Cytoplasmic sharing through apical membrane remodeling.

Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62 kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules < 1 kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.

Introduction

Throughout the tree of life, there are upper limits to the size of individual cells. This size limitation is imposed by genome content, which impacts biosynthetic capacity and cell growth (Conlon and Raff, 1999; Mueller, 2015). In diverse tissues and organisms, the existence of ‘giant cells’ is driven by polyploidy, the presence of greater than a diploid genome content (Van de Peer et al., 2017; Schoenfelder and Fox, 2015). Purposes of polyploidy across evolution remain largely unknown. However, one potential advantage of a tissue containing few, large polyploid cells vs. numerous, small diploid cells is the ability of cytoplasmic components to move over much larger distances.

A common form of polyploidy is multinucleation. Sharing of cytoplasm in a multinucleate tissue or organism is an important and recurring adaptation across evolution. Multinucleate cells can be large, metabolically-active cells with unique shapes and functions ranging from specialized force distribution to tissue barrier preservation. During organismal development, examples of multinucleation include animal skeletal muscle, mammalian osteoclasts, and mammalian syncytial placental trophoblasts (Deng et al., 2017; Gerbaud and Pidoux, 2015; Pereira et al., 2018). Multinucleation also arises in response to tissue stress, such as following injury to the Drosophila abdominal epithelium or the human corneal epithelium (Losick et al., 2013; Ikebe et al., 1986). A commonality of these numerous examples of multinucleation is the ability to exchange, over long distances, cytoplasmic components such as RNA, proteins, and even organelles (Rustom et al., 2004; McLean and Cooley, 2013).

The cellular mechanisms underlying multinucleation are diverse. During cell division, multinucleation can occur through incomplete cytokinesis, followed by formation of a stable cytoplasmic bridge between nuclei. This process occurs in diverse examples of germ cell development (Greenbaum et al., 2011) and also in some somatic cells such as the ring canal of the Drosophila ovary (McLean and Cooley, 2013) and the plasmodesmata of plants (Lůcas and Wolf, 1993). A second major mechanism of multinucleation involves plasma membrane breaches. Such breaches can involve distinct actin-based protrusive structures. Podosome-like structures facilitate multinucleation in Drosophila skeletal muscle and mammalian macrophages (Faust et al., 2019; Sens et al., 2010). While the mechanisms are diverse, one common feature of the above-discussed examples of multinucleation and cytoplasm sharing identified to date are clearly visible plasma membrane disruptions.

Here, we report a visual animal-wide screen, using multi-color lineage labeling approaches in the tractable animal model Drosophila melanogaster, for multinucleate tissues that share cytoplasm. We discover cytoplasm sharing in the rectal papilla, a common insect resorptive intestinal epithelium that is critical for maintaining ionic homeostasis (Wigglesworth, 1932; Cohen et al., 2020). Likely due to its extreme proximal location in the gut of many insect species, this epithelium is linked to the infiltration of diverse pathogens, such as those involved in African sleeping sickness and also viruses being pursued as insect control measures (Gu et al., 2010; Filosa et al., 2019). Here, we reveal that cytoplasm sharing onset in Drosophila papillae occurs during a short developmental window, indicating robust molecular regulation. We find that papillar cytoplasm sharing requires neither incomplete cytokinesis nor canonical actin-based membrane breach regulators. Using transmission electron microscopy, we further identify that this developmentally programmed process involves extensive remodeling of apical junctions and lateral membranes, but not clearly identifiable plasma membrane breaches. Using genetic screening, we implicate specific regulators of membrane remodeling, notably the GTPase Dynamin/Shibire, in the mechanism of papillar cytoplasmic sharing. From analysis of shibire mutants, we uncover a requirement for gap junction establishment and specific gap junction proteins in papillar cytoplasm sharing. Mutant animals defective in papillar cytoplasm sharing are intolerant of a high-salt diet, indicating a physiological role of long-range cytoplasm movement in this tissue. Unlike all known examples of multinucleation, our results show that cytoplasm sharing in rectal papillae requires developmentally programmed apical membrane remodeling, which creates a giant resorptive epithelial network of 100 nuclei. This tissue represents a new system to investigate the diversity of multicellular tissue organization and mechanisms and functions of cytoplasm sharing.

Results

Drosophila hindgut papillae undergo developmentally programmed cytoplasmic sharing

To identify new examples of adult tissues in Drosophila that share cytoplasm, we ubiquitously expressed Cre and UAS-dBrainbow (Hampel et al., 2011; Figure 1A), a Cre-Lox-based system that randomly labels cells with only one of three fluorescent proteins. We used animals heterozygous for UAS-dBrainbow to ensure single-labeling of cells. We ubiquitously expressed Cre, which does not require heat-shock induction, from early embryonic stages (before cells endocycle to become polyploid). Cre-mediated excision occurs independently of Gal4 expression and Gal80ts repression of dBrainbow. Therefore, we can ensure that multi-labeled cells only arise by cytoplasm sharing between cells not related by cell division or incomplete cytokinesis (Figure 1B). We examined a wide range of tissues (Figure 1—figure supplement 1A). From our screen, we discovered that the rectal papilla is a new example of a tissue with cytoplasm sharing. Adult Drosophila contain four papillae, each with 100 nuclei of genome content between 8 and 16C (Fox et al., 2010), that reside in the posterior hindgut (Figure 1C). Each papilla is a polarized epithelial cone with the apical region facing the gut lumen and the basal region surrounding a central canal that connects to the fly’s hemolymph (Figure 1D). The papillar structure supports its function to reabsorb water, ions, and small molecules from the gut lumen and recycle them back to the hemolymph (Cohen et al., 2020). Knowing that adult papillar cells share cytoplasm, we next used our dBrainbow system to identify when papillar cells begin to share relative to other developmental events that we previously identified (Figure 1E). Using both fixed and live imaging of whole organs, we found that at 62 hours post-puparium formation (HPPF), each papillar cell contains only one dBrainbow label (Figure 1F). By contrast, at 69HPPF, multi-labeled cells are apparent (Figure 1F’,H–H’). We quantitatively measured papillar sharing across the tissue (Figure 1—figure supplement 1B, Materials and methods) and found that cytoplasm sharing initiates over a narrow 6 hr period (68-74HPPF, Figure 1G). Our results suggested that at least RNA and possibly protein passes between papillar cells to facilitate cytoplasm sharing. To directly test if protein is shared, we photo-activated GFP (GFPPA) in single adult papillar cells and observed in real time whether GFPPA spreads to adjacent cells. We find the principal papillar cells, but not the secondary cells at the papillar base (Garayoa et al., 1999; Figure 1—figure supplement 1C), share protein across an area of at least several nuclei (Figure 1I–I’). We next tested whether a larger protein can be shared between papillar cells. We used rectal papillae RNA-sequencing data (Leader et al., 2018) to identify proteins that are endogenously expressed, cytoplasmic, and relatively large. We therefore generated flies expressing a UAS-inducible, photoactivatable GFP fused to Glyceraldehyde 3 phosphate dehydrogenase 2 (UAS-Gapdh2-GFP This construct should produce a tagged protein of 62.3 kDa. We found that Gapdh2-GFPPA protein is shared between cells, as it never stops at a papillar cell–cell boundary, though it may move at a slower rate than GFPPA (Figure 1—figure supplement 1D). Therefore, proteins as large as ~62 kDa (the size of GFP-tagged Gapdh2) can move across an area covered by multiple papillar nuclei. Additionally, the movement of our Gapdh2 transgenic protein indicates that papillar cells likely share endogenously expressed proteins. These results indicate that papillae undergo a developmentally programmed conversion from 100 individual cells to a single giant multinuclear cytoplasm that shares the products of ~1200 genomes.

Figure 1.

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.

(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) N background (p=0.8786). (H) Quantification of cytoplasmic sharing in adult WT, fzr RNAi, and N-expressing animals (N = 12–20, rep = 2).

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) N background (p=0.8786). (H) Quantification of cytoplasmic sharing in adult WT, fzr RNAi, and N-expressing animals (N = 12–20, rep = 2).

We next examined whether cytoplasm sharing requires either programmed endocycles or mitoses. We have previously shown that larval papillar cells first undergo endocycles, which increase cellular ploidy. Then, during metamorphosis, pupal papillar cells disassemble polytene chromosomes and undergo polyploid mitotic cycles, which increase cell number (Fox et al., 2010; Stormo and Fox, 2016; Stormo and Fox, 2019). Both endocycles and mitoses occur well prior to the start of papillar cytoplasm sharing (Figure 1E). Papillar endocycles require the Anaphase-Promoting Complex/Cyclosome regulator fizzy-related (fzr) while the papillar mitoses require Notch signaling (Schoenfelder et al., 2014). Knockdown of fzr significantly disrupts cytoplasm sharing (Figure 1—figure supplement 1E,F,H). We hypothesize that endocycles are required for differentiation of the papillae, which later enables these cells to trigger cytoplasm sharing. In contrast, blocking Notch signaling, which initiates papillar mitotic divisions (Fox et al., 2010), does not prevent sharing (Figure 1—figure supplement 1E,G,H). Thus, papillar cytoplasm sharing requires developmentally programmed endocycles but not mitotic cycles.

Cytoplasmic sharing requires membrane remodeling proteins

As our dBrainbow approach only identifies cytoplasm sharing events that do not involve incomplete division/cytokinesis, we examined whether sharing results from fusion pore formation, as in skeletal muscle. A well-studied model of such cell–cell fusion in Drosophila is myoblast fusion, which requires an actin-based podosome (Richardson et al., 2007; Sens et al., 2010). We conducted a candidate dBrainbow-based RNAi screen (77 genes, Figure 2A, Table 1) of myoblast fusion regulators and other plasma membrane components. Remarkably, 0/15 myoblast fusion genes from our initial screen regulate papillar cytoplasm sharing (Figure 2A, Figure 2—figure supplement 1A, Table 1). Furthermore, dominant-negative forms of Rho family GTPases have no impact on dBrainbow labeling (Figure 2—figure supplement 1B), providing additional evidence against actin-based cytoplasm sharing. Instead, we found 8/77 genes, including subunits of the vacuolar H+ ATPase (Vha16-1), ESCRT-III complex (Vps2), and exocyst (Exo84) (Figure 2A) are required for papillar cytoplasm sharing. Through additional screening, the only myoblast fusion regulator required for papillar cytoplasm sharing is singles bar (sing), a presumed vesicle trafficking gene (Estrada et al., 2007; Figure 2—figure supplement 1A). Given the enrichment of our candidate screen hits in membrane trafficking and not myoblast fusion, we further explored the role of membrane trafficking in cytoplasm sharing.

Figure 2.

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).

(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).

Table 1.

Cytoplasm sharing primary candidate screen gene results.

