RESEARCH ARTICLE
D p1-media ed mi ochond ial ission egula es calcium
and F-ac in dynamics du ing wound healing
Susana Pon e
1
, La a Ca alho
1
, Ma ia Gaglia di
1
, Isabel Campos
2
, Paulo J. Oli ei a
3
and An onio Jacin o
1,
*
ABSTRACT
Mi ochond ia adap o cellula needs by changes in mo phology
h ough usion and ission e en s, e e ed o as mi ochond ial
dynamics. Mi ochond ial unc ion and mo phology a e in ima ely
connec ed and he dys egula ion o mi ochond ial dynamics is linked
o se e al human diseases. In his wo k, we in es iga ed he ole o
mi ochond ial dynamics in wound healing in he D osophila
emb yonic epide mis. Mu an s o mi ochond ial usion and ission
p o eins ail o close hei wounds, indica ing ha he egula ion o
mi ochond ial dynamics is equi ed o wound healing. By li e-
imaging, we ound ha loss o unc ion o he mi ochond ial ission
p o ein Dynamin- ela ed p o ein 1 (D p1) comp omises he inc ease
o cy osolic and mi ochond ial calcium upon wounding and leads o
educed eac i e oxygen species (ROS) p oduc ion and F-ac in
de ec s a he wound edge, culmina ing in wound healing impai men .
Ou esul s highligh a new ole o mi ochond ial dynamics in he
egula ion o calcium, ROS and F-ac in du ing epi helial epai .
KEY WORDS: D p1, F-ac in, Calcium, Mi ochond ia, Mi ochond ial
dynamics, Wound healing
INTRODUCTION
Mi ochond ia pe o m c i ical cellula unc ions such as ene gy
p oduc ion, egula ion o calcium (Ca
2+
), edox homeos asis and
cell dea h (El-Ha ab and Scaglia, 2016). Mi ochond ial shape is
con olled by an agonizing usion and ission e en s (Lewis and
Lewis, 1914; Nunna i e al., 1997), desc ibed as mi ochond ial
dynamics, which allow mi ochond ia o adap o cellula demands
(Nunna i and Suomalainen, 2012).
Dynamin- ela ed p o eins egula e mi ochond ial dynamics
h ough hei GTPase ac i i y (Hoppins e al., 2007).
Mi ochond ial ission is accomplished by Dynamin- ela ed
p o ein 1 (D p1). Upon ac i a ion, D p1 is ec ui ed om he
cy osol o he mi ochond ia, oligome izes and cons ic s his
o ganelle un il i s di ision is achie ed (Bleaza d e al., 1999;
Lab ousse e al., 1999; Smi no a e al., 2001; Yoon e al., 2001).
Mi ochond ial usion equi es he me ging o bo h he ou e (OMM)
and he inne mi ochond ial memb anes (IMM). Mi o usin 1 (M n1)
and Mi o usin 2 (M n2) a e esponsible o OMM usion (Rojo
e al., 2002), while Op ic a ophy 1 (Opa1) media es usion o he
IMM (G ipa ic e al., 2004; Olichon e al., 2003).
Regula ion o mi ochond ial dynamics is essen ial o
de elopmen (Chen e al., 2003; Ishiha a e al., 2009; Wa e ham
e al., 2007) and dys egula ion o i s machine y is implica ed in a
wide ange o human diseases, including neu opa hies, ype II
diabe es and cance (Ande son e al., 2018; Ranie i e al., 2013;
Ro i a-Llopis e al., 2017). Howe e , he ole o mi ochond ial
dynamics in o he con ex s, such as epi helial epai , is s ill la gely
unknown.
Wound healing in simple epi helia is cha ac e ized by he
accumula ion o F-ac in and non-muscle myosin II (myosin) a
he cell bounda ies ha ace he wound, o ming an ac omyosin
cable ha con ac s and b ings cells oge he , he eby closing he
hole (Bemen e al., 1999; Danjo and Gipson, 1998; Kieha e al.,
2000; Xu and Chisholm, 2011). Addi ionally, wound healing
in ol es cell c awling media ed by ac in p o usions (Ab eu-Blanco
e al., 2012a; Ve boon and Pa khu s , 2015) and cellula
ea angemen s (Ca alho e al., 2018; Razzell e al., 2014).
Recen s udies sugges ha mi ochond ia migh be equi ed o
issue epai by p oducing eac i e oxygen species (ROS). They ha e
shown ha mi ochond ial ROS p omo e wound healing by egula ing
F-ac in and myosin a he wound edge, ei he by ac ing on Rho
GTPases (Muliyil and Na asimha, 2014; Xu and Chisholm, 2014) o
on cell–cell junc ion emodelling (Hun e e al., 2018). In his wo k,
we show ha he mi ochond ial dynamics machine y is essen ial o
epai , as mu an s o hese p o eins ail o close epi helial wounds. In
pa icula , he ission p o ein D p1 is equi ed o F-ac in
accumula ion a he wound edge, o p ope cy osolic and
mi ochond ial Ca
2+
dynamics and o ROS p oduc ion upon
wounding. Ou wo k e eals a no el ole o mi ochond ial ission
in egula ing ROS, Ca
2+
and F-ac in dynamics du ing epi helial epai .
RESULTS
Mi ochond ial dynamics p o eins a e equi ed o wound
healing
To es whe he he mi ochond ial dynamics machine y (Fig. 1A) is
equi ed o epi helial epai , we pe o med a p e iously desc ibed
wounding assay in he D osophila emb yonic epide mis (Campos
e al., 2010). We lase -wounded la e-s age emb yos bea ing
wild- ype and mu an alleles o mi ochond ial dynamics p o eins
and assessed he wound-healing pheno ype by he pe cen age o
non-healing wounds.
