper Polarizedgrowth CytochalasinD

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TheSpitzenkörperisadynamicandspecializedmulticomponentcellcomplexpresentin thetipsofhyphalcells.TheamphiphilicstyryldyeFM4-64wasfoundtobeidealforimaging thedynamicchangesoftheapicalvesicleclusterwithingrowinghyphaltips.Itiswidely usedasamarkerofendocytosisandtovisualizevacuolarmembranes.Hereweperformed uptakeexperimentsusingFM4-64tostudythedynamicoftheSpitzenkörperinTrichosporon asahii.WeobservedthatSpitzenkörperswerepresentatthetipofthebuddingsiteofthe spore,blastospore,andthegermtubeofT.asahii.WealsofoundthatSpitzenkörperswere presentatthetipofthehyphaeaswellasthesubapicalregions.CytochalasinD,aninhibitor ofactinpolymerization,leadstoabnormalSpitzenkörperformationandlossofcellpolarity. ©2015SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Studies have shown that vesicle trafﬁcking is fundamen-tal to numerous activities in eukaryotic organisms, and is involved in cell growth and differentiation. The vesicle trafﬁcking network includes exocytosis and endocytosis.1

In filamentous fungi, vesicle trafficking is concerned with the polarized tip growth.2,3 _Vesicle _trafficking _to _the _apex

during tip growth is highly organized and involves the activity of a speciﬁc, multicomponent organelle complex which, in most cases, is called the Spitzenkörper. The

∗ _{Corresponding}_author.

E-mail:wangwl66@163.com(W.Wang). ♦ _These_authors_contributed_equally_to_this_work.

Spitzenkörper is a vesicle-organizing center that is inti-mately involved in fungal cell morphogenesis, and it spatially and temporally coincides with polarity-related components. The Spitzenkörper was originally recognized as a dark region in phase-contrast microscopy at the tip of actively growing hyphae.4 _{Subsequently,} _electron

microscopy revealed that this structure is rich in secre-tory vesicles.5 _During _tip _growth, _the _dynamic _behavior

ofthe Spitzenkörper ispredominated bysecretoryvesicles that form an “apical vesicle cluster,” which is intimately associated with the precise growth pattern of the hyphal apex.6

http://dx.doi.org/10.1016/j.bjm.2015.11.002

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TheSpitzenkörperregulateshyphalapicalgrowth primar-ilyvia exocytosis. Therole ofSpitzenkörperinendocytosis andrecyclingiscurrentlynotclear.Herewereportthatthe Spitzenkörpercomprises early compartments ofthe endo-cytic pathway in Trichosporon asahii. We performed uptake experiments using FM4-64, a widely used dye for endocy-tosis, to study the vesicle trafﬁcking.7 _We _observed _that

cytochalasin D,an inhibitor ofactin polymerization, leads toabnormalSpitzenkörperformationandlossofcell polar-ity.

Methods

Strainsandgrowthconditions

ThetypestrainofT.asahii,strainCBS2479,waspurchased fromCentraalbureauvoorSchimmelcultures(Utrecht, Nether-lands) and used in this study. Cells were propagated by incubationinYPD(1%yeastextract,2%peptone,2%dextrose) mediumat37◦C.For FM4-64(Biotium)staining,5␮Lofthe stocksolution(1.64mmol/Lindimethylsulfoxide(DMSO))was addeddirectlyto20mLofculture,followedbyincubationfor anadditional10min.Thecellswereharvestedbybrief cen-trifugationandwashedwithphosphate-bufferedsaline(PBS, pH7.4).

Spitzenkörperinhibition

CytochalasinD (FermentekLtd.)wasdissolved inDMSO to obtaina concentrationof2mmol/L, andthis wasadded to culturetoaﬁnalconcentrationof0.2,0.5,1,2,or5␮mol/L. SameamountofDMSOwithoutCytochalasinDwasusedasa control.Theeffectsofthevariousconcentrationsofinhibitor were tested bypretreating the samples withthe indicated compoundfor15minpriortotheadditionofFM4-64.Insome experiments,thecellsweretreatedwiththeinhibitorfor2h, afterwhichtheinhibitorsolutionwasremoved,andthecells werewashed onceandthen resuspendedinfreshmedium withoutinhibitorordye.Thecellswerethenallowedtorecover forupto4h,afterwhichthedyewasaddedtothecellsfor analysis.

