Quantitative optical nanoscopy of mitochondrial-derived vesicles in neurons classifies pre-peroxisomal and clearing organelles

Domain: physics
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Healthymitochondriaarecrucialformaintainingneuronalhomeostasis.Theiractivitydependsonadynamiclipidandproteinexchangethroughfusion,fission,andvesiculartrafficking.Studyingvesiclesinneuronsischallengingwithconventionalmicroscopyduetotheirsmallsize,heterogeneity,anddynamics.Weusemulticolourstimulatedemissiondepletionnanoscopytouncovertheultrastructureofmitochondrial-derivedvesicles(MDVs)inliveneurons,biosensorstodefinetheirfunctionalstate,andapulse-chasestrategytoidentifytheirturnoverinsitu.Weidentifiedthreepopulationsofvesicularstructures:onetransportingdegradationproductsoriginatingfromoxidativestress,oneshuttlingcargoandnewlytranslatedproteinsforlocalorganellebiogenesisandoneconsistingofsmall,functionalmitochondria.Furthermore,weprovideevidencesupportingthatdenovoperoxisomesbiogenesisoccursviathefusionofendoplasmicreticulumandMDVsatmitochondrialsites.OurdataprovidemechanisticinsightintoorganellebiogenesisdrivenbysignificantdiversityinMDVmorphology,functionalstate,andmolecularcomposition.

Introduction
Mitochondriaarehighlydynamicstructuresthatcontinuallyaltertheir
morphologythroughfusionanddivisiontoensuremitochondrial
homeostasisandsupportcellularfunctions1–3.Inadditiontofusionand
fission,recentresearchhasrevealedkeymechanismsgoverning
mitochondrialmembraneplasticity.Thesemechanismsinvolveeither
directmembranecontactorvesiculartransporttofacilitatethe
exchangeoflipidsandproteinsbetweenmitochondriaandthesur-
roundingenvironment4–6.Theseprocessesareparticularlysignificant
inneurons,wheretheextendedandpolarizedcellularshapeposes
additionalchallengesincoordinatingthedistributionoforganelles
andnutrientsacrossvariouscompartments7.Amongthedifferent
pathways,mitochondrial-derivedvesicles(MDVs)haveemergedaskey
playersinmitochondrialsignalingwithinthecell,participatingina
diversearrayoffunctions8.MDVsplayapivotalroleinmitochondrial
qualitycontrol(MQC)bytransportingoxidizedcargothatistargeted
fordegradationwithintheendolysosomalsystem9–12. Thisprocess
operatesunderbasalconditionsandincreasesduringoxidativestress
whendamageislocalizedanddoesnotextendtotheentire
organelle13–15. Ultrastructuralanalysisviaelectronmicroscopyhas
revealedthatMDVsarecharacterizedbyeithersingleordouble
membranes16.Fluorescencemicroscopystudieshaveelucidatedtheir
diverseproteincargo,suggestingdifferentfunctionsincludingselec-
tivetransportofmitochondrialcargotoperoxisomes17,18,antigen
presentationontheplasmamembrane19,20, andpackagingof
mitochondrialproteinsinextracellularvesicles21. Despiteextensive
studiesofMDVsinimmortalizedcelllinesandothercelltypes,their
formation,dynamics,andfunctionalrolesinneuronsremainpoorly
understood. Neuronsarehighlypolarizedandcompartmentalized,
withaxonsanddendritesexhibitingdistinctmorphology,protein
composition,andfunctionalrequirements.Notably,inaxons,aspe-
cializedclassofvesiclesremovesthemitochondrion-anchoringpro-
teinSyntaphilin,facilitatingmitochondrialmotility22,highlightingthe
potentialforcompartment-specificregulationofmitochondrial
dynamics. TheseobservationssuggestthepossibilitythatMDVsin
neuronsareheterogeneous,differinginbiogenesis,cargocomposi-
tion,andfunctiondependingontheirsubcellularlocation.Investi-
gatingMDVsinprimaryneuronsisthereforecriticaltounderstanding
howthesevesiclescontributetocompartment-specificMQC,orga-
nelleturnover,andoverallneuronalhomeostasis—mechanistic
insightsthatcannotbefullycapturedinstandardcelllinemodels.
Moreover,whiletraffickingofmitochondrialproteinsandlipidsis
essentialforcellularhomeostasis,theprecisefunction,composition,
andsubcellularlocalizationofMDVsremainlargelyundefined,parti-
cularlyinneurons.Thisambiguitymaystemfromthedenseclustering
ofmitochondriaandMDVswithinconfinedspaceslikeaxonsand
synapses,whichposesachallengeforaccuratevisualizationusing
conventionalfluorescencemicroscopytechniques.Todirectlyobserve
specificproteinsassociatedwithMDVsinliveneuronalcells,we
employedstimulatedemissiondepletion(STED)nanoscopyincon-
junctionwithbrightcell-permeabledyes23 andgeneticallyencoded
self-labelingenzymes(SNAP/Halo)24,25. Thisapproachoffersseveral
advantages:firstly,multicolorSTEDprovidesthespatialresolution
necessarytoobserveandquantifythefinemorphologyofsingleMDVs
withintheintricateneuronalarchitecture,enablingustoresolvethe
budding-outprocessfrommitochondria;andsecondly,byemploying
adiversesetofsensors,wecanassessthefunctionalstateofMDVs
insitu.Astheimagingisperformedinlivingneurons,weadditionally
implementedasequentiallabelingstrategytotrackthelifetimeand
traffickingoftheproteinsintheMDVs.OurfindingsrevealMDVs
exhibitingsphericalandtubularshapesofdifferentsizes,distributed
acrossvariousneuronalcompartments.TheseMDVscontaineither
degradationproductsresultingfromoxidativestressornewlysyn-
thesizedproteinsenrichedinperoxisomalbiogenesismarkers.Our
imagingdatashowthepresenceofendoplasmicreticulum(ER)atMDV
formationsites,alsoforperoxisomal-positiveMDVs,aswellasthe
presenceofDynamin-relatedprotein1(DRP1),supportingthe
hypothesisthatfissionmachineryisrequiredforthebiogenesisofpre-
peroxisomalvesicles.Altogether,weshedlightonultrastructural
featuresofMDVsandelucidatetheirdiverseroleintheclearanceof
mitochondrialdebrisandperoxisomebiogenesiswithinprimary
neurons.

