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RESEARCH ARTICLE
Drosophila Wash and the Wash regulatory complex functionin
nuclear envelope buddingJeffrey M. Verboon, Mitsutoshi Nakamura,
Kerri A. Davidson, Jacob R. Decker, Vivek Nandakumar andSusan M.
Parkhurst*
ABSTRACTNuclear envelope (NE) budding is a recently described
phenomenonwherein large macromolecular complexes are packaged
inside thenucleus and extruded through the nuclear membranes.
Although ageneral outline of the cellular events occurring during
NE budding isnow in place, little is yet known about the molecular
machinery andmechanisms underlying the physical aspects of NE bud
formation.Using a multidisciplinary approach, we identify Wash, its
regulatorycomplex (SHRC), capping protein and Arp2/3 as new
molecularcomponents involved in the physical aspects of NE bud
formation in aDrosophila model system. Interestingly, Wash affects
NE budding intwo ways: indirectly through general nuclear lamina
disruption via anSHRC-independent interaction with Lamin B leading
to inefficient NEbud formation, and directly by blocking NE bud
formation along withits SHRC, capping protein and Arp2/3. In
addition to NE buddingemerging as an important cellular process, it
shares many similaritieswith herpesvirus nuclear egress mechanisms,
suggesting newavenues for exploration in both normal and disease
biology.
KEY WORDS: WASH, Wash regulatory complex, Nuclear
envelopebudding, Nuclear exit, Arp2/3, Actin nucleation,
Vesicle-mediatednucleocytoplasmic transport
INTRODUCTIONTransport of macromolecules from the nucleus to the
cytoplasm isessential for all developmental processes, including
the regulationof differentiation and aging, and, when
mis-regulated, is associatedwith diseases and cancer (Grünwald et
al., 2011; Siddiqui andBorden, 2012; Tran et al., 2014). This
indispensable process hasbeen thought to occur exclusively through
nuclear pore complexes(NPCs), channels that regulate what exits
(and enters) the nucleus(Daneholt, 2001; Grünwald et al., 2011).
Recently, nuclearenvelope (NE) budding was identified as an
alternative pathwayfor nuclear exit, particularly for large
developmentally requiredribonucleoprotein (megaRNP) complexes that
would otherwiseneed to unfold/remodel to fit through the NPCs
(Fradkin andBudnik, 2016; Hatch and Hetzer, 2014, 2012; Jokhi et
al., 2013; Liet al., 2016; Parchure et al., 2017; Speese et al.,
2012). In thispathway, large macromolecule complexes, such as
megaRNPs, areencircled by the nuclear lamina (type-A and -B lamins)
and the
inner nuclear membrane (INM), pinched off from the INM, fusewith
the outer nuclear membrane and release the megaRNPs into
thecytoplasm (Fig. 1A–C). Strikingly, NE budding shares
manyfeatures with the nuclear egress mechanism used by
herpesviruses(Bigalke and Heldwein, 2016; Hagen et al., 2015; Lye
et al., 2017;Parchure et al., 2017; Roller and Baines, 2017). As
viruses oftenutilize pre-existing host pathways, the parallel
between nuclear exitof herpesvirus nucleocapsids and that of
megaRNPs and/or otherlarge cargoes suggests that NE budding may be
a general cellularmechanism (Fradkin and Budnik, 2016; Mettenleiter
et al., 2013;Parchure et al., 2017; Roller and Baines, 2017).
Indeed, thispathway has also been implicated in the removal of
obsoletemacromolecular complexes or other material (i.e. large
proteinaggregates or poly-ubiquitylated proteins) from the nucleus
(Jokhiet al., 2013; Ramaswami et al., 2013; Rose and Schlieker,
2012).
NE budding was first demonstrated in Drosophila
synapsedevelopment, proving to be essential for neuromuscular
junction(NMJ) integrity. In this context, a C-terminal fragment of
theWingless receptor Fz2, Fz2C, was shown to associate withmegaRNPs
that formed foci at the nuclear periphery and exitedthe nucleus by
budding through the nuclear envelope (Speese et al.,2012). Failure
of this process resulted in aberrant synapsedifferentiation and
impaired NMJ integrity (Speese et al., 2012).In a subsequent study,
the NE budding pathway was shown to benecessary for the nuclear
export of megaRNPs containingmitochondrial RNAs: disruption of NE
budding led todeterioration of mitochondrial integrity and
premature agingphenotypes that were similar to those associated
with laminmutations (i.e. laminopathies) (Jokhi et al., 2013; Li et
al., 2016).Similar endogenous perinuclear foci/buds have been
observed inplants and vertebrates, as well as otherDrosophila
tissues (i.e. larvalsalivary gland nuclei; Fig. 1B,C), suggesting
that cellular NEbudding is a widely conserved process (Hadek and
Swift, 1962;Hochstrasser and Sedat, 1987; LaMassa et al., 2018;
Panagaki et al.,2018 preprint; Parchure et al., 2017; Speese et
al., 2012; Szollosiand Szollosi, 1988).
The spectrum of processes requiring this non-canonical
nuclearexit pathway and the molecular machineries needed for this
process,which encompasses membrane deformations, traversal across
amembrane bilayer and nuclear envelope remodeling for a return
tohomeostasis, are largely unknown. One class of proteins that
areinvolved in membrane–cytoskeletal interactions and organization
isthe Wiskott–Aldrich Syndrome (WAS) protein family (Takenawaand
Suetsugu, 2007). WAS protein subfamilies are involved in awide
variety of essential cellular and developmental processes, aswell
as in pathogen infection and disease (Burianek and Soderling,2013;
Campellone and Welch, 2010; Rottner et al., 2010; Rottyet al.,
2013; Takenawa and Suetsugu, 2007). WAS family proteinspolymerize
branched actin through the Arp2/3 complex, and oftenfunction as
downstream effectors of Rho family GTPases
Handling Editor: Daniel BilladeauReceived 5 January 2020;
Accepted 28 May 2020
Basic Sciences Division, Fred Hutchinson Cancer Research Center,
Seattle,WA 98109, USA.
*Author for correspondence ([email protected])
J.M.V., 0000-0002-4454-6043; M.N., 0000-0001-7879-3176; K.A.D.,
0000-0001-9432-6359; J.R.D., 0000-0002-8116-6402; V.N.,
0000-0003-1293-5607; S.M.P.,0000-0001-5806-9930
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© 2020. Published by The Company of Biologists Ltd | Journal of
Cell Science (2020) 133, jcs243576. doi:10.1242/jcs.243576
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https://jcs.biologists.org/content/editor-bios/#billadeaumailto:[email protected]://orcid.org/0000-0002-4454-6043http://orcid.org/0000-0001-7879-3176http://orcid.org/0000-0001-9432-6359http://orcid.org/0000-0001-9432-6359http://orcid.org/0000-0002-8116-6402http://orcid.org/0000-0003-1293-5607http://orcid.org/0000-0001-5806-9930
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(Campellone andWelch, 2010; Takenawa and Suetsugu, 2007).
