Cytoplasmic sharing through apical membrane remodeling

  1. Nora G Peterson
  2. Benjamin M Stormo
  3. Kevin P Schoenfelder
  4. Juliet S King
  5. Rayson RS Lee
  6. Donald T Fox  Is a corresponding author
  1. Department of Cell Biology, Duke University Medical Center, United States
  2. University Program in Genetics and Genomics, Duke University, United States
  3. Department of Pharmacology & Cancer Biology, Duke University Medical Center, United States
  4. Duke-NUS Medical School, Singapore
6 figures, 7 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Developmentally programmed cytoplasmic sharing in Drosophila papillae.

(A) The dBrainbow construct (Hampel et al., 2011). Cre recombinase randomly excises one pair of lox sites, and approximately 1/3 of cells express either EGFP, mKO2, or mTFP1. (B) Model of dBrainbow expression with no, partial, or complete cytoplasmic sharing. (C) Drosophila digestive tract with rectum containing four papillae labeled in magenta box. (D) Cartoon of a cross-section through an adult rectal papilla. The papilla consists of an epithelial cone with the apical region facing the gut lumen and the interior basal region facing a central canal leading to the fly hemolymph. The principal papillar cells have microvilli-like projections on the apical edge. One layer of larger, secondary cells forms the base of the papilla. The papilla is covered in a cuticle layer (dark gray). Nuclei are marked in blue. (E) Approximate timeline of ubiquitous Cre induction and cytoplasm sharing onset (68–74 HPPF) within papillar development (Fox et al., 2010). Cytoplasmic sharing is temporally separate from papillar mitoses. (F–F’’) Representative dBrainbow papillae at 62 (F), 69 (F’), or 80 (F’’) hours post-puparium formation (HPPF). (G) Cytoplasmic sharing quantification during pupal development. Lines = mean at each time, which differs significantly between 66 and 74 HPPF (p<0.0001). Each point = 1 animal (N = 9–18, rep = 2). (H) Live dBrainbow-labeled papillar cells during cytoplasmic sharing (69 HPPF). (H’) Fluorescence of neighboring cells in (H). (I–I’) Representative adult papilla expressing photo-activatable GFP (GFPPA). Single cells were photo-activated (yellow X) in secondary cells (I) and principal cells (I’). Time = seconds after activation.

Figure 1—figure supplement 1
The hindgut rectal papillae share cytoplasm independent of mitosis.

(A) Representative images of dBrainbow expression in the indicated adult tissues. (B) Schematic of cytoplasmic sharing quantification. The mKO2-positive papillar area is divided by the total papillar area to give a score of cytoplasmic sharing. Numbers close to one indicate near-complete sharing. (C) Schematic of principal cells (sharing) and secondary cells (non-sharing) at the papillar base that together form each papilla. (D) Gapdh2-GFPPA activated in single cells in an adult papilla and imaged every 15 s. (E–G) Representative adults expressing dBrainbow in a (E) wild-type (WT), (F) fzr RNAi (p<0.0001), or (G) NDN background (p=0.8786). (H) Quantification of cytoplasmic sharing in adult WT, fzr RNAi, and NDN-expressing animals (N = 12–20, rep = 2).

Figure 2 with 1 supplement
Cytoplasmic sharing requires membrane remodeling proteins.

(A) Primary dBrainbow candidate screen. RNAi and dominant-negative versions of 77 genes representing the indicated roles were screened for sharing defects, and eight genes were identified. (B) Secondary membrane trafficking screen. 36 genes were screened with 12 sharing genes identified. (C) Secondary screen of dominant-negative and constitutively-active Rab GTPases. (D–G) Representative dBrainbow in (D–D’) wild type (WT) (D) pre-sharing (48HPPF) and (D’) post-sharing (young adults), (E) adult shi RNAi, (F) adult Rab5 RNAi, (G) adult Rab11 RNAi. (H) Quantification of (D–G), including two RNAi lines for shi, Rab5, and Rab11. Pre-sharing and knock downs differ significantly from post-sharing WT (p<0.0001, N = 9–32, rep = 2–3).

Figure 2—figure supplement 1
Membrane trafficking genes expressed during a developmental window regulate cytoplasm sharing.

(A) Quantification of cytoplasmic sharing in animals expressing dsRNA for myoblast fusion regulators (N = 8–11, rep = 2). All knockdown lines are previously published (Bischoff et al., 2013; Xing et al., 2018; Linneweber et al., 2015; Johnson et al., 2011; Brunetti et al., 2015). Only sing RNAi significantly differs from WT (p<0.0001). (B) Quantification of cytoplasmic sharing in animals expressing dsRNA for Rho family GTPases (N = 6–8, rep = 2). (C) Cell counts in WT and knockdown rectal papillae (N = 11–23, rep = 2). Only Rab11 #1 RNAi had a significantly different cell number than WT (p=0.0323). (D–E) Representative animals expressing dBrainbow in either a WT (D) or shi RNAi (E) genetic background were raised at 18°C until 3–4 days PPF and shifted to 29°C to induce shi knockdown at a later timepoint than in Figure 2E and H. (F) Sharing quantification in late-induced animals (N = 10–11, rep = 2).

Figure 3 with 2 supplements
Gap junction establishment, but no membrane breaches, accompany cytoplasm sharing.

(A–A’) Endosome localization (GFP-myc-2x-FYVE), representative of (A) pre- and (A’) post-sharing onset. (B) Endosomes in shi RNAi post-sharing, see Methods. (C) Aggregated endosome line profiles for WT pre-sharing (N = 6, rep = 3), WT post-sharing (N = 7, rep = 2), and shi RNAi post-sharing (N = 10, rep = 2). Shaded area represents standard error. (D–D’) Shi-Venus localization pre- and post-sharing onset. (E) Line profiles as in (D–D’) (N = 4–5, rep = 3). (F–O) Representative Transmission Electron Micrographs (TEMs). (F–F’’) Microvillar-like structures (MV) pre- (F), mid- (F’), and post- (F’’) sharing onset. (G–G’’) Mitochondria and surrounding membrane pre- (G), mid- (G’), and post- (G’’) sharing onset. (H–J) Microvillar-like structures (MV) of adult papillae in WT (H), shi RNAi (I), and Rab5 RNAi (J). (K–M) Mitochondria and surrounding membranes of adult papillae in WT (K), shi RNAi (L), and Rab5 RNAi (M). Inset in (L) shows trapped vesicles. (N–O) WT and shi RNAi post-sharing. Adherens (orange), septate (green), and gap (blue) junctions are highlighted. (P) Quantification of the ratio of gap junction length to septate plus gap junction length (Fraction gap junction) (N = 3–4, rep = 2). p<0.0001 for the difference in gap junction ratio between WT and shi RNAi.

Figure 3—figure supplement 1
Changes in endosome polarity and apical junction shape accompany the onset of cytoplasm sharing.

(A) Quantification of the average endosome intensity difference between representative basal and apical areas across papillae in Figure 3A–C (N = 6–10, rep = 2). (B–B’) Representative localization of Rab5-YFP, green, before sharing onset (B) and after sharing onset (B’). (B’’) Aggregated line profiles of Rab5-YFP intensity before and after the beginning of sharing (N = 10, rep = 2). (C–C’’) Representative TEMs of apical (adherens, septate, and gap) junctions pre (C), mid (C’), and post (C’’) sharing onset. (D–F) Representative TEMs of apical junctions of post-sharing adult WT (D), shi RNAi (E), and Rab5 RNAi (F) papillar cells. (G–G’’) Apical junction electron micrograph measurements of post-sharing WT and shi RNAi pupal papillar cells (N = 3–4, rep = 2). Average gap junction (G) and septate junction (G’) widths were measured alongside gap and septate junction length. Width measurements were taken along the length of each cell–cell junction and averaged to give one point per cell–cell junction. (G’’) Raw septate and gap junction lengths (nm) that were used to calculate gap junction ratio in Figure 3P.

Figure 3—figure supplement 2
Extracellular spaces separate nuclei throughout much of the papillar lateral membrane.