Results
MDSheterogeneityanddistinctturnoverdynamicsinneurons
WeappliedSTEDnanoscopytofixedandliveprimaryrathippocampal
neurons to visualize the morphology of mitochondria in different
cellularcompartmentsduringneuronaldevelopment(DIV5–10)(Fig.1
andSupplementaryFig.1).TheC-terminallocalizationpeptideofthe
mitochondrion´s outer membrane protein 25 (OMM) was fused to
either the self-labeling Halo/SNAP-tags (Halo/SNAP-OMM) or the
rsEGFP2 protein26,27(rsEGFP2-OMM), for live- or fixed- mitochondria
labeling, respectively. With this strategy, all outer mitochondrial
membraneswerelabeledandcompatiblewithSTEDimaging23,28,pre-
servingmitochondrialmorphology29,andreducingoff-targetlabeling
effects30(Fig.1a,bandSupplementaryFig.1a–d).Themitochondrial
morphologywasquantifiedbythreemainparameters:area(A),width
(W),andlength(L)(SupplementaryFig.1a),whichwereextractedby
our custom analysis pipeline “Mitography” (Supplementary Fig. 1i).
Analyzing nearly1400mitochondria in STEDimages,mitochondrial
area, width, and length values were significantlysmaller than those
obtainedbyconfocalimaging(Fig.1ainsetandSupplementaryFig.1e,
f).Thus,STEDbutnotconfocalmicroscopycouldresolveindividual
mitochondria(SupplementaryFig.1c,d)anddefinetheirvesicularand
tubularfinestructures.Mitochondrialwidthvaluesweredistributed
narrowlyaroundamodeof139nm,with75%ofmitochondriahavinga
width of 100–200nm. However, mitochondria with widths below
100nm(3.2%)andabove300nm(5.1%)werealsopresent.Instead,the
widthdistributionobtainedbyconfocalmicroscopydisplayedamode
of 357nm, with 80% of values above 300nm and an overall wider
distribution. Similarly, mitochondrial length values obtained with
STED were shorter compared to confocal analysis, even for length
valueswellabovethediffractionlimit.Infact,whileconfocalimaging
showedelongatedstreakyshapesthatcouldbemisinterpretedasa
single long mitochondrion, STED imaging would resolve individual
mitochondria aligned in series along axons and dendrites (Supple-
mentaryFig.1d).Similartrendswerealsotrueforthemeasuredarea,
whereSTED,butnotconfocal,analysisrevealedasignificantpopula-
tionsmallerthan0.086µm2(A )(Fig.1ainset).Wethereforeestab-
th
lishedanareathresholdatthisvaluetofocusonthesefinestructures,
definedhereasmitochondrial-derivedstructures(MDSs).Atthispoint,
thismayincludeMDVs,fragments,andsmallfunctionalmitochondria.
In the subsequent experiments, we combined well-established MDV
markerswithprecisemeasurementsofsizeandmorphologytochar-
acterizethedifferentsubsetsofMDSspresentinneurons,withspecific
attentiontoMDVs.MDSswereassmallas80nmacross,andtheywere
oftenlocatednearlargermitochondria,makingitdifficulttoidentify
theminconfocalimaging(Fig.1b).Thedistributionoftheareawas
broad, with a mode of0.035µm2, corresponding, for example, to a
length and width of 233nm and 150nm, respectively (Fig. 1c and
SupplementaryFig.1g,h).Bycalculatingtheaspectratio(AR)ofMDSs
astheratiobetweenlengthandwidth,AR=W/L,wecouldinvestigate
theirultrastructureheterogeneityfurther(Fig.1d).CircularMDSswere
defined as having an AR of 0.5–1, while AR<0.5 represents tubular
MDSs,denotedassticks(Fig.1e).Wefurtherinvestigatedthespatial
distribution of vesicles and sticks across the neuronal cell, and we
found that both MDS types were equally distributed in axons and
dendrites(Fig.1f).
Profitingfromthespatio-temporalresolutionofSTEDnanoscopy,
we followed the formation ofMDSs in living neurons over time,con-
firming that these structures indeed originated from mitochondria
(SupplementaryMovies1–3).Wetrackedeventssuchasfissionfollowed
bytheadditionalformationofavesiclewithadiameterofapproximately
90nmatthesiteoffission(SupplementaryFig.1jandSupplementary
Movie 1). Furthermore, we observed MDSs budding out from lateral
protrusionsofthemitochondrialmembranethroughtheformationofa
thin constriction (vesicle neck, 72nm), followed by the excision of a
newly formed vesicle (Supplementary Fig. 1k, l and Supplementary
Movie2).Interestingly,atsitesofvesicleformation,weobservedtheER,
suggesting possible similarities with the mechanism of mitochondrial
fission(SupplementaryFig.1m,nandSupplementaryMovie3)31.
Consideringtheobservedvarietyinsizesandshapesandlitera-
ture data about the involvement of MDVs in mitochondrial protein
traffickingandrecycling,wecheckedwhethermitochondriaandMDSs
were associated with different turnover dynamics. We applied a
sequentiallabelingmethodbasedontheself-labelingSNAP-tag.Inthis
mitochondria experiment, mitochondrial membrane replacement over time and
spacecanbedetectedbyimagingthecolorvariationintheimages
(Fig.2).NeuronsweretransfectedwithaSNAP-tagplasmidfusedtothe
C-terminal localization peptide of OMP25 (SNAP-OMM). After 24h
(t=0),wepulsedthecellsfor1hwiththeredfluorescentligandtet-
ramethylrhodamine (TMR-Star-BG) to label all mitochondrial mem-
branes tagged with SNAP-OMM at time zero (t=0). After multiple
washingsteps,theculturewassentbacktotheincubator.Twenty-four
hourslater(t=24h),wepulsedthecellsoncemorewiththeligand
coupledwiththefar-redsiliconrhodaminedye(SiR-647-BG)(Fig.2a).
By imaging the cells after the second pulse, we could differentiate
between old and newly produced outer mitochondrial membranes,
labeledwithtwospectrallydistinctdyes.Wefoundthatthepopulation
was highly heterogeneous in content with newly formed and old
vesicles containing either one label or the other, but the majority
showed colocalization of the two dyes, highlighting a continuous
membraneturnoveroccurringwithin24h(Fig.2b–e).Toquantifythe
turnoverrate,wemeasuredthefluorescenceintensityratiobetween
thenewlylabeledandthetotalamountofproteins,i.e.,thesumofold
andnew.TakingintoconsiderationthatexogenousSNAP-OMMwasinte-
gratedbothinthesomaandinthedistalfilamentsalreadywithinthe
first4hofproteinexpressionandthatitsdistributionremainedcon-
stantwithin24h(SupplementaryFig.2a–d),wefoundthattheturn-
overdidnothappenatthesamerateineachcompartment;infact,
bothmitochondriaandMDSsrefreshedtheirmembranecontentfas-
ter near the cell soma than in distal filaments, as measured by the
progressive decrease in the intensity ratio between the SiR-647-BG
labelingandthetotallabelingintensityatincreasingdistancesfrom
thecellsoma(Fig.2f).
In conclusion, STED nanoscopy revealed significant diversity in
thesize,shape,andturnoverofMDSs,withfasterproteinturnoverfor
MDSslocatednearthecellsomathanforthoseindistalfilaments.

MDSarecharacterizedbydifferentfunctionalstates
ConsideringthevarietyofshapesandsizesobservedwithSTEDand
thenumerousfunctionstowhichMDVshavebeenassociatedwith,
we wanted to investigate whether sizes might be related to specific
functions. MDVs have been described as a possible mechanism for
MQC12,workingtogetherwithmitophagyandotherpathways32toclear
mitochondria from potentially damaging products in different cell
types.The formation of such vesicles is triggered by mitochondrial
stress, and they are enriched in oxidized proteins16. We used STED
imaging in combination with functional readouts to elucidate the
physiologicalstateofsuper-resolvedMDSsinneuronalcellstoidentify
MDVs involved in MQC. Mitochondria were labeled, as previously
described,tovisualizetheoverallpopulation,withtheadditionofa
spectrally separated signal generated from two markers of mito-
chondrialstate:(i)tetramethylrhodamineethylester(TMRE),tostain
the fully functional and metabolically active mitochondria, or (ii)
MitoSOX, to highlight mitochondria undergoing oxidative stress
(Fig.3).Themajority(73%)ofmitochondriawerelabeledbyTMREand
thesewerelargerascomparedtothosewithveryweaksignal(mean
area: A
TMRE+ =0.423µm2, A TMRE−=0.069µm2) (Fig. 3a, d). Similar
trendswereobservedforMDSs(A<A
th
,N=105):TMRE+werelarger
(meanarea:A
TMRE+ =0.062µm2,A TMRE−=0.040µm2)andlonger(mean
length: L
TMRE+ =0.36µm, L TMRE−=0.28µm) than TMRE− (Fig. 3b, e).
MostMDSswereround-shapedvesicles,ofwhich68%lackedmem-
branepotential.Similarresultswereobtainedforthesticks(71%with
nomembrane potential)(Fig. 3f,g),suggesting the possiblerole of
these membranous vesicles without membrane potential in MQC.
STEDilluminationhadminimalimpactonmitochondrialmembrane
potentialandmorphology,asneuronsshowedsimilarlossofTMRE
signal and mitochondrial stability under both STED and confocal
imaging conditions (Supplementary Fig. 3a–d and Supplementary
Note1).
Next, we investigated the presence of reactive oxygen species
(ROS)insideMDSsusingMitoSOX(Fig.3c).Mostmitochondriawere
labeled by MitoSOX (82%), indicating, as expected, the presence of
ROS. Mitochondria without MitoSOX were on average smaller than
those with (mean area: A MitoSOX−=0.10µm2, A
MitoSOX+ =0.32µm2, 
Fig. 3h). Focusing on MDSs, the majority were MitoSOX+ (69%)
andweresimilarinsize(meanarea:A
mitoSOX+ =0.045µm2,A mitoSOX−=
0.039µm2)(Fig.3i)buthaddifferentshapescomparedtotheMitoSOX-
MDVs. Vesicles were enriched in ROS, while sticks were not (mean
aspectratio:AR
mitoSOX+ =0.70,AR mitoSOX−=0.63)(Fig.3j,k).MitoSOX
alonedidnotaffectmitochondrialROSproduction,asitsfluorescence
remainedstableunlesscombinedwithphototoxicMitotrackerGreen,
which increased ROS through photo-bleaching (Supplementary
Fig.3h,iandSupplementaryNote1).
Together, these experiments showed that neuronal MDSs are
highly heterogeneous in size and function, suggesting a possible
relation between MDSs and MDVs. We observed small MDSs
(A=0.04μm2; median length and width: L=250nm, W=160nm)
lacking membrane potential but showing ROS accumulation. These
vesicles have characteristics similar to previously reported small
mitochondria and MDVs thataccumulate oxidized proteins and are
subject to autophagosome engulfment3,10,16, pointing to a potential
roleinMQC.Instead,largerMDSs(A=0.06μm2)havethesamefea-
turesasmitochondria,havingbothmembranepotentialandROS.
Tobetterunderstandthenatureofthesevesiclesandtodiffer-
entiate them from small functional mitochondria, we labeled the
mitochondrialtranscriptionfactorA(TFAM)(SupplementaryFig.3i),
the most abundant protein inside mitochondrial nucleoids, where
mitochondrialDNA(mtDNA)islocated33,34.UsingSTEDimaging,we
identifiedsinglenucleoidsofaround100nminsizeinsidemitochon-
dria(SupplementaryFig.3j).Wealsospottedsimilarstructuresinside
MDSsthatweresimilarinsizetothosewehadidentifiedwithmem-
branepotentialandROS(SupplementaryFig.3k).Wethereforecon-
cluded that nucleoids, together with mitochondria functional state,
size, and morphology, are pivotal for differentiating clearing MDSs
fromsmallmitochondria.
Altogether,weobservedsmallvesicularandtubularMDSsenri-
chedinROSandwithoutmembranepotential,suggestingtheirroleas
MDVsinnvolvedinMQC.Asecond,largerpopulation,withalargerarea,
stillbelowtheresolutionofaconfocalmicroscope,hasamembrane
potential and can also contain mtDNA, thus resembling small
mitochondria.