Weidentified Wash as a new WAS subfamily that is regulated in
acontext-dependent manner:Wash can bind directly to Rho1 GTPase(in
Drosophila) or it can function along with the multi-proteinWASH
regulatory complex [SHRC; comprised of SWIP,Strumpellin, FAM21 and
CCDC53 (also known as WASHC4,WASHC5, WASHC2 and WASHC3,
respectively, in mammals)](Derivery et al., 2009; Duleh and Welch,
2010; Gomez andBilladeau, 2009; Jia et al., 2010; Linardopoulou et
al., 2007; Liu
et al., 2009; Park et al., 2013; Veltman and Insall, 2010;
Verboonet al., 2018, 2015a,b). Wash regulation by Rho family
GTPasesoutside of Drosophila has not yet been described (Jia et
al., 2010);instead its regulation has been characterized in the
context of itsSHRC. WASH and its SHRC are evolutionarily conserved
and theirmis-regulation is linked to cancers and neurodegenerative
disorders(Leirdal et al., 2004; Linardopoulou et al., 2007; McGough
et al.,2014; Nordgard et al., 2008; Ropers et al., 2011; Ryder et
al., 2013;Türk et al., 2017; Valdmanis et al., 2007; Zavodszky et
al., 2014).
Fig. 1. Wash mutant nuclei lack NE buds. (A) Schematic of NE
budding steps: megaRNPs are assembled and Fz2C is incorporated (1),
the nuclearlamina is modified by aPKC (2), megaRNPs enter the
membrane deformation (3) and are encapsulated by inner nuclear
membrane (INM) (4), scission of the INM(5), NE bud fusion with the
outer nuclear membrane (ONM) (6) and megaRNP exit into cytoplasm
(7). (B–D″) Super-resolution micrograph projection (B) orsingle
slice (D) of wild-type larval salivary gland nucleus stained with
antibodies to Lamin B and Fz2C. Large Lamin B- and Fz2C-positive
puncta indicate NE buds(n, nucleus; c, cytoplasm). (B′) High
magnification view of highlighted region of B. (C) High
magnification view of NE buds. (D′,D″) High magnification view of
NEbuds in D. (E,F) TEM micrographs taken at 800× (E) and 8000× (F)
of wild-type larval salivary gland nucleus (cytoplasm false colored
in green). (G) TEMmicrographs (8000×) of NE buds (red arrowheads)
in wild-type larval salivary gland nuclei showing the distribution
of phenotypes observed (black arrows denoteNPCs). (H–I′) TEM
micrographs (800×; H,I or 8000×; H′,I′) of wash null (H,H′) and
wash RNAi (I,I′) larval salivary gland nuclei. (J–L″) Salivarygland
nuclei from wild-type (J–J″), wash null (K–K″), and wash RNAi
(L–L″) larvae stained for Lamin B and Fz2C. (M) Quantification of
the number of NEbuds per nucleus in larval salivary glands (the
size of the spots is proportional to number of nuclei with the
indicated number of buds). The mean±95% c.i.is shown. P-values are
indicated (Kruskal–Wallis test). Scale bars: 5 µm (B,D,E,H,I,J–L″);
0.5 µm in (B′,C,D′,D″,F,G,H′,I′).
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Importantly, we have shown that Wash is present in the
nucleuswhere it interacts directly with B-type lamins and, when
mutant,affects global nuclear organization/functions, as well as
causing anabnormal wrinkled nucleus morphology reminiscent of
thatobserved in diverse laminopathies (Verboon et al.,
2015b,c).Mammalian WASH proteins have also been shown to localize
tothe nucleus in developmental and cell-type specific
manners(Verboon et al., 2015c; Xia et al., 2014).Here, we show that
Wash, its SHRC, capping protein and Arp2/3
are also involved in the NE budding pathway, as mutants for any
ofthese components lack Fz2C foci/lamin buds and display the
NMJintegrity and premature aging phenotypes previously
associatedwith the loss of NE budding. In addition, we find that
CCDC53 andSWIP (SHRC subunits) colocalize with Fz2C foci/lamin
buds. Weshow that Wash is present in several independent
nuclearcomplexes. The nuclear interactions of Wash with its SHRC
areseparate from those with B-type Lamin, leading to effects
ondifferent subsets of nuclearWash functions.We also find
thatWash-dependent Arp2/3 actin nucleation activity is required for
proper NEbudding. We propose that Wash and its SHRC play a physical
and/or regulatory role in the process of NE budding.
RESULTSDrosophila Wash mutants lack NE budsDrosophila larval
salivary glands undergo NE budding; Fz2C focican be observed
surrounded by Lamin B at the nuclear peripheryusing confocal
microscopy (Fig. 1B–D″) (see also Fig. S1A) ortransmission electron
microscopy (TEM; Fig. 1E,F) (Hochstrasserand Sedat, 1987). Through
TEM, we observe that these megaRNPsare adjacent to a curved
evagination of the nuclear membrane,suggesting that there is likely
a membrane deformation event thatprecedes megaRNP encapsulation by
the INM (40%, n=78 NE-budding events) (Fig. 1G). Finally, Fz2C
antibody is a biomarkerfor nuclear buds (Speese et al., 2012), and
colocalizes with theseLamin foci (Fig. 1B–D″,J–J″).While staining
wash mutants for Lamin B, we observed notably
fewer Lamin ‘buds’ (for either A- or B-type Lamins) than in
wild-type nuclei (Verboon et al., 2015b). To determine whether
thisreduction of Lamin buds was a result of disrupted NE budding,
weco-labeled wash mutant larval salivary gland nuclei for Lamin
Band Fz2C. We generated wash mutant salivary glands in twodifferent
ways: (1) using an outcrossed homozygous null
allele,washΔ185hz(outX) (hereafter referred to as ‘wash null’;
Verboon et al.,2018), and (2) expressing an RNAi construct forwash
(HMC05339)specifically in the salivary gland using the GAL4-UAS
system(hereafter referred to as ‘wash RNAi’) (Fig. 1J–M). We find
thatwash null and wash RNAi larval salivary gland nuclei exhibit
anaverage of 0.17 (95% c.i. 0.1–0.25; n=101) and 0.1 (95% c.i.
0.04–0.16; n=104) Fz2C foci/NE buds, respectively, compared to
anaverage of 6.58 (95% c.i. 6.02–7.16) Fz2C foci/NE buds in
wildtype (n=101, P
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knockdown larval salivary glands are spherical
andmorphologically indistinct from wild-type nuclei, suggesting
thatthewrinkled nuclear phenotype observed inwashmutants (Verboonet
al., 2015b) is SHRC independent.Consistent with their loss of Fz2C
foci/NE buds, SHRC component
knockdowns also exhibit phenotypes associated with disrupted
NEbudding (Fig. 3S–V). Adult IFM from 21-day-old Strump orCCDC53
knockdown flies show a decrease in mitochondrialactivity, as
measured by determining ATP-Synthetase α levels
(Fig. 3S–U; Fig. S1G–H″). Strump RNAi1 and RNAi2 show a 3.6-and
3.5-fold decrease in activity compared to wild type (n=50 andn=50,
P
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nuclear lamina in Wash and SHRC mutants. Wild-type
larvalsalivary gland nuclei co-labeled with antibodies to Lamin B
andLamin C show tight Lamin B and Lamin C colocalization aroundthe
nuclear periphery (Fig. 4F,H,I,R). Strikingly, in nuclei fromwash
null and wash RNAi larval salivary glands, the signals fromthe two
Lamins are separated, with Lamin B lying closest to the
INM (consistent with it encoding a CAAX domain) and Lamin
Cpositioned closest to the chromatin (consistent with Lamin
Cassociation with chromatin) (Fig. 4G,G′,J–M,R). Importantly,nuclei
from SHRC subunit knockdowns (CCDC53 RNAi andStrump RNAi) have
Lamin B and Lamin C organization that isindistinguishable from wild
type (Fig. 4N–R).