Gene category	Gene	Annotation symbol	Gene ID	Sharing disrupted?
Autophagy	Atg1	CG10967	FBgn0260945	No
Autophagy	Atg7	CG5489	FBgn0034366	No
Autophagy	Atg8a	CG32672	FBgn0052672	No
Cell cycle/Chromosomes	blue	NA	FBgn0283709	No
Cell cycle/Chromosomes	CapD2	CG1911	FBgn0039680	No
Cell cycle/Chromosomes	Cdc2	CG5363	FBgn0004106	Yes
Cell cycle/Chromosomes	Clamp	CG1832	FBgn0032979	No
Cell cycle/Chromosomes	endos	CG6513	FBgn0061515	No
Cell cycle/Chromosomes	fzr	CG3000	FBgn0262699	Yes
Cell cycle/Chromosomes	Mi-2	CG8103	FBgn0262519	No
Cell cycle/Chromosomes	Rbp9	CG3151	FBgn0010263	No
Cell cycle/Chromosomes	SA-2	CG13916	FBgn0043865	No
Cell signaling	Chico	CG5686	FBgn0024248	No
Cell signaling	Egfr	CG10079	FBgn0003731	Yes
Cell signaling	grk	CG17610	FBgn0001137	No
Cell signaling	N	CG3936	FBgn0004647	No
Cell signaling	Ptp61F	CG9181	FBgn0267487	No
Cell signaling	rho	CG1004	FBgn0004635	Yes
Cell signaling	ru	CG1214	FBgn0003295	No
Cell signaling	spi	CG10334	FBgn0005672	No
Cell signaling	stet	CG33166	FBgn0020248	No
Cell signaling	wts	CG12072	FBgn0011739	No
Cell signaling	βggt-II	CG18627	FBgn0028970	No
Cytoskeleton	ALiX	CG12876	FBgn0086346	No
Cytoskeleton	Cdc42	CG12530	FBgn0010341	No
Cytoskeleton	DCTN1-p150	CG9206	FBgn0001108	No
Cytoskeleton	pav	CG1258	FBgn0011692	No
Cytoskeleton	wash	CG13176	FBgn0033692	No
Hindgut-enriched	dac	CG4952	FBgn0005677	No
Hindgut-enriched	Dr	CG1897	FBgn0000492	No
Hindgut-enriched	nrv3	CG8663	FBgn0032946	No
Membrane component	Flo1	CG8200	FBgn0024754	No
Membrane component	Flo2	CG32593	FBgn0264078	No
Membrane component	Iris	CG4715	FBgn0031305	No
Myoblast fusion	Arf51F	CG8156	FBgn0013750	No
Myoblast fusion	Arp2	CG9901	FBgn0011742	No
Myoblast fusion	Arp3	CG7558	FBgn0262716	No
Myoblast fusion	Ced-12	CG5336	FBgn0032409	No
Myoblast fusion	dock	CG3727	FBgn0010583	No
Myoblast fusion	hbs	CG7449	FBgn0029082	No
Myoblast fusion	Hem	CG5837	FBgn0011771	No
Myoblast fusion	mbc	CG10379	FBgn0015513	No
Myoblast fusion	Rac1	CG2248	FBgn0010333	No
Myoblast fusion	Rho1	CG8416	FBgn0014020	No
Myoblast fusion	rols	CG32096	FBgn0041096	No
Myoblast fusion	rst	CG4125	FBgn0003285	No
Myoblast fusion	SCAR	CG4636	FBgn0041781	No
Myoblast fusion	siz	CG32434	FBgn0026179	No
Myoblast fusion	WASp	CG1520	FBgn0024273	No
Polarity	Abi	CG9749	FBgn0020510	No
Polarity	CadN	CG7100	FBgn0015609	No
Polarity	cindr	CG31012	FBgn0027598	No
Polarity	cno	CG42312	FBgn0259212	No
Polarity	Gli	CG3903	FBgn0001987	No
Polarity	l(2)gl	CG2671	FBgn0002121	No
Polarity	Nrg	CG1634	FBgn0264975	No
Polarity	sdt	CG32717	FBgn0261873	No
Polarity	shg	CG3722	FBgn0003391	No
Vesicle trafficking	Atl	CG6668	FBgn0039213	No
Vesicle trafficking	Bet1	CG14084	FBgn0260857	No
Vesicle trafficking	Chmp1	CG4108	FBgn0036805	No
Vesicle trafficking	CHMP2B	CG4618	FBgn0035589	No
Vesicle trafficking	dnd	CG6560	FBgn0038916	No
Vesicle trafficking	Exo84	CG6095	FBgn0266668	Yes
Vesicle trafficking	lerp	CG31072	FBgn0051072	No
Vesicle trafficking	Rab11	CG5771	FBgn0015790	Yes
Vesicle trafficking	Rab23	CG2108	FBgn0037364	No
Vesicle trafficking	Rab4	CG4921	FBgn0016701	No
Vesicle trafficking	Rab7	CG5915	FBgn0015795	No
Vesicle trafficking	Rab8	CG8287	FBgn0262518	No
Vesicle trafficking	RabX4	CG31118	FBgn0051118	No
Vesicle trafficking	Vha16-1	CG3161	FBgn0262736	Yes
Vesicle trafficking	Vha55	CG17369	FBgn0005671	No
Vesicle trafficking	VhaAC39-1	CG2934	FBgn0285910	No
Vesicle trafficking	VhaAC39-2	CG4624	FBgn0039058	No
Vesicle trafficking	Vps2	CG14542	FBgn0039402	Yes
Vesicle trafficking	Vps33b	CG5127	FBgn0039335	No
Total screen results
Sharing disrupted	8
No sharing phenotype	69
Total	77
Screen results by category
Polarity	9
Vesicle trafficking	19
Myoblast fusion	15
Cell cycle/Chromosomes	9
Cell signaling	11
Autophagy	3
Cytoskeleton	5
Hindgut-enriched	3
Membrane component	3
Total	77

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).

We conducted two secondary dBrainbow screens to find specific membrane trafficking pathway components that regulate papillar sharing. First, a focused candidate membrane trafficking screen revealed additional components (12/36 genes screened, Figure 2B, Table 2) including three more vacuolar H+ ATPase subunits, five more exocyst components, and the Dynamin GTPase shibire (shi) (Figure 2B,D,E,H). Second, we screened constitutively-active and dominant-negative versions of all 31 Drosophila Rabs. Sharing requires only a small number of Rabs, specifically the ER/Golgi-associated Rab1, the early endosome-associated Rab5, and the recycling endosome-associated Rab11 (Figure 2C,D,F–H). Given our identification of the membrane vesicle recycling circuit involving shi, Rab5, and Rab11, we focused on these genes. Two unique RNAi lines for each gene show consistent sharing defects, and most of these knockdowns completely recapitulate the pre-sharing state (Figure 2H). Despite exhibiting strong cytoplasm sharing defects, shi, Rab5, and Rab11 RNAi papillae appear morphologically normal, with only minor cell number decreases (Figure 2—figure supplement 1C). These results suggest that membrane recycling GTPases regulate a specific developmental event associated with cytoplasm sharing, and not papillar morphogenesis. In agreement with these GTPases acting during development, rather than as part of an ongoing transport process, GTPase knockdown after sharing onset does not block cytoplasm sharing (Figure 2—figure supplement 1D–F). Together, our screens reveal that membrane trafficking, particularly Dynamin-mediated endocytosis and early/recycling endosome trafficking, regulates papillar cytoplasmic sharing.

Table 2.

Membrane trafficking primary and secondary candidate screen gene results.

Gene category	Gene subcategory	Gene	Annotation symbol	Gene ID	Sharing disrupted?	Screen
Membrane trafficking	ER	Atl	CG6668	FBgn0039213	No	Primary
Membrane trafficking	ESCRT	Chmp1	CG4108	FBgn0036805	No	Primary
Membrane trafficking	ESCRT	CHMP2B	CG4618	FBgn0035589	No	Primary
Membrane trafficking	ESCRT	lsn	CG6637	FBgn0260940	No	Secondary
Membrane trafficking	ESCRT	Vps2	CG14542	FBgn0039402	Yes	Primary
Membrane trafficking	ESCRT	Vps4	CG6842	FBgn0283469	No	Secondary
Membrane trafficking	Exocyst	Exo70	CG7127	FBgn0266667	No	Secondary
Membrane trafficking	Exocyst	Exo84	CG6095	FBgn0266668	Yes	Primary
Membrane trafficking	Exocyst	Sec10	CG6159	FBgn0266673	Yes	Secondary
Membrane trafficking	Exocyst	Sec15	CG7034	FBgn0266674	Yes	Secondary
Membrane trafficking	Exocyst	Sec5	CG8843	FBgn0266670	Yes	Secondary
Membrane trafficking	Exocyst	Sec6	CG5341	FBgn0266671	Yes	Secondary
Membrane trafficking	Exocyst	Sec8	CG2095	FBgn0266672	Yes	Secondary
Membrane trafficking	Lysosome	lerp	CG31072	FBgn0051072	No	Primary
Membrane trafficking	Rab-associated	CG41099	CG41099	FBgn0039955	No	Secondary
Membrane trafficking	Rab-associated	mtm	CG9115	FBgn0025742	No	Secondary
Membrane trafficking	Rab-associated	nuf	CG33991	FBgn0013718	No	Secondary
Membrane trafficking	Rab-associated	Rala	CG2849	FBgn0015286	No	Secondary
Membrane trafficking	Rab-associated	Rep	CG8432	FBgn0026378	No	Secondary
Membrane trafficking	Rab-associated	Rip11	CG6606	FBgn0027335	No	Secondary
Membrane trafficking	Vacuolar H+ ATPase	Vha16-1	CG3161	FBgn0262736	Yes	Primary
Membrane trafficking	Vacuolar H+ ATPase	Vha16-2	CG32089	FBgn0028668	No	Secondary
Membrane trafficking	Vacuolar H+ ATPase	Vha16-3	CG32090	FBgn0028667	No	Secondary
Membrane trafficking	Vacuolar H+ ATPase	Vha16-5	CG6737	FBgn0032294	Yes	Secondary
Membrane trafficking	Vacuolar H+ ATPase	Vha55	CG17369	FBgn0005671	No	Primary
Membrane trafficking	Vacuolar H+ ATPase	VhaAC39-1	CG2934	FBgn0285910	No	Primary
Membrane trafficking	Vacuolar H+ ATPase	VhaAC39-2	CG4624	FBgn0039058	No	Primary
Membrane trafficking	Vacuolar H+ ATPase	VhaPPA1-1	CG7007	FBgn0028662	Yes	Secondary
Membrane trafficking	Vacuolar H+ ATPase	VhaPPA1-2	CG7026	FBgn0262514	Yes	Secondary
Membrane trafficking	Vesicle trafficking	Bet1	CG14084	FBgn0260857	No	Primary
Membrane trafficking	Vesicle trafficking	Chc	CG9012	FBgn0000319	No	Secondary
Membrane trafficking	Vesicle trafficking	dnd	CG6560	FBgn0038916	No	Primary
Membrane trafficking	Vesicle trafficking	shi	CG18102	FBgn0003392	Yes	Secondary
Membrane trafficking	Vesicle trafficking	Vps29	CG4764	FBgn0031310	No	Secondary
Membrane trafficking	Vesicle trafficking	Vps33b	CG5127	FBgn0039335	No	Primary
Membrane trafficking	Vesicle trafficking	Vps35	CG5625	FBgn0034708	No	Secondary
Total screen results
Sharing disrupted	12
No sharing phenotype	24
Total	36
Screen results by category	Total	Hits
ER	1	0
ESCRT	5	1
Exocyst	7	6
Lysosome	1	0
Rab-associated	6	0
Vacuolar H+ ATPase	9	4
Vesicle trafficking	7	1
Total	36

Gap junction establishment, but no membrane breaches, accompany cytoplasm sharing

To better understand how membrane trafficking GTPases initiate cytoplasm sharing during development, we examined endosome and Shi localization during sharing onset. We imaged a GFP-tagged pan-endosome marker (myc-2x-FYVE), overexpression of which should not alter endosome shape or localization (Gillooly et al., 2000; Wucherpfennig et al., 2003), and a Venus-tagged shi before and after sharing. Endosomes are evenly distributed shortly before sharing, but become highly polarized at the basal membrane around the time of sharing onset (Figure 3A–A’,C, Figure 3—figure supplement 1A). This basal endosome repositioning requires Shi (Figure 3B–C, Figure 3—figure supplement 1A) and the change in endosome localization is attributed to Rab5-positive early endosomes (Figure 3—figure supplement 1B–B’’). Additionally, Shi localization changes from apical polarization to a uniform distribution during sharing onset (Figure 3D–E). These localization changes indicate that membrane trafficking factors which regulate cytoplasm sharing are highly dynamic during cytoplasm sharing onset.

Figure 3.

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.

(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.

(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 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.

To determine what membrane remodeling events underlie GTPase-dependent cytoplasm sharing, we turned to ultrastructural analysis. Adult ultrastructure and physiology of papillar cells has been examined previously in Drosophila (Wessing and Eichelberg, 1973) and related insects (Gupta and Berridge, 1966). These cells contain elaborate membrane networks that facilitate selective ion resorption from the gut lumen, facing the apical side of papillar cells, to the hemolymph, facing the basal side. Still, little is known about developmental processes or mechanisms governing the unique papillar cell architecture. We looked for changes in cell–cell junctions and lateral membranes that coincide with cytoplasm sharing, especially to determine if there is a physical membrane breach between cells. We identified several dramatic changes in membrane architecture. First, apical microvilli-like structures form during sharing onset (Figure 3F–F’’). Just basal to the microvilli, apical cell–cell junctions are straight in early pupal development and compress into a more curving, tortuous morphology around the time of cytoplasm sharing onset (Figure 3—figure supplement 1C–C’’). One of the most striking changes, coincident with Shi re-localization, is formation of pan-cellular endomembrane stacks surrounding mitochondria. These stacks are likely sites for active ion transport, such as that mediated by the P-type Na+/K+-ATPase, coupled to mitochondria for ATP (Figure 3G–G’’; Berridge and Gupta, 1967; Patrick et al., 2006). Thus, massive apical and intracellular plasma membrane reorganization coincides with both cytoplasm sharing and Shi/endosome re-localization. We next assessed whether the extensive membrane remodeling requires Shi, Rab5, and Rab11. In shi and Rab5 RNAi animals, microvilli protrude downward, instead of upward (Figure 3H–J). Additionally, apical junctions do not compress as in controls (Figure 3—figure supplement 1D–F). Notably, membrane stacks are greatly reduced (Figure 3K–M). shi RNAi animals exhibit numerous trapped vesicles, consistent with a known role for Dynamin in membrane vesicle severing (Damke et al., 1994; Hinshaw and Schmid, 1995; Figure 3L, inset). Together, we find that Shi and endosomes extensively remodel membranes during papillar cytoplasm sharing.