MDShavedifferentsizesandproteincompositions
Oneofthemajorfunctionsofmitochondriaistoprovideenergyinthe
formofATP,whichisproducedduringoxidativephosphorylation.To
further investigate MDSs composition and gain insight into their
function,weexaminedthenanoscaledistributionandabundanceof
oxidativephosphorylationcomplexes(OXPHOS)(Fig.4).Tovisualize
andquantifyOXPHOSweusedtwodifferentlabelingstrategies:for
live-cellimagingweexpressedfull-lengthcytochromeCoxidasesub-
unit8A(COX8A)fusedtoaSNAP-tag(SupplementaryFig.4a,b),while
forfixedcellsweusedanantibodycocktailtargetingmultipleOXPHOS
subunits(subunitsCI–V)(Fig.4a).WithSTED,weidentifiedtheinner
membrane ultrastructure, which was not distinguishable from the
outermitochondrialmembranebyconventionalmicroscopy(Fig.4b).
LiveneuronalimagingofCOX8Aenabledustovisualizemitochondrial
cristae as inner membrane invaginations of about 50nm wide and
100nm apart from each other (Supplementary Fig. 4a-c). Similar
valueswerereportedpreviouslyforHeLacells35.Apartfromlabeling
elongatedtubularmitochondria,COX8Arevealedafewpunctanear
mitochondrial tubules (Supplementary Fig. 4b inset), which could
indicatedoublemembraneMDSs.Time-lapseimagingofCOX8Aand
OMMshowedthedifferentdynamicsofinnerandoutermitochondrial
membranes, which are often asynchronously arranged: during the
formationofadoublemembranemitochondrialprotrusion,theinner
membranedetachedfirst,whiletheoutermembraneremainedcon-
nected to the mitochondrion (Supplementary Movie 4). OXPHOS-
cocktailstainedmitochondriaassmallclustersof60–100nminsize,
next to the OMM (Fig. 4b).The OXPHOS composition ofthe MDSs
was highly variable between cells (Supplementary Fig. 4d): only
some MDSs contained respiratory complexes, while most small
vesiclesandstickslackedOXPHOS(Fig.4c,d).Weadditionallyfound
thatareasofOXPHOS+MDSsweresignificantlylargerthanthoseof
OXPHOS−MDSs(meanarea:A
OX+ =0.048µm2,A OX−=0.027µm2;mode
area: A
OX+ =0.028 µm2, A OX−=0.021µm2) (Fig. 4d). The same trend
wastrueforlengthandwidth,whiletheirARswerenotsignificantly
different(SupplementaryFig.4e–g).Ourresultssuggestthatasmall
group of larger MDSs likelyrepresent functionallyactive mitochon-
dria, with membrane potential, mtDNA, and OXPHOS. However,
smaller MDSs (width <150nm) usually lack membrane potential,
accumulateROS,andmayormaynotcontainOXPHOS.Wetherefore
addedanadditionalstainingfortheoutermitochondrialmembrane,
theimportproteinTOM20,whichwasalsoshowntolocalizeexclu-
sivelyinclearingvesiclesdirectedtolysosomes11.Byimmunolabelling
TOM20,werevealedmitochondria,asexpected,butalsoMDSs,which
wereOXPHOS+.Moreover,weidentifiedasmallpopulationofTOM20+
and OXPHOS− (3%), likely representing MDVs involved in the MQC
directedtolysosomes(Fig.4e–gandSupplementaryFig.4h).These
vesicleswereonaveragesmallerinsizethantheothers(Supplemen-
taryFig.4i).Wefoundthat24%ofMDSswereOXPHOS+butTOM20−.
Wehypothesizedthatthispopulationcouldbeinvolvedinadifferent,
yetunknownfunction,sincetheycontainOXPHOScomplexes,butdo
nottargetlysosomes.Insupportofthis,Hazanetal.recentlyshowed
that isolated mitochondria from Saccharomyces cerevisiae release
MDVscarryingafunctionalATPsynthasecomplexandproposedthat
these vesicles fuse with naïve mitochondria and regenerate ATP-
deficientmitochondria36.
Overall,wefoundthatdoublemembraneMDSswithOXPHOSare
typicallylargerthanthosewithoutandcanbeinvolvedinorganelle-to-
organellecommunication.Withinthispopulation,asubsetcontaining
TOM20issignificantlylargerandlikelyrepresentssmall,intactmito-
chondria. Vesicles containing TOM20, but not OXPHOS, are the
smallestinsizeandpotentiallyrepresentMDVsinvolvedinMQC.

PerturbationofmitochondriarespirationaffectsMDS
WecharacterizedthesizeandmorphologyofMDSs,andfurtherana-
lyzedtheirfunctionality,togetherwithathoroughanalysisofOXPHOS
andmtDNAcontenttoidentifyMDVswithintheMDSpopulation.To
gain insights into their regulation, we perturbed mitochondria
respiration by treating neurons with antimycin A (AA), a drug that
inhibitsmitochondrialelectrontransportandleadstoROSproduction
(Fig.5andSupplementaryFig.5).Firstly,neuronsweretreatedwith
lowdosesofAA(5nM)foraprolongedtime(6h)tomimicthemild
andreversiblemitochondrialstresscharacteristicsoftheinitialstages
ofsomeneurodegenerativediseases22.Werefertothistreatmentas
mildstress.Afterapplyingmildstress,weobservedmitochondriawith
STED, counted them, and analyzed their morphologies (Fig. 5a, b).
With Mitography, we observed reduced area and length values in
treated cells compared to controls. The AR, however, showed
increased values (Supplementary Fig. 5a–h). These morphological
changeswereevenmorepronouncedwhenweinducedastrongstress
(40µMAA,1h)(SupplementaryFig.5i–l).Thestrongstress,however,
likelyinducedmitophagy,sinceweobservedmitochondriafragmen-
tation, and the AR shifted towards higher values (Supplementary
Fig.5m).Mildstress,incontrast,didnotaltermitochondrialnumberor
area,butinducedasignificantincreaseinMDS,furtherhighlighting
theexistenceofMDVsinvolvedinstress-inducedMQC(Fig.5c–eand
SupplementaryFig.5n–p).Wefurtherinvestigatedthecompositionof
theseMDSsandobservedthatAAtreatmentreducedthenumberof
thoseenrichedinOXPHOS(Fig.5f).Thisresultisinlinewithourpre-
vious observation that OXPHOS is excluded from smaller vesicles,
whichinsteadshowedROSaccumulationandlackedTMREstaining.
Altogether, these results agree with the proposed role of MDVs in
MQC.Tofurtherconfirmthesefindings,weperformedthemildAA
treatmentandexaminedinteractionswithlysosomes,thewastedis-
posalsystemwithinthecell(Fig.5g–j).Welabeledthemviaexogenous
taggingofthelysosomal-associatedmembraneprotein1(Lamp1)with
the Red Fluorescent Protein (RFP), followed by immunostaining
againstthefluorescenttagandquantifyingthedegreeofinteraction
with MDSs in controls and treated samples (5nM AA, 6h). Upon
mitochondrialstress,thenumberofMDSsinteractingwithlysosomes
increasedsignificantly(Fig.5g).Moreover,STEDmicroscopyallowed
ustoobservetheengulfmentofmitochondrialprotrusionsandMDSs
bylysosomalstructures(Fig.5i,j).
Overall,theseresultssuggestthatneuronsarelikelytoadaptto
metabolic stress by releasing MDSs in the form of MDVs targeting
lysosomes,tomitigatedamagedmitochondrialcomponentsandpre-
ventmitophagy.

DiverseMDSsconnectwithperoxisomesinneurons
WesawthattheMDSpopulationishighlyheterogeneousandclearing
MDVstargetlysosomestosupportmitochondriaduringstresscondi-
tions. Another group of MDVs was previously reported to shuttle
cargoes to a subpopulation of peroxisomes18 and to be involved in
their biogenesis37,38. Peroxisomes are membrane-bound oxidative
organellesthatshareimportantcellularfunctionswithmitochondria,
such as fatty acid oxidation and peroxide reduction39,40. However,
interactionsbetweenmitochondriaandperoxisomesinneuronshave
not been studied in detail. To investigate the relationship between
MDSs and peroxisomes, we labeled the peroxisome biogenesis
protein PEX14 together with the OMM (Fig. 6). We identified many
PEX14 puncta located close to mitochondria (Fig. 6a). STED micro-
scopy showed the distribution of PEX14 and its organization with
respecttothemitochondrialoutermembrane(Fig.6b).PEX14wasalso
found in mitochondrial protrusions, vesicles, and sticks (Fig. 6c).
Unexpectedly, most (86%) of the elongated sticks colocalized with
PEX14.Overall,76%ofMDSs,includingvesiclesandsticks,colocalized
with PEX14. PEX14+ MDSs were on average significantly larger than
PEX14−(meanarea:A
PEX+ =0.035µm2,A PEX–=0.028µm2)(Fig.6dand
SupplementaryFig.6a–c).MostoftheMDSscolocalizingwithPEX14
alsocontainedOXPHOS(Fig.6e–gandSupplementaryFig.6d),
which might represent double-membranevesiclestargetedtoperoxisomes.
However,therewasalsoafractionofMDSs(20%ofthepopulation)
thatcolocalizedwithPEX14,butnotwithOXPHOS.Uponlowdosesof
AA,theratioofPEX+MDSsremainedconstant,suggestingthatthese
vesiclesarenotaffectedbymitochondrialstressandthereforearenot
involvedinMQC(SupplementaryFig.6e–g).
Because of the shared functions in lipid metabolism and the
involvementofMDVsinperoxisomebiogenesis38,weinvestigatedthe
lipidcompositionofMDSs(SupplementaryFig.7).Bystainingboththe
OMMandlipids(NileRed),wedemonstratedthecoexistenceofpep-
tidesandlipidsinMDSs(SupplementaryFig.7a,b).Wefurtherused10-
N-nonyl acridine orange (NAO) to stain cardiolipin, which isalmost
exclusively found in the mitochondrial inner membrane41 (Supple-
mentaryFig.7c).Asexpected,NAOaccumulatedinsidemitochondria
but was not found in small vesicles and sticks (Supplementary
Fig.7d,e).Theabsenceofcardiolipinsupportsourobservervationthat
smallervesiclesandstickslackinnermitochondrialmembranecom-
ponentssuchasOXPHOS.
Altogether, these results indicate the existence of at least two
MDStypesinteractingwithperoxisomes,differingincompositionand
possiblyfunction.

