Accepted Article What’s already thisknown abouttopic? Statements Conflict ofinterest: The authors state no conflict interest.of Funding sources:IZKF RWTHAachen University 129/12). (Start [email protected]: 52074 Aachen,Germany Wendlingweg 2 RWTH Aachen University Institute of Molecular and Cellular Nicole Schwarz Correspondence: This article is article This hasThis article been accepted publicationfor andundergone peerfull but review not has 1 Article :Original type Article DR SCHWARZ NICOLE (Orcid : 0000-0001-7888-6970) ID article asarticle doi: 10.1111/bjd.18014 this cite Please Record. of Version the and version this between differences to lead may through the been paginationtypesetting,proofreading andcopyediting, process, which Institute ofMolecular and Cellular Anatomy, RWTH Aachen University, Aachen,Germany • •

epidermal layers. epidermal layers. Autophagy-impaired have been shown to result in thickening of of cellular components byautophagy. Terminal epidermalis differentiationa mult 6a mutations lead to impaired mitochondrial quality control p rotected b Running title: Mitochondrial quality incontrol PC y S.M. LehmannS.M. co py ri g ht. Allri ht.

1 , R.E.Leube g hts reserved. hts

istep thatistep process includes the elimination 1 , N.Schwarz , 1 Accepted Article This article is article This protection and maintenance of integritycellular Theypresent in are arefor critical filaments stress all cells. intermediate epithelial Keratin Introduction recycling. Conclusion: We propose an influenceof keratinson degradationautolysosomal and accumulatein and arenot sufficientlyrecycled. and autophagosome formation, however, are clearancedisturbedEarlyuncoupling. mitochondrial after ofmitochondria steps mitophagy Results: Pachyonychia congenita keratinocytes accumulate old mitochondria and display a mitophagy. used in fluorescence-based biochemical and toassaysthe dissectdifferent steps of keratinocytesPachyonychia ImmortalisedMethods: derived from congenitapatientswere at which stage. Objectives:Wetherefore asked ifmitophagydisturbed is inPachyonychia congenita andif so disease causedby mutations inkeratins6,16, and 17. thickening of the as it is encountered in Pachyonychia congenita, a rare lose theirincludingorganellestheir mitochondria by Disturbed autophagy. autophagyto leads Background:is Epidermal differentiation processina multi-level which need keratinocytes to Summary theWhat is translational message? What does this study add? disease Pachyonychia congenita (PC). inMutations keratin result than30 in more including diseases,human rare skin the netw cytoplasmic into assemble • • • • •

to Pachyonychia congenita pathogenesis. keratin inMutations 6ato lead severely impaired might which mitophagy contribute Pachyonychiacongenita.inOveraged andautolysosomes mitochondria accumulate influenceKeratins maturation autolysosomal or reformation. contact sites.mitochondria Keratinsmitochondrialto contribute as control quality well as of maintenance ER- mutations6, in keratins16, and 17. also occurs in Pachyonychia congenita, a rare skin disease caused by p rotected b y

co py orks that are anchored to desmosomes andhemidesmosomes. ri g ht. Allri ht. 1 g hts reserved. hts not affected. We find that autolysosomes . Equalamounts ofItype . typeand II Accepted Article rhomboid-like (PARL) rhomboid-like then degraded. and andusually is importedinto mitochondria where itis cleaved bypresenilin-associated autophagosomes autophagosomes andlysosomes is controlled by Rab , whichbelongthe to Ras-like ofdegradation mammalian In the cargo. theof autophagosome'scells, fusion is to This fusion for required form acidification autolysosomes. lysosomes the and autolysosomes. members, of Rab7, One superfamily. its GTPase autolysosomes and form andautolysosomes proto-lysoso form a processcalled lysosomeautophagic reformation inwhichtubules extrude from reformation. defect. Indeed, we could show that mitophagy differentiation, we asked whether PC-linked mutations tocontribute a mitophagy by phenotypeepidermal anin hyperproliferation conjunction delayedwith terminal This is accomplished by processes multiple Since includingmitophagy. PC is characterized sustains cellhealth itis also needed for organelle removal during keratinocytedifferentiation. Epidermal differentiation strongly differentiation onEpidermal depends macroautophagy. localization and function. Recently,filaments intermediatewere shown to mitochondrialinfluence structure, This article is article This acid inhibits autophagy. acid inhibits keratin networkfilament by of treatment cells phosphorylationstatevimentin’s and therefore its . around damaged mitochondria. as p62/SQSTM1(Sequestosome 1) optineurin or inorderto induceautophagosome formation proteins. recruitment ofubiquitinthe E3 ligase Parkin,whichubiquitinto OMM moieties attaches in(OMM) results PINK1 thestabilisationmembrane of subsequenton theOMMand as wounding UV and exposure. and 17expressionis upregulated whenstratified epithelia encounterstressful situationssuch partof mitochondrialqualitycontrol. Mi overallmitochondrial function. for is crucial characterised by and hypertrophic dystrophy. isPC incausedmutations by II keratins6a, type 6b and 6c,type and16I keratins 17.and Itis homolog (PTEN)-inducedhomolog kinase(PINK1) 1 Parkin. and best-studied mitophagy pathways relies on the that produce lessATP andrelease highof levels reactiveoxygenspecies One (ROS). of the results Disturbed mitochondrial control in quality of accumulationthe damagedmitochondria mitochondrialthe networkthrough constant fusion of fissionand mitochondria. existing dysfunctional mitochondria through selective macroautophagy (mitophagy) or by renewal of 8 This, ubiquitin- recruits in turn,LC3-bindingand adaptor autophagic proteins such p 12 rotected b hasRab7 shown been to directlyvimentin, with interact modulating 14 y 5 In order In torestore lysosomes, autolysosomes are recycled through Maintaining the cellular mitochondrial pool as healthy as possible co py 9–11 4 ri g Autophagosomes are transportedto and withfuse ht. Allri ht. 6 mes thatmaturelysosomes. into mes This is accomplished either by selective elimination of g hts reserved. hts with the with protein phosphataseinhibitor okadaic is impaired due impaired is disturbedto autolysosomal /threonine kinase phosphatase and tensin tochondrial turnoveris therefore an essential hasbeenof implicated inthe maturation 7 Depolarisation of Depolarisation theouter mitochondrial 6 PINK1 is targeted to PINK1 targeted mitochondria is 13 Furthermore, of disruption the 16,17 autophagyWhile per se 15