(A) Representative TEM cross-section of an adult WT papilla. The apical edge facing the gut lumen is at the top; the basal edge facing the papillar central canal is at the bottom of the image.

Figure 4 with 1 supplement
Gap junction proteins are required for cytoplasmic sharing.

(A–A’’) Representative apical junctions highlighted by junctional type in pre (A), mid (A’), and post (A’’) sharing onset. (B) Quantification of fraction gap junction (gap junction length / (gap + septate junction length)) in pre-, mid-, and post-sharing onset pupae (N = 3–4, rep = 2). (C) Drosophila innexin expression in the adult rectum (Methods). (D–D’) Adherens junctions in pre- (D) and post- (D’) sharing pupae visualized by NrxIV-GFP. (E–E’) WT pupae pre- and post-sharing onset stained with anti-Inx3. (F) Quantification of cytoplasm sharing in WT, ogreDN, Df(1)BSC867/+ (a 10-gene-deficiency covering ogre, Inx2, and Inx7), and ogre RNAi adult papillae (N = 13–14, rep = 2). (G) Representative adult rectal papilla expressing GFP-ogre and dBrainbow. (H) Survival of WT, shiDN, and ogreDN animals on a high-salt diet (N = 27–37, rep = 3). (I) Proposed model for cytoplasmic sharing in an intact papillar epithelium.

Figure 4—figure supplement 1
Gap junction formation coincides with cytoplasm sharing onset.

(A–A’’) Apical junction TEM measurements of pre-, mid-, and post-sharing onset pupal papillar cells (N = 3–4, rep = 2). Average gap junction (A) and septate junction (A’) widths were measured alongside gap and septate junction length. (A’’) Raw septate and gap junction lengths (nm) used to calculate gap junction ratio in Figure 4B. (B–B’) Gap junction localization visualized by UAS-GFP-ogre in pre (B) and post (B’) sharing onset pupae. (C–C’) Representative images of post-sharing WT and shi RNAi animals stained for anti-Inx3. (D) Representative image of byn-Gal4 driving GFPNLS expression throughout the pre-sharing hindgut. Arrows indicate the ileum. (D’) 60H12-Gal4 driving GFPNLS expression in pre-sharing papillae but not in the hindgut ileum or pylorus. Arrows indicate the ileum. (E) Representative image of 60H12-Gal4 driving dBrainbow in adult papillae. (E’) Representative image of 60H12-Gal4 driving shiDN expression in a dBrainbow background in adult papillae. (E’’) Quantification of cytoplasm sharing in 60H12-Gal4 and 60H12-Gal4 > shiDN animals (N = 11, rep = 2). (F) Model of membrane and junctional changes requiring membrane trafficking genes that coincide with the onset of cytoplasm sharing.