Gap junction proteins are required for cytoplasmic sharing

Our extensive ultrastructural analysis did not reveal any clear breaches in the plasma membrane, despite numerous membrane alterations. Adult papillae exhibit large extracellular spaces between nuclei that eliminate the possibility of cytoplasm sharing throughout much of the lateral membrane (Figure 3—figure supplement 2A; Wessing and Eichelberg, 1973; Gupta and Berridge, 1966). Instead, through our GTPase knockdown studies, we identified a striking alteration in the apical cell–cell interface that strongly correlates with cytoplasm sharing. Specifically, shi animals frequently lack apical gap junctions (Figure 3N–O) (p<0.0001) (Figure 3P, Figure 3—figure supplement 1H–H’’). Upon closer examination of control animal development, we find that apical gap junction-like structures arise at cytoplasm sharing onset. There is almost no gap junction-like structure before cytoplasm sharing (Figure 4A–B, Figure 2—figure supplement 1A–A’’). Given our electron micrograph results, we determined which innexins, the protein family associated with gap junctions in invertebrates (Bauer et al., 2005; Phelan et al., 1998), are expressed in rectal papillae. From RNA-seq data (Methods), we determined that ogre (Inx1), Inx2, and Inx3 are most highly expressed (Figure 4C). This combination of innexins is not unique to rectal papillae; the non-sharing brain and optic lobe (Figure 4—figure supplement 1A) also express high levels of all three (Leader et al., 2018). We examined localization of Inx3 (a gap junction component) (Curtin et al., 1999; Richard et al., 2017), and compared it to a septate junction component, NeurexinIV (NrxIV) (Laprise et al., 2009). NrxIV localizes similarly both pre and post-sharing onset (Figure 4D–D’), indicative of persistent septate junctions remaining between papillar cells. In contrast, Inx3 organizes apically only after cytoplasm sharing (Figure 4E–E’, Figure 4—figure supplement 1B–B’). Inx3 also does not localize to cell–cell boundaries in shi RNAi animals (Figure 4C–C'). We tested whether innexins are required for cytoplasm sharing. Knocking down these three genes individually causes mild yet significant cytoplasm sharing defects (Figure 4F). However, we see larger defects in animals expressing dominant-negative ogre (Figure 4F–G; Spéder and Brand, 2014), which contains a N-terminal GFP tag that interferes with channel passage. Also, heterozygous animals containing a ten gene-deficiency spanning ogre, Inx2, and Inx7 have more severe defects (Figure 4F, Df(1)BSC867). Finally, we tested whether cytoplasm sharing is essential for normal rectal papillar function. Rectal papillae selectively absorb water and ions from the gut lumen for transport back into the hemolymph, and excrete unwanted lumen contents (Cohen et al., 2020). One test of papillar function is viability following the challenge of a high-salt diet (Bretscher and Fox, 2016; Schoenfelder et al., 2014). However, with our pan-hindgut driver byn-Gal4 used for all previous experiments, we noted animal lethality with shi, Rab5, and Rab11 knockdown within a few days on control food. We observed melanization and necrosis throughout the hindgut (data not shown) which prevented us from attributing any phenotypes directly to papillar cytoplasm sharing. We therefore identified an alternative driver (60H12-Gal4) with rectum-specific expression during pupation and adulthood (Figure 4—figure supplement 1D–D’). We used this driver to express shiDN. These animals display similar sharing defects as we find with byn-Gal4 (Figure 4—figure supplement 1E–E’’). Reassuringly, 60H12-Gal4 > shiDN animals do not show lethality on a control food diet (Figure 4H) allowing us to test rectal papillar physiological function on a high-salt diet. Using either pan-hindgut or papillae-specific knockdown of cytoplasm sharing regulators, we find both shi and ogre animals are extremely sensitive to the high-salt diet (mean survival <1 day, Figure 4H). These results underscore an important function for gap junction proteins, as well as membrane remodeling by Dynamin/Shibire, in cytoplasm sharing.

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.

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, ogre, 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, shi, and ogre animals on a high-salt diet (N = 27–37, rep = 3). (I) Proposed model for cytoplasmic sharing in an intact papillar epithelium.

(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 GFP expression throughout the pre-sharing hindgut. Arrows indicate the ileum. (D’) 60H12-Gal4 driving GFP 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 shi expression in a dBrainbow background in adult papillae. (E’’) Quantification of cytoplasm sharing in 60H12-Gal4 and 60H12-Gal4 > shi animals (N = 11, rep = 2). (F) Model of membrane and junctional changes requiring membrane trafficking genes that coincide with the onset of cytoplasm sharing.

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 GFP expression throughout the pre-sharing hindgut. Arrows indicate the ileum. (D’) 60H12-Gal4 driving GFP 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 shi expression in a dBrainbow background in adult papillae. (E’’) Quantification of cytoplasm sharing in 60H12-Gal4 and 60H12-Gal4 > shi animals (N = 11, rep = 2). (F) Model of membrane and junctional changes requiring membrane trafficking genes that coincide with the onset of cytoplasm sharing.

Discussion

A distinctive mechanism and model of cytoplasm sharing

Our findings identify Drosophila rectal papillae as a new and distinctive example of cytoplasm sharing between multiple nuclei in a simple, genetically tractable system. One defining property of papillar cytoplasm sharing is the lack of an easily observable conduit in the lateral membrane through which cytoplasm can be exchanged. Cytoplasm sharing in a multinucleate tissue/organism frequently involves the creation of a large membrane breach associated with major actin cytoskeleton rearrangement (Kim et al., 2015; Deng et al., 2017; Martin, 2016). However, papillar cytoplasm sharing does not require canonical myoblast fusion regulators nor major actin remodeling factors such as Rho family GTPases. Aside from membrane breaches, other cell types are known to share cytoplasm through the formation of cytoplasmic bridges such as ring canals or plasmodesmata. Such bridge structures assemble as the result of incomplete cytokinesis (Mahowald, 1971; Lůcas and Wolf, 1993). In contrast, papillar cytoplasm sharing does not require mitosis or cytokinesis, and does not contain intercellular bridge structures visible by electron microscopy.

In addition to lacking a large, observable membrane breach, papillar cytoplasm sharing occurs within an intact, polarized epithelium, and apical cell–cell junctions and lateral membranes are retained after the onset of sharing. In contrast, other epithelia known to fuse cytoplasm, such as C. elegans epithelia fused by Epithelial Fusion Failure 1 (EFF-1), dismantle cell–cell junctions (Smurova and Podbilewicz, 2016). Further, cells with ring canals retain cell–cell junctions and lateral membranes (Peifer et al., 1993).

Given the retention of cell junctions and absence of clear intercellular bridges, channels, or breaches in lateral membrane, our data lead us to propose that a specialized function of gap junction proteins facilitates cytoplasm sharing between neighboring cells in an otherwise intact epithelium (Figure 4I). Although gap junctions typically transfer molecules of <1 kDa, elongated proteins up to 18 kDa are observed to pass through certain vertebrate gap junctions (Cieniewicz and Woodruff, 2010). Alternatively, gap junction-mediated cell to cell communication has been previously implicated in fusion of placental trophoblasts and osteoclasts (Firth et al., 1980; Dunk et al., 2012; Schilling et al., 2008), so we cannot rule out an indirect role for gap junctions in papillar cells, such as through regulation/recruitment of a fusogenic protein (Petrany and Millay, 2019). Future work beyond the scope of this study can determine if, for example, papillar gap junctions exhibit a specialized structure to directly facilitate exchange of large cytoplasmic contents. As for the connection between membrane remodeling and gap junction formation, Rab11 has been previously reported to recycle gap junction components in Drosophila brain and mammalian cell culture (Augustin et al., 2017). Dynamin2 was also implicated in gap junction plaque internalization in mammalian cells (Gilleron et al., 2011). However, neither of these factors has been previously implicated in gap junction establishment. We show that Dynamin is required for gap junction formation in papillar cells. Future studies will determine the exact role of Dynamin in gap junction establishment. Another clue for future study is that papillar cytoplasm sharing is developmentally regulated, occurring over a brief 6 hr window, and requires membrane remodeling by trafficking GTPases and gap junction establishment (Figure 4I, Figure 4—figure supplement 1H). Our results argue that papillar sharing is triggered by a permanent structural rearrangement rather than an active transport mechanism, as the membrane remodelers we identified are required specifically during developmental membrane remodeling.

The mechanisms we report here may be relevant to other emerging roles for membrane remodeling and cytoplasm sharing in the literature. Here, we identify a close relationship between the formation of membrane stacks and cytoplasm sharing. Basolateral membrane infoldings to expand cellular surface area are a common feature of absorptive cells (Pease, 1956). The mammalian kidney tubule cells exhibit similar basolateral membrane extensions to which ion transporters such as the Na+/K+-ATPase are localized (Maunsbach, 1966; Molitoris et al., 1992; Avner et al., 1992; Pease, 1955; Sjöstrand and Rhodin, 1953). Our results suggest that the same membrane remodeling factors that regulate cytoplasm sharing are required for the formation of membrane stacks. To our knowledge, this is the first study to reveal factors involved in basolateral membrane infolding biogenesis. Additionally, our results may also explain other examples of cytoplasm sharing where the underlying mechanism remains to be determined, such as transient cytoplasm sharing in the zebrafish myocardium (Sawamiphak et al., 2017). Together, our studies indicate that the Drosophila papillar epithelium represents a distinctive example of cytoplasmic sharing to generate giant multinucleate cells.

Functions and implications of transforming a multicellular tissue into a giant multinucleate cytoplasm

Our results have several implications for functions and regulation of multinucleation. Here we show that the membrane and junctional changes associated with cytoplasm sharing are required for normal Drosophila rectal papillar function. Papillae in other insects are known to undergo visible movement upon muscle contraction, which may facilitate cytoplasm movement (Lowne, 1869). Arthropod papillar structures are subject to peristaltic muscle contractions from an extensive musculature (Rocco et al., 2017), which aid in both excretion and movement of papillar contents into the hemolymph (Habas mantel and Mantel, 1968). Further, relative to other hindgut regions, the rectum appears to have specialized innervation (Cohen et al., 2020) and regulation by the kinin family of neuropeptides, which are hypothesized to provide additional input in to muscle activity in this critical site of reabsorption (Audsley and Weaver, 2009; Lajevardi and Paluzzi, 2020). We speculate that these muscle contractions aid in vigorous movement of papillar cytoplasm, which includes ions and water taken up from the intestinal lumen. The movement of these papillar contents may facilitate both cytoplasm exchange between papillar cells and the interaction of ions and ion transport machinery with intracellular membrane stacks. This idea is supported by our finding that animals lacking a large common papillar cytoplasm die when fed a high-salt diet.

Given the importance of insect papillae in pathogen biology, the knowledge that this common anatomical structure is a shared cytoplasm can impact both human disease intervention and agricultural pest control. Papillae occur in both primitive insect orders such as Zygentoma and Odonata and also in Lepidopterans, Hymenopterans, and Dipterans, the latter of which exhibit the most prominent and elaborate structures (Palm, 1949). Furthermore, electron micrographs of the hindgut of the mosquito, Aedes aegypti, and the ant, Formica nigricans, show striking ultrastructural similarity to Drosophila, and these studies leave open the possibility that multinucleation may be conserved in insect papillae (Hopkins, 1967; Wessing and Eichelberg, 1973; Garayoa et al., 1999). Cytoplasm sharing is a known mechanism that facilitates pathogen spread (Eugenin et al., 2009), and papillae are an avenue of entry for numerous pathogens including kinetoplastids and mosquito viruses (Gu et al., 2010; Filosa et al., 2019). Thus, our findings may impact strategies to prevent diseases such as African sleeping sickness, or to target agricultural pests that threaten agricultural production.