2,3 6, Accepted Article 95°C for 5 min and subsequently separated by 8-14% discontinuous sodium dodecyl withoff rubbera policeman. lysates were st blue) bromophenol were addedthe andlysed cells were scraped 250mercaptoethanol, µg/ml Primaryrabbit polyclonal against(1:1000 immunoblot; PINK1antibody for and 10% (v/v) human growth factor in a 5% CO Sigma-Aldrich) Fungizone, (penicillin,solution streptomycin, (v/v) antibiotic-antimycotic All lines supplemented were MediumFisher) (Thermocell EpiLife culturedin with 10% buffer 2x SDS of Cell wereprepared bywashing themwith first PBS in the µl 100 dish. culture analysisImmunoblot were obtained Dako. from immunoblot;(1:5000 and for guinea againstP0448) pig (1:5000 for immunoblot; P0141) fromBiotechnik. Progenhorse Secondary radish Polyclonal guineaantibodypig p62 against (1:1000 waspurchased immunoblot; GP62-C)for against optineurin (1:1000 for immunoblot; mAb #58981) was obtained from Cell Signaling. (1:2000 immunoblot;for A2066) was from Sigma-Aldrich. Monoclonal rabbit immunoblot;(1:1000 were for Cell from Signaling4292) andagainst antibody beta- obtained Abcam, from antibodies against PT to 1.2 mM Ca mM 1.2 to This article is article This For1:3. passaging, cellswashed were and incubated15 for min in PBSwithout Ca rat tail I (Corning). The cell lines were passaged once or twice per week at a ratio of 37°C. They were culturedinuncoated flasksor onglass coverslips coated with Haven). method. They were generously provided by Dr Leonard M. Milstone (Yale University, New patientscongenita also of byHPVimmortalization keratinocytes epidermal with E6/E7the TheSalzburg). PCcell lines K6aN171del K6aN171Kderived and were Pachyonychia from providedby DrsJulia Reichelt, Verena andThomasWally Lettner (EB houseAustria, normal human epidermalkeratinocytes using the methodE6/E7 HPV andkindly was humanwild-type The keratinocyte cell line K6a wtwasobtained by immortalization of Cells andmethods Materials conditions, cells wereswitched standard from culturemM medium(0.06 Ca cellhigher 10 lines and wild-type For undercontrol. for experiments differentiation solution(Thermo Fisher). Cells were usedwithin 10passages. Passageswere equalfor PC neutralizer trypsin in resuspended and accutase with min ~10 for incubated thereafter and ( 100mM 100mM Tris-HCl (pH 8), 10% (v/v) glycerol, 3% (w/v) SDS, 7.5% (v/v) 18

2+ p rotected b medium for (high calcium) three days. y co py ri g ht. Allri ht. g hts reserved. hts EN (1:10009559)EN for PI3K immunoblot; and oredat -20°C.Proteins were denatured at peroxidase-coupled antibodies rabbitagainst 2 humidified atmosphere at humidified atmosphere

2+ ab23707) was , lowcalcium), β - 2+ /Mg 2+

Accepted Article LightCycler 96 Real-Time PCR System (Roche). The cDNA was diluted 1:5 in H in 1:5 diluted was cDNA The (Roche). System PCR Real-Time 96 LightCycler EssentialProbes DNA Masterkit (Roche) accordingthe to manufacturer’s onprotocol a manufacturer’s protocol. Quantitative real usingperformed FirstStrand Transkriptor Fisher). A260/280ratios were between 1.74 Reverse 2.1. and transcription into wascDNA protocol. RNA concentration and qualitywas determined using NanoDrop1000 (Thermo a RNA was isolated usingthe RNeasy MiniKit (Qiagen) according tothe manufacturer´s PCR Quantitative Three independent were performed. experiments antibodies. Subsequent procedures started with a stepblocking and continued as described. was incubated in stripping buffer (0.1 M , pH 2) 3x for 20 min to remove the bound The following primers were used: HPRT (UPL probe #022) (5’- forward 1:4, 1:8,(1:2, 1:16, 1:32). standard was prepared by pooling cDNA of every sample and a dilution series was generated This article is article This (Roche). Foldfactor givenisas E For quantification, efficiency and CT values were determined using LightCycler 96 software analysed in duplicates for each condition. was three RNA from experiments independent (5’-GCCAGAACCTACCCTGCTC-3’). HDAC63’), (UPL #58) probe forward (5’-AGTTCACCTTCGACCAGGAC-3’) reverse and AACAGTGACCCTGAGCGAAG-3’) and reverse (5’-AAGTTGGGTTTACAGAGGCGTA- CAGAGAAGCCCATGGACAG-3’), optineurin probe #72)(UPL(5’- forward 3’), p62(UPL #14) forward probe (5’-AGCTGCCTTGTACCCACATC-3’) reverse and (5’- GGAGCTGAGGAATGACTGGA-3’) andreverse (5’-ACAATGTGAACAATGCTCTGCT- PARK2CTGAATCCGCATTTTTGTTG-3’), (UPL (Parkin) probe #85) forward (5’- PARL (UPL probe#17) forward (5’-GCTCACTGCGGTCCTAACC-3’) and reverse (5’- GCCATCAAGATGATGTGGAAC-3’) andrevers ##65) (5’- PINK1 forward (UPLprobe CAAGACATTCTTTCCAGTTAAAGTTG-3’), (5’- and reverse TGATAGATCCATTCCTATGACTGTAGA-3’) NIH). wasDensitometric evaluation performed using ImageJ (Fiji Solo.WL.4M. distribution, helptheof an enhanced chemiluminescence methoddetection and usingFusion- the hour. After repeatedTBS-T, washing visualization with ofbound antibodieswas done with incubatedwithsecondary the HRP-conjugated antibody diluted in10% Roti-Block 1 for TBS-T (130 mM NaCl, 50 mM Tris base, 0.1% (v/v) Tween-20). The membrane was in 10%Roti-Blockdiluted over at night 4°C, thewaswashed 3x 10 membrane for minwith BlockingBlock reagent (Carl Roth) 1h. Afterfor the incubationwithantibody primary glycine,20% at100methanol) V 1 for h. The PVDFwasmembranewith blocked 10%Roti- wasperformedmembrane in the of presence transferbuffer (25mM Tris-HCl,190 mM protein The onto (SDS-PAGE). PVDFelectrophoresis transfer gelsulphate-polyacrylamide reference ). gene). reference 19,20 To perform incubation of further antibodies on the same membrane, the membrane p rotected b y co py ri g goi ht. Allri ht. ( ∆ CTgoi) / E cDNA cDNA Synthesis kit (Roche) according tothe time PCRusingtime was performed the FastStart g hts reserved. hts ref ( ∆ CTref) e (5´-GACCAGCTCCTGGCTCATT-3’),e . (GOI = gene of interest; REF = (GOI= geneinterest; REF of .