Author response image 1
Author response image 2

Tables

Table 1
Cytoplasm sharing primary candidate screen gene results.
Gene categoryGeneAnnotation symbolGene IDSharing disrupted?
AutophagyAtg1CG10967FBgn0260945No
AutophagyAtg7CG5489FBgn0034366No
AutophagyAtg8aCG32672FBgn0052672No
Cell cycle/ChromosomesblueNAFBgn0283709No
Cell cycle/ChromosomesCapD2CG1911FBgn0039680No
Cell cycle/ChromosomesCdc2CG5363FBgn0004106Yes
Cell cycle/ChromosomesClampCG1832FBgn0032979No
Cell cycle/ChromosomesendosCG6513FBgn0061515No
Cell cycle/ChromosomesfzrCG3000FBgn0262699Yes
Cell cycle/ChromosomesMi-2CG8103FBgn0262519No
Cell cycle/ChromosomesRbp9CG3151FBgn0010263No
Cell cycle/ChromosomesSA-2CG13916FBgn0043865No
Cell signalingChicoCG5686FBgn0024248No
Cell signalingEgfrCG10079FBgn0003731Yes
Cell signalinggrkCG17610FBgn0001137No
Cell signalingNCG3936FBgn0004647No
Cell signalingPtp61FCG9181FBgn0267487No
Cell signalingrhoCG1004FBgn0004635Yes
Cell signalingruCG1214FBgn0003295No
Cell signalingspiCG10334FBgn0005672No
Cell signalingstetCG33166FBgn0020248No
Cell signalingwtsCG12072FBgn0011739No
Cell signalingβggt-IICG18627FBgn0028970No
CytoskeletonALiXCG12876FBgn0086346No
CytoskeletonCdc42CG12530FBgn0010341No
CytoskeletonDCTN1-p150CG9206FBgn0001108No
CytoskeletonpavCG1258FBgn0011692No
CytoskeletonwashCG13176FBgn0033692No
Hindgut-enricheddacCG4952FBgn0005677No
Hindgut-enrichedDrCG1897FBgn0000492No
Hindgut-enrichednrv3CG8663FBgn0032946No
Membrane componentFlo1CG8200FBgn0024754No
Membrane componentFlo2CG32593FBgn0264078No
Membrane componentIrisCG4715FBgn0031305No
Myoblast fusionArf51FCG8156FBgn0013750No
Myoblast fusionArp2CG9901FBgn0011742No
Myoblast fusionArp3CG7558FBgn0262716No
Myoblast fusionCed-12CG5336FBgn0032409No
Myoblast fusiondockCG3727FBgn0010583No
Myoblast fusionhbsCG7449FBgn0029082No
Myoblast fusionHemCG5837FBgn0011771No
Myoblast fusionmbcCG10379FBgn0015513No
Myoblast fusionRac1CG2248FBgn0010333No
Myoblast fusionRho1CG8416FBgn0014020No
Myoblast fusionrolsCG32096FBgn0041096No
Myoblast fusionrstCG4125FBgn0003285No
Myoblast fusionSCARCG4636FBgn0041781No
Myoblast fusionsizCG32434FBgn0026179No
Myoblast fusionWASpCG1520FBgn0024273No
PolarityAbiCG9749FBgn0020510No
PolarityCadNCG7100FBgn0015609No
PolaritycindrCG31012FBgn0027598No
PolaritycnoCG42312FBgn0259212No
PolarityGliCG3903FBgn0001987No
Polarityl(2)glCG2671FBgn0002121No
PolarityNrgCG1634FBgn0264975No
PolaritysdtCG32717FBgn0261873No
PolarityshgCG3722FBgn0003391No
Vesicle traffickingAtlCG6668FBgn0039213No
Vesicle traffickingBet1CG14084FBgn0260857No
Vesicle traffickingChmp1CG4108FBgn0036805No
Vesicle traffickingCHMP2BCG4618FBgn0035589No
Vesicle traffickingdndCG6560FBgn0038916No
Vesicle traffickingExo84CG6095FBgn0266668Yes
Vesicle traffickinglerpCG31072FBgn0051072No
Vesicle traffickingRab11CG5771FBgn0015790Yes
Vesicle traffickingRab23CG2108FBgn0037364No
Vesicle traffickingRab4CG4921FBgn0016701No
Vesicle traffickingRab7CG5915FBgn0015795No
Vesicle traffickingRab8CG8287FBgn0262518No
Vesicle traffickingRabX4CG31118FBgn0051118No
Vesicle traffickingVha16-1CG3161FBgn0262736Yes
Vesicle traffickingVha55CG17369FBgn0005671No
Vesicle traffickingVhaAC39-1CG2934FBgn0285910No
Vesicle traffickingVhaAC39-2CG4624FBgn0039058No
Vesicle traffickingVps2CG14542FBgn0039402Yes
Vesicle traffickingVps33bCG5127FBgn0039335No
Total screen results
Sharing disrupted8
No sharing phenotype69
Total77
Screen results by category
Polarity9
Vesicle trafficking19
Myoblast fusion15
Cell cycle/Chromosomes9
Cell signaling11
Autophagy3
Cytoskeleton5
Hindgut-enriched3
Membrane component3
Total77
Table 2
Membrane trafficking primary and secondary candidate screen gene results.
Gene categoryGene subcategoryGeneAnnotation symbolGene IDSharing disrupted?Screen
Membrane traffickingERAtlCG6668FBgn0039213NoPrimary
Membrane traffickingESCRTChmp1CG4108FBgn0036805NoPrimary
Membrane traffickingESCRTCHMP2BCG4618FBgn0035589NoPrimary
Membrane traffickingESCRTlsnCG6637FBgn0260940NoSecondary
Membrane traffickingESCRTVps2CG14542FBgn0039402YesPrimary
Membrane traffickingESCRTVps4CG6842FBgn0283469NoSecondary
Membrane traffickingExocystExo70CG7127FBgn0266667NoSecondary
Membrane traffickingExocystExo84CG6095FBgn0266668YesPrimary
Membrane traffickingExocystSec10CG6159FBgn0266673YesSecondary
Membrane traffickingExocystSec15CG7034FBgn0266674YesSecondary
Membrane traffickingExocystSec5CG8843FBgn0266670YesSecondary
Membrane traffickingExocystSec6CG5341FBgn0266671YesSecondary
Membrane traffickingExocystSec8CG2095FBgn0266672YesSecondary
Membrane traffickingLysosomelerpCG31072FBgn0051072NoPrimary
Membrane traffickingRab-associatedCG41099CG41099FBgn0039955NoSecondary
Membrane traffickingRab-associatedmtmCG9115FBgn0025742NoSecondary
Membrane traffickingRab-associatednufCG33991FBgn0013718NoSecondary
Membrane traffickingRab-associatedRalaCG2849FBgn0015286NoSecondary
Membrane traffickingRab-associatedRepCG8432FBgn0026378NoSecondary
Membrane traffickingRab-associatedRip11CG6606FBgn0027335NoSecondary
Membrane traffickingVacuolar H+ ATPaseVha16-1CG3161FBgn0262736YesPrimary
Membrane traffickingVacuolar H+ ATPaseVha16-2CG32089FBgn0028668NoSecondary
Membrane traffickingVacuolar H+ ATPaseVha16-3CG32090FBgn0028667NoSecondary
Membrane traffickingVacuolar H+ ATPaseVha16-5CG6737FBgn0032294YesSecondary
Membrane traffickingVacuolar H+ ATPaseVha55CG17369FBgn0005671NoPrimary
Membrane traffickingVacuolar H+ ATPaseVhaAC39-1CG2934FBgn0285910NoPrimary
Membrane traffickingVacuolar H+ ATPaseVhaAC39-2CG4624FBgn0039058NoPrimary
Membrane traffickingVacuolar H+ ATPaseVhaPPA1-1CG7007FBgn0028662YesSecondary
Membrane traffickingVacuolar H+ ATPaseVhaPPA1-2CG7026FBgn0262514YesSecondary
Membrane traffickingVesicle traffickingBet1CG14084FBgn0260857NoPrimary
Membrane traffickingVesicle traffickingChcCG9012FBgn0000319NoSecondary
Membrane traffickingVesicle traffickingdndCG6560FBgn0038916NoPrimary
Membrane traffickingVesicle traffickingshiCG18102FBgn0003392YesSecondary
Membrane traffickingVesicle traffickingVps29CG4764FBgn0031310NoSecondary
Membrane traffickingVesicle traffickingVps33bCG5127FBgn0039335NoPrimary
Membrane traffickingVesicle traffickingVps35CG5625FBgn0034708NoSecondary
Total screen results
Sharing disrupted12
No sharing phenotype24
Total36
Screen results by categoryTotalHits
ER10
ESCRT51
Exocyst76
Lysosome10
Rab-associated60
Vacuolar H+ ATPase94
Vesicle trafficking71
Total36
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (D. melanogaster)w1118Bloomington Drosophila Stock CenterBDSC:3605; FLYB:FBst0003605; RRID:BDSC_3605w1118