MDSsparticipateinformingpre-peroxisomesatmitochondria
Totestwhetherthispopulationofperoxisome-connectedMDSscould
beinvolvedinperoxisomedenovobiogenesis,wefirstperformeda
pulse-chaseexperiment,basedonenzymaticlabeling,andcharacter-
ized the nature and lifetime of this population of small vesicles
showing both mitochondrial and peroxisomal content. Neurons
transfected with OMP25-SNAP were pulsed for 1h with a non-
fluorescent ligand to cover all available SNAP-tag expressed at this
timepoint(t=0)andchasedfor24h.After24h(t=24h),cellswere
pulsed with the SNAP ligand SiR-647-BG to label newly synthesized
membranes.Afterfixation,neuronswerestainedforPEX14(Fig.7a).
An abundant population of labeled PEX14 puncta (38%) colocalized
with newly produced MDSs (Fig. 7b). In these newly formed small
vesicles,noTMREsignalwasdetectable,suggestingalteredmembrane
potential(Fig.7c–e).Overall,ourresultsdemonstratethepresenceofa
distinctand prominent population of MDSs (70–150nm), likely not
involvedinMQC,butinsteadbeingMDVscontributingtoperoxisome
biogenesis,assupportedbythediscoveryofnewlytranslatedprotein
contentinsidethesevesicles.
Another key protein involved in the de novo peroxisome
biogenesis is the peroxisomal biogenesis factor 3 (PEX3), a core
component of the machinery required for the assembly of peroxi-
somemembranevesiclespriortotheimportofmatrixproteins.PEX3
is alsoknown to be involved in the process of pre-peroxisome for-
mationviaMDVs38.TofurthertesttheMDS’sconnectionwithdenovo
biogenesis, we silenced PEX3 expression and examined the con-
sequencesinneuronalcells(SupplementaryFig.8andSupplementary
Note 2). Three commercially available siRNAs targeting distinct
regions of the PEX3 transcript were used. PEX3 protein levels were
assessed24and48hpost-transfectionviaimmunostaining.Quantifi-
cationrevealedasignificantreductioninthefluorescenceintensityof
PEX3punctae(p=7.68462×10−4)andadecreaseintheirlinearnum-
ber density (p=0.01075) relative to non-transfected controls (Sup-
plementary Fig. 8a–d). Similarly, mature peroxisomes labeled with
PMP70showedareduceddensity(p=0.00903),demonstratingthat
transient PEX3 knockdown effectively impairs de novo peroxisome
formation(SupplementaryFig.8e–g).
WethenemployedSTEDtoinvestigatedenovoperoxisomebio-
genesisandexaminedthedistributionofvesicularprotrusionswith
respecttothemitochondrialmembranetodeterminewhetherthere
wasapreferentialsiteofvesicleformation(Fig.8).Onlythoseattached
tomitochondriaandlabeledwithOMMwereconsidered.Mostofthe
identifiedprotrusionswerefoundatthetipoftubularmitochondria
(Fig. 8a, c), in accordance with the recently proposed model for
TOM20+ vesicle formation42. However, we also saw membrane pro-
trusionsbuddingoutfromthelateralsideofthemitochondrialtubule
(Fig.8b,c).WethenusedmulticolorSTEDtolocalizemarkersforpre-
andmatureperoxisomesandexaminedtheirdistributionwithinthe
protrusions.Denovoperoxisomebiogenesishasrecentlybeenshown
torequirebothER-andmitochondria-targetedproteins.Infact,inte-
gralperoxisomalmembraneproteinssuchasPEX3andPEX14localize
tomitochondriaincellslackingperoxisomesandarereleasedinthe
formofvesicles,which,uponfusionwithPEX16+ER-derivedvesicles,
lead to the generation of pre-peroxisomes38. However, the precise
locationofvesiclefusionandthemechanismofperoxisomebiogen-
esisarestillunknown.Here,pre-peroxisomeswereidentifiedbythe
double labeling of PEX14 and PEX16 (Fig. 8d), while mature peroxi-
someswerelabeledbythemembraneproteinPMP70(Fig.8e).Mostof
the vesicular protrusions were stained by two markers, either
PEX14+PEX16+ or PEX16+PMP70+, whereas single marker structures
were less common (Fig. 8f, g). When we measured the sizes of all
protrusions,thoselabeledbyPEX14+PEX16+wereclearlydistinctfrom
thoselabeledbyPEX16+PMP70+:PEX14+16+vesiclesweresignificantly
smaller(meansize:140nm)thanPEX16+PMP70+vesicles(meansize:
177nm)(Fig. 8h).The size differencelikelyreflects the “age” ofthe
peroxisomal structures identified here: immature pre-peroxisomes at
mitochondria (PEX14+16+)aresmallerthanthematureonesPEX16+PMP70+).Tofur-
ther confirm this observation, we measured the sizes of the overall
peroxisomepopulation,identifiedinthiscasebythedoublestaining
ofPEX16+PMP70+(Fig.8i).Peroxisomesshowedawiderangeofsizes
andshapes,withPMP70+structuresbeingbiggerthanPMP70−,likely
toindicatethepresenceofmatureperoxisomes(PEX16+PMP70+)and
smallvesicles,orpossibleperoxisomeprecursors,shuttlingbetween
mitochondriaandperoxisomes(PEX16+PMP70−)(Fig.8j).Theseresults
pointtothehypothesisthatpre-peroxisomevesiclegenerationhap-
pens at mitochondria sites, via the fusion of PEX14+ and PEX16+
vesicles.
PEX14+16+vesicles,stillattachedtoorincontactwiththemito-
chondrial membrane, may represent the sites where mitochondrial
andERproteinsfusetogethertogiverisetonewperoxisomes.How
doesthisprocesshappen?Whicharetheplayersinvolved?Toanswer
thesequestions,wefirstperformed two-color time-lapseSTED ima-
gingandmonitoredmitochondrialvesicleformationinthepresence
ofeitherERorperoxisomalmarkers(Fig.9).Weobservedmitochon-
drialvesiclesreleasedfromboththelateralsideandthetipofmito-
chondrialtubulesandinbothcasesERtubulesarepresentatthesites
ofvesiclerelease(Fig.9a,bandSupplementaryMovies5and6).These
time-lapses resemble the process happening during mitochondrial
fission,whereERtubuleswraparoundthesiteofconstrictiontofavor
fission31, suggesting a similar involvement of ER in MDS formation.
Moreover, the proximity of ER structures at sites of mitochondrial
protrusioncouldfacilitatetheencounterbetweenmitochondriaand
ER-derivedvesicles,aconditionrequiredforthedenovoperoxisome
biogenesis process. To specifically identify pre-peroxisomal protru-
sion,wethereforelabeledeitherPEX16(Fig.9c)orPEX14(Fig.9d)and
confirmed the presence of the ER, marking the site between the
elongatedtubuleandtheprotrusionconstriction.Theseresultsoverall
showedthatmostoftheanalyzedmitochondrialprotrusionsthatare
colocalizingwitheitherPEX16orPEX14alsohaveanERsignalnearby
(Fig. 9e, f). Time-lapse confocal and STED imaging revealed mito-
chondrialprotrusionscolocalizingwithPEX14-GFP.Theseprotrusions
detachedfrommitochondrialtubulestoformMDSsassociatedwith
peroxisomalmarkers(SupplementaryFig.9a,b).Anotherfundamental
playerinmitochondrialfissionisthedynamin-relatedprotein1(DRP1).
Therefore,welookedatthedistributionofDRP1proteininrelationto
mitochondrialprotrusions.WefoundDRP1atmostoftheidentified
protrusions,colocalizingwithERorPEX14(Fig.9g,h).Next,toperturb
DRP1 function, we expressed DRP1K38A mutant (Supplementary
Note2),whichharborsadominant-negativemutationintheguanosine
triphosphate (GTP)-binding pocket, thereby inhibiting GTP binding
andhydrolysis43.Previousstudieshaveshownthatthismutantexhibits
reduced efficiency in mediating mitochondrial constriction and
division44 Consistent with these reports, our results show that
DRP1K38Aoverexpressioninducestheformationofelongatedmito-
chondrial tubules and promotes an overall elongation of the mito-
chondrial network relative to cells expressing DRP1 wild type
(SupplementaryFig.9c–e).Analysisofthespatialrelationshipbetween
mitochondriaandpre-peroxisomalmarkers,suchasPEX3andPEX14,
revealed a significant reduction in the number of pre-peroxisomal
punctae contacting mitochondria in neuronal cells expressing
DRP1K38A compared to control cells (Supplementary Fig. 9f–h), as
measured by the linear number density of contact sites per mito-
chondrial length. In contrast, no differences were observed in the
contactsitesbetweenmitochondriaandmatureperoxisomeslabeled
withPMP70(SupplementaryFig.9i).Collectively,thesefindingsindi-
cate that disruption of DRP1 function compromises mitochondrial
dynamicsandselectivelyimpairstheassociationbetweenmitochon-
dria and pre-peroxisomal structures, without affecting mature
peroxisome interactions. They further suggest a potential role for
DRP1intheregulationofdenovopre-peroxisomebiogenesismedi-
atedbyMDSs.Altogether,theseresultsshowthatERandDRP1mark
sitesofmitochondrialvesicleprotrusions,whichmaydetachfromthe
mitochondrialtubuletoformpre-peroxisomes(Fig.9i).
In conclusion, the findings from both pulse-chase experiments
and STED imaging provide compelling evidence for a distinct and
significant population of small MDSs contributing to peroxisome
biogenesis,sheddinglightonthemechanismofdenovoperoxisome
biogenesis,andemphasizingtheroleofthemitochondria-ERcon-
nectionandfissionmachineryintheprocessofvesiclefusionandpre-
peroxisomeformation.