2 O and a Accepted Article cells pertreatmentwere imagedwith an63×/1.40-N.A.oil DICM27 objectiveimmersion (Carl Mowiol Roth).with UsingApoTome.2 the (Carl Zeiss), microscope NucleiRT. werestained with Hoechst33342 (Invitrogen)andthecoverslips were mounted further propagated for three days prior to fixatio low calcium medium was replaced with high calcium medium (1.2 mM Ca This article is article This with theAddgenewith pftLC3plasmid(gift #21074 Yoshimori).from Tamotsu wereCells seeded atday 0on collagen-coated coverslips.They weretransfected onday 3 LC3 Three independent with were performedexperiments substraction (rolling ball set5).todistributionParkin was scored as cytoplasmic ordotted. mitochondrial perarea cell was determined Parkin-positive for cells after background contrast, MitoTracker andParkin-eGFP. Using ImageJ (Fijidistribution, NIH), the performedthe onwas same day for allconditions,with equal exposure interference times for DIC63×/1.40-N.A. M27 oil objectiveimmersion (Carl Zeiss). Imaging of one experiment theusing magnification 63x were at imaged cells Zeiss) microscope (Carl ApoTome.2 andthe (Invitrogen) werecoverslips mounted with Mowiol(Carl Roth). Usingthe diethylpyrocarbonate water for 25 min RT,at nuclei were stained with Hoechst33342 Scientific) for30Fisher minat37°C. 4% with After fixation in warmPFA with medium at 37°C,cells werestainedwith100 nM MitoTracker Red(Thermo CMXRos at37°C hfor 2 18hand with or DMSOonly18 hfor as a After 2xcontrol. 15min washing the manufacturer’s protocol (Takara Bio). On day 2 the cells were treated CCCP with 10 µM (providedconstruct Sven by Geisler, Tübingen,Germany) on using 1 day accordingXfect to aParkin-eGFP with transfected and 0 day on coverslips on collagen-coated seeded were Cells Induction ofmitophagy 30 cells each percondition. Data were normalized tountreatedK6a wild-typecells. determined for every region of Threeinterest. independent wereexperiments performed with that showspositive signal ineither channel. Fluorescence intensitymean valueswere calculated was interest of regions of a mask and algorithm) (Otsu thresholded were channels red and green NIH), distribution, (Fiji ImageJ Using 0.2%. of intensity laser a and unit) (green500-540 signal)nm signal)and 580-640nm(red with apinhole set1–2 AUat (airy DIC63×/1.40-N.A. M27 oil objective.immersion lightThe emitted was between monitored 37°C. The 488nmline of an was argon/krypton laser used for fluorescence recording via a daysfour afterseeding. Live cell imagingwa pMitoTimerAddgenetheplasmid #52659(gift Zhen from Yan) were and imaged three or MatTek) coated with rat tail collagen I (Corning). After two days cells were transfected with wereCells seededon day 0on glass-bottom dishes (12mmglass-diameter, thickness1.5#, MitoTimer p rotected b y co py ri g ht. Allri ht. g hts reserved. hts s doneon aZeiss LSM710 Duomicroscope at n with 4%PFA in DEPC 25minwater for at ≥ 48 cells per condition. ≥ 21 2+ 16 transfected On day 4 the the 4 day On ) and cells were were cells and ) Accepted Article fluorescence shifts from green to to green from shifts fluorescence transfected with MitoTimer. MitoTimer is mitochondrialwhetheris quality inPC keratinocytes, control werecompromised cells cell line prepared from a healthy donor producing only wild-type K6a (K6a wt). To find out producinglineseither K6aN171K or K6aN171del were mutants with examined together a viral bytransformatio healthy E6/E7 individuals PC pathology, immortalizedpreparedTo investigate were keratinocytes PC and from patients mitochondriaOld accumulatePachyonychia in congenita keratinocytes Results p when Data are summarized and presented as mean ± SD. Differences were considered significant using Software) (GraphPad ANOVA one-way withDunnett’sor either Bonferroni post-test. for each experiment. Differences betweengroups Each wasexperiment performed leastat 3 times.number Theof analysed cells are indicated Statistical analysis circumference. the ratio of ER-covered mitochondrial ci pixels away frommitochondrial the andmembrane theirlength was determined to calculate to theircalculate circumference.Close were ER defined membranes as no15than more NIH), (Fiji distribution, ImageJimages. Using (Olympus) using software iTEM (Olympus). forsolution 3 min. wereImages taken on an EM10 (Carl with Zeiss) a digital camera contrast, sectionswere treatedwith 3% uranyl acetate for5 min and with0.08M lead citrate sectionswereprepared withanultramicrotome (Leica) usingdiamond a knife. To enhance wasas thePolymerization 48 performed at 60°C intermedium. h. Semi- for ultrathin and 5.2,pH for2 hindark the and thereafterdehydrated and embedded in aralditeusing acetone 1 h. Fixed samples were treated with 0.5% uranyl acetate in 0.05 M sodium maleate buffer, This article is article This Pelletedcells were fixed 4% in formaldehyde/1% glutaraldehydefor 2h in1%and OsO Electron microscopy wereperformedindependent with Threeyellow. experiments were scored as either greenor red andparticles positive in both channelswere scored as set(rolling ballafter background positivein substraction to 20). Particles only onechannel in the greenred and channels thewithhelp ofthe “analyseparticles” 5-50 function (size Px) LC3 NIH),(Fiji distribution, UsingImageJ punctae positive were automatically determined (CarlZeiss) with exposure timesequalinterference for contrast, green and red channels. ≤ 0.05. p rotected b y co py ri red over time as the protein ages. g ht. Allri ht. rcumference versus whole mitochondrial a mitochondria-targeted dsRed whosederivative g hts reserved. hts Analyses were done on 50000x magnification ≥ 50 mitochondria50were per line encircled cell n. For the current studytwo PC-derived cell were evaluated were usingGraphPad Prism5.0 ≥ 16 cells 16 percondition. 22 Aging of Aging MitoTimer 4 for Accepted Article known phosphorylation sites. accumulates onmitochondria PINK1dysfunction, due to its two onautophosphorylation presenilin-associated rhomboid- mitochondrial ligase Parkin. Under PINK1normal is imported into conditions, and mitochondria cleaved by kinase1 andon dysfunctional mitochondria of (PINK1) the recruitment subsequent ubiquitin- Mitophagy comprises a series of events starting with the accumulation of PTEN-induced keratinocytes Expression ofearly mitophagyrelated proteins for subsequent mitophagy are not disturbed. unaltered. These results suggestthat recognit reducedPC in independentlycells ofcalcium addition(Figs 3e, were althoughf), their ratios protein expression 3d),was unaltered (Fig. while itsand counterpartPI3KPTEN were mRNA expression in or either low high calciummedium (Figs3a-c).PINK1 Similarly, revealedanalysisnoQuantitative PCRin changes significant PINK1, PARLand Parkin undergo aseries of changesincluding loss theof their bymitophagy.mitochondria Cells wereCells eithergrown inlow calcium mMCa (0.06 medium Ca This article is article This mitophagy resulting inof accumulation mitophagy resulting old dysfunctionaland mitochondria. andimplicated aremitochondrial quality in Loss ofMAMs control. leads to reduced Mitochondria-ER contact sites are known as PCK6aN171del 2a). patient celllines(Fig. covered by endoplasmic reticulum, only 9.8% were covered in K6aN171K and 8% in arrangement. While 22.9% ofthe circumfere d). More carefulanalysis, however,revealed striking differences inER-mitochondrial obvious inmitochondrial difference be morphology could identifiedat first glance 2b- (Fig. Electron microscopy was performed next to examine mitochondrial ultrastructure. No contactMitochondria-ER sites are reduc control cells. control cells (Fig.1). We therefore conclude that becomemitochondria older in PC than in two days afterMitoTimer transfection in bothPC but cellspatient-derived in wild-type not derived cells. However, fluorescence microscopy revealed a significant shift towards red of couldratios notbe observed after 24h comparing transfection wild-type control patient- and takesapproximately 48h upon its Differe expression. 2+ ) medium for 3 days to induce differentiation. During differentiation, keratinocytes keratinocytes differentiation, During differentiation. induce to days 3 for ) medium p rotected b y co 24 py ri g ht. Allri ht. ed in Pachyonychiain congenita ed g hts reserved. hts nce of mitochondria in control cells were ion of dysfunctionaland marking mitochondria mitochondria-associated membranes (MAMs) membranes (MAMs) mitochondria-associated like protein Upon(PARL). mitochondrial is not altered in Pachyonychia congenita nces inred/green fluorescence intensity 2+ ) or in high (1.2 mM calcium 23