Fig. 3. See next page for legend.
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As an orthogonal means of determining whether Wash
functionsseparately to the SHRC to influence the nuclear lamina,
weseparated protein complexes from fly Kc cell nuclear lysates
usingBlue Native PAGE. We observed that Wash is present in
multiplenuclear complexes, suggesting it is involved in multiple
nuclearprocesses (Fig. 4S). We find that one major
Wash-containingcomplex (∼900 kDa) overlaps with a complex
containing CCDC53,Strumpellin and FAM21 (Fig. 4S), whereas a
separate Wash-containing complex (∼450 kDa) overlaps with a Lamin
B-containing complex (Fig. 4S). To confirm that Wash formsdistinct
complexes with the SHRC components and with Lamin Bin the nucleus,
we immunoprecipitated Wash, three SHRC subunits(CCDC53, Strumpellin
and FAM21), Lamin B and Lamin C fromfly cell nuclear lysates and
probed the resulting western blots forSHRC and Lamin B (Fig. 4T).
In each case, Wash pulls down thecorresponding SHRC subunit, the
SHRC subunit pulls itself down,and SHRC complex members
co-immunoprecipitated with eachother (Fig. 4T). However, while Wash
pulls down Lamin B, none ofthe SHRC subunits co-immunoprecipitated
with Lamin B or LaminC. These results are consistent with Wash
forming distinct nuclearcomplexes with Lamin B and with the SHRC,
and suggest thatWash may have two roles in NE budding: an indirect
role on LaminB to regulate the Lamin meshwork, and a direct role
with its SHRCin the physical formation of NE buds.
Wash–SHRC interactions are required for NE budding,whereas
Wash–Lamin B interaction is required for nuclearmorphologyTo
further delineate Wash–Lamin B versus Wash–SHRC functionin NE
budding, we mapped the sites on the Wash protein thatfacilitate
binding to Lamin B and CCDC53 (the interaction of Washwith this
SHRC subunit has been reported to facilitate stable Wash–SHRC
formation; Derivery et al., 2009; Jia et al., 2010; Rottneret al.,
2010; Verboon et al., 2018), then made specific pointmutations that
functionally block these interactions (see Materials
and Methods) (Fig. S2A). The final constructs, harboring
pointmutations in the context of the full-length Wash protein,
designatedwashΔSHRC and washΔΔLamB, respectively, were examined
forinteraction specificity using GST pulldown assays (Fig.
S2B).Transgenics generated with these Wash point mutations
(seeMaterials and Methods), as well as a wild-type Wash
rescueconstruct (washWT), were individually crossed into the wash
nullhomozygous background so that the onlyWash activity comes
fromthe transgene under control of the endogenous wash promoter.
Thewild-type version of these transgenics (washWT) is expressed in
boththe nucleus and cytoplasm (Verboon et al., 2015b) and
rescuespreviously described wash mutant phenotypes, including
itspremature ooplasmic streaming phenotype in oocytes (Fig.
S2C,C′,G)(Liu et al., 2009; Verboon et al., 2018). The other two
wash pointmutation transgenic lines are similarly functional; as
expected,washΔΔLamB rescues the premature ooplasmic streaming
phenotype,whereas washΔSHRC did not (Fig. S2D–G). Interestingly,
while theknockdown of Wash downregulates expression of the SHRC and
viceversa (Derivery et al., 2009; Jia et al., 2010; Verboon et al.,
2018),Washand CCDC53 are still present in washΔSHRCmutant nuclei
despite theirinability to bind to each other (Fig. S2H–K″).
Salivary gland nuclei from washWT larvae show a rescue of
NEbudding: nuclei from these mutants show 6.58 (95% c.i.
6.22–6.95)buds per nuclei (n=102; P=0.895 compared to wild-type)
andnuclear morphology is indistinguishable from that in wild
type(Fig. 5A–A″,D). However, nuclei from washΔSHRC point
mutantsshow 0.5±0.1 buds per nucleus (n=101, P
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construct, indicating mitochondrial damage (Fig. 5P). Western
blotsof IFM lysates from washΔSHRC show an increase in
poly-ubiquitinaggregates compared to in the washWT construct,
indicating
mitochondrial damage (Fig. 5P). IFM lysates from washΔΔLamB
show a less-pronounced intermediate increase in
poly-ubiquitinaggregates (Fig. 5P). Larval body wall muscle from
washΔΔLamB
Fig. 4. Alteration of the nuclear lamina structure can reduce,
but does not eliminate, NE budding. (A) Confocal micrograph
projection of Lamin BRNAi larvalsalivary gland nucleus co-labeled
with antibodies to Lamin C and Fz2C. (B) Quantification of number
of NE buds in control,wash RNAi, and Lamin B RNAi nucleisubunit
(the size of the spots is proportional to number of nuclei with the
indicated number of buds). (C) Confocal micrograph projection of
adult IFM fromLamin B RNAi flies aged 21 days stained with
ATP-Synthetase α (ATP-Syn) and with phalloidin. (D) Quantification
of ATP-Syn α fluorescence intensity in adultIFM. (E) Western blot
of adult IFM lysates from control and Lamin B RNAi flies aged 21
days showing poly-ubiquitin aggregate protein levels, and actin
loadingcontrol. (F–G′) Single slice confocal micrographs of nuclear
periphery from wild-type (F) and wash null (G,G′). In F and G,
arrows indicate inward projecting NEbuds and arrowheads indicate
outward projecting NE buds. Arrowheads in G′ indicate areas of
Lamin B/Lamin C separation. (H–Q) Single slice
super-resolutionmicrographs of larval salivary gland nuclei
(H,J,L,N,P) and line plots of regions indicated by dashed lines
(I,K,M,O,Q) from wild-type (H,I), wash null (J,K), washRNAi (L,M),
CCDC53 RNAi (N,O), and Strumpellin RNAi (P,Q) showing Lamin B and
Lamin C organization at the nuclear periphery. Arrowheads in J and
Lindicate areas of Lamin B/Lamin C separation. Lamin C also shows
lower level uniform distribution within the nucleus. (R)
Quantification of the percentage ofsalivary gland nuclei showing
Lamin B and Lamin C separation. (S) Western blots from Blue Native
PAGE of wild-type nuclear extracts probed with antibodies toWash,
SHRC subunits (CCDC53, Strumpellin and FAM21), and Lamin B. A
putative ∼900 kDa complex with Wash and SHRC (red line) and ∼450
kDa complexwithWash and Lamin B (blue lines) are indicated. (T)
Western blots of immunoprecipitations from wild-type nuclear
extracts with no primary antibody included (no1° AB), a
non-specific antibody (9e10), Wash, CCDC53, Strumpellin, FAM21,
Lamin B and Lamin C. Blots were probed with antibodies to CCDC53,
Strumpellin,FAM21 and Lamin B as indicated. P-values are indicated
[two-tailed Fisher’s exact test (R); two-tailed Student’s t-test
(D); Kruskal–Wallis test (B)]. The mean±95% c.i. is shown for B and
D. Scale bars: 5 µm (A–A″); 1 µm (F–H,J,L,N,P); 10 µm (C–C″). n,
nucleus; c, cytoplasm.