The sharing of cytoplasm also has the potential to neutralize detrimental genomic imbalances between nuclei caused by aneuploidy. Our prior work (Schoenfelder et al., 2014) revealed that papillae are highly tolerant of chromosome mis-segregation, and our work here suggests this tolerance may be due in part to neutralization of aneuploidies through cytoplasm sharing. This finding may also be relevant to the study of multinucleate tumors, such as those found in pancreas, bone, and fibrous tissues (Doane et al., 2015; Hasegawa et al., 2017; Mancini et al., 2017), or to conditions of aberrant organelle inheritance (Asare et al., 2017). Finally, we note that our study reveals that, even in a well-studied model organism such as Drosophila, we still have yet to appreciate the full diversity of tissue organization strategies. Our Brainbow-based approach could be applied to other contexts to identify other tissues with cytoplasm sharing, including those with gap junction-dependent but membrane breach-independent cytoplasm sharing. Collectively, our findings highlight the expanding diversity of multicellular tissue organization strategies.

Materials and methods

Fly stocks and genetics

Flies were raised at 25°C on standard media (Archon Scientific, Durham, NC) unless specified otherwise. See Table 4 for a list of fly stocks used. See Table 3 for a full list of fly lines screened in primary and secondary screens. See Table 5 for panel-specific genotypes.

Table 4.

Fly stocks used in addition to the screens.

Stock name	Stock number	Origin	References
w1118	3605	BDSC
tub-Gal4	5138	BDSC
tub-Gal80ts	NA	NA
UAS-dBrainbow	34513	BDSC	Hampel et al., 2011
UAS-dBrainbow	34514	BDSC	Hampel et al., 2011
Hsp70 > cre	851	BDSC
UAS-fzr RNAi	25550	VDRC	Fox et al., 2010; Schoenfelder et al., 2014
UAS-shi RNAi #1	28513	BDSC
UAS-shi RNAi #2	36921	BDSC
UAS-Rab5 RNAi #1	30518	BDSC
UAS-Rab5 RNAi #2	67877	BDSC
UAS-Rab11 RNAi #1	27730	BDSC
UAS-Rab11 RNAi #2	22198	VDRC
UAS-SCAR RNAi #1	36121	BDSC	Bischoff et al., 2013
UAS-SCAR RNAi #2	51803	BDSC	Xing et al., 2018
UAS-kirre RNAi	27227	VDRC	Linneweber et al., 2015
UAS-sns RNAi	877	VDRC	Linneweber et al., 2015
UAS-schizo RNAi	36625	VDRC	Johnson et al., 2011
UAS-sing RNAi	12202	VDRC	Brunetti et al., 2015
UAS-Cdc42DN	6288	BDSC
UAS-Rac1DN	6292	BDSC
UAS-Rho1DN	7328	BDSC
UAS-GFPNLS	4776	BDSC
UAS-GFP-Myc-2x-FYVE	42712	BDSC	Gillooly et al., 2000; Wucherpfennig et al., 2003
UAS-YFP-Rab5	9775	BDSC
60H12-Gal4	39268	BDSC
UAS-shiDN	5822	BDSC
NrxIV-GFP	50798	BDSC
Df(1)BSC867	29990	BDSC
UAS-ogre RNAi	7136	VDRC	Holcroft et al., 2013; Spéder and Brand, 2014
byn-Gal4	-	NA	Singer et al., 1996
UAS-GFPPA	-	Lynn Cooley	Datta et al., 2008
UAS-NDN	-	NA	Rebay et al., 1993
UAS-shi-Venus	-	Stefano De Renzis	Fabrowski et al., 2013
UAS-GFP-ogre	-	Andrea Brand	Spéder and Brand, 2014
UAS-Gapdh2-GFPPA	-	-	This paper

Table 3.

Primary and secondary candidate screen stock numbers used and results.