Accepted Article mitochondria mitochondria marking the site of initial autophagosome formation. facilitate autophagosome-lysosome fusion. facilitate autophagosome-lysosome This article is article This effector proteins LC3A LC3B.and mediates the aggregation of dysfunctional mitochondria and directly binds to the autophagic deacetylase 6(HDAC6) the and autophagyreceptors SQSTM1/p62 andoptineurin. p62 proteins become highly ubiquitinylatedwhich is recognized by the ubiquitin-binding histone expression ofmitophagy and autophagosome mark To down narrow further which stepof mitophagyPCin isimpairedwe investigated the keratinocytes forMarkers autophagosome formation are notaltered in Pachyonychia congenita of subsequentstepsmitochondria, in mitophagy are PC. disturbed dysfunctional mitochondria are marked for mitophagy andParkin is recruited to reduction becould observed between all time points. This leads to the conclusion, thatwhile signaldeclineonly occurred within the first 2hours of CCCPtreatment whereas in K6awt a showed less mitochondrial signal 4e). (Fig. Ofnote, in K6aN171Kmitochondrial the cells signal per cell was not reduced in K6aN171del while K6aN171K and wild-type controls panels; quantification Accordingly,in 4d). Fig. the percentageof the area ofmitochondrial fractionremaining of cells had still r Parkin displayedcells PCcytoplasmic a pattern Parkin again and theirlost mitochondria Inonlytheir mitochondria. contrast,K6aN171K 9.33% of cellsK6aN171delPC 9.8% and of 72.61% of the control cellsshowed arestored steadystate cytoplasmic Parkinpattern andlost ismitochondria not affected byK6a18 mutations. hours by after mitophagy CCCP, induction middle panels; quantification in Fig. 4d). Hence, Parkin recruitment to dysfunctional showed a co-local Parkin-eGFP dotted pattern of control cells and95.33%of K6aN171K PC ofcells and94.60% K6aN171del PC cells upper panels; quantification in Fig. 4d). 2 hours after mitophagy by induction CCCP, 93.77% 99.35% ofcontrol cells, K6aN171Kcells, and PC 100%K6aN171delPC cells) 4a-c (Figs cells. Untreated showed cells an even cytoplasmic of distribution of Parkin-eGFP (100% 4a).(Fig. Mitochondria were eliminatedby (CCCP). CCCP treatment led to the translocation2within ofParkin tomitochondria hours induced by adding the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone were transfected with fluorescent Parkin tovisualize itstranslocation. Mitophagy was outerstabilisation on the membrane. mitochondrial To this end, healthyPC and control cells not disposed of. We therefore asked if Parkin is recruited to mitochondria PINK1following Although present in bothis PINK1 PC oldmitochondriacell lines, stilland accumulate are Mitophagy is impaired in Pachyonychiacongenita keratinocytes lysosomes. autophagy bymediating the attachment ofautophagosomes to motors transporttofor 25 Furthermore, HDAC6 induces HDAC6 Furthermore, cortactin-dependentto actin-remodelling p rotected b y co py ri g ht. Allri ht. 9,10 Optineurin becomesrecruited to damaged 26 g ecruited to their mitochondria (Figs 4a-c lower mitophagy 4a)hours 18control (Fig. in after hts reserved. hts isingwith MitoTracker (Figs 4a-c ers. After translocation of Parkin, OMM 11 HDAC6 promotes HDAC6