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mutants exhibit an increased number of ghost boutons: 3.6
ghostboutons per 100 boutons (n=1004) compared to control
(washWT;1.4 ghost boutons per 100 boutons; n=960; P=0.0021),
consistentwith the intermediate Fz2C foci/NE bud phenotype
displayed bythese mutants (Fig. 5Q–S). Thus, these Wash point
mutationsclearly demonstrate that Wash has dual roles in NE
budding.
Wash is required for the initial steps of NE bud formationTo
understand how Wash and its SHRC fits into the physical NEbudding
pathway, we examined its relationship to previouslyproposed
cellular events comprising NE budding. Based on the lackof NE buds
observed in aPKC mutants and the requirement forlamin
phosphorylation in herpesvirus nuclear egress, aPKC hasbeen
proposed to phosphorylate Lamin thereby seeding sites thatcan
undergo NE budding (Speese et al., 2012). As both wash andaPKC
mutations almost completely abolish NE budding, epistasis
experiments are not feasible. In addition, knockdown
ofDrosophilaWash downregulates expression of the SHRC members and
viceversa (Fig. S1I–L″; Verboon et al., 2018). However, because
theSHRC member CCDC53 accumulates at budding sites, we lookedat
localization of CCDC53 in aPKC knockdown salivary glandnuclei.
Consistent with the proposed role for aPKC, CCDC53 doesnot
accumulate in foci, nor are NE buds present in this
background,which suggests that Wash and the SHRC function after
aPKC in thisprocess (Fig. 6A–A″).
Torsin, an AAA ATPase, has been proposed to function in NEbud
scission from the inner nuclear membrane (INM) as Torsinaccumulates
at sites of contact between the megaRNP and INM, andtorsin mutants
exhibit accumulation of INM-tethered megaRNPswithin the perinuclear
space (Jokhi et al., 2013). To determinewhether Wash and its SHRC
are required for the initial steps of NEbud formation, and in
particular before these buds pinch off from the
Fig. 5. Wash point mutants show separation of phenotypes for
specific Wash activities. (A–C″) Confocal micrograph projections of
larval salivarygland nuclei from washWT (A–A″), washΔΔLamB (B–B″),
and washΔSHRC (C–C″) stained for Lamin B and Fz2C. (D)
Quantification of NE buds per nucleus inlarval salivary gland
nuclei (the size of the spots is proportional to number of nuclei
with the indicated number of buds). (E–J) Single slice
super-resolutionmicrographs of larval salivary gland nuclei (E,G,I)
and line plots of regions indicated by dashed lines (F,H,J) from
washWT (E,F),washΔΔLamB (G,H), and washΔSHRC
(I,J) showing Lamin B and Lamin C organization at the nuclear
periphery. Arrowheads in G indicate areas of Lamin B/Lamin C
separation; n, nucleus; c, cytoplasm.(K) Quantification of the
percentage of salivary gland nuclei showing Lamin B and Lamin C
separation. (L–N″) Confocal micrograph projections of adult IFM
fromwashWT (L–L″), washΔΔLamB (M–M″) and washΔSHRC (N–N″) flies
aged 21 days stained for the activity-dependent mitochondrial
marker ATP-Synthetase α(ATP-Syn) and with phalloidin. (O)
Quantification of ATP-Syn α fluorescence intensity from adult IFMs.
(P) Western blot of adult IFM lysates from washWT,washΔΔLamB,
washΔSHRC and washΔArp2/3 flies aged 21 days showing poly-ubiquitin
aggregate protein levels, and actin loading control. (Q,R) Confocal
micrographprojection of synaptic boutons from washWT (Q) and
washΔΔLamB (R) larval body wall muscle co-stained for HRP and DLG.
Ghost boutons are indicated (arrows).(S)Quantification of ghost
bouton frequency in larval bodywallmuscle neurons.P-values are
indicated [two-tailed Fisher’s exact test (K,S); two-tailed
Student’s t-test(O); Kruskal–Wallis test (D)]. The mean±95% c.i. is
shown for D and O. Scale bars: 5 µm (A–C″); 1 µm (E,G,I); 10 µm
(L–N″); 20 µm (Q,R).
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INM, we also looked at the localization of CCDC53 in torsin
RNAiknockdown salivary gland nuclei. Consistent with
establishedtorsin phenotypes, we find increased numbers of NE buds,
andimportantly, these buds colocalizewith CCDC53 foci (Fig.
6B–B″),suggesting that torsin acts afterWash/SHRC role in this
pathway. Toverify this, we looked at epistasis by generating wash
and torsindouble RNAi knockdown larval salivary gland nuclei and
co-staining them for Lamin B and Fz2C.We found thatwash and
torsindouble RNAi larval salivary gland nuclei exhibited an average
of0.27 (95% c.i. 0.21−0.34) Fz2C foci/NE buds (n=220), comparedto
an average of 6.58 (95% c.i. 6.02−7.16) Fz2C foci/NE buds inwild
type (n=100, P
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(n=50), as assayed by determining the level of ATP-Synthetase
α(Fig. 7N–O). Additionally, western blots of IFM lysates
fromwashΔArp2/3 show an increase in poly-ubiquitin
aggregates,indicating mitochondrial damage (Fig. 5P). As might be
expected,washΔArp2/3 does not exhibit separated Lamin B and Lamin
C
meshes (Fig. 7P–R). Taken together, our data are consistent with
amodel in which Wash and the SHRC work upstream of the
Arp2/3complex to promote NE budding, and that Wash functions
withLamin B and independently of the SHRC and Arp2/3 to
affectnuclear morphology.
Fig. 7. Arp2/3 activity is required for NE budding. (A,B)
Western blot of Kc cell cytoplasmic and nuclear extracts probed
with antibodies to Arp2/3 subunit Arp3(A) or Arpc1 (B). (C–C″)
Confocal micrograph projections of wild-type larval salivary gland
nucleus showing Arpc1 colocalization with Lamin B puncta at
thenuclear periphery (arrows, box). (D–D″) High magnification views
of Arpc1 enrichment around NE bud boxed in C′ (arrows). (E–F″)
Confocal micrographprojections of Arp3 RNAi (E–E″) and Arpc1 RNAi
(F–F″) salivary gland nuclei stained for Lamin B and Fz2C. (G)
Quantification of NE buds per nucleusin larval salivary glands (the
size of the spots is proportional to number of nuclei with the
indicated number of buds). (H–I″) Confocal micrograph projections
ofadult IFM fromArp3 RNAi (H–H″) and Arpc1 RNAi (I–I″) flies aged
21 days stained for ATP-Synthetase α (ATP-Syn) and with phalloidin.
(J) Quantification of ATP-Syn α fluorescence intensity in adult
IFM. (K) Western blot of adult IFM lysates from control, Arp3 RNAi
and Arpc1 RNAi flies aged 21 days showingpoly-ubiquitin aggregate
protein levels, and actin loading control. (L–L″) Confocal
micrograph projections of washΔArp2/3 larval salivary gland nucleus
stained forLamin B and Fz2C. (M) Quantification of NE buds per
nucleus in larval salivary glands glands (the size of the spots is
proportional to number of nucleiwith the indicated number of buds).