Gene	Annotation symbol	Gene ID	Mutant or UAS transgene	Stock center	Stock number	Chr	Sharing disrupted?	Notes
Abi	CG9749	FBgn0020510	RNAi	BDSC	51455	2	No
ALiX	CG12876	FBgn0086346	RNAi	BDSC	33417	3	No
ALiX	CG12876	FBgn0086346	RNAi	BDSC	50904	2	No
Arf51F	CG8156	FBgn0013750	RNAi	BDSC	51417	3	No
Arf51F	CG8156	FBgn0013750	Mutant	BDSC	17076	2	No
Arf51F	CG8156	FBgn0013750	RNAi	BDSC	27261	3	No
Arp2	CG9901	FBgn0011742	RNAi	BDSC	27705	3	No
Arp3	CG7558	FBgn0262716	RNAi	BDSC	32921	3	No
Atg1	CG10967	FBgn0260945	RNAi	BDSC	44034	2	No
Atg1	CG10967	FBgn0260945	RNAi	BDSC	26731	3	No
Atg7	CG5489	FBgn0034366	RNAi	BDSC	34369	3	No
Atg7	CG5489	FBgn0034366	RNAi	BDSC	27707	3	No
Atg8a	CG32672	FBgn0052672	RNAi	BDSC	28989	3	No
Atg8a	CG32672	FBgn0052672	RNAi	BDSC	58309	2	No
Atg8a	CG32672	FBgn0052672	RNAi	BDSC	34340	3	No
Atl	CG6668	FBgn0039213	RNAi	BDSC	36736	2	No
Bet1	CG14084	FBgn0260857	RNAi	BDSC	41927	2	No
blue	NA	FBgn0283709	RNAi	BDSC	44094	3	No
blue	NA	FBgn0283709	RNAi	BDSC	41637	2	No
CadN	CG7100	FBgn0015609	RNAi	BDSC	27503	3	No
CadN	CG7100	FBgn0015609	RNAi	BDSC	41982	3	No
CapD2	CG1911	FBgn0039680	Mutant	BDSC	59393	3	No
Cdc2	CG5363	FBgn0004106	RNAi	VDRC	41838	3	Yes
Cdc2	CG5363	FBgn0004106	RNAi	BDSC	NA	3	No
Cdc42	CG12530	FBgn0010341	RNAi	BDSC	42861	2	No
Cdc42	CG12530	FBgn0010341	DN	BDSC	6288	2	No
Ced-12	CG5336	FBgn0032409	RNAi	BDSC	28556	3	No
Ced-12	CG5336	FBgn0032409	RNAi	BDSC	58153	2	No
Chc	CG9012	FBgn0000319	DN	BDSC	26821	2	No
Chc	CG9012	FBgn0000319	RNAi	BDSC	27350	3	No
Chc	CG9012	FBgn0000319	RNAi	BDSC	34742	3	No
Chico	CG5686	FBgn0024248	RNAi	BDSC	36788	2	No
Chmp1	CG4108	FBgn0036805	RNAi	BDSC	33928	3	No
CHMP2B	CG4618	FBgn0035589	RNAi	BDSC	28531	3	No
CHMP2B	CG4618	FBgn0035589	RNAi	BDSC	38375	2	No
cindr	CG31012	FBgn0027598	RNAi	BDSC	35670	3	No
cindr	CG31012	FBgn0027598	RNAi	BDSC	38976	2	No
Clamp	CG1832	FBgn0032979	RNAi	BDSC	27080	3	No
cno	CG42312	FBgn0259212	RNAi	BDSC	33367	3	No
cno	CG42312	FBgn0259212	RNAi	BDSC	38194	2	No
dac	CG4952	FBgn0005677	RNAi	BDSC	26758	3	No
dac	CG4952	FBgn0005677	RNAi	BDSC	35022	3	No
DCTN1-p150	CG9206	FBgn0001108	DN	BDSC	51645	2	No
dnd	CG6560	FBgn0038916	RNAi	BDSC	27488	3	No
dnd	CG6560	FBgn0038916	RNAi	BDSC	34383	3	No
dock	CG3727	FBgn0010583	RNAi	BDSC	27728	3	No
dock	CG3727	FBgn0010583	RNAi	BDSC	43176	3	No
dock	CG3727	FBgn0010583	Mutant	BDSC	11385	2	No
Dr	CG1897	FBgn0000492	RNAi	BDSC	26224	3	No
Dr	CG1897	FBgn0000492	RNAi	BDSC	42891	2	No
Egfr	CG10079	FBgn0003731	DN	BDSC	5364	2	Yes
Egfr	CG10079	FBgn0003731	RNAi	VDRC	43267	3	Yes
endos	CG6513	FBgn0061515	RNAi	BDSC	53250	3	No
endos	CG6513	FBgn0061515	RNAi	BDSC	65996	3	No
Exo70	CG7127	FBgn0266667	RNAi	BDSC	28041	3	No
Exo70	CG7127	FBgn0266667	RNAi	BDSC	55234	3	No
Exo84	CG6095	FBgn0266668	RNAi	BDSC	28712	3	Yes
Flo1	CG8200	FBgn0024754	RNAi	BDSC	36700	3	No
Flo1	CG8200	FBgn0024754	RNAi	BDSC	36649	2	No
Flo2	CG32593	FBgn0264078	RNAi	BDSC	55212	3	No
Flo2	CG32593	FBgn0264078	RNAi	BDSC	40833	2	No
fzr	CG3000	FBgn0262699	RNAi	VDRC	25550	2	Yes
Gli	CG3903	FBgn0001987	RNAi	BDSC	31869	3	No
Gli	CG3903	FBgn0001987	RNAi	BDSC	58115	2	No
grk	CG17610	FBgn0001137	RNAi	BDSC	38913	3	No
hbs	CG7449	FBgn0029082	RNAi	BDSC	57003	2	No
Hem	CG5837	FBgn0011771	Mutant	BDSC	8752	3	No
Hem	CG5837	FBgn0011771	Mutant	BDSC	8753	3	No
Hem	CG5837	FBgn0011771	RNAi	BDSC	29406	3	No
Hem	CG5837	FBgn0011771	RNAi	BDSC	41688	3	No
Hsc70Cb	CG6603	FBgn0026418	RNAi	BDSC	33742	3	No
Hsc70Cb	CG6603	FBgn0026418	DN	BDSC	56497	2	No
Iris	CG4715	FBgn0031305	RNAi	BDSC	50587	2	No
Iris	CG4715	FBgn0031305	RNAi	BDSC	63582	2	No
l(2)gl	CG2671	FBgn0002121	RNAi	BDSC	31517	3	No
lerp	CG31072	FBgn0051072	RNAi	BDSC	57436	2	No
lilli	CG8817	FBgn0041111	RNAi	BDSC	26314	3	No
lilli	CG8817	FBgn0041111	RNAi	BDSC	34592	3	No
mbc	CG10379	FBgn0015513	RNAi	BDSC	32355	3	No
mbc	CG10379	FBgn0015513	RNAi	BDSC	33722	3	No
Mi-2	CG8103	FBgn0262519	RNAi	BDSC	16876	3	No
mtm	CG9115	FBgn0025742	RNAi	BDSC	38339	3	No
N	CG3936	FBgn0004647	DN	Rebay Lab	NA	2	No
N	CG3936	FBgn0004647	RNAi	Sara Bray	NA	1	No
Nrg	CG1634	FBgn0264975	RNAi	BDSC	28724	3	No
Nrg	CG1634	FBgn0264975	RNAi	BDSC	38215	2	No
Nrg	CG1634	FBgn0264975	RNAi	BDSC	37496	2	No
nrv3	CG8663	FBgn0032946	RNAi	BDSC	29431	3	No
nrv3	CG8663	FBgn0032946	RNAi	BDSC	50725	3	No
nuf	CG33991	FBgn0013718	RNAi	BDSC	31493	3	No
pav	CG1258	FBgn0011692	RNAi	BDSC	35649	3	No
pav	CG1258	FBgn0011692	RNAi	BDSC	43963	2	No
Ptp61F	CG9181	FBgn0267487	RNAi	BDSC	32426	3	No
Ptp61F	CG9181	FBgn0267487	RNAi	BDSC	56036	2	No
Rab1	CG3320	FBgn0285937	CA	BDSC	9758	3	No
Rab1	CG3320	FBgn0285937	DN	BDSC	9757	3	Yes	Requires 60H12-Gal4
Rab1	CG3320	FBgn0285937	RNAi	BDSC	27299	3	Yes
Rab1	CG3320	FBgn0285937	RNAi	BDSC	34670	3	No
Rab2	CG3269	FBgn0014009	CA	BDSC	9761	2	No
Rab2	CG3269	FBgn0014009	DN	BDSC	9759	2	No
Rab3	CG7576	FBgn0005586	CA	BDSC	9764	3	No
Rab3	CG7576	FBgn0005586	DN	BDSC	9766	2	No
Rab4	CG4921	FBgn0016701	CA	BDSC	9770	3	No
Rab4	CG4921	FBgn0016701	DN	BDSC	9768	2	No
Rab4	CG4921	FBgn0016701	DN	BDSC	9769	3	No
Rab5	CG3664	FBgn0014010	CA	BDSC	9773	3	Yes
Rab5	CG3664	FBgn0014010	DN	BDSC	42704	3	Yes	Requires 60H12-Gal4
Rab5	CG3664	FBgn0014010	RNAi	BDSC	67877	2	Yes
Rab5	CG3664	FBgn0014010	RNAi	BDSC	30518	3	Yes
Rab5	CG3664	FBgn0014010	RNAi	BDSC	51847	2	No
Rab6	CG6601	FBgn0015797	CA	BDSC	9776	3	No
Rab6	CG6601	FBgn0015797	DN	BDSC	23250	3	No
Rab7	CG5915	FBgn0015795	CA	BDSC	9779	3	No
Rab7	CG5915	FBgn0015795	DN	BDSC	9778	3	No
Rab7	CG5915	FBgn0015795	DN	BDSC	9778	3	No
Rab8	CG8287	FBgn0262518	DN	BDSC	9780	3	No
Rab8	CG8287	FBgn0262518	CA	BDSC	9781	2	No
Rab8	CG8287	FBgn0262518	DN	BDSC	9780	3	No
Rab9	CG9994	FBgn0032782	CA	BDSC	9785	3	No
Rab9	CG9994	FBgn0032782	DN	BDSC	23642	3	No
Rab10	CG17060	FBgn0015789	CA	BDSC	9787	3	No
Rab10	CG17060	FBgn0015789	DN	BDSC	9786	3	No
Rab11	CG5771	FBgn0015790	CA	BDSC	9791	3	No
Rab11	CG5771	FBgn0015790	DN	BDSC	23261	3	Yes
Rab11	CG5771	FBgn0015790	RNAi	BDSC	27730	3	Yes
Rab11	CG5771	FBgn0015790	RNAi	VDRC	108382	2	Yes
Rab11	CG5771	FBgn0015790	RNAi	VDRC	22198	3	Yes
Rab11	CG5771	FBgn0015790	Mutant	BDSC	42708	3	Yes
Rab14	CG4212	FBgn0015791	CA	BDSC	9795	2	No
Rab14	CG4212	FBgn0015791	DN	BDSC	23264	3	No
Rab18	CG3129	FBgn0015794	CA	BDSC	9797	3	No
Rab18	CG3129	FBgn0015794	DN	BDSC	23238	3	No
Rab19	CG7062	FBgn0015793	CA	BDSC	9800	3	No
Rab19	CG7062	FBgn0015793	DN	BDSC	9799	3	No
Rab21	CG17515	FBgn0039966	CA	BDSC	23864	2	No
Rab21	CG17515	FBgn0039966	DN	BDSC	23240	3	No
Rab23	CG2108	FBgn0037364	RNAi	BDSC	36091	3	No
Rab23	CG2108	FBgn0037364	RNAi	BDSC	55352	2	No
Rab23	CG2108	FBgn0037364	CA	BDSC	9806	3	No
Rab23	CG2108	FBgn0037364	DN	BDSC	9804	3	No
Rab26	CG34410	FBgn0086913	CA	BDSC	23243	3	No
Rab26	CG34410	FBgn0086913	DN	BDSC	9808	3	No
Rab27	CG14791	FBgn0025382	CA	BDSC	9811	2	No
Rab27	CG14791	FBgn0025382	DN	BDSC	23267	2	No
Rab30	CG9100	FBgn0031882	CA	BDSC	9814	2	No
Rab30	CG9100	FBgn0031882	DN	BDSC	9813	3	No
Rab32	CG8024	FBgn0002567	CA	BDSC	23280	3	No
Rab32	CG8024	FBgn0002567	DN	BDSC	23281	2	No
Rab35	CG9575	FBgn0031090	CA	BDSC	9817	3	No
Rab35	CG9575	FBgn0031090	DN	BDSC	9820	3	No
Rab39	CG12156	FBgn0029959	CA	BDSC	9823	3	No
Rab39	CG12156	FBgn0029959	DN	BDSC	23247	3	No
Rab40	CG1900	FBgn0030391	CA	BDSC	9827	3	No
Rab40	CG1900	FBgn0030391	DN	BDSC	9829	2	No
Rab9D	CG32678	FBgn0067052	CA	BDSC	9835	3	No
Rab9D	CG32678	FBgn0067052	DN	BDSC	23257	2	No
Rab9E	CG32673	FBgn0052673	CA	BDSC	9832	2	No
Rab9E	CG32673	FBgn0052673	DN	BDSC	23255	3	No
Rab9Fb	CG32670	FBgn0052670	CA	BDSC	9844	3	No
Rab9Fb	CG32670	FBgn0052670	DN	BDSC	9845	2	No
RabX1	CG3870	FBgn0015372	CA	BDSC	9839	2	No
RabX1	CG3870	FBgn0015372	DN	BDSC	23252	3	No
RabX2	CG2885	FBgn0030200	CA	BDSC	9842	3	No
RabX2	CG2885	FBgn0030200	DN	BDSC	9843	2	No
RabX4	CG31118	FBgn0051118	RNAi	BDSC	28704	3	No
RabX4	CG31118	FBgn0051118	RNAi	BDSC	44070	2	No
RabX4	CG31118	FBgn0051118	CA	BDSC	23277	2	No
RabX4	CG31118	FBgn0051118	DN	BDSC	9849	3	No
RabX5	CG7980	FBgn0035255	CA	BDSC	9852	X	No
RabX5	CG7980	FBgn0035255	DN	BDSC	9853	2	No
RabX6	CG12015	FBgn0035155	CA	BDSC	9855	2	No
RabX6	CG12015	FBgn0035155	DN	BDSC	9856	3	No
CG41099	CG41099	FBgn0039955	RNAi	BDSC	34883	3	No
Rac1	CG2248	FBgn0010333	RNAi	BDSC	28985	3	No
Rac1	CG2248	FBgn0010333	DN	BDSC	6292	3	No
Rala	CG2849	FBgn0015286	DN	BDSC	32094	2	No
Rala	CG2849	FBgn0015286	RNAi	BDSC	34375	3	No
Rbp9	CG3151	FBgn0010263	RNAi	BDSC	42796	3	No
Rep	CG8432	FBgn0026378	RNAi	BDSC	28047	3	No
rho	CG1004	FBgn0004635	Mutant	BDSC	1471	3	Yes
rho	CG1004	FBgn0004635	RNAi	BDSC	38920	3	Yes
rho	CG1004	FBgn0004635	RNAi	BDSC	41699	2	Yes
Rho1	CG8416	FBgn0014020	DN	BDSC	7328	3	No
Rho1	CG8416	FBgn0014020	DN	BDSC	58818	2	No
Rho1	CG8416	FBgn0014020	RNAi	BDSC	32383	3	No
Rip11	CG6606	FBgn0027335	RNAi	BDSC	38325	3	No
rols	CG32096	FBgn0041096	RNAi	BDSC	56986	2	No
rols	CG32096	FBgn0041096	RNAi	BDSC	58262	2	No
rst	CG4125	FBgn0003285	RNAi	BDSC	28672	3	No
ru	CG1214	FBgn0003295	RNAi	BDSC	41593	3	No
ru	CG1214	FBgn0003295	RNAi	BDSC	58065	2	No
SA-2	CG13916	FBgn0043865	RNAi	VDRC	108267	2	No
SCAR	CG4636	FBgn0041781	RNAi	BDSC	31126	3	No
SCAR	CG4636	FBgn0041781	RNAi	BDSC	51803	2	No
SCAR	CG4636	FBgn0041781	Mutant	BDSC	8754	2	No
sdt	CG32717	FBgn0261873	RNAi	BDSC	33909	3	No
sdt	CG32717	FBgn0261873	RNAi	BDSC	35291	3	No
Sec10	CG6159	FBgn0266673	RNAi	BDSC	27483	3	Yes
Sec15	CG7034	FBgn0266674	RNAi	BDSC	27499	3	Yes
Sec5	CG8843	FBgn0266670	RNAi	VDRC	28873	3	Yes
Sec5	CG8843	FBgn0266670	RNAi	BDSC	50556	3	No
Sec6	CG5341	FBgn0266671	RNAi	VDRC	105836	2	Yes
Sec6	CG5341	FBgn0266671	RNAi	BDSC	27314	3	Yes
Sec8	CG2095	FBgn0266672	RNAi	BDSC	57441	2	Yes
shg	CG3722	FBgn0003391	RNAi	BDSC	27689	3	No
shi	CG18102	FBgn0003392	DN	BDSC	5822	3	Yes	Requires 60H12-Gal4
shi	CG18102	FBgn0003392	RNAi	BDSC	28513	3	Yes
shi	CG18102	FBgn0003392	RNAi	BDSC	36921	3	Yes
siz	CG32434	FBgn0026179	RNAi	BDSC	39060	2	No
spi	CG10334	FBgn0005672	RNAi	BDSC	28387	3	No
spi	CG10334	FBgn0005672	RNAi	BDSC	34645	3	No
stet	CG33166	FBgn0020248	RNAi	BDSC	57698	3	No
Vha16-1	CG3161	FBgn0262736	RNAi	BDSC	40923	2	Yes
Vha16-1	CG3161	FBgn0262736	RNAi	VDRC	104490	2	Yes
Vha16-1	CG3161	FBgn0262736	RNAi	VDRC	49291	2	Yes
Vha16-2	CG32089	FBgn0028668	RNAi	BDSC	65167	2	No
Vha16-3	CG32090	FBgn0028667	RNAi	BDSC	57474	2	No
Vha16-5	CG6737	FBgn0032294	RNAi	BDSC	25803	3	Yes
Vha55	CG17369	FBgn0005671	RNAi	BDSC	40884	2	No
VhaAC39-1	CG2934	FBgn0285910	RNAi	BDSC	35029	3	No
VhaAC39-2	CG4624	FBgn0039058	Mutant	BDSC	62725	3	No
VhaAC39-2	CG4624	FBgn0039058	RNAi	VDRC	34303	2	No
VhaPPA1-1	CG7007	FBgn0028662	RNAi	BDSC	57729	2	Yes
VhaPPA1-2	CG7026	FBgn0262514	RNAi	BDSC	65217	2	Yes
Vps2	CG14542	FBgn0039402	RNAi	VDRC	24869	3	Yes
Vps2	CG14542	FBgn0039402	RNAi	BDSC	38995	2	Yes
lsn	CG6637	FBgn0260940	RNAi	BDSC	38289	2	No
Vps29	CG4764	FBgn0031310	RNAi	BDSC	53951	2	No
Vps33b	CG5127	FBgn0039335	RNAi	BDSC	44006	2	No
Vps35	CG5625	FBgn0034708	RNAi	BDSC	38944	2	No
Vps4	CG6842	FBgn0283469	RNAi	BDSC	31751	3	No
wts	CG12072	FBgn0011739	RNAi	BDSC	41899	3	No
wash	CG13176	FBgn0033692	RNAi	BDSC	62866	2	No
WASp	CG1520	FBgn0024273	RNAi	BDSC	25955	3	No
WASp	CG1520	FBgn0024273	RNAi	BDSC	51802	2	No
βggt-II	CG18627	FBgn0028970	RNAi	BDSC	50516	2	No
βggt-II	CG18627	FBgn0028970	RNAi	BDSC	34902	3	No

Table 5.

Additional Methods.

Panel	Additional 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 1G	Hsp70 > 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 1H	Hsp70 > 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 2A	UAS-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 2B	Secondary 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 2C	Secondary 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 2D	Hsp70 > 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 2E	Young 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 2F	Young 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 2G	Young 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 2H	Animals 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 3B	Pupae 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 3C	Aggregated 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 3E	Aggregated 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 3H	Electron micrograph of microvillar-like structures of WT (w1118) young adult papillar cells.
Figure 3I	Electron micrograph of microvillar-like structures of young adult byn-Gal4, Gal80ts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C).
Figure 3J	Electron micrograph of microvillar-like structures 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 3K	Electron micrograph of mitochondria and surrounding membrane material of WT (w1118) young adult papillar cells.
Figure 3L	Electron micrograph of mitochondria and surrounding membrane material of young adult byn-Gal4, Gal80ts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C).
Figure 3M	Electron 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 3N	Electron 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 3O	Electron 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 3P	Gap 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 4B	Gap 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 4C	Relative 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 4F	Young 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 4G	See Figure 4F.
Figure 4H	60 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 1A	Hsp70 > cre; UAS-dBrainbow; tubulin-Gal4 animals raised at 29°C. Tissues dissected at adulthood.
Figure 1—figure supplement 1D	byn-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 1E	Hsp70 > cre; UAS-dBrainbow; byn-Gal4 animals were shifted from 25 to 29°C during L3 and dissected at adulthood.
Figure 1—figure supplement 1F	Hsp70 > 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 1G	Hsp70 > 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 1A	Hsp70 > 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 1B	Hsp70 > 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 1C	Papillar 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 1D	Hsp70 > 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 1E	Hsp70 > 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 1A	See 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 1D	Electron micrograph of apical junctions (adherens, septate, and gap) of WT (w1118) young adult papillar cells.
Figure 3—figure supplement 1E	Electron 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 1F	Electron 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 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. 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 2A	TEM of young adult (w1118) papilla.
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. 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 1C	byn-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 1D	byn-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 1E	Hsp70 > 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'.