while the Accepted Article turnover. turnover. or recycling,of suggestingcompromised maturation rolea 6a keratin in autolysosomal While autolysosomeformation is notaffected,autolysosomes accumulate becauseof delayed.and Still, mitochondrial isclearance early late mitophagy not markers are changed. accumulatemitochondria over peritimea different inkeratin mutations 6a: K6aN171K and K6aN171del. We show that in PC old Pachyonychia Tothis endcongenita. we used two patient-derived keratinocyte linescell with paperIn this we addressed the possibleof mitophagy role thepathogenesisin of Discussion patients. recycling severelyin impairedautolysosomal from is Pachyonychia derived cells congenita that indicate Taken together, results au these 71.13% in K6aN171K cells, PC vs.82.6% 63.8% K6aN171delinand cells) PC 6d). (Fig. percentage ofautolysosomesin cells PC 56.82% (57.86% invs. controlcells, 55.09% vs. control elevationwild-type thecells, ofextracellular led calciumto increasedsignificantly a autolysosomes (only red LC3 puncta) was not alte (Figs 6a-ccells) panels;quantification lower 6e). Likewise, while the percentage of autolysosomes/cell in K6aN171K PCcells, and152.5 autolysosomes/cellinK6aN171del PC 138.00 in cells, control both in increased cell(69.65lines autolysosomes/cell PC significantly amountthe of autolysosomesin controlcells, the number of autolysosomes perwas cell (Figscells) 6a-c upper panels; 6e). While calcium did notaddition alter autolysosomes/cell in K6aN171K PCcells, and80.88 autolysosomes/cellinK6aN171del PC keratinocyteshealthy control (61.99 au state low calcium conditions in K6aN171K andK6aN171delkeratinocytes compared to This article is article This conditions. autolysosomes andlose theirgreen fluorescen autophagosomesmitophagy with fuse mRFP-GFP-LC3-containing to lysosomes form fusion protein fluoresces bothgreen andred und specifically localises to autophagic membranes ofautophagosomes and autolysosomes. of autolysosome be formation can monitoredusing by aLC3tandem LC3 fluorophore. process The PCkeratinocytes. in affected is formation autolysosome whether asked next We Autolysosome maturation is perturbed in Pachyonychia congenita keratinocytes. likely not the fordisturbedcause PC mitophagy in keratinocytes. differential expression of key players of autophagosome formation and maturation are most Accordingly, optineurin were p62 unaffectedand atthe levelTherefore, protein 5d-e). (Figs aswell inPCkeratinocytesdifferentiationafterby of induction calcium addition(Figs5a-c). Optineurin, HDAC6 and p62mRNA expression was not inhealthychanged controlcells as 21 The total The numberof autolysosomes per wascell already elevated under steady- p rotected b y co py ri g ht. Allri ht. tolysosomes/cell in 105.6control cells, odof 48 hoursand that CCCP-induced g tolysosome generationbuttolysosome isunaffected that hts reserved. hts ce due tolysosomal acidic degradativeand er steady-stateer inductionofconditions. After red in low red incalcium to conditions compared 27 The Accepted Article autolysosomal recycling.autolysosomal KIF5B, andof aregulator, member the 1 important two family, are factors for Those tubules thenbecomeacidic andmature into lysosomes. Rab7,vesicle a transport lack initially membrane which andautolysosomal lysosomal acidity. protolysosomes form recycledorderinare Duringthis lysosomes. totubulesreformation the reform protrude from Autolysosomal reformation is the process in which lysosomal components of autolysosomes disturbing autolysosomal recycling. handling, calciummitochondria-ER whichresults in autophagy, enhanced ( and autophagosomes. structuredamaged thataengulfsorder mitochondriain membranous form to matrix. domainsERshuttling for calcium intracellular from to mitochondrial transfer, calcium the autophagosome seemedformation to be unaffected. Additionally, MAMs specialized are proliferation. cell increased the autophagy-inhibitors 3-MA or wortmannin pr reports (reviewed in (reviewed reports ofroleA intermediate mitochondrial in filaments has homeostasis been pointedoutseveralin inhibition ofinhibition MTORC1 byrapamycin enhanced autophagy reduced and cellmigration. on wound healingsowas it shownthatfar, stimulated ATG7 knockdown cell migration while autophagy and show epidermal acanthosis and hyperkeratosis. mice lackingATG7,whichis important autophagosome for assembly, havea lowlevel of reports which confirm therole ofautophagydifferentiation.keratinocyte Forexample, in seen after calcium-induced differentiationof in theoutermostepidermalof layer. Accumulation beonly could autolysosomes keratinocytes who lose theirorganelles during of the keratinizationcourse tobecomedead mitochondrial mitochondrial andcalcium increase an in cytoplasmic calcium leads to autophagy. therefore autophagy.activates Loss ofmitochondri weakened stress responseresulting inDNAdamage, cell cycle arrest andpremature aging. This article is article This migration andmigration healing. wound woundingofupregulation isthe keratins6,16 17. and organelles restores thusand cell health.Partof the stressinkeratinocytes response after Autophagyvital is for cells tosurvive stresses as it damagedeliminates proteins and distance. example, itbeen has shownthatmutations lead ato mitochondria-ER disturbed diseases. a number ofhuman diseases such neurodegenerative and as cardiomyophathies, skin and viamitochondria autophagy is called mitophagy mitophagy. Disturbed autophagylead and to components such as proteins organelles.or ofoldand Elimination dysfunctional Autophagya conserved is process which leads to the lysosomal degradationof cellular treatment. explainingwhy the effects ofPC mutations on autophagy beseen can after calcium Elevation ofintracellular ofkeratinocytescalcium additionally inducestheir differentiation, 37 Intracellular calcium actsas a regulatorof autophagyMTORC1by inhibiting and 35 28,29 40 Mitochondria-ER contact sites are importantthe for omegasome,of theformation Keratin 6a mutations may onact two levels different ( Functional autophagyespecially is inof terminal important differentiation p rotected b 36 Although contact mitochondria-ER sites were reduced inPCcells, patient 5 ), but no clear functional connection has been established yet. For 12,41 y co 30 KIF5Bhas been implicated in thecontrol of intermediate Furthermore, Furthermore, keratinocytesautophagy-deficient display a py 4,33 ri Whilenot much is knownabout the of influence autophagy g ht. Allri ht. g hts reserved. hts PC keratinocytes. There have been several several been have There keratinocytes. PC esent enhanced inflammatory responses andesentresponses inflammatory enhanced a-ER a-ER contact sites as well as a decrease in 32 Keratin 6 has been linked to cell cell to linked been 6 has Keratin 16 Keratinocytes treated with i ) by disturbing ii ) by 38,39