Methods
Primaryhippocampalneuroncultures
All experiments were performed in accordance with animal welfare
guidelinessetforthbyKarolinskaInstitutetandwereapprovedunder
theEthicalPermitnr2645-2021bytheSwedishBoardofAgriculture
(Jordbruksverket).Ratswerehousedwithfoodandwateravailablead
libitumina12-hlight/darkenvironment.Primaryhippocampalcultures
were prepared from embryonic day 18 (E18) Sprague Dawley rat
embryos. Embryos were collected randomly from the pregnant rats
without prior determination of sex. The pregnant mothers were
sacrificed with CO inhalation and aorta cut; brains were extracted
2
fromtheembryos.Hippocampiweredissectedandmechanicallydis-
sociatedinMinimumEssentialMedium(MEM)(ThermoFisherScien-
tific,21090022).40×103cellsperwellwereseededin12-wellplateson
a poly-D-ornithine (Sigma-Aldrich, P8638) coated #1.5 18mm glass
coverslips(Marienfeld,0117580)andletthemattachinMEMwith10%
Horse Serum (Thermo Fisher Scientific, 26050088), 2 mM L-Glut
(Thermo Fisher Scientific, 25030-024) and 1mM Sodium pyruvate
(Thermo Fisher Scientific, 11360-070), at 37°C at an approximate
humidityof95–98%with5%CO .After3hthemediawaschangedto
2
Neurobasal Medium (Thermo Fisher Scientific, 21103-049) supple-
mentedwith2%B-27(ThermoFisherScientific,17504-044),2mMl-
Glutamine,and1%Penicillin-Streptomycin(Sigma-Aldrich,P4333).The
cultureswerekeptat37°Catanapproximatehumidityof95–98%with
5%CO forupto24days.Themediumwaschangedtwiceperweek.
2
TheexperimentswereperformedonculturesfromDIV5uptoDIV10.

Neuronaltransfectionandlive-cellstaining
NeuronsweretransfectedbetweenDIV5andDIV10usingLipofecta-
mine2000TransfectionReagent(ThermoFisherScientific,11668019),
accordingtotheinstructionsofthemanufacturer.
Plasmidsused:OMP25-rsEGFP227;Halo-OMP2528;SNAP-Sec-61β28;
SNAP-OMP2523;PEX16-rsEGFP250;mCherry-DRP131wasagiftfromGia
Voeltz(Addgeneplasmid#49152;http://n2t.net/addgene:49152;RRI-
D:Addgene_49152); mCherry-DRP1K38A44 was a gift from Samantha
Lewis (Addgene plasmid # 232350; http://n2t.net/addgene:232350;
RRID:Addgene_232350);Lamp1-RFP51wasagiftfromWaltherMothes
(Addgene plasmid # 1817; http://n2t.net/addgene:1817; RRI-
D:Addgene_1817);PEX14-GFPwasakindgiftfromtheEggelingLab.To
generate SNAP-tagged TFAM and COX8A, the plasmid pAAV-EF1a-
mCherry-IRES-WGA-Cre52,agiftfromKarlDeisseroth(Addgeneplas-
mid#55632;http://n2t.net/addgene:55632;RRID:Addgene_55632),was
digestedwithBamHIandEcoRI.SNAPtagwasinsertedasaBamHI-
EcoRIfragment,anditwasamplifiedwiththefollowingoligos:
BamHI-SNAP-FatgactggatccGACAAAGACTGCGAAATGAAGCG
EcoRI-SNAP-RagtcatgaattcTTAACCCAGCCCAGGCTTGCC
TheobtainedpAAV_002-EF1a-SNAPplasmidwasfurtherdigested
withKpnIandBamHI.TheCDSsequencesofTFAMandCOX8Afrom
rat(NM_031326;NM134345,respectively)wereinsertedasKpnI-BamHI
fragments.TFAMwasamplifiedwiththefollowingoligos:
KpnI- atgactggtaccggattggccaccATGGCGCTGTTCCGGGGAAT
TFAM-F
BamHI- agtcatggatccgctaccgctgccATTCTCAGAGATGTCTCCCGG
TFAM-R
COX8Awasamplifiedwiththefollowingoligos:
KpnI-COX8A-F
atgactggtaccggattggccaccATGTCTTCCCTGACGCCGC
BamHI-COX8A-R
agtcatggatccgctaccgctgccCTCCCGCTTCTTGTAGGTTTC
For labelingorganelles fused withHalo-Tag andSNAP-Tag pro-
teins,24haftertransfection,neuronswerewashedinartificialcere-
brospinalfluid(ACSF)andlabeledwith200nMoftheSNAPorHalo
substrates (New England BioLabs, SNAP-Cell 647-SiR, 590CP-Halo-
ligand)for1hat37°C.Then,theneuronswerewashedthreetimeswith
ACSFfor5mineachtime.ThecultureswerewashedagaininACSFand
then either processed for immunostaining or imaged in the same
bufferatroomtemperature(RT).Forlabelingtheaxoninitialsegment
(AIS), neuronal cultures were incubated with anti-pan-neurofascin
primary antibody (UC Davis/NIH NeuroMab Facility clone A12/18,
1:100),for5minatRT,subsequentlywashedthreetimeswithACSF
buffer,thenincubatedwithanti-mouse-Alexa488secondaryantibody
(Thermo Fisher Scientific, A-11001) for 5min at 37°C and finally
washedthreetimeswithACSFbuffer.
For labeling mitochondria membrane potential, Tetra-
methylrhodamine Ethyl Ester Perchlorate (TMRE) (Thermo Fisher
Scientific,T669)wasusedaloneorincombinationwithMitoTracker™
Green FM (Thermo Fisher Scientific, M7514). The cells were loaded
withTMREatafinalconcentrationof100nM,incubatedat37°Cfor
1h,andthenimagedinACSFmedia.WhenMitoTracker™GreenFM
wasrequired,cellswereloadedwith20nMofthedyefor15minat
37°C,shortlybeforeimaging.
To label mitochondria ROS, the fluorescent indicator MitoSOX
(Thermo Fisher Scientific, M36008) was added to the neurons at a
concentrationof200nMandleftfor20mininincubation.Thecells
werewashedtwotimeswithACSFandthenimaged.Tostainallcellular
membranes,neuronswerefirstwashedinACSFandthenimagedinthe
samebuffersupplementedwith300nMofthelipophilicdyeNileRed
(dissolved in MeOH) (Sigma-Aldrich, 72485). For cardiolipin-specific
staining,10-N-nonylacridineorange(NAO)(ThermoFisherScientific,
A1372)wasaddedtothecellat10nMconcentrationfor30minat37°C.

siRNA
Three siRNAs (s222855; s222856; s222857) were purchased from
Thermo Fisher Scientific and used to transfect neuronal cells using
Lipofectamine™RNAiMAXTransfectionReagent(ThermoFisherSci-
entific, 13778100), following the manufacturer’s instructions. The
downregulationofPEX3proteinwasmonitoredat24and48hafter
transfectionviaimmunostainingwithaspecificPEX3antibody
(HPA042830,AtlasAntibodies).

Immunofluorescencestaining
NeuronswerewashedinACSFandthenfixedinpre-warmed4%par-
aformaldehyde(PFA)inphosphate-bufferedsaline(PBS)(pH7.4)atRT
for 15min and permeabilized for 5min in 0.1% Triton-X-100 buffer,
followedbyablockingstepwith5%bovineserumalbumin(A8022,
Sigma-Aldrich)inPBS,for30minatRT.Incubationwithprimaryand
secondary antibodies was performed in PBS solution for 1h at RT.
Samples were mounted in custom-made Mowiol mounting media,
supplementedwithDABCO(ThomasScientific,C966M75).
Antibodiesandnanobodiesarelistedandusedasfollows:anti-
PEX14 (Biosite, 10594-1-AP-20, 1:200); Total OXPHOS Rodent WB
AntibodyCocktail(Abcam,ab110413,1:200);anti-TOM20(SantaCruz
Biotechnology, sc-11415, 1:50 dilution); anti-MAP2 (Abcam, ab5392,
1:2000); anti-TFAM (Abcam, ab13160, 1:200); FluoTag®-X4 anti-GFP
(NanoTag, N0304-Ab580 or N0304-Ab635P-L); anti-PMP70 (Abcam,
ab85550, 1:200); anti-mCherry (Abcam, ab167453, 1:200); anti-Drp1
(Cell Signaling Tech, D6C7 #8570); anti-PEX3 (Atlas Antibodies,
HPA042830);anti-rabbitAlexa594(ThermoFisherScientific,A-11037,
1:200 dilution); anti-mouse Alexa594 (Thermo Fisher Scientific, A-
21203,1:200dilution);anti-rabbitSTARRED(Abberior,2-0012-011-9,
1:200dilution);anti-mouseSTARRED(Abberior,2-0002-011-2,1:200
dilution); anti-mouse AlexaFluor488 (Thermo Fisher Scientific, A-
11001,1:200dilution),anti-ChickenAlexaFluor488(Abcam,ab150173,
1:200dilution).