Fig. 1A shows a scheme o mi ochond ial dynamics wi h all he
es ed p o eins ep esen ed. Rega ding usion, we es ed ou mu an
alleles and one miRNA o Mi ochond ial assembly egula o y
ac o (Ma ,aD osophila M n homolog); as well as wo Opa1
mu an alleles and wo he e oallelic combina ions. Conce ning
mi ochond ial ission, we es ed h ee D p1 alleles and h ee
Recei ed 7 Oc obe 2019; Accep ed 9 Ma ch 2020
1
CEDOC, Ch onic Diseases Resea ch Cen e , NOVA Medical School/Faculdade
de Ciências Medicas, Uni e sidade NOVA de Lisboa, 1169-056 Lisboa, Po ugal.
2
Animal Pla o ms, Champalimaud Cen e o he Unknown, 1400-038 Lisboa,
Po ugal.
3
CNC, Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a,
UC Bio ech Building, 3060-197 Can anhede, Po ugal.
*Au ho o co espondence ([email p o ec ed])
S.P., 0000-0002-4297-1265; L.C., 0000-0001-6350-3279; M.G., 0000-0001-
6411-0901; I.C., 0000-0002-1541-8552; P.J.O., 0000-0002-5201-9948; A.J., 0000-
0002-4193-6089
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License (h ps://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use,
dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed.
1
© 2020. Published by The Company o Biologis s L d
|
Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
he e oallelic combina ions. We also es ed o he ission egula o s:
Fission p o ein 1 (Fis1), which ac s as a ecep o o D p1 a he
OMM (Losón e al., 2013), and Ganglioside-induced di e en ia ion
associa ed p o ein 1 (GDAP1), whose unc ion is no well
unde s ood (Hube e al., 2013).
We obse ed h ee ypes o wound closu e pheno ypes: open,
in e media e and closed wounds (Fig. 1B). Closed wounds a e
iden i iable by a small melanized spo . Open wounds show a
melanized ing a ound he hole. In he in e media e pheno ype,
melaniza ion occu s in a la ge ci cula a ea bu a clea hole is absen ,
making i unce ain whe he he wound is open o closed. Con ol
emb yos (w
1118
) ha e an ou s anding capaci y o epi helial epai , as
94.7% o he wounds a e closed (Fig. 1C). Mu a ions in ei he
mi ochond ial ission o usion genes inc eased he equency o
open and in e media e wounds (Fig. 1C).
As i is unclea whe he he in e media e wounds ep esen a
closu e impai men o jus a melaniza ion de ec , we excluded hese
wounds om he s a is ical analysis o he wound healing
pheno ype. Compa ing only closed e sus open wounds, all
mi ochond ial ission mu an s showed highe pe cen age o open
wounds han con ols (Fig. 1D). Rega ding mi ochond ial usion,
om he ou es ed Ma alleles and he miRNA, only Ma
J
showed an inc eased pe cen age o open wounds compa ed o
con ols. Opa1 mu an s showed a signi ican wound closu e
pheno ype (Fig. 1D).
As we obse ed wound-closu e de ec s o mu a ed e sions o
bo h usion and ission p o eins, hese da a sugges ha he egula ion
o mi ochond ial dynamics is necessa y o wound healing.
D p1 mu an s show delayed wound healing
Mi ochond ial ission mu an s showed a mo e consis en wound
healing pheno ype han usion mu an s. The e o e, we decided o
explo e he ole o mi ochond ial ission in epi helial epai by
ocusing on he unc ion o D p1.
Fig. 1. Mi ochond ial dynamics p o eins a e equi ed o wound healing. (A) Scheme o he p o eins in ol ed in mi ochond ial dynamics used in he
wounding assay sc een. (B) Rep esen a i e images o ha ching la ae, 16 h a e wounding, showing he h ee obse ed wound pheno ypes: closed,
in e media e and open. Closed wounds p esen a small scab, while open wounds show a ing o melaniza ion a ound he hole. In e media e wounds ha e
mo e melaniza ion han closed and open wounds bu no a clea hole. A owheads poin o he wound. Scale ba : 200 µm. (C) G aph o pe cen age o
closed, in e media e and open wounds in con ols (w
1118
) and mu an alleles o mi ochond ial dynamics p o eins. (D) G aph o pe cen age o open wounds
in con ols and mu an alleles o mi ochond ial dynamics p o eins. Rega ding usion, all Opa1 alleles and he e oallelic combina ions showed inc eased
pe cen age o open wounds compa ed o con ols; o Ma , only he Ma
J
mu a ion shows signi ican ly inc eased pe cen age o open wounds compa ed o
con ols. All he es ed ission genes and he e oallelic combina ions showed highe pe cen age o open wounds compa ed o con ols. Fishe ’s exac es was
used o es o signi ican di e ences be ween g oups. UAS-Ma miRNAi was exp essed unde he con ol o he da-Gal4 d i e . The g aph in D shows he
same emb yos om C, excluding hose wi h in e media e wounds. ns, no signi ican (P>0.05), **P≤0.01, ***P≤0.001, ****P<0.0001. The numbe o emb yos
o each condi ion is shown below he ba s in C and D.
2
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
To unde s and he ole o D p1 in wound healing, we used
spinning-disk mic oscopy o image con ol and D p1 mu an emb yos
exp essing GFP::Moesin (Kieha e al., 2000), an F-ac in ma ke , and
ollowed he dynamics o closu e (Mo ie 1). Con ol emb yos
accumula e F-ac in a he wound edge (Fig. 2A) and he wound a ea
p og essi ely dec eases un il he hole is closed (Fig. 2A,F). Al hough
he ini ial a ea was simila in bo h condi ions (Fig. 2D), D p1 mu an
wounds ook on a e age 128±34 min o close, signi ican ly longe
han con ols (56±17 min) (Fig. 2E). In milde cases, D p1 mu an
wounds closed a a slowe a e (Fig. 2B,F). In o he cases ( h ee ou o
13 D p1 mu an emb yos), he pheno ype was s onge ; al hough he
wound con ac ed o abou 40 min pos -wounding (mpw), i s a ea
began o inc ease again un il 120–130 mpw (Fig. 2C,F). A e his
expansion phase, wounds con ac ed again, and in one case i was
almos closed by he end o imaging (Fig. 2C 180 mpw, F). We
quan i ied he wound a ea o con ol and D p1 mu an s in he i s
30 mpw and ound signi ican di e ences in he i s minu es a e
wounding (4 mpw and 10 mpw o mild and s ong condi ions,
espec i ely) (Fig. 2G).