Fluorescencemicroscopy

Mountedslides were photographed using aLeica DM 3000 fluorescencemicroscopewithaLeicaDFC300FXcameraand an LP560 filter (Ex543/Em560). The viability of the single-celled propagules was confirmed by taking aliquots and determiningthepercentagethatgerminatedafterovernight incubation in the medium. Viability of hyphae was con-firmedbytheirgeneralappearance,presenceofcytoplasmic streaming,andobservationofcontinuedtipelongationand growth. Images were recorded using a real-time digital imaging setup. At least 30 individual cells were exam-ined for each experiment, and all the experiments were performed using at least three independent batches of cells.

Results

UptakeofFM4-64atdifferentstagesofcellgrowthand development

FM4-64isalipophilicprobethatﬂuorescesstronglyafter bind-ingtotheouterplasmamembrane.TenminutesafterFM4-64 wasaddedtotheculturemedium,ﬂuorescencewasobserved inbothapicalandsubapicalhyphalcompartments.To deter-minethekineticsofFM4-64staining,cellswereculturedat room temperature inthe presence ofFM4-64and samples wereexaminedatvarioustime-points.

At8h,asinglefluorescentspotwithadiameterof0.5–1␮m wasobservedatthetipofthebuddingsiteofspores(Fig.1A). Similarfluorescentspotswereobservedatthetipofthegerm tube(Fig.1B)at16h.Dimbackgroundstainingwasobserved in the cytoplasm (Fig. 1A and B). After 24h of culture, a largerandbrighterfluorescentspot(1–2␮mindiameter)was observed at the apical or subapical hyphae (Fig. 1C). This fluorescentspotresemblestheSpitzenkörperstructure.8

Flu-orescentstainingwasalsoobservedatseptins(Fig.1D),which could bethesitesforbranchprojection.Inaddition, strong ﬂuorescencewasobservedintheelongatedsporesofthe pseu-dohyphae(Fig.1E),aswellasthechain-likespores(Fig.1F).No Spitzenkörperswerenotedinthesestructures.

InhibitionofSpitzenkörperformation

FM4-64uptakewasassessedinthepresenceofCytochalasin D,aninhibitorofﬁlamentousactin.T.asahiicellsweretreated withdifferentconcentrationofCytochalasinD(0.2,0.5,1,2,or 5␮mol/L)for15minpriortotheadditionoftheFM4-64,and theinhibitorwaskeptinthesolutionthroughoutthecourse oftheexperiment.

Lightmicroscopyobservationrevealedthatastheincrease ofCytochalasinD concentration(from 0.2to5␮mol/L), the inhibition effects also increased. Hyphal growth became slowerandhyphaebecameshorterbutwider(Fig.2).

When treated with a low dose of Cytochalasin D (0.2␮mol/L), the fluorescence was scattered in the central portion of the hyphae, but no obvious fluorescence was observedatthetopofthehyphae (Fig. 3B).Asthe concen-trationincreased,fluorescencewasscatteredfurtherintothe entirecellbody(Fig.3C–E).At5␮mol/L,thehyphalgrowthwas completelyblockedandonlyalowbackgroundfluorescence wasobserved(Fig.3F).NoSpitzenkörperwasobservedwhen cellsweretreatedwithanyconcentrationofCytochalasinD.In theabsenceofCytochalasinD,FM4-64-stainedapicalvesicle clustersatthetipofhyphae(Fig.3A).