Drugsandmetabolictreatments
To inhibit mitochondria respiration and specifically inhibit mito-
chondrial electron transport chain complex III and favors ROS pro-
duction,antimycinA(Sigma-Aldrich,A8674)wasused,followingtwo
different protocols: to induce a strong mitochondria stress (strong
stress),thedrugwasaddedtothecellsfor1hat40μMor50nMfinal
concentration;toinduceamilderbutprolongedstress(mildstress),
neurons werecultured for 6h inthe presenceof 5nM of the same
drug.Aftertreatments,neuronswerewashedinACSFandplacedin
freshmediaat37°Cfor1htoequilibrate,beforetheimaging.
TodeterminethecolocalizationbetweenMDSsandLysosomes,in
both control and treated samples, an automated decision was made
throughaMATLABscriptusingthebinarymitochondriaandlysosome
images. The script measures the average Lysosome signal in each
detectedmitochondrioninthebinaryimage.Itthenfindsthethreshold
Lysosome value by plotting the histogram of the average Lysosome
signalforallthemitochondriainanimage,andfittingthehistogram
countswithanexponentialdecay(background)+Gaussian(truesignal).
Thelocalminimumbetweenthetwopeaksoftheextractedfunction,
i.e.,theinterceptionofthetwocurves,istakenasthethresholdLyso-
some value to which the individual mitochondria average Lysosome
signal is compared. The threshold decision is done on an image-to-
image basis, as different images have different signal intensities dueto
varyingimageacquisitionparametersandlabelingefficiency.

MDStraffickingandturnover
MDStraffickingandturnovermeasurementswereperformedwitha
SNAP tagpulse-chase approach. In detail: neurons were transfected
withaSNAP-tagplasmid;forthecontrolexperiments,0,4,13,16,18,
20and24haftertransfection(T=0)cellswereloadedwithSNAP-Cell®
TMR-Starligand(NEB,S9105S)for1hat3μM;subsequentlywashed3
timesinACSF;letequilibrateinfreshmediafor1h;fixedin4%PFAfor
20minandimmunostainedforMap2,usedhereasaneuronalmarker.
Fortheturnoverexperiment,24haftertransfection(T=0),cellswere
loaded with SNAP-Cell®TMR-Star ligand for 1h at3 μM to label all
proteinsthatwereproduceduntilthattime;subsequentlywashed3
timesinACSF;letequilibrateinfreshmediafor1h,andthenletthemin
theincubatorinfreshmediafor24h(T=1).Afterthis,cellswereloa-
ded witha second dye,SNAP-Cell® 647-SiR(NEB,S9102S) for 1h at
200nM,followingthesamewashingprocedureasbefore.Thecells
were thenfixedin 4%PFAfor20min andimagedin aconfocaltile
recordingmodality.Thisway,bothold(SNAP-Cell®TMR-Star)andnew
(SNAP-Cell®647-SiR)OMP25wereidentified.
To image the new mitochondrial membrane and measure the
mitochondriamembranepotentialatthesametime,asimilarapproach
was used,butinstead ofSNAP-Cell® TMR-Star,SNAP cell-block (NEB,
S9106S)wasaddedtocellsfor1hat10μMconcentration,toblockall
the SNAP-tag proteins expressed at T=0. 24h later (T=1) cells were
incubatedwith200nMSNAP-Cell®647-SiRandTMRE(ThermoFisher
Scientific, T669), at 100nM concentration. After 1h, the cells were
washed, as above, and directly imaged in ACSF. This way, the newly
producedproteinswerefirstlocalizedbySiR-647fluorescence,andthe
membranepotential wasfurtherinvestigatedbyrecordingtheTMRE
signal.Fortheexperimentsonmembranerefreshmentinconnectionto
peroxisomes,thesameapproachwasused,andafterfixation,thePEX14
antibodywasfurtherusedtodetectperoxisomesandinvestigatethe
presence of new OMP25 membranes at peroxisome sites, possibly
indicatingperoxisomedenovobiogenesis.

STEDimagingandacquisitionsettings
STED nanoscopy has been performed at two different setups; a
custom-built3D-STEDandacommercialLeicaTCSSP83XSTED.The
custom-built STED setup is previously described in detail53. Briefly,
excitationofthedyeswasdonewithpulseddiodelasers:oneat561nm
(PDL561,AbberiorInstruments),oneat640nm(LDH-D-C-640,Pico-
Quant),andoneat510nm(LDF-D-C-510,PicoQuant).Alaserat775nm
(KATANA08HP,OneFive)wasusedasthedepletionbeam.Thelaser
beamswerefocusedonthesampleusinganHCPLAPO100×/1.40Oil
STEDWhiteobjective(15506378,LeicaMicrosystems),throughwhich
alsothefluorescencesignalwascollected.Theimagingwasdonewith
a561nmexcitationlaserpowerof8–20µW,a640nmexcitationlaser
power of 4–10µW, and a 775nm depletion laser power of 128mW,
measuredatthefirstconjugatebackfocalplaneoftheobjective.Both
oneandtwo-colorSTEDimagingweredoneinaline-by-linescanning
modality.Thepixelsizeforallimageswas20–30nm.Thepixeldwell
timewas50µs.ThecommercialLeicaTCSSP83XSTEDisequipped
withaHCPLAPO100×/1.40OilSTEDWhiteobjective.Theimageswere
recorded by exciting 590-CA/Alexa594 and 647-SiR/STAR RED with
590nmand650nmlaserlines,respectively.ASTEDbeamat775nm
hasbeenusedtodepletebothlaserlines.Two-colorSTEDimageswere
recorded line-by-line, averaging over 16 or 32 lines. The spectral
detection windows were adjusted to 600–645nm for 590-CA/
Alexa594andto665–750nmfor647-SiR/STARRED.

Mitography:automatedmitochondriamorphologydata
analysis
TheautomatedimageanalysisofmitochondriaandMDVmorphology,
Mitography, is carried out in two parts. Image processing and data
gatheringareperformedinFiji/ImageJ,whiledatahandling,fitting,and
visualization are performed in MATLAB (The Mathworks) (Supple-
mentary Fig. 1i). This analysis can further analyze the position of
individual mitochondria in relation to other fluorescent markers or
neuronalcompartments.TheImageJMitographyscriptstakeinputimagesofmitochondria
in theneuritesofneurons labeledthroughtheOMM(e.g.,OMP25).
Input images can be raw, smoothed, or deconvolved, where the
smoothing or deconvolution can help with the robustness of the
analysis and likely result in an increased number of successfully
extracted data points. Tests have been performed to ensure that
deconvolutionorsmoothingisnotaffectingtheoveralldistributionof
extractedandfittedmorphologicalparameters.Allmorphologyana-
lysis presented here is performed on Richardson-Lucy deconvolved
images.PriortorunningtheImageJMitographyanalysisscript,abin-
arymaskofthemitochondriamustbeextracted.Thisisperformed
through thresholdingandbinarizationsteps,anddepending onthe
labelingtype,signallevel,anduniformityofthestructureinthefieldof
view,differenttypesofbinarizationareused.Providedinthescripts
aretwovariants:oneusesglobalthresholding,andtheothermainly
uses local Bernsen thresholding (local radius <10 pixels, contrast
threshold<15).Thethresholdingisfollowedbymorphologicalfiltering
(e.g., erosion, dilation, filling holes) to create a binary mask of the
mitochondriaandMDVsthatiswell-fittedtotheactualsizeandshape
of the mitochondriainthe originalimage. Additionally, an inverted
soma mask is extracted and used in the analysis when the soma is
presentintheimage,toleaveonlymitochondriaintheneuritesforthe
analysis. In the main Mitography script,a well-dilated mitochondria
binarymaskismultipliedwiththeoriginalOMMimage,removingthe
backgroundoutsidethemitochondria,andthusallowingforabetter
line profile extraction and subsequently a more robust fitting. The
binary mitochondria image itself is alsoused initially for parameter
extraction.AnalyzeparticlesandcorrespondingellipsoidalfittinginFiji
is used to gather data such as ellipse centroid position, ellipsoidal
length(ellipsemajoraxislength),ellipsoidalwidth(ellipseminoraxis
length),area,andpositioningangle(ellipseangle).Togetameasureof
the mitochondrial length that is more accurate for non-ellipsoidal-
shaped mitochondria, a skeletonization is performed on the binary
mitochondria representation. The Analyze skeleton plugin54 and the
summationofthebranchlengthsoftheindividualskeletonsresultina
second measurement of the mitochondrial length. For small mito-
chondria(A<0.2µm2),theellipsoidallengthisalwayschosen,asitis
moreaccurateforsmallermitochondriathatarenon-branched,non-
curved,andwheretheskeletondoesnotcoverthefulllnotcoverthefulllength,while
thelongerofthetwomeasurementsistakenforlargermitochondria.
Line profiles (0.75µm long, 5 pixels wide) are drawn across all
binarymitochondria,inadirectionperpendiculartothepositioning
angle,atthreedifferentpoints:thecenterandclosetotheendpoints
along the long axis of the mitochondria. For shorter mitochondria
(L<2.1µm),thesepointsarechosentobeatL/6and5×L/6,whilefor
longermitochondria(L≥2.1µm),thepointsarechosenat350nmand
L−350nm.The fullset of extracted data and line profiles per mito-
chondrion are matched to the numbered mitochondria in the
binaryimage.
InMATLAB,extractedparameters,lineprofiles,andbinarymaps
areimported.Here,lineprofilesofmitochondriaarefittedtoextract
the width at the center and outer positions. Firstly, the number of
peaksofthelineprofileisextracted.Ifthenumberofpeaksis1or2,a
fittingisperformedwithacombinationofGaussianfunctions,eitheras
asingle(nooutermembrane visible,asin confocalimagesofmost
mitochondria and STED images of narrow MDVs) or double (outer
membranevisible,asinSTEDimagesofmostmitochondriaandcon-
focal images of wide mitochondria). While fitting a single-Gaussian
function across two unresolved membranes will overestimate the
widthofthemitochondrion,itisthebestestimationofthewidththat
canbeextractedfromsuchacase.Furthermore,disregardingthese
casesentirelywouldresultinafarlargershiftoftheoveralldistribution
ofwidths.Ifthreeorfourpeaksarelocated,itislikelythatwehavetwo
mitochondriaside-by-sidewithoutermembranesvisible.Inthiscase,
thepositionsofthepeaksareusedtocalculatethedistancebetween
theindividualpeaks,andthesevaluesareusedasthemitochondria
widths. These mitochondria are generally disregarded later in the
analysis, due to a more uncertain and likely erroneous parameter
extraction for non-width parameters, but they can be investigated
further if of interest. If more than four peaks are found in the line
profile,ortheR-squarevaluesofthefitsarebelowsetthresholds,the
mitochondrion is immediately disregarded due to any parameter
extractionbeingfartooerroneous,andthemitochondrionlikelynot
beingaccuratelydetected.Inmostcases,however,theFWHMofthe
single-Gaussianfitinthecaseofasingledetectedpeak,orthedistance
betweenthetwopeaksofthedouble-Gaussianfitinthecaseoftwo
detectedpeaks,isextractedasthemitochondrialwidth,assumingthe
R-square value of the respective fit is higher than a set threshold.
Following the fitting, the center position of each mitochondrion is
checkedagainsttheprovidedbinaryimagesofinterest(e.g.,dendrite
marker,axoninitialsegmentmarker).Thebinarypixelvalueofeach
positionissavedasaBooleanvariableforeachmitochondrion,gen-
eratingaregisterofBooleanparametersdefiningthepositionofthe
mitochondriawithrespecttovariousneuronalcompartments.Addi-
tionally,secondarymorphologicalparametersarealsocalculatedfrom
theextractedmorphologicalparameters.TheseincludetheAR,L/W ,
m
andthewidthratiobetweentheouterandcenterwidth,W /W .All
o m
extractedparametersandfittingparameters,permitochondrion,are
savedinatab-delimitedtextfile.