Ou esul s sugges ha inhibi ion o mi ochond ial ission
impai s wound closu e. We nex asked whe he he induc ion o
mi ochond ial ission has he opposi e e ec , accele a ing he
wound healing p ocess. A e alida ing ha he o e exp ession o
D p1 leads o induc ion o mi ochond ial ission, esul ing in a
agmen ed mi ochond ial ne wo k (Fig. S1), we compa ed con ols
and emb yos o e exp essing D p1 (UAS-D p1) and exp essing he
F-ac in ma ke mChe y::Moesin (Milla d and Ma in, 2008) unde
he con ol o a ubiqui ous d i e (da-Gal4) and ollowed he wound
closu e dynamics o e ime (Fig. 2H,I). We ound no signi ican
di e ences in ei he he ime o wound closu e (Fig. 2J) o in he
wound a ea o e ime (Fig. 2K). This sugges s ha inc eased ission
does no ha e an impac on wound closu e dynamics.
These esul s show ha , while D p1 o e exp ession has no e ec
on wound closu e, D p1 loss-o - unc ion impai s wound healing,
sugges ing ha mi ochond ial ission is necessa y o wound epai
egula ion.
Wounding induces no majo changes in mi ochond ial
mo phology
Ou p e ious esul s sugges ha mi ochond ial ission is equi ed
o p ope wound healing, so we wonde ed whe he wounding
igge s changes in mi ochond ial mo phology, owa ds a mo e
agmen ed mi ochond ial ne wo k.
To analyse mi ochond ial mo phology, we used emb yos
exp essing mi ochond ia (EYFP::mi o, Lajeunesse e al., 2004)
and memb ane (PLCγPH::ChFP, He sz e g e al., 2013) ma ke s
and compa ed con ol and D p1 mu an emb yos. D p1 mu an s
showed longe mi ochond ia han con ols bo h be o e and upon
wounding (Fig. 3A,B). Mi ochond ial mo phology quan i ica ion
con i med ha he mi ochond ial leng h (Fig. 3C) was highe in
D p1 mu an s han in con ols, while he numbe o b anches was
simila (Fig. 3D), bo h be o e and upon wounding. In con ol
emb yos, wounding led o a educ ion in he numbe o
mi ochond ial b anches (Fig. 3D) bu he o e all mi ochond ial
leng h was una ec ed (Fig. 3C), sugges ing ha wounding does no
lead o majo mi ochond ial mo phology changes.
Rega ding localiza ion and apicobasal dis ibu ion o
mi ochond ia inside he cell, we did no obse e majo di e ences
be ween con ol and D p1 mu an s, ei he be o e o upon wounding
(Fig. 3A–Bi, XZ and YZ sec ions).
Ou esul s sugges ha , al hough wounding does no s ongly
in luence mi ochond ial mo phology, an elonga ed mi ochond ial
ne wo k such as ha seen in D p1 mu an s is de imen al o wound
healing.
D p1 mu an s ha e F-ac in de ec s du ing wound closu e
Al hough cells can compensa e o he loss o he ac omyosin cable
(Ducuing and Vincen , 2016), his s uc u e is one o he main
d i ing o ces o wound healing (Zulue a-Coa asa and Fe nandez-
Gonzalez, 2017). The e o e, we checked whe he he wound healing
pheno ype in D p1 mu an s was associa ed wi h ac omyosin cable
de ec s. We imaged con ol and D p1 mu an emb yos exp essing
GFP::Moesin (Kieha e al., 2000) and Zip::GFP (Lye e al., 2014)
o compa e hei F-ac in and myosin le els.
Bo h con ols and D p1 mu an emb yos accumula ed F-ac in
(Fig. 4A,B) and myosin (Fig. 4C,D) a he wound edge. Howe e ,
F-ac in le els we e lowe in D p1 mu an s when compa ed o
con ols (Fig. 4E). We ound no signi ican di e ences in myosin
le els be ween D p1 mu an and con ol emb yos (Fig. 4F). These
esul s sugges ha he wound healing pheno ype in D p1 mu an s
migh be caused by de ec s in F-ac in bu no in myosin le els.
The o ma ion o he ac omyosin cable depends on emodelling
o he adhe ens junc ions (AJs) (Ab eu-Blanco e al., 2012a;
Ca alho e al., 2014; Hun e e al., 2015; Ma subayashi e al.,
2015). A e wounding, he AJ p o ein E-cadhe in (E-cad) is
down egula ed a he cell bounda ies acing he wound, emaining
only a he la e al junc ions o leading-edge cells. To es whe he he
F-ac in de ec s obse ed in D p1 mu an s we e associa ed wi h
E-cad emodelling de ec s, we imaged con ol and D p1 mu an
emb yos exp essing ubi-E-cad::GFP (Oda and Tsuki a, 1999) and
mChe y::Moesin (Milla d and Ma in, 2008) be o e and upon
wounding. We obse ed no signi ican di e ences in E-cad le els o
con ol and D p1 mu an emb yos, ei he be o e o a e wounding
(Fig. S2).
In summa y, we p opose ha D p1 egula es F-ac in dynamics
du ing wound closu e, independen ly o AJs emodelling.
D p1 mu an s ha e al e ed cy osolic and mi ochond ial
calcium dynamics
The i s signal o be de ec ed upon wounding is an in acellula
Ca
2+
bu s (An unes e al., 2013; Razzell e al., 2013; Sammak e al.,
1997; Xu and Chisholm, 2011; Cla k e al., 2009). This Ca
2+
inc ease egula es many wound-healing s eps, including ac omyosin
cable o ma ion (An unes e al., 2013; Xu and Chisholm, 2011).
Mi ochond ia a e known egula o s o Ca
2+
homeos asis (Finkel
e al., 2015; Gio gi e al., 2008; Rizzu o e al., 2012), so we asked
whe he he F-ac in de ec s obse ed upon D p1 loss-o - unc ion
could esul om impai ed Ca
2+
dynamics.