The UAS-Gapdh2-GFP construct was generated by gene synthesis (Twist Biosciences). The GFP was placed at the C-terminus with a 12-amino acid fusion linker (GSAGSAAGSGEF) (Waldo et al., 1999) codon-optimized for Drosophila. This insert was then cloned into the pBID-UASC-FG vector modified to remove the FLAG tag and extraneous cloning sites. Transgenic flies were generated at Duke University. brachyenteron (byn)-Gal4 was the driver for all UAS transgenes with the exception of the screen in Figure 1—figure supplement 1A, which used tub-Gal4, and the shi knockdown in Figure 4H, which used 60H12-Gal4. 60H12-Gal4 expresses only in the papillar cells and not the rest of the hindgut, and use of this driver blocks cytoplasm sharing using UAS-shi (Figure 4—figure supplement 1D–E’’). For all Gal4 experiments, UAS expression was at 29°C, except in Figure 1F–H, where it was at 25°C. If byn-Gal4 expression of a given UAS-transgene was lethal, the experiment was repeated with a temperature-sensitive Gal80 repressor transgene and animals were kept at 18°C until shifting to 29°C at an experimentally-determined time point that would both result in viable animals and permit time to express the transgene prior to sharing onset.

For salt feeding assays, age- and sex-matched siblings were transferred into vials containing 2% NaCl food made with Nutri-Fly MF food base (Genesee Scientific) or control food (Schoenfelder et al., 2014). Flies were monitored for survival each day for 10 days.

Tissue preparation

For fixed imaging, tissues were dissected in PBS and immediately fixed in 3.7% formaldehyde + 0.3% Triton-X for 15 min. Immunostaining was performed in 0.3% Triton-X with 1% normal goat serum (Fox et al., 2010). The following antibodies were used: Rabbit anti-GFP (Thermo Fisher Scientific, Cat#A11122, 1:1000), Rat anti-HA (Roche, Cat#11867423001, 1:100), Rabbit anti-Inx3 (generous gift from Reinhard Bauer, 1:75), [Lehmann et al., 2006], 488, 568, 633 secondary antibodies (Thermo Fisher Scientific, Alexa Fluor, 1:2000). Tissue was stained with DAPI at 5 μg/ml and mounted in VECTASHIELD Mounting Media on slides.

Microscopy

Light microscopy

For fixed imaging, images were obtained on either a Leica SP5 inverted confocal with a 40X/1.25NA oil objective with emission from a 405 nm diode laser, a 488 nm argon laser, a 561 nm Diode laser, and a 633 HeNe laser under control of Leica LAS AF 2.6 software, or on an Andor Dragonfly Spinning Disk Confocal plus. Images were taken with two different cameras, iXon Life 888 1024 × 1024 EMCCD (pixel size 13 um) and the Andor Zyla PLUS 4.2 Megapixel sCMOS 2048 x 2048 (pixel size 6.5 um) depending on imaging needs. Images were taken on the 40x/1.25–0.75 oil 11506250: 40X, HCX PL APO, NA: 1.25, Oil, DIC, WD: 0.1 mm, coverglass: 0.17 mm, Iris diaphragm, Thread type: M25, 63x/1.20 water 11506279: 63X, HCX PL APO W Corr CS, NA: 1.2, Water, DIC, WD: 0.22 mm, Coverglass: 0.14–0.18mm, thread type: M25, and 100x/1.4–0.70 oil 11506210: HCX PL APO, NA: 1.4, Oil, DIC, WD: 0.09 mm, Coverglass: 0.17 mm, Iris Diaphragm, Thread type: M25. The lasers used were: 405 nm diode laser, 488 nm argon laser, 561 nm diode laser, and HeNe 633 nm laser.

For live imaging, hindguts were dissected and cultured based on previous protocols (Fox et al., 2010). Live imaging of cell fusion was performed on a spinning disc confocal (Yokogawa CSU10 scanhead) on an Olympus IX-70 inverted microscope using a 40x/1.3 NA UPlanFl N Oil objective, a 488 nm and 568 nm Kr-Ar laser lines for excitation and an Andor Ixon3 897 512 EMCCD camera. The system was controlled by MetaMorph 7.7.

Photo-activation was carried out using Leica SP5 and SP8 microscopes and the FRAP Wizard embedded in the Leica AS-F program. An initial z-stack of the tissue was acquired both before and after activation to examine the full extent of GFPPA movement in three dimensions. GFPPA transgenes were activated by either point activation or region of interest activation with the 405 nm laser set to between 5 and 20%, depending on the microscope and sample of interest. For each imaging session, test activations on nearby tissues were performed prior to quantify experiments to ensure that only single cells were being activated. After activation, the wizard software was used to acquire time lapses of 15 s to 2min of a single activation plane in order to capture protein movement. Extremely low 488 nm and 405 nm laser powers were used in acquisition of the time lapse images of GFP and Hoechst respectively. Low level 405 nm scanning did not significantly activate GFPPA, and control experiments were performed without the use of 405 nm time lapses and showed the same protein movement results (data not shown).

Transmission electron microscopy

Hindguts were dissected into PBS and fixed in a solution of 2.5% glutaraldehyde in 0.1% cacodylate buffer, pH 7.2. Post-fix specimens were stained with 1% osmium tetroxide in 0.1M cacodylate buffer, dehydrated, soaked in a 1:1 propylene oxide:Epon 812 resin, and then embedded in molds with fresh Epon 812 resin at 65°C overnight. The blocks were cut into semi-thin (0.5 µm) sections using Leica Reichert Ultracuts and the sections were stained with 1% methylene blue. After inspection, ultra-thin sections (65−75 nm) were cut using Leica EM CU7 and contrast stained with 2% uranyl acetate, 3.5% lead citrate solution. Ultrathin sections were visualized on a JEM-1400 transmission electron microscope (JEOL) using an ORIUS (1000) CCD 35 mm port camera.

Image analysis

All image analysis was performed using ImageJ and FIJI (Rueden et al., 2017; Schindelin et al., 2012).

Cytoplasm sharing calculation

Cytoplasmic sharing was quantified by manually tracing the total papillar area by morphology and the area marked by mKO2 signal in one z-slice of the papillar face of each animal. The area marked by mKO2 was summed and divided by the sum of the total papillar area to yield the papillar fraction marked by mKO2 which indicates the degree of cytoplasmic sharing within each animal. Papillae without mKO2 signal were excluded from the area measurements.

Line profiles

For line profile data collection, fixed and mounted hindguts were imaged on a Zeiss Apotome on the 40Xoil objective. Once moved into ImageJ, the images were rotated with no interpolation so that the central canal was perpendicular to the bottom of the image. From the midline of the central canal, a straight line (width of 300) was drawn out to one edge of the papillae. One papilla was measured per animal. Papillae were measured at the widest width. Next, the Analyze > Plot Profile data was collected from this representative 300 width line and moved into Excel. In Excel, the data was first was normalized to the maximum length of the papillae and the maximum GFP intensity per animal. Each data point is a % of the total length of the papillae and a % of the maximum GFP intensity. Next, the X values were rounded to its nearest 1% value. Next, all the Y-values were averaged per X value bins (average % GFP intensity per rounded % distance value). % GFP intensity values were plotted from 1–100% total distance of papilla.

Statistical analysis

Statistical analysis was performed in GraphPad Prism 8. Detailed statistical tests and methods are reported in Table 6.

Table 6.

Additional statistics.

Panel	N (animals) per group	Bio. reps	Statistical test	P-value
Figure 1G	9–18	2	Unpaired t-test	66HPPF:74HPPF < 0.0001
Figure 2H	9–32	2–3	One-way ANOVA with Tukey's multiple comparisons test	ANOVA:<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 3C	6–10	2–3	see 3-S1A	see Figure 3—figure supplement 1A
Figure 3E	4–5	3	Unpaired t-test	Apical region: Pre:Post < 0.0001
Figure 3P	3–4	2	Unpaired t-test	WT:shi RNAi < 0.0001
Figure 4B	3–4	2	Unpaired t-test	Pre:Post < 0.0001
Figure 4F	13–14	2	One-way ANOVA with Tukey's multiple comparisons test	ANOVA:<0.0001 WT:ogreDN < 0.0001 WT:Df < 0.0001 WT:ogre RNAi 0.0007
Figure 4H	27–37	3	One-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 1H	12–20	2	Unpaired t-test	WT:fzr RNAi < 0.0001 WT:NDN ns, 0.1786
Figure 2—figure supplement 1A	8–11	2	One-way ANOVA with Tukey's multiple comparisons test	ANOVA:<0.0001 Sing RNAi:all others < 0.0001 All others: ns
Figure 2—figure supplement 1B	6–8	2	One-way ANOVA	ANOVA: ns, 0.3692
Figure 2—figure supplement 1C	11–23	2	One-way ANOVA with Tukey's multiple comparisons test	ANOVA: 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 1F	10–11	2	Unpaired t-test	ns, 0.0782
Figure 3—figure supplement 1A	6–10	2	One-way ANOVA with Tukey's multiple comparisons test	ANOVA:<0.0001 Pre:Post < 0.0001 Pre:shi RNAi ns, 0.7882 Post:shi RNAi < 0.0001
Figure 3—figure supplement 1B''	10	2	Unpaired t-test	Apical basal difference (see 1-S3A) Pre:Post 0.0007
Figure 3—figure supplement 1G	3–4	2	Unpaired t-test	ns, 0.2203
Figure 3—figure supplement 1G'	3–4	2	Unpaired t-test	ns, 0.4754
Figure 3—figure supplement 1G''	3–4	2	Multiple unpaired t-tests	Septate: WT:shi RNAi ns, 0.1547 Gap: WT:shi RNAi < 0.0001
Figure 4—figure supplement 1A	3–4	2	One-way ANOVA	ns, 0.8973
Figure 4—figure supplement 1A'	3–4	2	One-way ANOVA	ns, 0.3994
Figure 4—figure supplement 1A''	3–4	2	Multiple unpaired t-tests	Septate: all ns Gap: Pre:Post 0.0004 Gap: all others, ns
Figure 4—figure supplement 1E''	11	2	Unpaired t-test	WT:shiDN < 0.0001

Genotype and experiment-specific method notes

Some additional methodological details, including animal genotype, applied to only a specific figure panel. Please see Table 6 for this information.

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This report of a mechanism for the sharing of cytoplasmic contents in multinucleated cells was both distinct from previously reported mechanisms of failed cytokinesis and plasma membrane breaches and of interest to a broad readership. The authors use an elegant approach for identifying cells with shared cytoplasms by screening brainbow flies for the appearance of cells with "mixed" labels. The current mechanisms of cytoplasmic sharing are scant, and this paper goes quite a distance in rectifying this without the additional gap junction studies.

Decision letter after peer review:

Thank you for submitting your article "Cytoplasmic sharing through apical membrane remodeling" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Utpal Banerjee as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yukiko M Yamashita (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

The manuscript by Petersen and colleagues reports a novel mechanism for the sharing of cytoplasmic contents in multinucleated cells. This mechanism is seemingly distinct from previously reported mechanisms of failed cytokinesis and plasma membrane breaches. The authors use an elegant approach for identifying cells with shared cytoplasms by screening brainbow flies for the appearance of cells with "mixed" labels. They both identify a novel population in the Drosophila rectal papillae, and go on to use this approach to screen for genes essential for the mixing phenotype. Once they fail to find hits for canonical actin-based cytoskeletal remodelling, they go on to conduct secondary screens for endocytosis and membrane trafficking factors, which are critical for cytoplasmic sharing. Finally, they examine cellular ultrastructure by EM and fail to find evidence of plasma membrane breaches, consistent with their genetic manipulations, but they do observe pronounced apical membrane remodelling and the presence of gap junctions coinciding with the time of sharing. Pursing this avenue further, they genetically perturb gap junction proteins, and while not as significant as membrane remodelling factors (likely due to their redundancy), the authors demonstrate that gap junctions are important for cytoplasmic sharing. This process of cytoplasmic sharing seems physiologically important, as flies with failed sharing are not able to cope with a high-salt diet challenge and die.