Genetic reagent (D. melanogaster)tub-Gal4Bloomington Drosophila Stock CenterBDSC:5138; FLYB:FBst0005138; RRID:BDSC_5138y1 w*; P{tubP-GAL4}LL7/TM3, Sb1 Ser1
Genetic reagent (D. melanogaster)tub-Gal80tsNANANA
Genetic reagent (D. melanogaster)UAS-dBrainbowBloomington Drosophila Stock Center; (Hampel et al., 2011)BDSC:34513; FLYB:FBst0034513; RRID:BDSC_34513w1118; P{UAS-Brainbow}attP2
Genetic reagent (D. melanogaster)UAS-dBrainbowBloomington Drosophila Stock Center; (Hampel et al., 2011)BDSC:34514; FLYB:FBst0034514; RRID:BDSC_34514w1118; P{UAS-Brainbow}attP40
Genetic reagent (D. melanogaster)Hsp70>creBloomington Drosophila Stock CenterBDSC:851; FLYB:FBst0000851; RRID:BDSC_851y1 w67c23 P{Crey}1b; D*/TM3, Sb1
Genetic reagent (D. melanogaster)UAS-fzr RNAiVienna Drosophila Resource CenterVDRC:25550; FLYB:FBst0455950w1118; P{GD9960}v25550
Genetic reagent (D. melanogaster)UAS-shi RNAi #1Bloomington Drosophila Stock CenterBDSC:28513; FLYB:FBst0028513; RRID:BDSC_28513y1 v1; P{TRiP.JF03133}attP2
Genetic reagent (D. melanogaster)UAS-shi RNAi #2Bloomington Drosophila Stock CenterBDSC:36921; FLYB:FBst0036921; RRID:BDSC_36921y1 sc* v1 sev21; P{TRiP.HMS00154}attP2
Genetic reagent (D. melanogaster)UAS-Rab5 RNAi #1Bloomington Drosophila Stock CenterBDSC:30518; FLYB:FBst0030518; RRID:BDSC_30518y1 v1; P{TRiP.JF03335}attP2
Genetic reagent (D. melanogaster)UAS-Rab5 RNAi #2Bloomington Drosophila Stock CenterBDSC:67877; FLYB:FBst0067877; RRID:BDSC_67877y1 sc* v1 sev21; P{TRiP.GL01872}attP40
Genetic reagent (D. melanogaster)UAS-Rab11 RNAi #1Bloomington Drosophila Stock CenterBDSC:27730; FLYB:FBst0027730; RRID:BDSC_27730y1 v1; P{TRiP.JF02812}attP2
Genetic reagent (D. melanogaster)UAS-Rab11 RNAi #2Vienna Drosophila Resource CenterVDRC:22198; FLYB:FBst0454467w1118; P{GD11761}v22198
Genetic reagent (D. melanogaster)UAS-SCAR RNAi #1Bloomington Drosophila Stock CenterBDSC:36121; FLYB:FBst0036121; RRID:BDSC_36121y1 sc* v1 sev21; P{TRiP.HMS01536}attP40
Genetic reagent (D. melanogaster)UAS-SCAR RNAi #2Bloomington Drosophila Stock CenterBDSC:51803; FLYB:FBst0051803; RRID:BDSC_51803y1 v1; P{TRiP.HMC03361}attP40
Genetic reagent (D. melanogaster)UAS-kirre RNAiVienna Drosophila Resource CenterVDRC:27227; FLYB:FBst0456824w1118; P{GD14476}v27227
Genetic reagent (D. melanogaster)UAS-sns RNAiVienna Drosophila Resource CenterVDRC:877; FLYB:FBst0471238w1118; P{GD65}v877/TM3
Genetic reagent (D. melanogaster)UAS-schizo RNAiVienna Drosophila Resource CenterVDRC:36625; FLYB:FBst0461775w1118; P{GD14895}v36625
Genetic reagent (D. melanogaster)UAS-sing RNAiVienna Drosophila Resource CenterVDRC:12202; FLYB:FBst0450437w1118; P{GD3396}v12202/TM3
Genetic reagent (D. melanogaster)UAS-Cdc42DNBloomington Drosophila Stock CenterBDSC:6288; FLYB:FBst0006288; RRID:BDSC_6288w*; P{UAS-Cdc42.N17}3
Genetic reagent (D. melanogaster)UAS-Rac1DNBloomington Drosophila Stock CenterBDSC:6292; FLYB:FBst0006292; RRID:BDSC_6292y1 w*; P{UAS-Rac1.N17}1
Genetic reagent (D. melanogaster)UAS-Rho1DNBloomington Drosophila Stock CenterBDSC:7328; FLYB:FBst0007328; RRID:BDSC_7328w*; P{UAS-Rho1.N19}2.1
Genetic reagent (D. melanogaster)UAS-GFPNLSBloomington Drosophila Stock CenterBDSC:4776; FLYB:FBst0004776; RRID:BDSC_4776w1118; P{UAS-GFP.nls}8
Genetic reagent (D. melanogaster)UAS-GFP-Myc-2x-FYVEBloomington Drosophila Stock CenterBDSC:42712; FLYB:FBst0042712; RRID:BDSC_42712w*; P{UAS-GFP-myc-2xFYVE}2
Genetic reagent (D. melanogaster)UAS-YFP-Rab5Bloomington Drosophila Stock CenterBDSC:9775; FLYB:FBst0009775; RRID:BDSC_9775y1 w*; P{UASp-YFP.Rab5}Pde808b
Genetic reagent (D. melanogaster)60H12-Gal4Bloomington Drosophila Stock CenterBDSC:39268; FLYB:FBst0039268; RRID:BDSC_39268w1118; P{GMR60H12-GAL4}attP2
Genetic reagent (D. melanogaster)UAS-shiDNBloomington Drosophila Stock CenterBDSC:5822; FLYB:FBst0005822; RRID:BDSC_5822w*; TM3, P{UAS-shi.K44A}3-10/TM6B, Tb1
Genetic reagent (D. melanogaster)NrxIV-GFPBloomington Drosophila Stock CenterBDSC:50798; FLYB:FBst0050798; RRID:BDSC_50798y1 w*; P{PTT-GA}Nrx-IVCA06597
Genetic reagent (D. melanogaster)Df(1)BSC867Bloomington Drosophila Stock CenterBDSC:29990; FLYB:FBst0029990; RRID:BDSC_29990Df(1)BSC867, w1118/Binsinscy
Genetic reagent (D. melanogaster)UAS-ogre RNAiVienna Drosophila Resource CenterVDRC:7136; FLYB:FBst0470569w1118; P{GD3264}v7136
Genetic reagent (D. melanogaster)byn-Gal4Singer et al., 1996FLYB:FBal0137290P{GawB}bynGal4
Genetic reagent (D. melanogaster)UAS-GFPPALynn Cooley; McLean and Cooley, 2013FLYB:FBti0148163P{20XUAS-IVS-Syn21-mC3PA-GFP-p10}
Genetic reagent (D. melanogaster)UAS-NDNRebay et al., 1993NANA
Genetic reagent (D. melanogaster)UAS-shi-VenusStefano De Renzis; Fabrowski et al., 2013NANA
Genetic reagent (D. melanogaster)UAS-GFP-ogreAndrea Brand; Spéder and Brand, 2014FLYB:FBtp0127574ogreUAS.N.GFP
Genetic reagent (D. melanogaster)UAS-Gapdh2-GFPPAThis paperNATransgenic line created through gene synthesis and embryo injection. Codon-optimized D. melanogaster Gapdh2 fused to GFPPAunder UAS control.
Antibodyanti-GFP(Rabbit polyclonal)Thermo Fisher ScientificCat# A11122; RRID:AB_221569IF (1:1000)
Antibodyanti-HA (Rat monoclonal)RocheCat# 11867423001; RRID:AB_390918IF (1:100)
Antibodyanti-Inx3(Rabbit polyclonal)Reinhard Bauer; Lehmann et al., 2006RRID:AB_2568555IF (1:75)
AntibodyAnti-Rabbit Alexa Fluor 488 (Goat)Thermo Fisher ScientificCat# A32731; RRID:AB_2633280IF (1:2000)
AntibodyAnti-Rabbit Alexa Fluor 568 (Goat)Thermo Fisher ScientificCat# A-11011; RRID:AB_143157IF (1:2000)
AntibodyAnti-Rat Alexa Fluor 633 (Goat)Thermo Fisher ScientificCat# A-21094; RRID:AB_2535749IF (1:2000)
OtherDAPI stainSigma-AldrichCat# D9542(1:5000)
Table 3
Primary and secondary candidate screen stock numbers used and results.
GeneAnnotation
symbol
Gene IDMutant or UAS
transgene
Stock centerStock numberChrSharing disrupted?Notes
AbiCG9749FBgn0020510RNAiBDSC514552No
ALiXCG12876FBgn0086346RNAiBDSC334173No
ALiXCG12876FBgn0086346RNAiBDSC509042No
Arf51FCG8156FBgn0013750RNAiBDSC514173No
Arf51FCG8156FBgn0013750MutantBDSC170762No
Arf51FCG8156FBgn0013750RNAiBDSC272613No
Arp2CG9901FBgn0011742RNAiBDSC277053No
Arp3CG7558FBgn0262716RNAiBDSC329213No
Atg1CG10967FBgn0260945RNAiBDSC440342No
Atg1CG10967FBgn0260945RNAiBDSC267313No
Atg7CG5489FBgn0034366RNAiBDSC343693No
Atg7CG5489FBgn0034366RNAiBDSC277073No
Atg8aCG32672FBgn0052672RNAiBDSC289893No
Atg8aCG32672FBgn0052672RNAiBDSC583092No
Atg8aCG32672FBgn0052672RNAiBDSC343403No
AtlCG6668FBgn0039213RNAiBDSC367362No
Bet1CG14084FBgn0260857RNAiBDSC419272No