We imaged emb yos exp essing he GCaMP6 Ca
2+
senso (Chen
e al., 2013) and measu ed Ca
2+
le els be o e and upon wounding
(Mo ie 2). As p e iously desc ibed (Razzell e al., 2013), wounding
induces a d ama ic and ansien inc ease in cy osolic Ca
2+
(cy Ca
2+
) le els in he cells a ound he wound, ha p opaga es in
a wa e-like manne eaching abou wo o i e cell laye s away om
he wound, depending on he wound size (Fig. 5A). In D p1 mu an
emb yos, he cy Ca
2+
bu s was less p onounced han in con ols
(Fig. 5B,C). Mo eo e , he a ea in which Ca
2+
inc ease was
obse ed was signi ican ly educed in D p1 mu an s compa ed o
con ols (Fig. 5D), sugges ing ha impai ing D p1 unc ion a ec s
no only Ca
2+
le els bu also he in e cellula Ca
2+
p opaga ion.
Mi ochond ia can up ake Ca
2+
om he cy osol, he eby
modula ing cy Ca
2+
(Szabadkai and Duchen, 2008). As
mi ochond ial mo phology in luences mi ochond ial Ca
2+
(mi Ca
2+
) le els (Bianchi e al., 2006; Ge encse and Adam-Vizi,
3
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
Fig. 2. D p1 emb yos show delayed wound healing. (A–C) Maximum Z p ojec ions o he epide mis o con ol (A), D p1 mild (B) and D p1 s ong (C)
mu an emb yos exp essing an F-ac in ma ke (GFP::Moesin) du ing wound closu e. In D p1 mild mu an s (B) wounds close slowe han in con ols (compa e
B wi h A). In D p1 s ong mu an s (C), al hough he wound con ac s in he i s 30–40 mpw, i hen s a s o expand (see 60–120 mpw). La e on, he wound
con ac s again and by 180 mpw i is almos closed. (D) G aph o a e age ini ial wound a ea in con ol and D p1 mu an emb yos (s ong and mild). (E) G aph
o wound closu e ime in con ol and D p1 mu an emb yos. Al hough he ini ial wound a ea o con ol and D p1 mu an s is simila (D), D p1 mu an s ake
longe o close hei wounds (E). Unpai ed - es wi h Welch’s co ec ion was pe o med o es o signi ican di e ences be ween g oups in D and E. ns, no
signi ican (P>0.05), ****P≤0.0001. (F) G aph o a e age wound a ea in con ol, D p1 mild and D p1 s ong mu an s o e ime. D p1 mild mu an wounds
close slowe han con ols. D p1 s ong mu an wounds ini ially con ac bu s a o expand a e 40 mpw. A 120–130 mpw wounds s a o con ac again.
(G) G aph o a e age wound a ea in con ol, D p1-mild and D p1-s ong mu an s in he i s 30 mpw, co esponding o he g ey egion in F. Signi ican
di e ences be ween con ol and D p1 mu an s s a a 4 mpw in D p1-mild mu an s and a 10 mpw in D p1-s ong mu an s. A wo-way ANOVA wi h a
Tukey’s co ec ion o mul iple compa isons was used o es o signi ican di e ences be ween g oups in G. As e isks (*) e e o con ol and D p1-mild
mu an s’compa isons. Numbe signs (#) e e o con ol and D p1-s ong mu an compa isons. Dashed lines depic an in e al o poin s in which he
compa ison be ween g oups gi es he same deg ee o s a is ical signi icance, gi en by he symbols abo e. #, P≤0.05, **; ##, P≤0.01; ***P≤0.001;
****P≤0.0001. E o ba s ep esen s.e.m. Numbe o emb yos pe condi ion is shown in each g aph. (H,I) Maximum Z p ojec ions o he epide mis o
con ol (H) and D p1-o e exp essing (UAS-D p1) (I) emb yos exp essing an F-ac in ma ke (mChe y::Moesin) ubiqui ously unde he con ol o he da-
Gal4 d i e du ing wound closu e. The wound-closu e dynamics a e simila be ween he wo g oups. (J) G aph o wound closu e ime in con ol and
UAS-D p1 emb yos. Unpai ed - es wi h Welch’s co ec ion was pe o med o es o signi ican di e ences be ween g oups. (K) G aph o a e age wound
a ea in con ol and UAS-D p1 emb yos o e ime. No signi ican di e ence was ound be ween con ol and D p1-o e exp essing emb yos, nei he in he
ime o wound closu e no he wound closu e dynamics. A wo-way ANOVA wi h a Sidak co ec ion o mul iple compa isons was used o es o
signi ican di e ences be ween g oups. ns, no signi ican (P>0.05). E o ba s ep esen s.e.m. Numbe o emb yos pe condi ion is shown in
each g aph. Scale ba s: 20 µm.
4
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
2005; Szabadkai e al., 2004), we examined con ol and D p1
mu an emb yos exp essing a mi ochond ia- a ge ed GCaMP3 Ca
2+
senso (mi o::GCaMP3, Lu as e al., 2012) be o e and upon
wounding (Mo ie 3). Simila o wha was de ec ed o cy Ca
2+
,we
obse ed an inc ease in mi Ca
2+
a ound he wound in bo h con ol
(Fig. 5E) and D p1 mu an (Fig. 5F) epide mis. Quan i ica ion o
mi Ca
2+
in ensi y showed a educed esponse upon wounding in
D p1 mu an emb yos compa ed o con ols (Fig. 5G, 0 mpw). This
educ ion is no as d ama ic as ha seen o cy Ca
2+
, which may be
due o he di e en sensi i i y o he cy Ca
2+
senso . No di e ences
we e ound in he a ea o inc eased mi Ca
2+
(Fig. 5H), sugges ing
ha only he cy Ca
2+
p opaga ion is a ec ed.