Reviewer #1:

This work is interesting and broadly relevant, as mechanisms of cytoplasmic sharing are scant, although the presence of multinucleated cells is common throughout the animal kingdom. The work is elegant, well controlled, and accurately described. In particular, the authors should be praised for their clear communication of the results and their implications. Although some comments need to be addressed, which are listed below, the paper is a good fit for eLife and warrants publication pending some minor changes to the manuscript.

1) The paragraph beginning "We next examined whether cytoplasm sharing requires the distinctive papillar cell cycle program, which completes prior to sharing onset (Figure 1—figure supplement 1D). Larval papillar…" is hard to understand for a general reader. Can the authors better place their current results in the context of their previous studies (Fox et al, 2010)?

2) The authors conduct photo-activation experiments to show that protein at least the size of GFP (~27kDa) can be shared between multi-nucleated regions. This extremely interesting observation suggests that large macromolecules may pass through gap junctions. Further data to test this would be interesting, including conducting photo-activation/macromolecule mobility experiments with macromolecules of different size and physicochemical properties (e.g. fluorescent proteins with different size or charge, e.g. tdTomato, supercharged-GFP).

3) The quality of EM images should be improved. This may be mostly due to reproduction in the.pdf, however some of the false coloring could be adjusted and labelled better to understand what the authors are trying to communicate. Cartoons of the orientation and region of cell/tissue being imaged would also help.

4) The following sentence appears to be an oxymoron "a straight to a more tortuous morphology around the time of cytoplasm sharing onset".

5) Further speculation of how ion transport in the gut may be affected by a lack of cytoplasmic sharing would be interesting, in addition to their discussion of the potential role of formation of intracellular membrane stacks. Is there evidence for transluminal transport affected by cytoplasmic properties in other systems? Why might this be advantageous for the animal?

6) In general, a clearer cartoon/schematic of the rectal papilla as well as the experimental flow would be helpful in the main figure, especially for readers without expertise in Drosophila models. It is a bit difficult to discern exactly when the heat-shock to induce Cre expression was being carried out, especially in the case that the Gal80ts fly line was also being used to repress Gal4 expression. Perhaps this information could be added to the timeline in Figure 1—figure supplement 1D and included in a main figure rather than in the supplement.

7) The data on localization of Rab5 endosomes could be strengthened. It would be nice to see other markers, that don't rely on over-expression of a transgenic construct. Alternatively, could the authors also assess from their EM data whether they see redistribution of vesicles pre- and post- sharing?

Reviewer #2:

In this manuscript, Peterson et al. describe cytoplasmic share across a large number of cells in Drosophila rectal papillae. Employing the dBrainbow system, the authors identify the rectal papilla as a novel tissue that undergoes cytoplasmic sharing. They show that this is a regulated process occurring 68 hours post puparium formation. Interestingly, none of the proteins involved in myoblast fusion seem to be essential for cytoplasmic sharing in the rectal papillae. Instead, the authors show that various proteins involved in vesicle trafficking are necessary for cytoplasm sharing. In particular, they implicate a role for the membrane vesicle recycling circuit consisting of Shibire, Rab5 and Rab11 in the process of cytoplasmic sharing. Knockdown of these components leads to defective cytoplasmic sharing. Furthermore, the authors show that cytoplasmic sharing is accompanied by extensive membrane reorganization. Electron micrographs reveal that cytoplasmic sharing is not accompanied by any membrane breaches but rather formation of gap-junction like structures. Knockdown of the gap-junction proteins, the Inxs, results in defects in cytoplasmic sharing, further supporting a role for gap junctions in the process. It is interesting to note that animals defective in cytoplasmic sharing are intolerant of a high-salt diet implicating a physiological role for this process during development.

This is an interesting study that identifies a novel tissue undergoing cytoplasmic sharing in the absence of plasma membrane breaching. The identification of Shi, Rab5, Rab11 and gap junction proteins in this process based on their mutant phenotypes is also intriguing, although the mechanisms by which these proteins promote cytoplasmic sharing remain unclear.

Specific Comments:

1) The authors made interesting observations that massive membrane reorganization in apical microvilli-like structures, apical cell–cell junctions, and endomembrane stacks surrounding mitochondria coinciding with cytoplasm sharing. However, it is unclear how/which of these membrane reorganization events would lead to cytoplasmic sharing.

2) The authors showed the appearance of gap junctions during cytoplasmic sharing. Do the plasma membranes for these gap junctions come from exocytosis? If so, why would inhibiting an endocytosis protein Shi inhibit gap junction?

3) Increased gap junctions and endomembrane stacks are quite separate observations. Is there any connection between these membrane structures, e.g. similar origin?

4) Could proteins larger than GFP pass through the papillar cells, presumably through the gap junctions?

5) What is the physiological significance of the association between endomembrane stacks and mitochondria?

6) How does the change of Shi localization (Figure 3D and D') contribute to cytoplasmic sharing?

7) Are Inx1-3 expressed in the Shi, Rab5 and Rab11 knockdown animals which show defective cytoplasmic sharing? If yes, is their localization altered?

8) Does fzr knockdown affect the levels of Shi, Rab5 and Rab11?

9) Do fzr mutants phenocopy the membrane architecture observed in the shi mutants?

10) What does the endosomal distribution look like in the rab5 and rab11 mutants at the onset of cytoplasmic sharing? (68HPPF)

Reviewer #3:

This study by Peterson et al. reveals a novel mechanism of cytoplasmic sharing through apical membrane remodeling (through gap junction, which allows for sharing of large molecules, much larger (>27kD) than canonical gap junction dependent diffusion (<1kD)) in Drosophila rectal papillae. They find that membrane trafficking pathway involving Shi, Rab5, Rab11 are involved in cytoplasmic sharing. This is a novel discovery on interesting biology of cytoplasmic sharing, with likely physiological relevance (as inhibiting cytoplasmic sharing leads to high-salt diet sensitivity). Overall, this is a high quality study that provides a novel biology: but I do have several concerns to be addressed.

Recombination can happen independently to sister chromatids if cells are in G2 phase when the recombination was induced. Plus, rectal papillae cells are polyploid, which increases the chance that a single cell can have multiple recombination events). If so, there should be a considerable number of cells that express multiple colors without cytoplasmic sharing. How do they access this possibility? Was recombination titrated such that recombination can happen only to one chromatid? The image in Figure 1D is so clear, so I don't doubt that each cell is labelled with one color, but if you think about the logic, we have to wonder why. Some discussion/explanation is necessary.

Figure 3: formation of endomembrane surrounding mitochondria during cytoplasmic sharing is interesting, but is there any evidence that this indeed contributes to cytoplasmic sharing? It's unclear how endomembrane would lead to cytoplasmic sharing.

Reviewer #1:

1) The paragraph beginning "We next examined whether cytoplasm sharing requires the distinctive papillar cell cycle program, which completes prior to sharing onset (Figure S1D). Larval papillar…" is hard to understand for a general reader. Can the authors better place their current results in the context of their previous studies (Fox et al, 2010)?

Thank you for this opportunity to clarify. In response, we added clarifying text to provide more context to this paragraph and have moved the timeline from the supplemental figure to the main figure:

The text previously read:

“We next examined whether cytoplasm sharing requires the distinctive papillar cell cycle program, which completes prior to sharing onset (Figure 1—figure supplement 1D). Larval papillar cells first undergo endocycles, which increase cellular ploidy, and then pupal papillar cells undergo polyploid mitotic cycles, which increase cell number (Fox et al., 2010).”

The text now reads:

“We next examined whether cytoplasm sharing requires either programmed endocycles or mitoses. We have previously shown that larval papillar cells first undergo endocycles, which increase cellular ploidy, and then pupal papillar cells undergo polyploid mitotic cycles, which increase cell number (Fox et al., 2010). Both endocycles and mitoses occur well prior to the start of papillar cytoplasm sharing (Figure 1E). Papillar endocycles require the Anaphase-Promoting Complex/Cyclosome regulator fizzy-related (fzr) while the papillar mitoses require Notch signaling (Schoenfelder et al., 2014).”

2) The authors conduct photo-activation experiments to show that protein at least the size of GFP (~27kDa) can be shared between multi-nucleated regions. This extremely interesting observation suggests that large macromolecules may pass through gap junctions. Further data to test this would be interesting, including conducting photo-activation/macromolecule mobility experiments with macromolecules of different size and physicochemical properties (e.g. fluorescent proteins with different size or charge, e.g. tdTomato, supercharged-GFP).

Thank you for your interest- in response, we designed and made a transgenic UAS-Gapdh2-GFPphotoactivatable (PA) fly line in order to test the sharing of a larger protein. We chose Gapdh2 as it is endogenously expressed in the rectal papillae, is relatively large (35.4 kDa), and cytosolic. We used this transgenic fly to test whether a protein more than twice the size of GFPPA alone can be shared between papillar cells. We found that the 62.3 kDa Gapdh2-GFPPA protein is shared between papillar cells, though much more slowly as would be expected for a larger protein. We never observe it to stop at a cell–cell boundary.

3) The quality of EM images should be improved. This may be mostly due to reproduction in the.pdf, however some of the false coloring could be adjusted and labelled better to understand what the authors are trying to communicate. Cartoons of the orientation and region of cell/tissue being imaged would also help.

We apologize for the image quality issue. We believe that the file conversion and compression decreased the image quality of the EM images. We have now included higher resolution images and that should improve the image quality in the final pdf.

4) The following sentence appears to be an oxymoron "a straight to a more tortuous morphology around the time of cytoplasm sharing onset".

We have re-written this sentence to address this comment.

The text now reads:

“Just basal to the microvilli, apical cell–cell junctions are straight in early pupal development and compress into a more curving, tortuous morphology around the time of cytoplasm sharing onset.”

5) Further speculation of how ion transport in the gut may be affected by a lack of cytoplasmic sharing would be interesting, in addition to their discussion of the potential role of formation of intracellular membrane stacks. Is there evidence for transluminal transport affected by cytoplasmic properties in other systems? Why might this be advantageous for the animal?

Thank you for the opportunity to further speculate on this topic. At this point, we truly can only speculate.

In the previous manuscript version, we hypothesized:

“We speculate that papillar cytoplasm movement across a giant multinuclear structure enhances resorption by facilitating interaction of ions and ion transport machinery with intracellular membrane stacks.”

In the revised version, we expand upon this hypothesis. The revised text now states:

“Arthropod papillar structures are subject to peristaltic muscle contractions from an extensive musculature (Rocco et al., 2017), which aid in both excretion and movement of papillar contents into the hemolymph (Mantel, 1968). Further, relative to other hindgut regions, the rectum appears to have specialized innervation and regulation by the kinin family of neuropeptides, which are hypothesized to provide additional input in to muscle activity in this critical site of reabsorption (Audsley and Weaver, 2009, Lajevardi and Paluzzi, 2020). We speculate that these muscle contractions aid in vigorous movement of papillar cytoplasm, which includes ions and water taken up from the intestinal lumen. The movement of these papillar contents may facilitate both cytoplasm exchange between papillar cells and the interaction of ions and ion transport machinery with intracellular membrane stacks.”

Regarding the reviewer’s question about transluminal transport and the advantage to the animal, these remain open questions that we look forward to addressing in the future.

6) In general, a clearer cartoon/schematic of the rectal papilla as well as the experimental flow would be helpful in the main figure, especially for readers without expertise in Drosophila models. It is a bit difficult to discern exactly when the heat-shock to induce Cre expression was being carried out, especially in the case that the Gal80ts fly line was also being used to repress Gal4 expression. Perhaps this information could be added to the timeline in Figure S1D and included in a main figure rather than in the supplement.

We thank the reviewer for this suggestion. We moved the timeline in Figure 1—figure supplement 1 to Figure 1, modified the timeline to include Cre, and clarified Cre and Gal4-expression in writing.

The text now reads:

“We used animals heterozygous for UAS-dBrainbow to ensure single-labeling of cells. We ubiquitously expressed Cre, which does not require heat-shock induction, from early embryonic stages, before cells endocycle to any great degree. Cre-mediated excision occurs independently of Gal4 expression and Gal80ts repression of dBrainbow. Therefore, we can ensure that multi-labeled cells only arise by cytoplasm sharing between cells not related by cell division or incomplete cytokinesis.”