blueNAFBgn0283709RNAiBDSC440943No
blueNAFBgn0283709RNAiBDSC416372No
CadNCG7100FBgn0015609RNAiBDSC275033No
CadNCG7100FBgn0015609RNAiBDSC419823No
CapD2CG1911FBgn0039680MutantBDSC593933No
Cdc2CG5363FBgn0004106RNAiVDRC418383Yes
Cdc2CG5363FBgn0004106RNAiBDSCNA3No
Cdc42CG12530FBgn0010341RNAiBDSC428612No
Cdc42CG12530FBgn0010341DNBDSC62882No
Ced-12CG5336FBgn0032409RNAiBDSC285563No
Ced-12CG5336FBgn0032409RNAiBDSC581532No
ChcCG9012FBgn0000319DNBDSC268212No
ChcCG9012FBgn0000319RNAiBDSC273503No
ChcCG9012FBgn0000319RNAiBDSC347423No
ChicoCG5686FBgn0024248RNAiBDSC367882No
Chmp1CG4108FBgn0036805RNAiBDSC339283No
CHMP2BCG4618FBgn0035589RNAiBDSC285313No
CHMP2BCG4618FBgn0035589RNAiBDSC383752No
cindrCG31012FBgn0027598RNAiBDSC356703No
cindrCG31012FBgn0027598RNAiBDSC389762No
ClampCG1832FBgn0032979RNAiBDSC270803No
cnoCG42312FBgn0259212RNAiBDSC333673No
cnoCG42312FBgn0259212RNAiBDSC381942No
dacCG4952FBgn0005677RNAiBDSC267583No
dacCG4952FBgn0005677RNAiBDSC350223No
DCTN1-p150CG9206FBgn0001108DNBDSC516452No
dndCG6560FBgn0038916RNAiBDSC274883No
dndCG6560FBgn0038916RNAiBDSC343833No
dockCG3727FBgn0010583RNAiBDSC277283No
dockCG3727FBgn0010583RNAiBDSC431763No
dockCG3727FBgn0010583MutantBDSC113852No
DrCG1897FBgn0000492RNAiBDSC262243No
DrCG1897FBgn0000492RNAiBDSC428912No
EgfrCG10079FBgn0003731DNBDSC53642Yes
EgfrCG10079FBgn0003731RNAiVDRC432673Yes
endosCG6513FBgn0061515RNAiBDSC532503No
endosCG6513FBgn0061515RNAiBDSC659963No
Exo70CG7127FBgn0266667RNAiBDSC280413No
Exo70CG7127FBgn0266667RNAiBDSC552343No
Exo84CG6095FBgn0266668RNAiBDSC287123Yes
Flo1CG8200FBgn0024754RNAiBDSC367003No
Flo1CG8200FBgn0024754RNAiBDSC366492No
Flo2CG32593FBgn0264078RNAiBDSC552123No
Flo2CG32593FBgn0264078RNAiBDSC408332No
fzrCG3000FBgn0262699RNAiVDRC255502Yes
GliCG3903FBgn0001987RNAiBDSC318693No
GliCG3903FBgn0001987RNAiBDSC581152No
grkCG17610FBgn0001137RNAiBDSC389133No
hbsCG7449FBgn0029082RNAiBDSC570032No
HemCG5837FBgn0011771MutantBDSC87523No
HemCG5837FBgn0011771MutantBDSC87533No
HemCG5837FBgn0011771RNAiBDSC294063No
HemCG5837FBgn0011771RNAiBDSC416883No
Hsc70CbCG6603FBgn0026418RNAiBDSC337423No
Hsc70CbCG6603FBgn0026418DNBDSC564972No
IrisCG4715FBgn0031305RNAiBDSC505872No
IrisCG4715FBgn0031305RNAiBDSC635822No
l(2)glCG2671FBgn0002121RNAiBDSC315173No
lerpCG31072FBgn0051072RNAiBDSC574362No
lilliCG8817FBgn0041111RNAiBDSC263143No
lilliCG8817FBgn0041111RNAiBDSC345923No
mbcCG10379FBgn0015513RNAiBDSC323553No
mbcCG10379FBgn0015513RNAiBDSC337223No
Mi-2CG8103FBgn0262519RNAiBDSC168763No
mtmCG9115FBgn0025742RNAiBDSC383393No
NCG3936FBgn0004647DNRebay LabNA2No
NCG3936FBgn0004647RNAiSara BrayNA1No
NrgCG1634FBgn0264975RNAiBDSC287243No
NrgCG1634FBgn0264975RNAiBDSC382152No
NrgCG1634FBgn0264975RNAiBDSC374962No
nrv3CG8663FBgn0032946RNAiBDSC294313No
nrv3CG8663FBgn0032946RNAiBDSC507253No
nufCG33991FBgn0013718RNAiBDSC314933No
pavCG1258FBgn0011692RNAiBDSC356493No
pavCG1258FBgn0011692RNAiBDSC439632No
Ptp61FCG9181FBgn0267487RNAiBDSC324263No
Ptp61FCG9181FBgn0267487RNAiBDSC560362No
Rab1CG3320FBgn0285937CABDSC97583No
Rab1CG3320FBgn0285937DNBDSC97573YesRequires 60H12-Gal4
Rab1CG3320FBgn0285937RNAiBDSC272993Yes
Rab1CG3320FBgn0285937RNAiBDSC346703No
Rab2CG3269FBgn0014009CABDSC97612No
Rab2CG3269FBgn0014009DNBDSC97592No
Rab3CG7576FBgn0005586CABDSC97643No
Rab3CG7576FBgn0005586DNBDSC97662No
Rab4CG4921FBgn0016701CABDSC97703No
Rab4CG4921FBgn0016701DNBDSC97682No
Rab4CG4921FBgn0016701DNBDSC97693No
Rab5CG3664FBgn0014010CABDSC97733Yes
Rab5CG3664FBgn0014010DNBDSC427043YesRequires 60H12-Gal4
Rab5CG3664FBgn0014010RNAiBDSC678772Yes
Rab5CG3664FBgn0014010RNAiBDSC305183Yes
Rab5CG3664FBgn0014010RNAiBDSC518472No
Rab6CG6601FBgn0015797CABDSC97763No
Rab6CG6601FBgn0015797DNBDSC232503No
Rab7CG5915FBgn0015795CABDSC97793No
Rab7CG5915FBgn0015795DNBDSC97783No
Rab7CG5915FBgn0015795DNBDSC97783No
Rab8CG8287FBgn0262518DNBDSC97803No
Rab8CG8287FBgn0262518CABDSC97812No
Rab8CG8287FBgn0262518DNBDSC97803No
Rab9CG9994FBgn0032782CABDSC97853No
Rab9CG9994FBgn0032782DNBDSC236423No
Rab10CG17060FBgn0015789CABDSC97873No
Rab10CG17060FBgn0015789DNBDSC97863No
Rab11CG5771FBgn0015790CABDSC97913No
Rab11CG5771FBgn0015790DNBDSC232613Yes
Rab11CG5771FBgn0015790RNAiBDSC277303Yes
Rab11CG5771FBgn0015790RNAiVDRC1083822Yes
Rab11CG5771FBgn0015790RNAiVDRC221983Yes
Rab11CG5771FBgn0015790MutantBDSC427083Yes
Rab14CG4212FBgn0015791CABDSC97952No
Rab14CG4212FBgn0015791DNBDSC232643No
Rab18CG3129FBgn0015794CABDSC97973No
Rab18CG3129FBgn0015794DNBDSC232383No
Rab19CG7062FBgn0015793CABDSC98003No
Rab19CG7062FBgn0015793DNBDSC97993No
Rab21CG17515FBgn0039966CABDSC238642No
Rab21CG17515FBgn0039966DNBDSC232403No
Rab23CG2108FBgn0037364RNAiBDSC360913No
Rab23CG2108FBgn0037364RNAiBDSC553522No
Rab23CG2108FBgn0037364CABDSC98063No
Rab23CG2108FBgn0037364DNBDSC98043No
Rab26CG34410FBgn0086913CABDSC232433No
Rab26CG34410FBgn0086913DNBDSC98083No
Rab27CG14791FBgn0025382CABDSC98112No
Rab27CG14791FBgn0025382DNBDSC232672No
Rab30CG9100FBgn0031882CABDSC98142No
Rab30CG9100FBgn0031882DNBDSC98133No
Rab32CG8024FBgn0002567CABDSC232803No
Rab32CG8024FBgn0002567DNBDSC232812No
Rab35CG9575FBgn0031090CABDSC98173No
Rab35CG9575FBgn0031090DNBDSC98203No
Rab39CG12156FBgn0029959CABDSC98233No
Rab39CG12156FBgn0029959DNBDSC232473No
Rab40CG1900FBgn0030391CABDSC98273No
Rab40CG1900FBgn0030391DNBDSC98292No
Rab9DCG32678FBgn0067052CABDSC98353No
Rab9DCG32678FBgn0067052DNBDSC232572No
Rab9ECG32673FBgn0052673CABDSC98322No
Rab9ECG32673FBgn0052673DNBDSC232553No
Rab9FbCG32670FBgn0052670CABDSC98443No
Rab9FbCG32670FBgn0052670DNBDSC98452No
RabX1CG3870FBgn0015372CABDSC98392No
RabX1CG3870FBgn0015372DNBDSC232523No
RabX2CG2885FBgn0030200CABDSC98423No
RabX2CG2885FBgn0030200DNBDSC98432No
RabX4CG31118FBgn0051118RNAiBDSC287043No
RabX4CG31118FBgn0051118RNAiBDSC440702No
RabX4CG31118FBgn0051118CABDSC232772No
RabX4CG31118FBgn0051118DNBDSC98493No
RabX5CG7980FBgn0035255CABDSC9852XNo
RabX5CG7980FBgn0035255DNBDSC98532No
RabX6CG12015FBgn0035155CABDSC98552No
RabX6CG12015FBgn0035155DNBDSC98563No
CG41099CG41099FBgn0039955RNAiBDSC348833No
Rac1CG2248FBgn0010333RNAiBDSC289853No
Rac1CG2248FBgn0010333DNBDSC62923No
RalaCG2849FBgn0015286DNBDSC320942No
RalaCG2849FBgn0015286RNAiBDSC343753No
Rbp9CG3151FBgn0010263RNAiBDSC427963No
RepCG8432FBgn0026378RNAiBDSC280473No
rhoCG1004FBgn0004635MutantBDSC14713Yes
rhoCG1004FBgn0004635RNAiBDSC389203Yes
rhoCG1004FBgn0004635RNAiBDSC416992Yes
Rho1CG8416FBgn0014020DNBDSC73283No
Rho1CG8416FBgn0014020DNBDSC588182No
Rho1CG8416FBgn0014020RNAiBDSC323833No
Rip11CG6606FBgn0027335RNAiBDSC383253No
rolsCG32096FBgn0041096RNAiBDSC569862No
rolsCG32096FBgn0041096RNAiBDSC582622No