(N–N″) Confocal micrograph projections of adult IFM from
washΔArp2/3 flies aged 21 days stained for ATP-Syn α and
withphalloidin. (O) Quantification of ATP-Syn α fluorescence
intensity in adult IFM. (P,Q) Single slice super-resolution
micrographs of larval salivary gland nucleus (P)and line plot of
region indicated (Q) from washΔArp2/3 showing Lamin B and Lamin C
organization at the nuclear periphery. n, nucleus; c, cytoplasm.(R)
Quantification of the percentage of salivary gland nuclei showing
Lamin B and Lamin C separation. P-values indicated [two-tailed
Fisher’s exact test (P);two-tailed Student’s t-test (H,M);
Kruskal–Wallis test (E,K)]. The mean±95% c.i. is shown for G, J, M
and O. Scale bars: 5 µm (C–C″,E–F″,L–L″); 1 µm (P);10 µm
(H–I″,N–N″); 0.5 µm (D–D″).
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Capping protein is required for NE buddingA subset of Wash–SHRC
complexes are known to associate withthe barbed end-binding
heterodimeric capping protein (CapZα andCapZβ), which in turn have
been shown to exhibit context-dependent functions ranging from
promoting Arp2/3-dependentactin assemblies to inhibiting FAM21
activity (Amândio et al.,2014; Derivery et al., 2009; Edwards et
al., 2014; Park et al., 2013;Rottner et al., 2010). To determine
whether capping proteins (Cpaand Cpb in Drosophila) play a role in
NE budding, we generatedRNAi knockdowns of cpa and cpb in larval
salivary glands. Wefind that these knockdown nuclei show a decrease
in NE buds withon average 1.87 (95% c.i. 1.61−2.13; n=134) and 1.11
(95% c.i.0.92−1.28; n=133) Fz2C foci/NE buds, respectively,
compared to6.58 (95% c.i. 6.02−7.16) Fz2C foci/NE buds per nucleus
in wildtype (n=102; P
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have been associated with the accumulation of
RNA–proteinaggregates in the nucleus, NE budding may be part of
theendogenous cellular pathway for removing such
aggregates/megaRNPs from the nucleus in normal cells (Laudermilch
et al.,2016; Parchure et al., 2017; Ramaswami et al., 2013).
Nuclear buds or NPCs?The parallels between the mechanism of NE
budding and herpesvirusnuclear egress, as well as the presence of
similar endogenousperinuclear foci/buds in other plant and animal
nuclei, has suggestedthat NE budding is a conserved endogenous
cellular pathway fornuclear export (Fradkin and Budnik, 2016;
Panagaki et al., 2018preprint; Parchure et al., 2017; Speese et
al., 2012). Indeed, INM-encapsulated electron-dense granules have
been identified in yeastand Torsin-deficient HeLa cells, and these
show similarities to theFz2C foci/NE buds observed in Drosophila
muscle and salivarygland nuclei (Laudermilch et al., 2016; Parchure
et al., 2017;Websteret al., 2014). While the full relationship
between NPCs and NE budsis not yet known, one important difference
is that the yeast and HeLanuclear granules observed are much
smaller (∼120 nm) than Fz2Cfoci/NE buds (∼500 nm) (see Fig. 1F,G).
Our identification of Washand SHRC, proteins with the capability of
remodeling corticalcytoskeleton and/or membranes, in the physical
aspects of NEbudding lend support for NE budding being an
alternativeendogenous nuclear exit pathway.
The nuclear lamina and NE buddingNE budding has been proposed to
occur at sites along the INMwhere the nuclear lamina is modified by
aPKC phosphorylation(Fradkin and Budnik, 2016; Parchure et al.,
2017; Speese et al.,2012). Both A- and B-type lamins play a role in
NE budding and arethought to be the target of aPKC phosphorylation
within the nuclearlamina, similar to the PKC-mediated
phosphorylation of lamins thatprecedes lamina disassembly in
mitotic NE breakdown (Güttingeret al., 2009), apoptosis (Cross et
al., 2000) or during viral capsidnuclear egress (Park and Baines,
2006). Viral NE budding requires avirus-encoded nuclear egress
complex (NEC), which has beenimplicated in the recruitment of
kinases to the INM (Bigalke andHeldwein, 2016; Mettenleiter et al.,
2013; Zeev-Ben-Mordehaiet al., 2015). Cellular counterparts for
these virally encoded NECproteins have not yet been identified. It
is also not yet known howthis kinase activity is restricted to
specific sites along the nuclearlamina or how those specific sites
are selected.We have previously shown that Wash interacts directly
with
Lamin B and that loss of nuclear Wash results in a wrinkled
nuclearmorphology reminiscent of that observed in
laminopathies(Verboon et al., 2015b). We reasoned that
Wash-mediateddisruption of the nuclear lamina may account for its
NE-buddingphenotypes. Consistent with this idea, we find Lamin B
knockdownnuclei and nuclei from a wash point mutant that disrupts
theinteraction of Wash with Lamin B (washΔΔLamB) exhibit a
wrinklednuclear morphology, reduced Fz2C foci/NE buds and
NE-budding-associated phenotypes, albeit not as strong as those
observed inWash or SHRC mutants.Lamin A/C and Lamin B isoforms form
homotypic meshworks
that interact among themselves (in as yet unknown ways), and
thatare somehow linked to integral membrane proteins of the INM
andto the chromatin adjoining the INM (Shimi et al.,
2015).Intriguingly, our data suggests that these lamin homotypic
meshesare likely layered, rather than interwoven, and that Wash
affects theanchoring of these lamin homotypic meshes to each other
and/orthe INM. Lamin knockdown or disruption of the Wash–Lamin
B
interaction leads to separated lamin isoform meshes andwrinkled
nuclear morphology that are not observed in SHRCand Arp2/3
knockdown nuclei, suggesting that Wash can alsoaffect NE budding by
a means independent of disrupted globalnuclear lamina integrity.
Interestingly, the functions of Washmediated with the SHRC and with
Lamin B involve separatenuclear complexes. Consistent with this, we
have shownpreviously that Drosophila Wash encodes several
independentbiochemical activities (actin nucleation, actin
bundling, MTbundling and actin–MT crosslinking) and that the use of
theseactivities is context dependent (Liu et al., 2009; Verboon et
al.,2018, 2015a,b). In particular, when Wash interacts with
Lamin,it does not require an association with SHRC or
Arp2/3(Verboon et al., 2015b). We suggest that the interaction
ofWash with Lamin B is required for organizing the nuclear
laminaand likely requires the actin bundling and/or
cross-linkingactivities of Wash rather than its actin nucleation
activity, suchthat wash mutants that cannot bind Lamin result in
separation ofthe Lamin isoform meshes from each other. Taken
together, ourdata suggest that loss of the interaction between Wash
and LaminB makes NE budding inefficient by generally disrupting
thenuclear lamina, rather than directly disrupting NE budformation.
The role of aPKC in NE budding may also besomewhat indirect by
generally disrupting the nuclear laminathereby reducing the
efficiency of NE bud formation.Alternatively, aPKC may target Wash:
WASH phosphorylationby Src kinases has been shown to be necessary
for regulating NKcell cytotoxicity (Huang et al., 2016).
