journal of cell science • accepted manuscript · 04.06.2020 · proteins polymerize branched...

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

(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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RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243576. doi:10.1242/jcs.243576

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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

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

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

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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https://jcs.biologists.org/lookup/doi/10.1242/jcs.243576.supplemental

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

(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

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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https://jcs.biologists.org/lookup/doi/10.1242/jcs.243576.supplementalhttps://jcs.biologists.org/lookup/doi/10.1242/jcs.243576.supplemental

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 using R-3.6.1. For count data(number of nuclear buds and number of 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 versus not swirling, and lamin separated versusoverlapping) a two-tailed Fisher’s exact test was used. For all otheranalyses, a two-tailed Student’s t-test was used to test for significancebetween conditions. For plots, the mean+95% c.i. was calculated using theHmisc-4.2-0 package using 1000 bootstrap resamples. P

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