rstCG4125FBgn0003285RNAiBDSC286723No
ruCG1214FBgn0003295RNAiBDSC415933No
ruCG1214FBgn0003295RNAiBDSC580652No
SA-2CG13916FBgn0043865RNAiVDRC1082672No
SCARCG4636FBgn0041781RNAiBDSC311263No
SCARCG4636FBgn0041781RNAiBDSC518032No
SCARCG4636FBgn0041781MutantBDSC87542No
sdtCG32717FBgn0261873RNAiBDSC339093No
sdtCG32717FBgn0261873RNAiBDSC352913No
Sec10CG6159FBgn0266673RNAiBDSC274833Yes
Sec15CG7034FBgn0266674RNAiBDSC274993Yes
Sec5CG8843FBgn0266670RNAiVDRC288733Yes
Sec5CG8843FBgn0266670RNAiBDSC505563No
Sec6CG5341FBgn0266671RNAiVDRC1058362Yes
Sec6CG5341FBgn0266671RNAiBDSC273143Yes
Sec8CG2095FBgn0266672RNAiBDSC574412Yes
shgCG3722FBgn0003391RNAiBDSC276893No
shiCG18102FBgn0003392DNBDSC58223YesRequires 60H12-Gal4
shiCG18102FBgn0003392RNAiBDSC285133Yes
shiCG18102FBgn0003392RNAiBDSC369213Yes
sizCG32434FBgn0026179RNAiBDSC390602No
spiCG10334FBgn0005672RNAiBDSC283873No
spiCG10334FBgn0005672RNAiBDSC346453No
stetCG33166FBgn0020248RNAiBDSC576983No
Vha16-1CG3161FBgn0262736RNAiBDSC409232Yes
Vha16-1CG3161FBgn0262736RNAiVDRC1044902Yes
Vha16-1CG3161FBgn0262736RNAiVDRC492912Yes
Vha16-2CG32089FBgn0028668RNAiBDSC651672No
Vha16-3CG32090FBgn0028667RNAiBDSC574742No
Vha16-5CG6737FBgn0032294RNAiBDSC258033Yes
Vha55CG17369FBgn0005671RNAiBDSC408842No
VhaAC39-1CG2934FBgn0285910RNAiBDSC350293No
VhaAC39-2CG4624FBgn0039058MutantBDSC627253No
VhaAC39-2CG4624FBgn0039058RNAiVDRC343032No
VhaPPA1-1CG7007FBgn0028662RNAiBDSC577292Yes
VhaPPA1-2CG7026FBgn0262514RNAiBDSC652172Yes
Vps2CG14542FBgn0039402RNAiVDRC248693Yes
Vps2CG14542FBgn0039402RNAiBDSC389952Yes
lsnCG6637FBgn0260940RNAiBDSC382892No
Vps29CG4764FBgn0031310RNAiBDSC539512No
Vps33bCG5127FBgn0039335RNAiBDSC440062No
Vps35CG5625FBgn0034708RNAiBDSC389442No
Vps4CG6842FBgn0283469RNAiBDSC317513No
wtsCG12072FBgn0011739RNAiBDSC418993No
washCG13176FBgn0033692RNAiBDSC628662No
WASpCG1520FBgn0024273RNAiBDSC259553No
WASpCG1520FBgn0024273RNAiBDSC518022No
βggt-IICG18627FBgn0028970RNAiBDSC505162No
βggt-IICG18627FBgn0028970RNAiBDSC349023No
Table 4
Fly stocks used in addition to the screens.
Stock nameStock numberOriginReferences
w11183605BDSC
tub-Gal45138BDSC
tub-Gal80tsNANA
UAS-dBrainbow34513BDSCHampel et al., 2011
UAS-dBrainbow34514BDSCHampel et al., 2011
Hsp70 > cre851BDSC
UAS-fzr RNAi25550VDRCFox et al., 2010; Schoenfelder et al., 2014
UAS-shi RNAi #128513BDSC
UAS-shi RNAi #236921BDSC
UAS-Rab5 RNAi #130518BDSC
UAS-Rab5 RNAi #267877BDSC
UAS-Rab11 RNAi #127730BDSC
UAS-Rab11 RNAi #222198VDRC
UAS-SCAR RNAi #136121BDSCBischoff et al., 2013
UAS-SCAR RNAi #251803BDSCXing et al., 2018
UAS-kirre RNAi27227VDRCLinneweber et al., 2015
UAS-sns RNAi877VDRCLinneweber et al., 2015
UAS-schizo RNAi36625VDRCJohnson et al., 2011
UAS-sing RNAi12202VDRCBrunetti et al., 2015
UAS-Cdc42DN6288BDSC
UAS-Rac1DN6292BDSC
UAS-Rho1DN7328BDSC
UAS-GFPNLS4776BDSC
UAS-GFP-Myc-2x-FYVE42712BDSCGillooly et al., 2000; Wucherpfennig et al., 2003
UAS-YFP-Rab59775BDSC
60H12-Gal439268BDSC
UAS-shiDN5822BDSC
NrxIV-GFP50798BDSC
Df(1)BSC86729990BDSC
UAS-ogre RNAi7136VDRCHolcroft et al., 2013; Spéder and Brand, 2014
byn-Gal4-NASinger et al., 1996
UAS-GFPPA-Lynn CooleyDatta et al., 2008
UAS-NDN-NARebay et al., 1993
UAS-shi-Venus-Stefano De RenzisFabrowski et al., 2013
UAS-GFP-ogre-Andrea BrandSpéder and Brand, 2014
UAS-Gapdh2-GFPPA--This paper
Table 5
Additional Methods.
PanelAdditional methods
Figure 1—figure supplement 1F-F''Hsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae dissected at 62 (D), 69 (D’), or 80 (D’’) hours post-puparium formation (HPPF) at 25°C. Hindguts were stained with Rabbit anti-GFP (Thermo-Fisher, A11122, 1:1000), Rat anti-HA (Sigma, 3F10, 1:100), and DAPI at 5 μg/ml.
Figure 1GHsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae dissected at various HPPF at 25°C. The area labeled by mKO2 was divided by total papillar area.
Figure 1HHsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae live-imaged at 69HPPF at 25°C.
Figure 1H'Fluorescence intensity measured in neighboring cells during sharing onset (1H).
Figure 1I-I'byn-Gal4/UAS-GFPPA, live-imaged during adulthood. Single secondary and principal cells were photoactivated and imaged every 3 s.
Figure 2AUAS-RNAis and dominant-negative versions of 77 genes representing a wide range of cellular roles were screened (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) for sharing defects. Animals expressing both UAS-dBrainbow and an UAS-driven RNAi or mutant gene were raised at 25°C and shifted to 29°C at L3. If a given RNAi or DN line was lethal when expressed with the byn-Gal4 driver, a Gal80ts was crossed in and the animals raised at 18°C with a shift to 29°C at pupation. Given the robustness of cytoplasmic sharing in WT animals, gene knockdowns or mutants with even single cell defects in sharing were considered ‘hits’.
Figure 2BSecondary screen of 36 genes representing various categories of membrane trafficking (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) for sharing defects. Animals expressing both UAS-dBrainbow and an UAS-driven RNAi were raised at 25°C and shifted to 29°C at L3. If a given RNAi line was lethal when expressed with the byn-Gal4 driver, a Gal80ts was crossed in and the animals raised at 18°C with a shift to 29°C at pupation. Given the robustness of cytoplasmic sharing in WT animals, gene knockdowns with even single cell defects in sharing were considered ‘hits’.
Figure 2CSecondary screen (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) of dominant-negative and constitutively-active variants of the Drosophila Rab GTPases. UAS-Rab11DN and UAS-Rab14DN required a Gal80ts repressor and temperature shifts from 18 to 29°C at pupation. UAS-Rab1DN and UAS-Rab5DN required papillar-specific expression using an alternative Gal4 driver (60 H12-Gal4), Gal80ts repressor, and temperature shifts from 18 to 29°C at pupation.
Figure 2DHsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals dissected pre-sharing (48 HPPF at 29°C).
Figure 2D'Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals raised at 18°C and shifted to 29°C at pupation and dissected post-sharing (young adult).
Figure 2EYoung adult animals expressing UAS-shi RNAi #1 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at pupation to maximize RNAi and minimize animal lethality.
Figure 2FYoung adult animals expressing UAS-Rab5 RNAi #1 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at 1–2 days PPF to maximize RNAi and minimize animal lethality.