Accepted Article harming autophagy. treatment inducesalso autophagyproviding a keratinocytes. which in turntriggers autophagy releaseupon its into the ofsuprabasalcytoplasm metabolismpromote mitochondrial the and associatedof mitochondrialROS, production important role in keratinocyte differentiation in organotypic where lysosomes it Furthermore, has recently beenshown thatautophagy lysosomal and play functions an signalling and becan by blocked the mTOR inhibitor rapamycin. reformationisAutolysosomal upregulated during situations.stress ItdependsmTOR on Research was byfunded IZKFRWTH AachenUniversity 129/12).(Start cell line.WeYoshimoriwild-type Zhen thankandGeisler, Sven Tamotsu plasmids. Yan for Milstone, providingkindlyfor theused celllines. thankWe EB the house for theAustria We are especially grateful to the Pachyonychia congenita community and M.Leonard Acknowledgments and epidermaldifferentiation are necessary to understand PCpathogenesis. patient’sPC keratinocytes. Further investigations clarifyinginterplaythe between autophagy we show Here, the indisturbed that are time turnover first for mitophagy and autolysosomal havefilamentsbeen showntomovealong withtheKIF5B. help of This article is article This keratinocytes and ame . intermediate filament muscle heart in while localization filament Rab7 hasbeen showndirectlytointeract with the 44 p rotected b

45 Rapamycin blocksKrt6a furthermore expression in human liorates PC symptoms. y co py 13,42 ri g ht. Allri ht. Additionally, vimentin as well vimentin Additionally, intermediate as askeratin g hts reserved. hts 46 feedbackmechanism to avoid excessive cell-

15,45 In contrast, rapamycin rapamycin In contrast,


Accepted Article 15 Yu L, McPhee CK, Zheng L, L, Zheng CK, McPhee L, Yu 15 4 BlanksonH, Holen Seglen I, Disruption PO. of the and 14 Cogli L, C,R, Bramato Progida Bucci C. Vimentin phosphorylationassembly and are 13 12 Kuchitsu Y, Kuchitsu Y,N,M. Homma Fukuda Fujita regulated knockoutRab7 unveils 12 YC, WongOptineurin Holzbaur is an ELF. autophagy damagedfor receptor 11 NarendraDP, Kane LA, HauserDN, 10 9 S,Pankiv Clausen Lamark TH, T, 8 Jin SM, Lazarou M, Wang C, 7 Meissner C,H, Lorenz Weihofen A, 6 Ni H-M,Williams JA, Ding Mitochondrial W-X. dynamicsand quality mitochondrial 5 N,of LeubeSchwarzOrganizersSpace:RE. How Filaments as Cellular Intermediate They 4 P,WongPA.6keratin Coulombe (K6) of Loss reveals intermediate function proteins afor 3 Leachman SA, Kaspar RL, Fleckman P, 2 McLean WHI, Rugg LunnyEL, DP, This article is article This 1 Omary MB. ‘IF-pathies’: a broad spectrum of References Inhibition of Hepatocytic Autophagy by Okadaic Acid. regulated by the small GTPase Rab7a. autolysosome maturation induced maturationglutamine by autolysosome starvation. NatlAcadProc Sci US A in parkin-mediated mitochondria mitophagy that is disrupted byALS-linked anmutation. 2007; Facilitate Degradationof UbiquitinatedProtein Aggregates byAutophagy. control. StructureAffect Mitochondrial Function. and wound during filaments repair. Pachyonychia Congenita. pachyonychia congenita. Autophagy induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable both. for proteolyticimport and destabilization byPARL. cleavesPARL human Pink1to Pink1 regulate trafficking. Clin Invest regulated by mTOR. mTOR. by regulated 282 Redox Biol :24131–45. 2010; 2009; p rotected b 6 119 :1090–106. 2015; Nature :1756–62. y co Nat Genet 2014; J Investig Dermatol Symp Proc Symp Dermatol J Investig 4 :6–13. py 2010; et ri J Cell Biol J Cell g 111 ht. Allri ht. et al. 465 Mitochondrial Mitochondrial membrane potential regulates PINK1 et al. et 1995; :E4439–48. et al. et al. :942–6. Biochim Biophys Acta p62/SQSTM1 BindsDirectly to Atg8/LC3 to Autophagy termination and lysosome reformation reformation lysosome and termination Autophagy et al. g The intramembranemitochondrial protease Keratin 16 17 keratinand mutations cause 2003; 9 hts reserved. hts et al. :273–8. Clinical and Pathological Features of of Features Pathological and Clinical intermediate filament-associated diseases. Cells p62/SQSTM1 is required p62/SQSTM1 Parkin- is for J Cell BiolJ Cell 163 2016; :327–37. :327–37. Exp Cell Res Cell Exp 2005; 5 J Neurochem J Cell Sci . doi:10.3390/cells5030030.. 2010; 2013; 10 191 :3–17. 1833 1995; 2018; :933–42. 2011; :1283–93. J Biol Chem J Biol 218 131 117 :522–30. :jcs215442. :856–67.