To u he in es iga e he impac o mi ochond ial ission
impai men in wound closu e, we es ed how ano he componen
o he ission machine y egula es wound healing. We knocked
down Fis1, a D p1 ecep o (Losón e al., 2013), by ubiqui ously
exp essing RNAi agains Fis1 and analysing i s e ec on Ca
2+
and
F-ac in dynamics upon wounding (Fig. S3). Bo h he cy Ca
2+
and
mi Ca
2+
bu s s upon wounding we e educed in Fis1 RNAi-
exp essing emb yos compa ed o con ols (Fig. S3A,B,C,E,F,G),
al hough no e ec on Ca
2+
p opaga ion ac oss he epide mis was
de ec ed (Fig. S3D,H). On he o he hand, no signi ican di e ences
in F-ac in le els we e ound be ween Fis1 knockdown and con ol
emb yos (Fig. S3I–K). These esul s suppo he hypo hesis ha
mi ochond ial ission egula es Ca
2+
dynamics du ing wound
closu e.
D p1 mu an s show educed mi ochond ial ROS p oduc ion
upon wounding
Ca
2+
-dependen ROS p oduc ion upon wounding has been shown o
egula e he F-ac in cy oskele on and he wound healing esponse
bo h in Caeno habdi is elegans and D osophila (Hun e e al.,
2018; Xu and Chisholm, 2014). Mi ochond ia a e known sou ces o
ROS (Mu phy, 2009) and hei abili y o p oduce hem can be
egula ed by mi ochond ial dynamics. F agmen a ion o he
mi ochond ial ne wo k is associa ed wi h inc eased ROS
p oduc ion and inhibi ion o mi ochond ial ission can educe
oxida i e s ess (Galloway e al., 2012). As ou esul s show ha
D p1 egula es Ca
2+
and F-ac in dynamics du ing wound healing,
we hypo hesized ha ROS p oduc ion migh be he link be ween
hese wo wound closu e e en s. To in es iga e whe he D p1
mu an s ha e al e ed ROS p oduc ion, we imaged con ol and D p1
mu an emb yos exp essing a gene ically-encoded a iome ic
mi ochond ial g een luo escen p o ein ha shi s i e e sibly o
ed luo escence when oxidized (Lake e al., 2014).
Upon wounding, we obse ed an inc ease in he ed signal, an
indica o o ROS p oduc ion, bo h in con ol and in D p1 mu an s
(Fig. 6A,B). To quan i y ROS p oduc ion, we calcula ed he ed:
g een luo escence in ensi y a io and obse ed ha he ROS le els
p oduced upon wounding we e signi ican ly lowe in D p1 mu an s
compa ed o con ol emb yos (Fig. 6C).
Ou esul s show ha impai ing mi ochond ial ission leads o
educed mi ochond ial ROS p oduc ion in esponse o wounding.
Fig. 3. Wounding does no induce majo changes in mi ochond ial mo phology. (A–Bi) Maximum Z p ojec ions o he epide mis o con ol (A,Ai) and
D p1 (B,Bi) mu an emb yos exp essing ubiqui ous mi ochond ial (EYFP::mi o, g een) and memb ane (PLCγPH::ChFP, magen a) ma ke s. XZ and YZ
sec ions a e shown below and on he igh , espec i ely. Inse s show a zoom o he dashed egion o he espec i e image. Scale ba : 10 µm. Inse scale ba :
5 µm. (C) G aph o a e age numbe o b anches in con ol and D p1 mu an s, be o e and upon wounding. Con ol and D p1 mu an s show simila numbe s o
mi ochond ial b anches. Wounding leads o a educ ion o b anching in con ols bu no in D p1 mu an s. (D) G aph o a e age mi ochond ial leng h in con ol
and D p1 mu an s, be o e and upon wounding. D p1 mu an mi ochond ial ne wo k has inc eased leng h, compa ed o con ols, bo h be o e and a e
wounding. Mi ochond ia om wounded epide mis show a simila leng h compa ed o unwounded, in bo h con ol and D p1 mu an s. A Mann-Whi ney U es
was used o es o signi ican di e ences be ween g oups. ns, no signi ican (P>0.05); *P=0.0275; ***P=0.0001; ****P<0.0001. n(con ol)=22 cells om nine
emb yos, n(D p1)=29 cells om 12 emb yos. E o ba s ep esen s.d., bw, be o e wounding; mpw, minu es pos wounding.
5
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open

The Rho GTPase e ec o Pkn is down egula ed a he wound
edge in D p1 mu an s
To u he in es iga e he mechanisms h ough which mi ochond ial
ission egula es he wound-healing esponse, we assessed whe he
known F-ac in modula o s we e dys egula ed in D p1 mu an s.
F-ac in dynamics and myosin con ac ili y a he wound edge ha e
been shown o be modula ed by membe s o he Rho amily o
GTPases, Rho1, Rac and Cdc42 (Ve boon and Pa khu s , 2015).
Mo eo e , he egula ion o hese GTPases is media ed by Ca
2+
and
ROS igge ed upon wounding (So o e al., 2013; Xu and Chisholm,
2014). Rho and i s e ec o s coo dina e ac omyosin cable o ma ion
and con ac ili y, while Rac and Cdc42 a e mo e impo an o o m
F-ac in p o usions ha coo dina e cell mig a ion a he leading edge
and he inal kni ing o he epi helium a he end o closu e (Ab eu-
Blanco e al., 2012b; Ve boon and Pa khu s , 2015).
As we obse ed de ec s in he F-ac in accumula ion a he wound
edge in D p1 mu an s, we decided o ocus on Rho e ec o s: Rho
kinase (Rok), Diaphanous (Dia) and P o ein kinase N (Pkn). Rok
ac i a es he myosin egula o y ligh chain, di ec ly by
phospho yla ion o by inac i a ion o myosin phospha ases, hus
p omo ing ac omyosin con ac ili y (Kimu a e al., 1996; Ueda
e al., 2002). Dia is a o min ha p omo es he polyme iza ion o
unb anched F-ac in (Na umiya e al., 1997). Pkn has been
implica ed in he egula ion o cell mig a ion, bu how i exe s i s
unc ion is no ully unde s ood (Lachmann e al., 2011; Lim e al.,
2004). We imaged emb yos exp essing GFP- agged e sions o he
Rok, Dia and Pkn (Fig. 7).