Figure 2GYoung adult animals expressing UAS-Rab11 RNAi #2 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at 1–2 days PPF to maximize RNAi and minimize animal lethality.
Figure 2HAnimals were shifted and dissected as in 2D-G. Additionally, Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing UAS-shi RNAi #2 were raised at 18°C and shifted to 29°C at pupation, animals expressing UAS-Rab5 RNAi #2 were raised at 18°C and shifted to 29°C at L3, and animals expressing UAS-Rab11 RNAi #1 were raised at 18°C and shifted to 29°C at 1–2 days PPF.
Figure 3A-A'Pupae expressing the early and late endosome marker UAS-GFP-myc-2x-FYVE were dissected pre (A, 48HPPF at 29°C) and post (A’, 72HPPF at 29°C) sharing onset.
Figure 3BPupae expressing UAS-GFP-myc-2x-FYVE in a UAS-shi RNAi #1 background at a post-sharing time point (24HPPF at 18°C + 72 hr at 29°C).
Figure 3CAggregated line profiles of UAS-GFP-myc-2x-FYVE intensity across papilla.
Figure 3D-D'Pupae expressing UAS-shi-Venus were dissected pre (D, 48HPPF at 29°C) and post (D’, 72HPPF at 29°C) sharing onset.
Figure 3EAggregated line profiles of Shi-Venus intensity from the basal (0% distance) to the apical (100% distance) edges of the papilla. See 3C.
Figure 3F-F''Transmission electron micrographs of the microvillar-like structures of pupal papillae pre (F, 60HPPF at 25°C), mid (F’, 66HPPF at 25°C), and post (F’’, 69HPPF at 25°C) cytoplasm sharing onset.
Figure 3G-G''Electron micrographs of mitochondria and surrounding membrane material pre (G, 60HPPF at 25°C), mid (G’, 66HPPF at 25°C), and post (G’’, 69HPPF at 25°C)
Figure 3HElectron micrograph of microvillar-like structures of WT (w1118) young adult papillar cells.
Figure 3IElectron micrograph of microvillar-like structures of young adult byn-Gal4, Gal80tsUAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C).
Figure 3JElectron micrograph of microvillar-like structures of young adult byn-Gal4, Gal80tsUAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C).
Figure 3KElectron micrograph of mitochondria and surrounding membrane material of WT (w1118) young adult papillar cells.
Figure 3LElectron micrograph of mitochondria and surrounding membrane material of young adult byn-Gal4, Gal80tsUAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C).
Figure 3MElectron micrograph of mitochondria and surrounding membrane material of young adult byn-Gal4, Gal80ts, UAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C).
Figure 3NElectron micrograph of post-sharing WT (TM3/UAS-shi RNAi #1) pupa (24HPPF at 18°C, shifted to 29°C for 50 hr, then dissected)
Figure 3OElectron micrograph of post-sharing byn-Gal4, Gal80ts,UAS-shi RNAi #1 pupa (24HPPF at 18°C, shifted to 29°C for 50 hr, then dissected)
Figure 3PGap junction length / (gap junction length + septate junction length) measured in WT and UAS-shi RNAi #1 pupae (see 3N-3O). Each point represents an image of a junction.
Figure 4A-A''Electron micrographs of apical junctions (adherens, septate, and gap) pre (A, 60HPPF at 25°C), mid (A’, 66HPPF at 25°C), and post (A’’, 69HPPF at 25°C)
Figure 4BGap junction length / (gap junction length + septate junction length) measured in pupae pre (60HPPF at 25°C), mid (66HPPF at 25°C), and post (69HPPF at 25°C) sharing onset. Each point represents an image of a junction.
Figure 4CRelative innexin transcript abundance (innexin X transcripts/total innexin transcripts) using data from Fly Atlas 2 (Leader et al., 2018) and RNA-seq of adult w1118 rectums performed in the Fox Lab.
Figure 4D-D'Pupae with endogenously GFP-tagged NrxIV (NrxIV-GFP) dissected pre (D, 48HPPF) and post (D', 72HPPF) sharing onset.
Figure 4E-E'Pupae stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75) pre (E, 48HPPF) and post (E', 58HPPF, papillae do not stain well at later timepoints) sharing onset.
Figure 4FYoung adult animals expressing no transgene (WT), UAS-ogreDN, UAS-ogre RNAi, or containing a deficiency covering ogre, Inx2, and Inx7 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were raised at 25°C until L3 and then shifted to 29°C until dissection at young adulthood.
Figure 4GSee Figure 4F.
Figure 4H60 H12-Gal4, Gal80ts driving UAS-shiDN and WT siblings were shifted from 18 to 29°C at pupation. byn-Gal4, Gal80ts driving UAS-ogreDN animals and WT siblings were raised at 25°C and shifted to 29°C at L3. Animals 1–3 days post-eclosion were sorted into sex-matched groups and fed a control diet or a high salt (2% NaCl) diet. Survival was assessed once per day for 10 days.
Figure 1—figure supplement 1AHsp70 > cre; UAS-dBrainbow; tubulin-Gal4 animals raised at 29°C. Tissues dissected at adulthood.
Figure 1—figure supplement 1Dbyn-Gal4/UAS-Gapdh2-GFPPA raised at 29°C and live-imaged during adulthood. Principal cells were photoactivated and imaged every 15 s.
Figure 1—figure supplement 1EHsp70 > cre; UAS-dBrainbow; byn-Gal4 animals were shifted from 25 to 29°C during L3 and dissected at adulthood.
Figure 1—figure supplement 1FHsp70 > cre; UAS-dBrainbow/UAS-fzr RNAi; byn-Gal4 animals were shifted from 25 to 29°C during L2 to maximize fzr knock down during endocycling. Animals were dissected at adulthood.
Figure 1—figure supplement 1GHsp70 > cre; UAS-dBrainbow; byn-Gal4/UAS-NDN animals were shifted from 25 to 29°C during L3 to ensure maximum UAS-NDN expression during mitoses. Animals were dissected at adulthood.
Figure 2—figure supplement 1AHsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing various previously published myoblast fusion RNAis raised at 25°C and shifted to 29°C at L3 and dissected post-sharing (young adult).
Figure 2—figure supplement 1BHsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing various previously published UAS-dominant-negative active regulators raised at 18°C and shifted to 29°C at L3 and dissected post-sharing (young adult).
Figure 2—figure supplement 1CPapillar cells were identified using byn-Gal4, Gal80ts, driving UAS-GFPNLS expression. Cells were counted in one, z-sectioned half of the papillae and multiplied by two to give an approximate cell count.
Figure 2—figure supplement 1DHsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals were raised at 18°C until 3–4 days PPF and shifted to 29°C and dissected at young adulthood.
Figure 2—figure supplement 1EHsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing UAS-shi RNAi #1 were raised at 18°C until 3–4 days PPF and shifted to 29°C and dissected at young adulthood.