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Accepted Article 5 Diokmetzidou A,SoumakaE, Kloukina I, 35 A, A,Greffard Hamaï V, Tuloup-Minguez 34 This article is article This 7 Rizzuto R, Pinton P, Carrington W, 37 AxeEL, WalkerSA, ManifavaM, 36 SM, Wojcik Bundman DS, Roop DR.Delayed WoundHealing in Keratin 6a 33 re-epithelialization Onset of after skininjury correlates with areorganizationof 32 Song X, Narzt MS, Nagelreiter IM, 31 4 MonteleonT,Agnihotri CL, Dahal A, 44 9 Gomez-Suaga PaillussonS, Stoica P, R, 39 Cardenas C, FoskettJK. signalsMitochondrial Ca2+ in autophagy. 38 5 ZhangZhou J, Lin W, J, 45 0 Hennings H, MichaelD, Cheng C, 40 43 Robert A, Tian P, RobertA,Tian Adam SA, 43 WangZ, Cui J,Wong WM, 42 Du W, SuChen QP, Y, 41 129 the maintenancemitochondrialhomeostasis of and cardiomyocyte survival. and Knockout Mice. Cell Biol woundin filamentskeratin keratinocytes: edge defining a potentialrole for 16. keratin unified mechanism for intermediate transport. filament 2012; Reticulum as Determinants of Mitochondrial Ca2+ Responses. the endoplasmic reticulum. compartments inphosphatidylinositolenriched 3-phosphate anddynamically to connected vivo. in and vitro DNAincreased damage, senescence and lipidaberrant composition oxidativeafter stress in J Invest Dermatol J Invest Signaling, and Mitochondrial Metabolism Necessary for Human Epidermal Differentiation. reactivation inH2O2-induced autophagy. Complex VAPB-PTPIP51 Regulates VAPB-PTPIP51 Complex Autophagy. 2013; componentstheir for assembly and linkage to the junctions.myotendinous 2016; ofmouse differentiation cellsin epidermal culture. :3705–20. :3705–20. β 1 integrin membrane recycling. 52 37 140 :44–51. :326–36. 1996; :617–26. p rotected b 132 Mol Cell Biol Mol Cell Redox Biol 2018; :381–97. y 138 co J CellBiol py :1945–54. :1945–54. 2016; et al. 2000; ri et al. g ht. Allri ht. et al. Kinesin 1 DrivesTubulation. Autolysosome et al. 11 Autophagic lysosomalreformation dependson mTOR 20 Cell Cycle :219–30. 2008; Kif5bcontrols the localization of myofibril :5248–55. Kinesin-dependenttransportof keratin filaments: a et al. et et al. et et al. et g et al. Int J Biochem Cell Biol hts reserved. hts et al. 182 Autophagosome formation from membrane Calcium regulation of growth Calcium regulation of and et al. Autophagy deficient keratinocytes display Close Contacts with the Endoplasmic 2013; et al. et et al. :685–701. LysosomesDegradation, Supportthe Curr Biol Cell The ER-Mitochondria Tethering Desmin and and Desmin Autophagy cell modulates migration 12 FASEB J 1980; :3317–28. 2017; 19 Science 2018; :fj.201800604R. :245–54. 2016; 27 α B-crystallin interplay in interplay B-crystallin :371–85. 1998; 70 Cell Calcium :76–81. :76–81. Development J Cell Sci J Cell 280 Dev Cell :1763–6. 2016;