Discussion

TheSpitzenkörperisadynamicstructureandcloselyrelated topolarizedgrowth.Itiscontinuouslypresentatthehyphal tipatallstagesofthecell cycle.Inthis study,werevealed thepresenceofSpitzenkörpersatthetipofthebuddingsite ofthespore,blastospore,andthegermtubeofT.asahii.We alsofoundthatSpitzenkörperswerepresentatthetipofthe

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Fig.1–FM4-64stainingofT.asahiicellsatdifferenttimepoints.Cellswereculturedatroomtemperatureinthepresenceof FM4-64andwereimagedafter8h(A),16h(B)and24h(C–F).(A)Fluorescencespotatthetipofthebuddingsiteofaspore (arrow).Backgroundstainingwasseenincytoplasm(orange).(B)Fluorescencespotatthetipofagermtube(arrow). Backgroundstainingwasseenincytoplasm(orange).(C)StainingofSpitzenkörperstructureatthesubapicalhyphae (arrow).(D)Stainingofseptins(arrow).(EandF)Stainingofelongatedsporesofthepseudohyphaeandchain-likespores(F). Scalebar:1␮m.

hyphaeaswellasthesubapicalregions.Inaddition,thespores atthetipofthe hyphae showedFM4-64ﬂuorescence, sug-gestingthatthesporemayhaveproducedgermtubesinthe oppositedirection.Interestingly,someotherstructuresofT. asahii,suchashyphaeandpseudohyphaewerealsopresented atbothendsoftheSpitzenkörper(Fig.1E),suggestingthat pseudohyphaecouldgrowbi-directionallyandcontinuetobe regulatedbytheSpitzenkörper.

Usingtransmissionelectronmicroscopy,weshowed previ-ouslythatpseudohyphaeproducedgermtubesandhyphae.9

Thestrongﬂuorescenceattheelongatedsporesofthe pseu-dohyphaefurthersuggeststhatT.asahiipseudohyphaecould formtruehyphae.ThephenomenonconﬁrmedthatT.asahii

pseudohyphaedidnotlosepolargrowthmomentum,andthey continuedthebuddingtransitiontohyphae.

Polarizedgrowth,whichenablescellstoassumeashape typicalforthespecies,isafundamentalfeatureforall fun-galcells.InSaccharomycescerevisiae,growthisconﬁnedtothe daughterbuds,whichinitiallygrowinapolarizedfashionand then switchtoisotropicgrowth.10–14 _In_pseudohyphal_cells,

polarizedgrowth persists,resultinginlonger buds.15

Polar-izedgrowthdependsontheactincytoskeleton,whichconsists ofcorticalactinpatchesandactincables.14_In_both_Candida

albicansand T.asahii,disruptionofactin cablesby cytocha-lasinA(forC.albicans)orcytochalasinD(forT.asahii)causes the hyphal tipsswell and the growth mode switches from

0.2 µmol/L

A

B

C

D

E

0.5 µmol/L 1 µmol/L 2 µmol/L 5 µmol/L

Fig.2–CytochalasinDtreatmentinhibitedhyphalgrowth.CellsweretreatedwithvariousconcentrationofCytochalasinD (0.2,0.5,1,2,or5␮mol/L)andgrewfor72h.Cellwereimagedwithabrightﬁledlightmicroscope.Scalebar:1␮m.

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0 µmol/L

A

B

C

D

E

F

0.2 µmol/L 0.5 µmol/L 1 µmol/L 2 µmol/L 5 µmol/L

Fig.3–CytochalasinDtreatmentblockedtheformationofSpitzenkörper.Cellsweretreatedwithvariousconcentrationof CytochalasinD(0,0.2,0.5,1,2,or5␮mol/L)thenaddedFM4-64andgrewfor72h.Cellswereimagedwithaﬂuorescence microscope.The0␮mol/LCytochalasinD(A)wasusedasacontrol.Scalebar:1␮m.

polarizedtoisotropic.Consistenttothesereports,ourcurrent studyshowedthatCytochalasinDtreatmentcausedtheloss ofpolarity(Fig.3),whichcouldbecaused,atleastpart,the diminishingoftheSpitzenkörper.16

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

We thank the staff of the Dermatology and Venereology Department, Beijing Military Command General Hospital of PLA for their assistance. This project was supported by the National Natural Science Foundation of China (No. 81271764) and the 11th Plan Project of PLA of China (No.06MB078).

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