AutomaticTMREandMitoSOX+/−detection
TodetermineifeachmitochondrionisTMRE+/MitoSOX+orTMRE−/
MitoSOX−, an automated decision was made through a MATLAB
scriptusingthepreviouslyextractedbinarymitochondriaimageand
theTMRE/MitoSOXimage.ThescriptmeasurestheaverageTMRE/
MitoSOXsignalineachdetectedmitochondrioninthebinaryimage.
It then finds the threshold TMRE/MitoSOX value by plotting the
histogram of the average TMRE/MitoSOX signal for all the mito-
chondria in an image, and fitting the histogram counts with an
exponentialdecay(background)+Gaussian(truesignal).Thelocal
minimumbetweenthetwopeaksoftheextractedfunction,i.e.,the
interception of the two curves, is taken as the threshold TMRE/
MitoSOXvaluetowhichtheindividualmitochondriaaverageTMRE/
MitoSOXsignaliscompared.Thethresholddecisionisdoneonan
image-to-image basis, as different images have different signal
intensitiesduetovaryingimageacquisitionparametersandlabeling
efficiency.

Mitochondriaprocessesnumberdensityanalysis
To calculate the mitochondria number density in processes, the
lengthsoftheprocesseswereneeded.Thesewerefoundbyusingthe
background fluorescence in the images of the outer mitochondrial
membranelabeledwiththeOMP25localizationpeptide,andthiswas
doneinaPythonscript.Theimagewasprocessedbyremovingthe
mitochondriainthesomathroughmultiplicationwithabinarysoma
map,settingallpixelsaboveathresholdtothethresholdvalue,per-
formingaglobalbinarizationoftheimagebasedonathresholdcal-
culated with the triangle method, performing morphological
operationssuchasremovingsmallobjects(<20pixels),dilation(20×),
and erosion (15×), and removing everything except the largest con-
nectedsegment,abinarymapoftheprocesseswasfound.Thiswas
skeletonized,andbysummingthelengthofalltheskeletalbranches,
thetotallengthoftheprocessesfortheimagewasfound.Eachimage
containedeitheranaxonordendrites(adecisionbasedonanimage
of,forexample,map2labeling),andhencethetotalnumberdensity
foraxonsanddendritescouldbefoundonanimage-to-imagebasis.
The triangle method, morphological operations, and skeletonization
wereallperformedusingtheimplementationsinthePythonpackage
scikit-ImageJ55.

QuantificationofMDStraffickingandturnoverdynamics
Quantification oftheturnover dynamicswasperformed bya com-
binationofmitochondrialmorphologythroughtheabove-described
analysisscriptsandanalysisofthesignalofthetwolabelsfromdif-
ferent timings for each mitochondrion. The refreshment rate was
calculatedasthesumofthesignalfromSNAP-Cell®SiR-Starligand
insidethemitochondrialarea,dividedbythesumofthesignalfrom
SNAP-Cell®TMR-Star ligand and thesum of the signal from SNAP-
Cell®SiR-Starligandinsidethemitochondrialarea.Thisratiometric
ratetakesvaluesbetween0and1,where1indicates100%refreshed
proteinand0indicates0%refreshedproteinintheinvestigatedtime
interval.Intheseexperiments,theratiooffluorescencesignaldoes
not increase linearly with the ratio of refreshed proteins, due to
different properties of the dyes and different excitation powers,
leadingtodifferentexcitationanddetectionefficiencies.Hence,the
ratiometric value cannot be translated directly into the ratio of
refreshed proteins. An automatic radial analysis was performed,
startingfromthecellsomaandmovingtowardthedistalfilaments,
dividingthecellintoconcentricringsof30μmthickness.Quantifi-
cation of the control turnover experiments was done by a combi-
nation of automated and manual analysis. Ilastik56 was used to
automatically segment the neuronal cell basedon theMAP2 fluor-
escent signal, followed by a manual correction to remove small
particlesandclosedholes,andtoensurethatonlyonesuccessfully
transfectedcellperimagewasdisplayed.Thecellsomawasfurther
removedfromthe segmentationtofocustheanalysis onneuronal
filaments exclusively. A mitochondrial binary map was made with
Mitography. Subsequently, an automatic radial analysis was per-
formed, starting from the cell soma and moving toward the distal
filaments,dividingthecellintoconcentricringsof30μmthickness.
The fluorescence intensity of mitochondrial structures throughout
thecellwasmeasuredasthelevelsofSNAP-Cell®TMR-Starintensity,
normalized by its intensity in the most proximal mitochondria to
compensateforvaryingexpressionlevelsindifferentcells,foreach
ring, for four different time points: 0h, (data not shown), 4–5h,
13–18hand20–24h.

Manualmitochondrialprotrusionsquantification
Mitochondrial protrusions were selected manually, based on the
OMP25channel.Todetermineiftheprotrusionwas“tip”or“side,”the
lengthofthemitochondrialtubulewasdividedintoquarters:protru-
sions in the two quarters at the extremity of the tubules were con-
sidered“tip”,whileprotrusionsfoundinthetwomiddlequarterswere
considered“side”.Todeterminewhethereachprotrusionwaspositive
or negative (+/−) forthe additional markers (PEX14, PEX16, PMP70,
DRP1), fluorescence intensity was quantified at the corresponding
positionineachchannel.Themeasuredsignalwasthencomparedwith
background intensity, defined as the average fluorescence intensity
within the neuronal filaments but outside any labeled structure. To
accesstheERassociation,fluorescenceintensitywasmeasuredbothat
thesamepositionoverlappingtheprotrusionandalongtheprotrusion
boundaryfacingthemitochondrialside.

QuantificationofPEX3RNAisilencing
PEX3punctawereidentifiedbasedonPEX3immunofluorescence,and
abinarymaskwasgenerated.Thenumberandfluorescenceintensity
ofPEX3puncta,aswellasthelinearnumberdensityofmatureper-
oxisomes (PMP70 immunostaining), were measured in cells trans-
fected with siRNA for 48h and compared with those in non-
transfectedcells.

DRP1analysis
DRP1WT and DRP1K38A transfected neurons were identified by the
fluorescentsignalofthemCherryfusiontag,andabinarymaskwas
generatedtoensurethatonlytransfectedneuronswereincludedin
theanalysis.PEX14punctaandmitochondriawereidentifiedbasedon
PEX14andATPbimmunofluorescence,respectively,andcorrespond-
ingbinarymaskswerecreated.Mitochondriallengthwasquantified
with Mitography, and to assess the ability of DRP1K38A to impair
mitochondrial fission, the percentage of mitochondria longer than
10μm was compared between DRP1WT and DRP1K38A expressing
neurons.Toinvestigatetheeffectsofmitochondrialfissionperturba-
tiononperoxisomebiogenesis,mitochondria-PEX14ormitochondria-
PMP70 contact sites were quantified in DRP1WT and DRP1K38A
expressingcells.