Fo he analysis o Rok and Pkn, we compa ed con ols wi h D p1
mu an emb yos, while o Dia we used RNAi o knockdown D p1.
Ou o he h ee Rho1 e ec o s, only Pkn showed a signi ican
educ ion in i s accumula ion a he wound edge in D p1 mu an s
when compa ed o con ols (Fig. 7D–F). The accumula ion o Rho1
e ec o s a he wound edge is a consequence o hei ac i a ion by
Rho1, so ou esul s sugges ha D p1 loss-o - unc ion leads o a
educ ion in Rho1 ac i i y upon wounding.
In summa y, we ha e iden i ied mi ochond ial-dynamics p o eins
as no el emb yonic wound-healing egula o s. Ou da a show ha
inhibi ion o mi ochond ial ission by D p1 loss-o - unc ion leads o
de ec s in Ca
2+
, ROS and F-ac in dynamics upon wounding,
culmina ing in wound-healing impai men .
DISCUSSION
Epi helial issues a e c i ical o p o ec us om he ex e nal
en i onmen (Lowe and Ande son, 2015). Unde s anding how
Fig. 4. D p1 mu an s show ac in de ec s
du ing wound closu e. (A–D) Maximum Z
p ojec ions o he epide mis o con ol (A,C)
and D p1 (B,D) mu an emb yos exp essing
an F-ac in (GFP::Moesin) (A,B) and a
Myosin (Zip::GFP) (C,D) ma ke be o e and
a e wounding. Images a e pseudo-colo ed
wi h a g adien o luo escence in ensi y,
anging om blue (low) o yellow (high).
Al hough no di e ences be ween con ols
and D p1 mu an s a e e iden be o e
wounding, D p1 mu an emb yos
accumula e less F-ac in a he wound edge
han con ols (compa e A,B). Myosin
accumula ion a he wound edge seems
simila be ween con ol and D p1 mu an
emb yos (compa e C,D). Scale ba : 20 µm.
(E) G aph o a e age F-ac in in ensi y a he
cell co ex be o e wounding and a he
wound edge. F-ac in le els a e signi ican ly
educed in D p1 mu an s a 10 and 20 mpw.
(F) G aph o a e age Myosin in ensi y a he
cell co ex be o e wounding and a he
wound edge. No signi ican di e ences
we e ound be ween con ol and D p1
mu an s. A wo-way ANOVA wi h a Sidak
co ec ion o mul iple compa isons was
used o es o signi ican di e ences
be ween g oups in E and F. Only signi ican
di e ences (P≤0.05) a e ep esen ed.
*P<0.05. E o ba s ep esen s.e.m.
Numbe o emb yos pe condi ion is shown
in each g aph. a.u., a bi a y uni s; bw,
be o e wounding; mpw, minu es pos
wounding.
6
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
epi helial issues d i e e icien wound epai is o he upmos
impo ance o he biomedical ield. In his s udy, we used a model
o simple epi helial wound closu e, he emb yonic epide mis o he
ui ly, o unco e he ole o mi ochond ial dynamics in he
wound-healing esponse. Li le is known abou how mi ochond ia
con ibu e o issue epai , besides hei in ol emen in he
p oduc ion o ROS, which in u n can egula e he wound-healing
p ocess (Hun e e al., 2018; Sanchez e al., 2018; Xu and Chisholm,
2014). Some da a suppo he conclusion ha mi ochond ial
dynamics can egula e cell mig a ion, an essen ial p ocess in
wound epai (Ko e al., 2017; Zhao e al., 2013), bu whe he his is
ele an o emb yonic wound epai had ne e been add essed.
We s a ed by pe o ming a gene ic sc een o unde s and whe he
he mi ochond ial dynamics machine y is equi ed o wound
closu e. Mi ochond ial dynamics is media ed by la ge GTPases,
namely D p1, ha media es mi ochond ial ission; and Opa1 and
M ns (Ma in D osophila) ha con ol he usion o he
mi ochond ial memb anes. Ou o he es ed ou Ma alleles and
miRNA-media ed knockdown, only Ma
J
mu an s showed an
inc eased numbe o open wounds compa ed o con ols. Mos o
he Ma alleles ha e no been cha ac e ized, so i is unclea why
only Ma
J
mu an s showed a wound-healing pheno ype, being
possible ha his is an unspeci ic e ec o his allele. I is also
concei able ha he pe du ance o ma e nal con ibu ion masks he
e ec s o Ma loss-o - unc ion in he case o he emaining Ma
alleles. On he o he hand, bo h Opa1 mu an alleles showed a
signi ican wound-healing pheno ype, a ou ing he hypo hesis ha
mi ochond ial usion is necessa y o p ope emb yonic wound
healing. Fu u e s udies ocusing on mi ochond ial usion a e
impo an o complemen ou esul s. Rega ding mi ochond ial
Fig. 5. D p1 mu an s show al e ed cy osolic and mi ochond ial Ca
2+
dynamics. (A,B) Maximum Z p ojec ions o he epide mis o con ol (A) and D p1
(B) mu an emb yos exp essing a cy osolic Ca
2+
senso (GCaMP6 ) be o e and a e wounding. Bo h con ol and D p1 mu an cells a ound he wound
d ama ically inc ease cy osolic Ca
2+
le els immedia ely upon wounding (0 mpw). In ensi y e u ns o p e-wound le els a e 15 min. Ca
2+
le els and a ea o
cells ha espond o he wound a e lowe in D p1 mu an s (B, 0 mpw) compa ed o con ols (A, 0 mpw). (C) G aph o cy osolic Ca
2+
in ensi y shows ha
cy osolic Ca
2+
is lowe in D p1 mu an s compa ed o con ols in he i s 2.5 mpw. (D) G aph o a e age a ea o ele a ed cy osolic Ca
2+
shows ha he Ca
2+
bu s a ea is lowe in D p1 mu an s compa ed o con ols om 0 o 1 mpw. (E,F) Maximum Z p ojec ions o he epide mis o con ol (E) and D p1 mu an
(F) emb yos exp essing a mi ochond ial Ca
2+
senso (mi o::GCaMP3) be o e and a e wounding. Wounding igge s an inc ease in mi ochond ial Ca
2+
le els
in bo h con ol and D p1 mu an cells a ound he wound (E,F a 0 mpw). (G) G aph o mi ochond ial Ca
2+
in ensi y in con ol and D p1 mu an s. D p1 mu an s
ha e a educed mi ochond ial Ca
2+
bu s a 0 mpw, compa ed o con ols. (H) G aph o a e age a ea o ele a ed mi ochond ial Ca
2+
in con ols and D p1
mu an emb yos. No signi ican di e ences we e ound be ween con ol and D p1 mu an s. Images a e pseudo-colo ed wi h a g adien o luo escence
in ensi y, anging om blue (low) o yellow (high). Dashed lines show he wound bounda ies. Scale ba : 20 µm. A wo-way ANOVA wi h a Sidak co ec ion o
mul iple compa isons was used o es o signi ican di e ences be ween g oups in C, D, G and F. Only signi ican di e ences a e ep esen ed: *P≤0.05,
**P≤0.01, ****P≤0.0001. E o ba s ep esen s.e.m. Numbe o emb yos pe condi ion is shown in each g aph. bw, be o e wounding; mpw, minu es pos
wounding.