Accepted Article This article is article This mitochondria inmitochondria K6aN171K,K6aN171del and cellscontrol (n ER. andmitochondria (a)shows aquantitativeof the histogramanalysis of withER contact microscopyinvestigateElectron to the wasperformed spatial relationship between Figure Mitochondria-ERis reduced inPachyonychia congenita keratinocytes. using one-wayANOVA with Dunnett’s post-test.p<0.05, * *** p<0.001. Comparisonsto24transfection hours afterK6a wt cells wereScale bars10 performed =µm. significantly increased red/green ratio of fluorescence intensity compared to K6a wt cells (d). contrast, Intransfection. afterhours 48K6aN171K and K6aN171del keratinocytesdisplay a coursethe of48hours. No differences inred/greenratios could beobserved24 hours after K6a wtcells 24 after hoursMitoTimer transfection. changes its colour greenfrom toredover independentof experiments 30cells per cell line greenthe andred channel.The histogram shows the ofthree ratiometric quantification and K6aN171del keratinocytes48h after transfection. Eachdepicts micrograph of overlayan representative fluorescence micrographs taken from wild type K6a and mutant K6aN171K Fluorescence imageswere recorded 24and 48hours aftertransfection. (a-c)show shifts whose green fluorescence toredwithfrom time increasing synthesis. after wereCellswith transfected enc MitoTimer accumulateFigure 1. Oldin Pachyonychiamitochondria keratinocytes. congenita Figure legends HickersonRP, Leake D, Pho LN, 46 mutations. Figure 3. Expression levels of early mitophagy markers are not affected by K6a Bonferroni post-test. *** p<0.001. bars = 100nm.Comparisons between groups wereperformedusing one-way ANOVA with arrowheads). Note the significant reduction of co contactsites inwt K6a (b), K6aN171KK6aN171del and(d) (c) keratinocytes(indicated by presentline). selected (b-d) micrographs electron depicting examples ofmitochondria-ER ±SD)themRNA the determining levels PINK1 of mitophagy early markers PINK1- (a), Thehistogram(a-c) depicts the results of qPCR (threeindependent meanexperiments each; congenita patients. keratininduciblean(K6a) in human keratinocytessymptomsimproves and inpachyonychia p rotected b J Dermatol SciJ Dermatol y co py ri g ht. Allri ht. 2009; et al. 56 oding a mitochondria-targeted dsRedderivative oding amitochondria-targeted g hts reserved. hts :82–8. Rapamycin selectively inhibits expression of each (d). Ratios were to normalized control ntact sites in the mutant (b-d).cells Scale ≥ 50 mitochondria 50 percell

Accepted Article This article is article This e) Immunoblot analysescell lysatesof the fromsame lines cell demonstratefurther p62 that levelsmRNA inmutant K6aN171Kand K6aN171del compared to K6a wtkeratinocytes. (d- p62conditions. Neither (a)nor(b) or optineurin analyses of three independent experiments from cells grown at low and high calcium (mean ±qPCR mRNA from levels prepared SD)were ofautophagosomal Histograms (a-c) K6a mutations. Figure 5. Expression oflevels markers forautophagosome arenotaffected formation by ANOVA Bonferroni post-test. with * p<0.05,** p<0.01, ***p<0.001. Comparisons Scaletreatment. = bars 20 µm. cell lines only changes within2 hof CCCP treatment butnot further after18 hCCCP cells, 2 after 2.84% hof CCCP, 0.58% 18after h ofCCCP)area butthatmitochondrial PC in that mitochondrial areasignificantly is reducedin K6a wtcellsCCCP by(4.87% untreatedin histogram depictspercentage± the (mean of SD)the cell covered byNotearea mitochondria. ofK6aN171del(e)K6aN171Kdisplay cells 90.2% dotted still and The (b, Parkin c, e). keratinocytes display a cytoplasmic Parkin distribution again(a, whereas e), 90.67% of dysfunctional mitochondria. After 18hofCCCP treatment, however,onlyK6a of 72.61% wt mitochondrial-targeted Parkin after two hours of CCCP treatment as expected for undercytoplasmicdistribution Parkin shifting control conditions d), (a-c; todotted, i.e. displaying eitherdotted or diffuse All cytoplasmic an display Parkin. cell lines even cells per condition each. (d)shows a histogra representative fluorescence micrographs of thr DMSOas a 18control for h, stained withMi 2 for h withh 18after seedingand or and treated thewith mitophagy(10 inducer CCCP µM) withwere a transfected Keratinocytes fluores Figure 4. Mitophagyis severely keratinocytes.impaired inPachyonychia congenita post-test. Bonferroni with p<0.05. * compared to K6awt. Comparisonsbetween groups wereusing performed one-wayANOVA calcium conditions and in K6aN171del keratinocytesunder high calcium conditions PTENthe antagonist PI3Kis significantlydecreased inunder K6aN171K low cells and high also notsignificantly(d) altered butthatits slightly inductorPTENis decreased (e) and that resultsthe of independentthree experiments; me from same the celllines(top: examples of bottom: histogramsimmunoblot images; depicting alterationssignificant Resultsimmunoblot detectable.analysis (d-f) are ofof lysates prepared K6aN171del keratinocyteswerewhich under low grownhighand calcium No conditions. cleavingprotease PARLandParkin (b) (c) inwt K6a and mutantK6aN171K and Corresponding histograms ofthe protein levels (mean±SD; three independent experiments) lines. cell mutant in the altered not also are levels protein (e) optineurin and (d) p rotected b y co py ri g ht. Allri ht. g hts reserved. hts toTracker and subsequentoTracker and between groups were performed using one-way m of the percentage (mean ± SD) of cells cent Parkin reporter (Parkin-eGFP) one day cent reporter (Parkin-eGFP) Parkin ee independent experiments with at least 48least at independent with experiments ee HDAC6 (c)show significant alterations of an ±SD)an revealPINK1 that protein levels are tly fixed. (a-c) show Accepted Article This article is article This micrographs (three independent experimentsof n autolysosomesshow only red fluorescence.(a-c) The representative fluorescence tandem Autophagosomes fluorophore. are represented byand green red puncta, whereas To monitor autophagosomeand autolysosome formation cells were transfected with aLC3 recycling. and maturation autolysosome lead toimpaired mutations 6. K6a Figure post-test. Bonferroni with shownare below.between Comparisons gr test. *p<0.05, ** p<0.01, ***p<0.001. Comparisons between groups were performed using one-way ANOVA with Bonferroni post- cell lines butnotinwt K6a keratinocytesin (quantificationScale e). = bars 20 µm. is significantly increasedhigh at to comparedcalcium low calciumconditions inboth mutant cells grown athigh(histogram calcium ind). the Furthermore, percentage ofonlypuncta red red puncta (autolysosomes) is significantly increased in PC cell lines compared to K6a wt keratinocytes grown atlowandhigh calcium showconditions thatthe total only number of p rotected b y co py ri g ht. Allri ht. oups were performed using oups wereperformed one-way ANOVA g hts reserved. hts ≥ 16 cells per condition each) taken from

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts

Accepted Article This article is article This p rotected b y co py ri g ht. Allri ht. g hts reserved. hts