Imageanalysis,codeavailability,andstatisticaltests
RawimageswereprocessedandvisualizedusingtheImSpectorsoft-
ware(Max-PlanckInnovation,Gottingen,Germany)andFiji/ImageJ57,58.
When necessary, images and movies were deconvolved using the
Richardson-Lucy algorithm, implemented in Imspector. The PSF
was modeled as a Gaussian function with a FWHM of 40–60nm.
Theregularizationparameter(10−5or10−10)andnumberofiterations
(max 10) varied depending on the quality of the output image.
Movies 1–3 are instead smoothed using a low-pass Gaussian filter,
implementedinImspector.Whennecessary,movieswerecorrected
for bleaching using the “Bleach correction” plugin and for drift
using the “MultiStackReg” (based on the “StackReg”) plugin in Fiji.
Plotted line profiles in the figures were averaged over 2–4 pixels
andfittedwiththesoftwareOriginPro2020.Allautomatedanalysishas
beenperformedmainlyinFiji/ImageJandMATLAB(TheMathworks),
with some auxiliary data handling performed in Python. Code
andscriptsforthevariouspartsoftheanalysisinImageJandMATLAB,
also described above, together with example data for running the
scripts, are available online at https://github.com/jonatanalvelid/
mitography-public and as a versioned release in Zenodo at https://
doi.org/10.5281/zenodo.17831561. See documentation in the readme
andinfofilesforfurtherinformationonthespecificscriptsandhow
to run them. For comparison of distributions of parameters for
differentconditions,aStudent’st-test(normallydistributeddata)ora
Kolmogorov–Smirnov test (non-normally distributed data) was
chosen.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript produced by Coceano et al. provides an excellent characterization of MDVs in neurons utilizing powerful\nmicroscopy techniques. However, data do not strongly support much of the functional roles described regarding these\nvesicles, and some major concerns brought up by the reviewers were not addressed. Unfortunately, because the current\ndriving take-home narrative of the manuscript relies on these immature data, I am not confident that the state of this\nmanuscript is ready for publication in Nature Communications. Further details can be found below:\nMajor Concerns:\n1. Two reviewers had major concerns with the experimental setup and interpretation that MDVs in different neuronal\ncompartments refresh their membrane content at different rates. However, the authors did not experimentally show that\nnewly synthesized proteins in the Halo/Snap-tag system are equally integrated in somal vs distal organelles. The authors\nshould experimentally address this concern or, at minimum, discuss alternative interpretations within the text.\na. There is also a statement about existing MDVs potentially incorporating newly synthesized OMP25-Snap, to which they\nprovide a logical argument in lieu of experimental data that is potentially not grounded in current literature.\nb. Since interpreting their results relies on understanding these dynamics, the authors should put pronounced effort into\ngenerating argument-supporting data.\n2. In agreement with Reviewer 2, I am unclear of the novelty of peroxisomes and MDV biogenesis reported in the\nmanuscript. Multiple studies describe MDV involvement in the de novo biogenesis of peroxisomes, providing mechanistic\ncharacterization (DOI: 10.1038/nature21375, and more recently DOI: 10.1016/j.devcel.2024.09.029). However, as stated by\nthe authors, none of these studies were performed in neurons. Therefore, it is critical to do some characterization to support\nthe novelty and arguments made within this study. For example, could Pex3 or another marker be depleted in neurons to\nlook at overall effects? As the manuscript currently stands, this point represents a significant gap in the study.\n3. The authors make claims about the functional roles of proteins and organelles in forming pre-peroxisomes. They make\nthese statements almost entirely from localization data without perturbing key proposed players, as multiple reviewers\nrequested (e.g., MDV biogenesis and DRP1). Additionally, they include exciting ER data visualizing tubules at MDV/pre-\nperoxisome scission sites in the updated manuscript. However, they do not acknowledge that these data are inconsistent\nwith the model that ER vesicles fuse with the maturing pre-peroxisome.\na. They also generally do not follow through on investigating the fate of these mitochondrial protrusions through timelapse.\nb. The representative images for ER and Drp1 are nice, but quantification is necessary to support the conclusions within the\ntext.\nc. Experiments highlighting the role of Drp1 through perturbations, such as Drp1 KD or overexpression of Drp1 mutants,\nwere again suggested by reviewers but not conducted.\nMinor Concerns:\n1. The authors should include novelty and motivation for using neuronal cultures in the background, as suggested by\nReviewer 2. This would differentiate the study and support their claims of neuronal novelty.\n2. Reviewer 3’s comments state that size cannot be used alone to determine an MDV, and the authors agree with the\ncomments. However, the text was not altered to address these changes. Lines 121-123 still suggest using size to define an\nMDV.\n3. The authors state that “only those attached to mitochondria and labelled with OMP25 were considered” for quantification\nin Figure 8. Could the authors state what percentage of vesicles were used in this quantification? If it is a small population,\nthis is important to know for the interpretation of the data. It is unclear whether this minor concern was addressed based on\nthe revised manuscript.\n4. There are no error bars on the normalized frequency plots (example, Fig. 4G).", "reviewer_2": "The authors have done a fair job in addressing my previous comments, except for specific comment No. 4, which they could\naddress with finding/acquiring pairs of images (confocal vs. STED) where the potential confounder of structural movement is\nabsent.\nOtherwise, I find the paper suitable for Nat. Communications.", "reviewer_4": "The concerns have been addressed, and the manuscript is well organized for publication.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point by point response to Reviewers of the manuscript NCOMMS-24-82932-T:\nQuantitative optical nanoscopy of mitochondrial-derived vesicles in neuronal cells classify\npre-peroxisomal and clearing organelles\nCoceano et al.\nWe thank the editor for the interest in our work and the reviewers for the critical assessment\nof our study which led us to further develop the connection between Mitochondrial-Derived\nVesicles (MDVs) and peroxisome biogenesis in the revised version.\nWe performed new experiments 1- to support the pulse-chase experiments and 2- to provide\nnew insights about the connection between MDVs and peroxisome biogenesis. The new data\nabout mitochondrial membrane refreshment is summarized in the new Supplementary Fig.\n2. Additionally, new experiments aimed at altering the process of peroxisome biogenesis are\nsummarized in the new Supplementary Fig. 8 and 9.\nWe modified the text to explain and discuss the new findings, including the motivation for\nand the novelties of using neuronal cultures. We clarified in the text the different parameters\nused to identify and characterize MDVs in neuronal cells. The term “mitochondrial-derived\nstructures (MDSs)” was chosen instead of “mitochondrial-derived vesicles (MDVs)” to\ncharacterize the diverse nano-sized structures, vesicular and non-vesicular, derived by\nmitochondrial outer membranes and here resolved by super-resolution light microscopy.\nWe updated Fig. 1, 4, 6, 8, 9 following the reviewer's suggestions and modified the text\naccordingly.\nWe modified the text and figures to adhere to the Nature Communication formatting\nrequirements.\nPlease find below a point-to-point response (blue) to the reviewers’ questions (black).\nReviewer #1 (Remarks to the Author):\n[... content omitted for brevity in this example, but would be full text ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript by Coceano et al. provides novel insights into mitochondrial-derived structures within neurons. The\nuse of STED super-resolution microscopy provides a powerful platform to investigate structural characteristics. The authors\nhave taken the reviewers' comments into account and provided new experiments and analyses that support their arguments\nand strengthen their functional model. Overall, this work will benefit the field, and I believe this manuscript is now ready for\npublication in Nature Communications.\nMinor Comments:\n• In Figure 9e-f, the authors mention \"nearby” ER signal as part of their quantification criteria, but I do not see where this\ncharacteristic was defined. Was there a specific distance that the ER signal needed to fall within?\n• Occasionally, there are grammatical errors in the new text (for example, Line 387 says “this mutant exhibit reduced\nefficiency” instead of “this mutant exhibits reduced efficiency”; Line 217, the word few is misspelled), so I recommend a\nquick grammar check before finalizing the paper."}, "author_response": "Point by point response to Reviewers of the manuscript NCOMMS-24-82932A:\nQuantitative optical nanoscopy of mitochondrial-derived vesicles in neurons classify pre-\nperoxisomal and clearing organelles\nCoceano et al.\nWe thank the editor for the interest in our work and the reviewer for the final critical assessment\nof our study.\nWe revised the paper to address the editorial requests (following the Author Checklist) and we\naddressed the last reviewer questions.\nPlease find below a point-to-point response (blue) to the reviewers’ questions (black).\nMinor Comments:\n• In Figure 9e-f, the authors mention \"nearby” ER signal as part of their quantification criteria,\nbut I do not see where this characteristic was defined. Was there a specific distance that the\nER signal needed to fall within?\nWe thank the reviewer for the comment. We updated the relative paragraph in the Methods\nsection as following: “To access ER association, fluorescence intensity was measured both at\nthe same position overlapping the protrusion and along the protrusion boundary facing the\nmitochondrial side. “\nBasically, to evaluate if the ER was associated to the protrusion or not, we measured the\nfluorescence intensity both at the same position overlapping the protrusion and at the border\nof the protrusion facing the mitochondrial side.\n• Occasionally, there are grammatical errors in the new text (for example, Line 387 says “this\nmutant exhibit reduced efficiency” instead of “this mutant exhibits reduced efficiency”; Line\n217, the word few is misspelled), so I recommend a quick grammar check before finalizing\nthe paper.\nWe thank the reviewer for the thorough check. We performed a grammar check of the\nmanuscript and corrected the errors found."}]

Decision letter