A physical role for Wash in NE buddingFor bud
formation/envelopment of a megaRNP or macromolecularcargo to occur,
the INM must interact with its underlying corticalnucleoskeleton to
allow the INM deformation/curvature necessaryto form the physical
NE bud. Force must also be generated thatallows the bud to extend
into the perinuclear space, as well as for thescission of the
nascent bud. In the cytoplasm, WAS family proteinsare often
involved in membrane–cortical cytoskeleton-coupledprocesses,
including both ‘inward’ membrane deformations (i.e.endocytosis) and
‘outward’membrane deformations (i.e. exocytosisand cell
protrusions), that are required for signal/environmentsensing and
cell movement during normal development, as well asduring
pathological conditions (Burianek and Soderling, 2013;Campellone
and Welch, 2010; Rottner et al., 2010; Stradal et al.,2004;
Takenawa and Suetsugu, 2007). Mammalian WASH, inparticular, has
been implicated in endosome biogenesis and/orsorting in the
cytoplasm, where it, along with its SHRC, drivesArp2/3-dependent
actin assembly to influence endosometrafficking, remodels membrane,
and facilitates membranescission (Derivery et al., 2009; Duleh and
Welch, 2010; Gomezand Billadeau, 2009; Rottner et al., 2010;
Simonetti and Cullen,2019). Thus, Wash encodes the biochemical
properties needed toregulate the membrane deformation/curvature
necessary to form theNE bud and/or play a role in generating the
forces necessary to pinchoff the NE bud from the INM. Consistent
with Wash playing a rolein the physical production of a NE bud, we
find that Wash acts priorto Torsin, a protein that is implicated in
NE bud scission from theINM, and it requires its actin nucleation
activity.
We have identified Wash and its SHRC as new players in
thecellular machinery required for the newly described endogenous
NEbudding pathway. Our data suggest that Wash is involved in
twonuclear functions that can affect NE budding. (1) Wash is
required tomaintain the organization of Lamin isoforms relative to
each other
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and the INM through its direct physical interaction with LaminB.
This Wash activity is SHRC and Arp2/3 independent, and is likelya
non-specific mechanism because global disruption of the
nuclearlamina/nuclear envelope would indirectly affect many
nuclearprocesses, including NE budding. (2) Wash is specifically
requiredfor NE bud formation. This Wash activity is SHRC and
Arp2/3dependent. While the focus of NE budding research to date
hascentered on the composition of the megaRNPs and the spectrum
ofcellular/developmental processes requiring NE budding, Washand
the SHRC are likely involved in the physical aspects of NEbudding.
Thus,Wash and SHRC provide a molecular entry into thephysical
machinery that underlies NE budding. In the future, it willbe
exciting to further explore the roles of Wash in NE budding, andto
determine how it functions to get macromolecular complexesthrough
the INM, and how closely these nuclear roles parallelthose in the
cytoplasm.
MATERIALS AND METHODSReagents and resourcesSpecific information
for all of the reagents and resources used in this studyare given
in Table S1.
Fly stocks and geneticsFlies were cultured and crossed at 25°C
on yeast-cornmeal-molasses-maltextract medium. Flies used in this
study are listed in Table S1. All fly stockswere treated with
tetracycline and then tested by PCR to ensure that they didnot
harbor Wolbachia. RNAi knockdowns were driven in the salivaryglands
by the GAL4-UAS system using the P{Sgs3-GAL4.PD} driver(Bloomington
Drosophila Stock Center, stock #6870). RNAi knockdownswere driven
in the indirect flight muscle by the GAL4-UAS system usingthe
P{w[+mC]=Mhc-GAL4.K}2 driver (Bloomington DrosophilaStock Center,
stock #55133). RNAi knockdowns were driven in thelarval body wall
muscle by the GAL4-UAS system using theP{w[+mW.hs]=GawB}BG487
driver (Bloomington Drosophila StockCenter, stock #51634). The
washΔ185 deletion allele was kept as acontinuously outcrossed stock
(Verboon et al., 2018).
Construction of Wash point mutant transgenic linesThe residues
required for the interaction of Wash with Lamin and CCDC53were
mapped by successive GST pulldown assays using fragments of
Washprotein, followed by specific point or substitution mutations
in the context ofthe full-length Wash protein (as detailed in Figs
6A and 7J). Point and/orsubstitution mutations were confirmed by
sequencing.
A 2.9-kb genomic fragment encompassing the entire wash gene
wasamplified by PCR, then subcloned into the Casper 4
transformation vectorby adding KpnI (5′) and BamHI (3′) restriction
sites. GFP was insertedN-terminal to theWash ATG by PCR
(GFP–WashWT). TheWash portion ofthis construct was swapped with the
point and/or substitution mutationsdescribed above to generate
GFP–WashΔSHRC, GFP–WashΔΔLamB andGFP–WashΔArp2/3.
These constructs were used to make germline transformants as
previouslydescribed (Spradling, 1986). Transgenic lines that mapped
to chromosome 2and that had non-lethal insertions were kept. The
resulting transgeniclines (P{w+; GFP-WashWT}, P{w+; GFP-WashΔSHRC},
P{w+; GFP-WashΔΔLamB}, and P{w+; GFP-WashΔArp2/3}) were recombined
onto thewashΔ185 null chromosome to assess the contribution of the
particular Washtransgene. The resulting recombinants (washΔ185
P{w+; GFP-WashWT}) areessentially gene replacements, as wash
activity is only provided by thetransgene. These transgenes do not
rely on overexpression, but rather on thespatial and temporal
expression driven by the endogenous wash promoteritself. We
analyzed a minimum of three lines per construct and checked
alllines to confirm that the levels and spatial distribution of
their expression isindistinguishable from that in wild type. The
wild-type version of thistransgene (washΔ185 P{w+; GFP-WashWT})
rescues the phenotypesassociated with the outcrossed washΔ185
mutation (Liu et al., 2009;Verboon et al., 2018, 2015a,b).
Antibody production and characterizationGuinea pig antibodies
were raised against bacterially double-tagged Fz2Cprotein at Pocono
Rabbit Farm & Laboratory (Canadensis, PA) using theirstandard
protocols. For expression of Fz2C, a DNA fragment coveringamino
acids 612–694 of Fz2 was generated by PCR and cloned into amodified
‘double-tag’ pGEX vector (Liu et al., 2009). Protein was purifiedas
described previously (Rosales-Nieves et al., 2006). Western
blotting wasused to confirm antibody specificity using Fz2 purified
protein, Kc cell,ovary and S2 whole-cell extracts. Antibody
generation for Wash and SHRCsubunits was described previously
(Rodriguez-Mesa et al., 2012; Verboonet al., 2015a).
Lysate preparationDrosophila cytoplasmic and nuclear extracts
were made from Kc167 cells.Briefly, cells were grown to confluence
in 500 ml spinflasks, pelleted for5 min at 500 g, resuspended in
100 ml cold 1× PBS and re-pelleted for 5 minat 500 g. Cell pellets
were flash frozen in liquid nitrogen. Cells wereresuspended in
sucrose buffer (0.32 M sucrose, 3 mM CaCl2, 2 mMMgAc,0.1 mMEDTA,
1.5%NP40) with 2× protease inhibitors [Complete proteaseinhibitor
(EDTA free; Sigma, St Louis, MO), 2 mM PMSF and 1 mMNa3VO4] and 2×
phosphatase inhibitors [PhosSTOP; Sigma, St Louis, MO]at 100 µl per
105 cells and incubated on ice for 30 min. Lysate was
douncehomogenized 10× on ice. Lysate was then centrifuged 10 min at
2900 g at4°C, nuclei formed a pellet and supernatant was
cytoplasmic extract. Lipidswere removed from the top of cytoplasmic
extract using a sterile swab, thenthe cytoplasmic fraction was
removed and centrifuged for 10 min at 3300 gat 4°C. Cytoplasmic
supernatant was removed and one-tenth of thesupernatant volume of
11× RIPA was added. Cytoplasmic extract wasaliquoted and flash
frozen. Nuclear pellet was resuspended with sucrosebuffer with
protease/phosphatase inhibitors and NP40 and re-dounced.Nuclear
lysate was then centrifuged for 10 min at 3300 g at 4°C,
andsupernatant was discarded. The nuclear pellet was resuspended in
sucrosebuffer without NP40 and centrifuged for 20 min at 3300 g at
4°C. Thesupernatant was discarded and nuclear pellet was
resuspended in 2.5 ml ofbuffer (20 mM HEPES pH 7.9, 0.5 mM EDTA,
100 mM KCl and 10%glycerol) per liter of cells used. DNAwas
degraded usingMNase in a 37°Cwater bath for 10 min. 20 μl of 500
mMEDTA per 500 μl lysate was addedand incubated on ice for 5 min.