7
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
ission, D p1
KG03815
, a loss o unc ion allele, showed he s onges
pheno ype compa ed wi h he o he D p1 mu an alleles. No much
de ail is a ailable abou how he di e en mu a ions a ec D p1
unc ion. Howe e , all di e en he e oallelic combina ions esul ed
in an inc ease in open wounds, which indica es ha he D p1 loss-
o - unc ion pheno ype is consis en . O he playe s o mi ochond ial
ission, Fis1 and Gdap1, a e also equi ed o wound healing, as
hei mu a ion leads o an inc eased numbe o unhealed wounds.
Al oge he , hese da a s ongly implica e mi ochond ial ission in
he egula ion o epi helial epai .
We ound ha he wound-closu e dynamics o D p1 mu an s is
signi ican ly a ec ed, as hese emb yos ake mo e han wo imes
longe o close he wounds han con ol emb yos. We obse ed wo
deg ees o wound closu e pheno ypes: a mild pheno ype,
cha ac e ized by a slowe wound closu e a e han in con ols;
and a s ong pheno ype, in which he wound a ea expands. I is
unclea why some wounds p esen his expansion phase, bu a
simila pheno ype has been desc ibed in mu an s o a componen
o he in e eb a e occluding junc ions (OJs). OJ dis up ion leads
o de ec s in he ac omyosin cable, cellula shapes and
ea angemen s as well as in issue mechanical p ope ies
(Ca alho e al., 2018). I would be in e es ing o explo e he
link be ween mi ochond ial dynamics and OJs in wound healing in
u u e s udies.
A ecen s udy in he C. elegans epide mis showed ha inc eased
mi ochond ial ission, ei he by impai ing usion o by d ug-
media ed ission induc ion, led o accele a ed wound closu e (Fu
e al., 2020). In e es ingly, we obse ed no di e ence in wound-
healing dynamics be ween D p1-o e exp essing emb yos and
con ols. Mo eo e , we obse ed no majo di e ences in
mi ochond ial mo phology be ween wounded and unwounded
emb yos, while in C. elegans wounding igge s mi ochond ial
ission (Fu e al., 2020). I seems ha mi ochond ial dynamics can
ha e di e en e ec s on wound healing, and ice e sa, depending
on he con ex . In he case o he D osophila emb yonic epide mis,
an elonga ed mi ochond ial ne wo k, such as obse ed in D p1
mu an s, is p ejudicial o wound healing, while inc easing he
agmen a ion o mi ochond ia has no impac on he wound-closu e
a e. We hus p opose ha he e is an op imal mi ochond ial
mo phology ha a ou s p ope wound closu e.
To unde s and he mechanism h ough which mi ochond ial
ission egula es wound healing, we cha ac e ized he known
wound-healing e en s in D p1 mu an s, such as he o ma ion o he
ac omyosin cable a he wound edge (Kieha e al., 2000; Wood
e al., 2002; Xu and Chisholm, 2011). D p1 mu an s show de ec s in
he F-ac in accumula ion a he wound edge, which may be he cause
o he obse ed wound-healing impai men .
The o ma ion o he ac omyosin cable is known o depend on
se e al ac o s, such as an in acellula Ca
2+
inc ease upon
wounding (An unes e al., 2013; Razzell e al., 2013; Xu and
Chisholm, 2011), he emodelling o he AJs (Ab eu-Blanco e al.,
2012a; Ca alho e al., 2014; Hun e e al., 2015), ROS p oduc ion
(Hun e e al., 2018; Xu and Chisholm, 2014) and he ac i a ion o
he Rho amily o GTPases and hei a ge s (B ock, 1996; Ve boon
and Pa khu s , 2015; Wood e al., 2002).