Figure 3—figure supplement 1ASee Figure 3A-C. Basal and apical membrane defined as 10–20% and 90–100% total distance of papillae, respectively.
Figure 3—figure supplement 1B-B'byn-Gal4 > UAS-Rab5-YFP animals dissected pre (48HPPF, 29°C) and post (72HPPF, 29°C) sharing onset.
Figure 3—figure supplement 1B''See Figure 3—figure supplement 1B-B' and Figure 3C.
Figure 3—figure supplement 1C-C''Electron micrographs of apical junctions (adherens, septate, and gap) pre (D, 60HPPF at 25°C), mid (D’, 66HPPF at 25°C), and post (D’’, 69HPPF at 25°C)
Figure 3—figure supplement 1DElectron micrograph of apical junctions (adherens, septate, and gap) of WT (w1118) young adult papillar cells.
Figure 3—figure supplement 1EElectron micrograph of apical junctions (adherens, septate, and gap) of young adult byn-Gal4, Gal80tsts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C).
Figure 3—figure supplement 1FElectron micrograph of apical junctions (adherens, septate, and gap) of young adult byn-Gal4, Gal80tsts, UAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C).
Figure 3—figure supplement 1GSee Figure 3N-O. Junction width was measured throughout and averaged per image. Each point represents one image of a junction.
Figure 3—figure supplement 1G'See Figure 3N-O. Junction width was measured throughout and averaged per image. Each point represents one image of a junction.
Figure 3—figure supplement 1G''See Figure 3N-O. Raw lengths shown were used to calculate ‘fraction gap junction’ in 3P. Each point represent one image of a junction.
Figure 3—figure supplement 2ATEM of young adult (w1118) papilla.
Figure 4—figure supplement 1ASee Figure 4A-B. Junction width was measured throughout and averaged per image. Each point represents one image of a junction.
Figure 4—figure supplement 1A'See Figure 4A-B. Junction width was measured throughout and averaged per image. Each point represents one image of a junction.
Figure 4—figure supplement 1A''See Figure 4A-B. Raw lengths shown were used to calculate ‘fraction gap junction’ in 3P. Each point represent one image of a junction.
Figure 4—figure supplement 1B-B'Pupae expressing byn-Gal4, Gal80tsts, UAS-ogreDN (UAS-GFP-ogre) dissected pre (B, 48HPPF, 29°C) and post (B', 72HPPF, 29°C) sharing onset.
Figure 4—figure supplement 1Cbyn-Gal4, Gal80ts pupae raised at 18°C until 0HPPF and then shifted to 29°C until dissection at 58HPPF. Pupal rectums were stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75).
Figure 4—figure supplement 1C'byn-Gal4, Gal80tsts, UAS-shi RNAi #2 pupae raised at 18°C until 0HPPF and then shifted to 29°C until dissection at 58HPPF. Pupal rectums were stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75).
Figure 4—figure supplement 1Dbyn-Gal4 > UAS-GFPNLS dissected pre (48HPPF, 29°C) sharing onset.
Figure 4—figure supplement 1D'60H12-Gal4 > UAS-GFPNLS dissected pre (48HPPF, 29°C) sharing onset. The pan-hindgut driver used in previous experiments, brachyenteron (byn-Gal4), causes animal lethality with shi, Rab5, and Rab11 knockdown within a few days. We therefore screened for and identified an alternative, papillae-specific driver (60H12-Gal4), derived from regulatory sequences of the hormone receptor gene Proctolin Receptor. 60H12-Gal4 > shiDN animals are viable on a control diet allowing us to test papillar function on a high-salt diet.
Figure 4—figure supplement 1EHsp70 > cre; UAS-dBrainbow; 60H12-Gal4 animals raised at 18°C and shifted to 29°C at pupation and dissected as young adults.
Figure 4—figure supplement 1E'Hsp70 > cre; UAS-dBrainbow; 60H12-Gal4 / UAS-shiDN animals raised at 18°C and shifted to 29°C at pupation and dissected as young adults.
Figure 4—figure supplement 1E''See Figure 4—figure supplement 1E-E'.
Table 6
Additional statistics.
PanelN (animals) per groupBio. repsStatistical testP-value
Figure 1G9–182Unpaired t-test66HPPF:74HPPF < 0.0001
Figure 2H9–322–3One-way ANOVA with Tukey's multiple comparisons testANOVA:<0.0001 Pre:WT < 0.0001 WT:shi #1 < 0.0001 WT:shi #2 < 0.0001 WT:Rab5 #1 < 0.0001 WT:Rab5 #2 < 0.0001 WT:Rab11 #1 < 0.0001 WT:Rab11 #2 < 0.0001 shi #1:Rab5 #2 0.0181 shi #1:Rab11 #2 0.0428 shi #2:Rab5 #2 0.0263 Rab5 #1:Rab5 #2 0.0009 Rab5 #1:Rab11 #2 0.0020 all others, ns
Figure 3C6–102–3see 3-S1Asee Figure 3—figure supplement 1A
Figure 3E4–53Unpaired t-testApical region: Pre:Post < 0.0001
Figure 3P3–42Unpaired t-testWT:shi RNAi < 0.0001
Figure 4B3–42Unpaired t-testPre:Post < 0.0001
Figure 4F13–142One-way ANOVA with Tukey's multiple comparisons testANOVA:<0.0001 WT:ogreDN < 0.0001 WT:Df < 0.0001 WT:ogre RNAi 0.0007
Figure 4H27–373One-way ANOVA with Tukey's multiple comparisons test (mean death at 10 days in each group)ANOVA:<0.0001 WTsalt:shiDNreg ns, 0.7173 WTsalt:shiDNsalt < 0.0001 shiDNsalt:shiDNreg < 0.0001 ANOVA:<0.0001 WTsalt:ogreDNreg < 0.0001 WTsalt:ogreDNsalt < 0.0001 ogreDNsalt:ogreDNreg < 0.0001
Figure 1—figure supplement 1H12–202Unpaired t-testWT:fzr RNAi < 0.0001 WT:NDN ns, 0.1786
Figure 2—figure supplement 1A8–112One-way ANOVA with Tukey's multiple comparisons testANOVA:<0.0001 Sing RNAi:all others < 0.0001 All others: ns
Figure 2—figure supplement 1B6–82One-way ANOVAANOVA: ns, 0.3692
Figure 2—figure supplement 1C11–232One-way ANOVA with Tukey's multiple comparisons testANOVA: 0.0044 shi RNAi #1:Rab11 RNAi #1 0.0244 Rab5 RNAi #2:Rab11 RNAi #1 0.0193 All others: ns
Figure 2—figure supplement 1F10–112Unpaired t-testns, 0.0782
Figure 3—figure supplement 1A6–102One-way ANOVA with Tukey's multiple comparisons testANOVA:<0.0001 Pre:Post < 0.0001 Pre:shi RNAi ns, 0.7882 Post:shi RNAi < 0.0001
Figure 3—figure supplement 1B''102Unpaired t-testApical basal difference (see 1-S3A) Pre:Post 0.0007
Figure 3—figure supplement 1G3–42Unpaired t-testns, 0.2203
Figure 3—figure supplement 1G'3–42Unpaired t-testns, 0.4754
Figure 3—figure supplement 1G''3–42Multiple unpaired t-testsSeptate: WT:shi RNAi ns, 0.1547 Gap: WT:shi RNAi < 0.0001
Figure 4—figure supplement 1A3–42One-way ANOVAns, 0.8973
Figure 4—figure supplement 1A'3–42One-way ANOVAns, 0.3994
Figure 4—figure supplement 1A''3–42Multiple unpaired t-testsSeptate: all ns Gap: Pre:Post 0.0004 Gap: all others, ns
Figure 4—figure supplement 1E''112Unpaired t-testWT:shiDN < 0.0001

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  1. Nora G Peterson
  2. Benjamin M Stormo
  3. Kevin P Schoenfelder
  4. Juliet S King
  5. Rayson RS Lee
  6. Donald T Fox
(2020)
Cytoplasmic sharing through apical membrane remodeling
eLife 9:e58107.
https://doi.org/10.7554/eLife.58107