Lysate was then nutated for 2 h at 4°C.Lysate was sonicated using a
Sonic Dismembrator (Model 60; FisherScientific) at setting 3.5 with
10 s per pulse for 15 min. Lysate wasclarified with a 15 min
centrifugation at 25,000 g, aliquoted and flashfrozen.
Adult indirect flight muscle (IFM) lysate was made from
21-day-oldflies in RIPA buffer (HEPES 50 mM pH 7.5, NaCl 150 nM, 1%
NP40,0.5% deoxycholate sodium salt, EDTA 5 mM). IFMs were dissected
fromflies in cold PBS. PBS was removed and 200 μl of 1× RIPA buffer
with 2×protease inhibitors (Complete EDTA free protease inhibitor;
Sigma) per 20IFMs was added. Lysates were homogenized with an
Eppendorfmicrocentrifuge homogenizing pestle on ice. Lysates were
thensonicated with a probe sonicator on setting 3 for three 10-s
pulses andcentrifuged at 25,000 g for 30 min at 4°C. Supernatant
was removed andMgCl2 was added to 2 mM and 8 μl of Benzonase
(Millipore) was addedper 200 μl of lysate. Lysates were nutated at
room temperature for 15 min.Lysates were centrifuged at 16,000 g
for 30 min at 4°C. Supernatant wasaliquoted and flash frozen.
Western blottingCytoplasmic and nuclear purity of lysates was
assayed using β-Tubulin (E7,1:2000, Developmental Studies Hybridoma
Bank) and Lamin B (monoclonal67.10, 1:1000, Developmental Studies
Hybridoma Bank) antibodies. Lysatesamples were normalized to a
loading control (Actin Clone C4, 1:2500; MPBiochemicals) and then
blotted according to standard procedures. Thefollowing antibodies
were used: anti-Rho1 monoclonal (P1D9, 1:50,Developmental Studies
Hybridoma Bank), anti-Arp3 (1:500; Stevensonet al., 2002), and
anti-Arpc1 (1:500; Stevenson et al., 2002), and anti-monoand
poly-ubiquitylated conjugates (FK2, 1:1000, Enzo Life Sciences,
EastFarmingdale, NY). Quantification of actin loading controls and
Ubiquitin-FK2 expression was performed using ImageJ (NIH).
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ImmunoprecipitationNuclear lysate was incubated with primary
antibody overnight at 4°C.Protein G–Sepharose (20 μl) was then
added in 0.5 ml Carol buffer (50 mMHEPES pH 7.9, 250 mM NaCl, 10 mM
EDTA, 1 mM DTT, 10% glycerol,0.1% Triton X-100) plus 0.5 mg/ml
bovine serum albumin (BSA) andprotease inhibitors (Complete
EDTA-free Protease Inhibitor cocktail;Sigma] and the reaction
allowed to proceed for 2 h at 4°C. The beadswere washed 1× with
Carol buffer plus BSA and 2× with Carol buffer alone.Analysis was
conducted using SDS-PAGE followed by western blotting.Antibodies
used for immunoprecipitations are as follows: anti-9e10
(1:9;Developmental Studies Hybridoma Bank), anti-Wash monoclonal
(1:6;P3H3; Rodriguez-Mesa et al., 2012), anti-CCDC53 (1:1000;
Verboon et al.,2015a), anti-Strumpellin (1:1000; Verboon et al.,
2015a), anti-FAM21(1:1000; Verboon et al., 2015a), anti-Lamin B
monoclonal (1:8; AD67.10,Developmental Studies Hybridoma Bank) and
anti-Lamin C monoclonal(1:10; LC28.26, Developmental Studies
Hybridoma Bank). Antibodiesused for the IP western blots are as
follows: mouse anti-CCDC53 polyclonal(1:1000), mouse
anti-Strumpellin polyclonal (1:400), mouse anti-FAM21polyclonal
(1:400), and anti-Lamin B monoclonal 67.10 (1:200).
GST pulldown assays and Blue Native PAGEGST pulldown assays were
performed as previously described (Magie andParkhurst, 2005; Magie
et al., 2002; Rosales-Nieves et al., 2006). BlueNative Page was
performed using a Novex Native PAGE Bis-Tris GelSystem (Invitrogen)
following manufacturer protocols. Briefly,Drosophila Kc cell
nuclear extract was resuspended in 1× NativePAGESample Buffer
(Invitrogen) with 1% digitonin and protease inhibitors,
andincubated for 15 min on ice. Samples were centrifuged at 16,200
g for30 min at 4°C, and supernatant was resuspended with G250
sampleadditive and Native PAGE sample buffer. These prepared
samples wereloaded on 3–12% Bis-Tris Native PAGE gels and
electrophoresed using a1× native PAGE running buffer system
(Invitrogen). The cathode bufferincluded 1× cathode buffer additive
(Invitrogen). Native mark proteinstandard (Invitrogen) was used as
the molecular mass marker. Proteinconcentrations of adult fly
mitochondrial preps were determined with aBCA protein assay kit
(Thermo Scientific, USA) following themanufacturer’s instructions.
The following antibodies were used: mouseanti-Wash monoclonal
(P3H3, 1:2; Rodriguez-Mesa et al., 2012), mouseanti-CCDC53
polyclonal (1:400; Verboon et al., 2015a), mouse anti-Strumpellin
polyclonal (1:400; Verboon et al., 2015a), mouse anti-FAM21
polyclonal (1:400; Verboon et al., 2015a) and rabbit anti-Lamin
Bpolyclonal (L6, 1:2500; Stuurman et al., 1996).
Electron microscopyDrosophila third-instar larva salivary glands
were processed for electronmicroscopy essentially as previously
described (Pitt et al., 2000). Glandswere dissected in 1× PBS then
placed directly in fixative solutions [2.2%glutaraldehyde, 0.9%
paraformaldehyde, 0.05 M cacodylate (pH 7.4),0.09 M sucrose, 0.9 mM
MgCl2]. Glands were fixed for 2.5 h at roomtemperature, followed by
several rinses with 0.09 M sucrose and 0.05 Mcacodylate (pH 7.4).
Glands were post-fixed in 1% osmium, 0.8%potassium ferricyanide,
0.1 M cacodylate (pH 7.2) for 45 min at 4°C,followed by several
rinses in 0.05 M cacodylate (pH 7.0). Glands were thentreated with
0.2% tannic acid in 0.05 M cacodylate (pH 7.0) for 15 min atroom
temperature, followed by several rinses in distilled H2O. Glands
wereplaced in 1% uranyl acetate in 0.1 M sodium acetate (pH 5.2)
for 1 h atroom temperature, and rinsed three times with 0.1 M
sodium acetate (pH5.2), followed by three rinses with distilled
H2O. Specimens weredehydrated in a graded acetone series, embedded
in Epon, and sectionedfollowing standard procedures. Grids were
viewed with a JEOL JEM-1230transmission electron microscope and
photographed with a GatanUltraScan 1000 CCD camera.