In addi ion o he es ablished cy Ca
2+
bu s , we also obse ed a
apid inc ease in mi Ca
2+
le els upon wounding, consis en wi h
wha has been seen in C. elegans wound epai (Xu and Chisholm,
2014), sugges ing ha , simila ly o he cy Ca
2+
bu s , his is a
conse ed esponse o issue inju y. Rema kably, bo h wound-
induced cy Ca
2+
and mi Ca
2+
bu s s we e educed upon D p1 loss-
o - unc ion, sugges ing ha mi ochond ial ission s ongly impac s
on Ca
2+
dynamics du ing wound closu e. I is well es ablished ha
mi ochond ia can ake up Ca
2+
om he cy osol, he eby modula ing
cy Ca
2+
le els (Szabadkai and Duchen, 2008). Howe e , he
ela ionship be ween cy Ca
2+
and mi Ca
2+
in he con ex o
wound healing is no clea . Inju y igge s Ca
2+
in lux om he
ex acellula en i onmen (An unes e al., 2013; Razzell e al., 2013;
Xu and Chisholm, 2011). The ele a ed cy Ca
2+
le els induce Ca
2+
elease om he endoplasmic e iculum (ER) media ed by he
inosi ol-3-phospha e (IP3) ecep o (IP3R), ollowed by
p opaga ion o Ca
2+
and IP3 o neighbou ing cells h ough gap
Fig. 6. D p1 mu an emb yos show educed mi ochond ial ROS p oduc ion upon wounding. (A,B) Maximum Z p ojec ions o he wound egion o
con ol (A) and D p1-mu an (B) emb yos exp essing he mi ochond ial ROS senso Mi oTime ubiqui ously, be o e and upon wounding (0 mpw). This
epo e gene encodes a p o ein ha i e e sibly changes i s luo escence spec um om g een o ed upon oxida ion. Images show he g een and ed
channels o each emb yo, as well as he ed:g een a io a e image p ocessing. Red:g een a io images a e pseudo-colo ed wi h a g adien o luo escence
in ensi y, anging om blue (low) o yellow (high). Scale ba : 10 µm. (C) G aph o he a e age ed:g een a io o con ol and D p1 mu an emb yos, be o e and
a e wounding (0 mpw). Red:g een a io is a measu e o ROS le els. P e-wound ROS le els a e simila be ween con ol and D p1 mu an s. Wounding
inc eases ROS le els, bo h in con ols and D p1 mu an s bu his inc ease is lowe in D p1 mu an emb yos compa ed o con ols. A wo-way ANOVA wi h a
Sidak co ec ion o mul iple compa isons was used o es o signi ican di e ences be ween g oups. ns, no signi ican , *P=0.0170, **P=0.0049,
****P<0.0001. E o ba s ep esen s.e.m. Numbe o emb yos pe condi ion is shown in he g aph.
8
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open
junc ions (Na ciso e al., 2015; Razzell e al., 2013; Res epo and
Basle , 2016). In o he cellula con ex s, mi ochond ia localize close
o he ER, o ming Ca
2+
signalling mic odomains. Ca
2+
up ake by
mi ochond ia educes he cy Ca
2+
le els close o he open ER
channels (local cy Ca
2+
), p e en ing hei Ca
2+
-dependen
inac i a ion. By con olling ER Ca
2+
channels ac i i y, mi Ca
2+
up ake a ec s global cy Ca
2+
(Billups and Fo sy he, 2002; Rizzu o
e al., 2012). Ou esul s lead us o specula e ha in D p1 mu an s
he Ca
2+
bu e ing capaci y o mi ochond ia is comp omised,
leading o IP3R inhibi ion and lowe global cy Ca
2+
le els. This
could also a ec he Ca
2+
wa e p opaga ion, as less Ca
2+
and/o IP3
would c oss gap junc ions. Indeed, D p1 mu an s showed a
educ ion in he a ea o Ca
2+
inc ease, which may indica e ha he
Ca
2+
wa e p opaga ion is a ec ed.
Mi ochond ial shape, numbe and dis ibu ion can a ec hei
con ac s wi h he ER and impac on mi Ca
2+
p opaga ion. Based on
p e ious s udies, he inhibi ion o D p1 wi h consequen elonga ed
mi ochond ial ne wo k should a ou he p oximi y wi h he ER and
acili a e mi Ca
2+
up ake (Cie i e al., 2018; Szabadkai e al., 2006).
He e, we obse e he opposi e e ec , sugges ing ha he ole o
D p1 on mi Ca
2+
egula ion may be con ex dependen . Fu he
wo k is needed o unde s and how D p1 egula es mi ochond ial
Ca
2+
up ake in he D osophila epide mis.
The wound-induced Ca
2+
bu s igge s he p oduc ion o
mi ochond ial ROS, which hen egula es he ac omyosin cable
o ma ion, ei he by ac i a ing RHO-1 o by egula ing AJ
emodelling (Hun e e al., 2018; Xu and Chisholm, 2014). Ou
esul s show ha he absence o unc ional D p1 leads o educed
Fig. 7. D p1 mu an s show educed
accumula ion o Pkn a he wound
edge, bu no o Rok o Dia. (A,B)
Maximum Z p ojec ions o con ol
(A) and D p1-mu an (B) emb yos
exp essing he Rok::GFP, be o e
wounding and a 10 and 30 mpw.
(C) G aph o he a e age Rok::GFP
in ensi y a he wound edge in
con ols and D p1 mu an s a 10 and
30 mpw, no malized o p e-wound
le els. No signi ican di e ences in
Rok accumula ion a he wound edge
we e ound be ween con ols and
D p1 mu an s. (D,E) Maximum Z
p ojec ions o con ol (D) and D p1
mu an s (E) exp essing he Dia::GFP,
be o e wounding and a 20 mpw.
(F) G aph o he a e age Dia::GFP
in ensi y a indi idual cell junc ions a
he wound edge in con ol and
D p1-mu an emb yos a 20 mpw,
no malized o hei espec i e
in ensi y be o e wounding. No
signi ican di e ences in Dia
localiza ion a he wound edge we e
ound be ween con ols and D p1
mu an s. (G,H) Maximum Z
p ojec ions o con ol (G) and
D p1-mu an (H) emb yos exp essing
he Pkn-GFP a 10 and 30 mpw.
(I) G aph o he a e age Pkn-GFP
in ensi y a he wound edge in
con ols and D p1 mu an s a 10 and
30 mpw, no malized o backg ound
le els. D p1 mu an s show a educed
accumula ion o Pkn a he wound
edge compa ed o con ols. E o
ba s ep esen s.e.m. A wo-way
ANOVA wi h a Sidak co ec ion o
mul iple compa isons was used o
es o signi ican di e ences
be ween g oups in C and I. A
Wilcoxon es was used o es
o signi ican di e ences
be ween g oups in F.
The numbe o emb yos pe condi ion
is shown in C and I. In F,
n(Con ol)=82 junc ions om nine
emb yos and n(D p1)=99 cell
junc ions om en emb yos. Scale
ba s: 20 µm. mpw, minu es pos
wounding.
9
RESEARCH ARTICLE Biology Open (2020) 9, bio048629. doi:10.1242/bio.048629
Biology Open