Immunostaining of larval salivary glandsSalivary glands were
dissected, fixed, stained and mounted as previouslydescribed
(Verboon et al., 2015a). Primary antibodies were added at
thefollowing concentrations: mouse anti-Wash monoclonal (P3H3,
1:200;Rodriguez-Mesa et al., 2012), mouse anti-CCDC53 polyclonal
(1:300;
Verboon et al., 2015a), mouse anti-Strumpellin polyclonal
(1:300; Verboonet al., 2015a), mouse anti-SWIP polyclonal (1:300;
Verboon et al., 2015a),mouse anti-FAM21 polyclonal (1:300; Verboon
et al., 2015a), mouse anti-Lamin B monoclonal (AD67.10 1:200,
Developmental Studies HybridomaBank), mouse anti-Lamin C monoclonal
(LC 28.26, 1:200, DevelopmentalStudies Hybridoma Bank), guinea pig
anti-Fz2C (1:2500, this study), ratanti-Arpc1 (1:500; Stevenson et
al., 2002), and rat anti-Cpa (1:200;Amândio et al., 2014).
Immunostaining of larval body wall muscleFlies were transferred
daily and wandering third-instar larvae were collectedand
subsequently fileted in cold PBS. Body wall muscle filets were
fixed for10 min. The fixative used was: 16.7 mMKPO4 pH 6.8, 75
mMKCl, 25 mMNaCl, 3.3 mM MgCl2 and 6% formaldehyde. After three
washes with 1×PBS with 0.1% Tween-20 (PTW), larval filets were
permeabilized in 1×PBS plus 1% Triton X-100 for 2 h at room
temperature, then blocked using1× PBS, 0.1% Tween-20, 1% BSA and
0.05% azide (PAT) for 2 h at 4°C.Antibodies were added at the
following concentrations: mouse anti-DLG(1:50) and rabbit anti-HRP
(1:1400). The larval filets were incubated 48 h at4°C. Primary
antibody was then removed and filets were washed three timeswith 1×
PBS, 0.1% Tween-20, 0.1%BSA, 2% normal goat serum (XNS) for30 min
each. Alexa Fluor-conjugated secondary antibodies
(Invitrogen)diluted in 1× PBS, 0.1% Tween-20, 0.1% BSA (PbT)
(1:1000) were thenadded and the filets were incubated overnight at
4°C. Larval filets werewashed ten times with PTW at room
temperature for 10 min each and weremounted on slides in SlowFade
Gold medium (Invitrogen, Carlsbad, CA)and visualized using a Zeiss
confocal microscope as described below. Thetotal number of boutons
was quantified in third-instar larval preparationsdouble labeled
with antibodies to HRP and DLG, at segments A2–A3muscles 6–7. The
number of ghost boutons was assessed by countingHRP-positive and
DLG-negative boutons.
Actin visualization and immunostaining of indirect flight
muscleUAS controlled RNAi-expressing flies were crossed to
MHC-GAL4driver flies and female RNAi/MHC-GAL4 trans-heterozygous
flies werecollected and aged 21 days at 25°C. Fly thoraxes were
dissected and cutalong the ventral side in cold PBS. Thoraxes were
fixed using 1:6fixative and heptane for 15 min. The fixative used
was: 16.7 mM KPO4pH 6.8, 75 mM KCl, 25 mM NaCl, 3.3 mM MgCl2 and
6%formaldehyde. After three washes with PTW thoraxes were then
cutalong the dorsal side resulting in two halves and fixed again
for 10 minusing 1:6 fix/heptane for 15 min. After three washes with
PTW, thoraxeswere permeabilized in 1× PBS plus 1% Triton X-100 for
2 h at roomtemperature, then blocked using PAT for 2 h at 4°C.
Antibodies wereadded at the following concentrations: mouse
anti-ATP-Synthase α(15H4C4 1:100, Abcam, Cambridge, UK), and mouse
anti-mono andpoly-ubiquitylated conjugates (FK2 1:200, Enzo Life
Sciences, EastFarmingdale, NY). The thoraxes were incubated 48 h at
4°C. Primaryantibody was then removed and thoraxes were washed
three times with XNSfor 30 min each. Alexa Fluor-conjugated
secondary antibodies (Invitrogen,Carlsbad, CA) diluted in PbT
(1:1000) andAlexa Fluor-conjugated phalloidin(1:50) were then added
and the thoraxes were incubated overnight at 4°C.Thoraxes were
washed ten times with PTW at room temperature for 10 mineach and
were mounted on slides in SlowFade Gold medium
(Invitrogen,Carlsbad, CA) and visualized using a Zeiss confocal
microscope as describedbelow. To quantify ATP-Synthase α
expression, we measured 512×512 pixelregions of the IFM and
measured total fluorescence using ImageJ (NIH).Fluorescence
measurements from separate experiments were normalized tothe
control genotype.
Confocal and super-resolution microscopyImages of fixed tissues
were acquired using a Zeiss LSM 780 spectralconfocal microscope
(Carl Zeiss Microscopy GmbH, Jena, Germany) fittedwith a Zeiss
40×/1.0 oil Plan-Apochromat objective and a Zeiss 63×/1.4
oilPlan-Apochromat objective. FITC (Alexa Fluor 488) fluorescence
wasexcited with the 488 nm line of an argon laser, and detection
was between498 and 560 nm. Red (Alexa Fluor 568) fluorescence was
excited with the561 nm line of a DPSS laser and detection was
between 570 and 670 nm.
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The pinhole was set to 1.0 Airy Units. Confocal sections were
acquired at0.2–1.0 µm spacing. Super-resolution images were
acquired using anAiryscan detector in Super Resolution mode and
captured confocal imageswere then processed using the Airyscan
Processing feature on the Zensoftware provided by the manufacturer
(Carl Zeiss Microscopy GmbH,Jena, Germany).
Live image acquisitionTo obtain live time-lapse images of
oocytes, female flies were first fattenedon yeast for 2 days.
Females were then injected in the abdomen with 0.4%Trypan Blue
(Thermo Fisher Scientific) diluted 1:5 in PBS, and allowed tosit
for 1–2 h. Ovaries were dissected into individual egg chambers
inhalocarbon 700 oil (Halocarbon Products, River Edge, NJ) on a
cover slip.Images were acquired on a RevolutionWD system (Andor
Technology Ltd.,Concord, MA)mounted on a Leica DMi8
(LeicaMicrosystems Inc., BuffaloGrove, IL) with a 63×/1.4 NA
objective lens with a 2× coupler andcontrolled by MetaMorph
software. Images were acquired with 561 nmexcitation using an Andor
iXon Ultra 888 EMCCD camera (AndorTechnology Ltd., Concord, MA).
Time-lapse images were obtained bytaking one single frame
acquisition every 10 s for either 5 or 30 min.
Statistical analysisAll statistical analyses were performed
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ubiquitin puncta), geneknockdowns and point mutations were compared
to the appropriatecontrol, and statistical significance was
calculated using a Kruskal–Wallistest for independence. For
frequency of observation (ghost versus matureboutons, swirling
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