We have also added a small diagram of a papilla to Figure 1 (Figure 1D).

7) The data on localization of Rab5 endosomes could be strengthened. It would be nice to see other markers, that don't rely on over-expression of a transgenic construct. Alternatively, could the authors also assess from their EM data whether they see redistribution of vesicles pre- and post- sharing?

Thank you for this opportunity to discuss and address our endosome localization data further. In the revised manuscript, we address this point by clarifying and emphasizing the GFP-myc-FYVE marker is not, in fact, an overexpression of a protein that would affect endosome localization with the following additional text:

“GFP-tagged pan-endosome marker (myc-2x-FYVE), overexpression of which should not alter endosome shape or localization (Gillooly et al., 2000, Wucherpfennig et al., 2003),”

Further, in response to this comment, we also used a Rab5 antibody to show Rab5 localization without using transgenic markers. The Rab5 antibody does not mark full endosomes and has a punctate appearance in papillar cells, as shown In Author response image 1. As it does not mark full endosomes, we do not observe the same degree of polarization observed with the GFP-myc-FYVE and Rab5-GFP transgenes. This is in agreement with other literature (Wucherpfennig et al., 2003). We also note that we cannot look at the same time point with the antibody as with transgenes, as a thick cuticle layer forms on papillae in late development, which causes technical issues with antibody staining. We therefore use an earlier timepoint soon after cytoplasm sharing instead.

Author response image 1.

Reviewer #2:

Specific Comments:

1) The authors made interesting observations that massive membrane reorganization in apical microvilli-like structures, apical cell–cell junctions, and endomembrane stacks surrounding mitochondria coinciding with cytoplasm sharing. However, it is unclear how/which of these membrane reorganization events would lead to cytoplasmic sharing.

Thank you for the opportunity to expand on this point, which falls beyond the scope of the current manuscript. The reviewer is absolutely correct- at this time it is unclear to us how the multiple membrane reorganization events which we report here to be directed by Dynamin, Rab5, and Rab11, are inter-related. We show here that they all occur within a succinct developmental window, in conjunction with the relocalization of Dynamin and endosomes. We speculate that each reorganization event is critical to transform this epithelium into a highly specialized reabsorptive structure. Future work can hopefully identify separation of function mutants that perturb only specific aspects of papillar membrane remodeling, so that we can individually evaluate the contribution of each to cytoplasmic sharing and papillar physiology. We note that a similar reviewer comment from a 2014 publication led us to first consider cytoplasm sharing as a possibility in papillar cells. We really value such input, and we absolutely intend to pursue these questions in the future!

2) The authors showed the appearance of gap junctions during cytoplasmic sharing. Do the plasma membranes for these gap junctions come from exocytosis? If so, why would inhibiting an endocytosis protein Shi inhibit gap junction?

This is an interesting question that we hope to answer in future studies. We speculate that plasma membrane and septate junction that exists in the apical region prior to sharing is sculpted and perhaps removed by endocytic factors. If this region is not remodeled, then there is no place for gap junction establishment. This is supported by staining of Inx3, a gap junction protein, that appears to be expressed in shi RNAi animals (Figure 4—figure supplement 1C-C’) but does not localize to cell–cell boundaries. However, we cannot entirely rule out that Shi has an indirect effect on gap junction establishment, such as through papillar cell differentiation or signaling.

3) Increased gap junctions and endomembrane stacks are quite separate observations. Is there any connection between these membrane structures, e.g. similar origin?

We agree with the reviewer. This comment is highly related to the above comment #1 from reviewer 2. Please see our response to that comment regarding this question, which is beyond the scope of our current manuscript.

4) Could proteins larger than GFP pass through the papillar cells, presumably through the gap junctions?

Please see our response to comment #2 from reviewer #1.

5) What is the physiological significance of the association between endomembrane stacks and mitochondria?

Thank you for the opportunity to address this interesting question. In response, we added text to expand on the significance of mitochondrion-endomembrane stack association. Berridge and Gupta, 1967, hypothesized that the mitochondria provide ATP to active ion transport ATPases. Patrick et al. (2006) found P-type Na+/K+-ATPase localized to the basal edge of Aedes aegypti rectal pads. We hypothesize that mitochondria supply ATP to P-type Na+/K+-ATPase among other ATP-dependent ion transporters located in the endomembrane stacks. The endomembrane stacks and associated mitochondria therefore support ion recycling from the rectal lumen back into the hemolymph.

6) How does the change of Shi localization (Figure 3D and D') contribute to cytoplasmic sharing?

Thank you for this question. At this time, we do not know if the change in Shi localization directly contributes to cytoplasmic sharing. We do show that Shi is required for changes in endosome positioning but that knock down of Rab5 does not affect Shi localization which suggests that Shi localization is upstream of the endosome positioning that occurs concurrently with cytoplasm sharing. This is certainly something to explore in the future.

7) Are Inx1-3 expressed in the Shi, Rab5 and Rab11 knockdown animals which show defective cytoplasmic sharing? If yes, is their localization altered?

Thank you for your interest- in response, we stained shi knockdown animals with anti-Inx3 antibody and found that Inx3 does not localize to cell–cell boundaries as in age-matched WT animals, which is consistent with our shi RNAi electron micrographs. We added Panel C-C’ to Figure 4—figure supplement 1 and the following text:

“Inx3 also does not localize to cell–cell boundaries in shi RNAi animals (Figure 4—figure supplement 1C-C’).”

We note that we looked at localization but not overall protein levels (by Western blot, for example) due to very limited antibody, so at this time we cannot conclude if shi RNAi affects Innexin expression as well as Innexin localization.

8) Does fzr knockdown affect the levels of Shi, Rab5 and Rab11?

Thank you for your interest- in response, we stained post-sharing WT and fzr RNAi animals for Rab5 and found that Rab5 looks similar in localization and level in WT and fzr RNAi animals, as shown In Author response image 2. This suggests that fzr RNAi is not acting directly through Rab5.

Author response image 2.

9) Do fzr mutants phenocopy the membrane architecture observed in the shi mutants?

We share the reviewer’s interest in this question. However, due to COVID-19, we do not have regular access to the EM facility, and therefore we cannot address this point in a timely manner. We do note that we have previously shown that fzr RNAi blocks papillar endocycles in larval development, and therefore we speculate that these endocycles are important for papillar cell identity and differentiation. As such, we would expect that the membrane architecture in fzr RNAi animals is not wild-type. However, given how early the endocycles occur in development, we expect that adult fzr RNAi animals have ultrastructure similar to larval papillar cells while shi RNAi animals are more like WT adult cells with grossly impaired membrane reorganization.

10) What does the endosomal distribution look like in the rab5 and rab11 mutants at the onset of cytoplasmic sharing? (68HPPF)

This is certainly an interesting question. In response, we used a Rab5 antibody to examine early endosomes and did not see an obvious difference in Rab5 distribution between WT and Rab11 RNAi animals around the time of sharing (not shown). The caveat is that we used Rab5 staining instead of the GFP-Myc-2x-FYVE or Rab5-YFP overexpression to mark endosomes. This is certainly a question to explore in more detail in the future.

Reviewer #3:

[…] Recombination can happen independently to sister chromatids if cells are in G2 phase when the recombination was induced. Plus, rectal papillae cells are polyploid, which increases the chance that a single cell can have multiple recombination events). If so, there should be a considerable number of cells that express multiple colors without cytoplasmic sharing. How do they access this possibility? Was recombination titrated such that recombination can happen only to one chromatid? The image in Figure 1D is so clear, so I don't doubt that each cell is labelled with one color, but if you think about the logic, we have to wonder why. Some discussion/explanation is necessary.

Please see our response to reviewer #1, comment #6.

Figure 3: formation of endomembrane surrounding mitochondria during cytoplasmic sharing is interesting, but is there any evidence that this indeed contributes to cytoplasmic sharing? It's unclear how endomembrane would lead to cytoplasmic sharing.

Please see our response to comment #1 from reviewer #2.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (D. melanogaster)	w1118	Bloomington Drosophila Stock Center	BDSC:3605; FLYB:FBst0003605; RRID:BDSC_3605	w1118
Genetic reagent (D. melanogaster)	tub-Gal4	Bloomington Drosophila Stock Center	BDSC:5138; FLYB:FBst0005138; RRID:BDSC_5138	y1 w*; P{tubP-GAL4}LL7/TM3, Sb1 Ser1
Genetic reagent (D. melanogaster)	tub-Gal80ts	NA	NA	NA
Genetic reagent (D. melanogaster)	UAS-dBrainbow	Bloomington Drosophila Stock Center; (Hampel et al., 2011)	BDSC:34513; FLYB:FBst0034513; RRID:BDSC_34513	w1118; P{UAS-Brainbow}attP2
Genetic reagent (D. melanogaster)	UAS-dBrainbow	Bloomington Drosophila Stock Center; (Hampel et al., 2011)	BDSC:34514; FLYB:FBst0034514; RRID:BDSC_34514	w1118; P{UAS-Brainbow}attP40
Genetic reagent (D. melanogaster)	Hsp70>cre	Bloomington Drosophila Stock Center	BDSC:851; FLYB:FBst0000851; RRID:BDSC_851	y1 w67c23 P{Crey}1b; D*/TM3, Sb1
Genetic reagent (D. melanogaster)	UAS-fzr RNAi	Vienna Drosophila Resource Center	VDRC:25550; FLYB:FBst0455950	w1118; P{GD9960}v25550
Genetic reagent (D. melanogaster)	UAS-shi RNAi #1	Bloomington Drosophila Stock Center	BDSC:28513; FLYB:FBst0028513; RRID:BDSC_28513	y1 v1; P{TRiP.JF03133}attP2
Genetic reagent (D. melanogaster)	UAS-shi RNAi #2	Bloomington Drosophila Stock Center	BDSC:36921; FLYB:FBst0036921; RRID:BDSC_36921	y1 sc* v1 sev21; P{TRiP.HMS00154}attP2
Genetic reagent (D. melanogaster)	UAS-Rab5 RNAi #1	Bloomington Drosophila Stock Center	BDSC:30518; FLYB:FBst0030518; RRID:BDSC_30518	y1 v1; P{TRiP.JF03335}attP2
Genetic reagent (D. melanogaster)	UAS-Rab5 RNAi #2	Bloomington Drosophila Stock Center	BDSC:67877; FLYB:FBst0067877; RRID:BDSC_67877	y1 sc* v1 sev21; P{TRiP.GL01872}attP40
Genetic reagent (D. melanogaster)	UAS-Rab11 RNAi #1	Bloomington Drosophila Stock Center	BDSC:27730; FLYB:FBst0027730; RRID:BDSC_27730	y1 v1; P{TRiP.JF02812}attP2
Genetic reagent (D. melanogaster)	UAS-Rab11 RNAi #2	Vienna Drosophila Resource Center	VDRC:22198; FLYB:FBst0454467	w1118; P{GD11761}v22198
Genetic reagent (D. melanogaster)	UAS-SCAR RNAi #1	Bloomington Drosophila Stock Center	BDSC:36121; FLYB:FBst0036121; RRID:BDSC_36121	y1 sc* v1 sev21; P{TRiP.HMS01536}attP40
Genetic reagent (D. melanogaster)	UAS-SCAR RNAi #2	Bloomington Drosophila Stock Center	BDSC:51803; FLYB:FBst0051803; RRID:BDSC_51803	y1 v1; P{TRiP.HMC03361}attP40
Genetic reagent (D. melanogaster)	UAS-kirre RNAi	Vienna Drosophila Resource Center	VDRC:27227; FLYB:FBst0456824	w1118; P{GD14476}v27227
Genetic reagent (D. melanogaster)	UAS-sns RNAi	Vienna Drosophila Resource Center	VDRC:877; FLYB:FBst0471238	w1118; P{GD65}v877/TM3
Genetic reagent (D. melanogaster)	UAS-schizo RNAi	Vienna Drosophila Resource Center	VDRC:36625; FLYB:FBst0461775	w1118; P{GD14895}v36625
Genetic reagent (D. melanogaster)	UAS-sing RNAi	Vienna Drosophila Resource Center	VDRC:12202; FLYB:FBst0450437	w1118; P{GD3396}v12202/TM3
Genetic reagent (D. melanogaster)	UAS-Cdc42DN	Bloomington Drosophila Stock Center	BDSC:6288; FLYB:FBst0006288; RRID:BDSC_6288	w*; P{UAS-Cdc42.N17}3
Genetic reagent (D. melanogaster)	UAS-Rac1DN	Bloomington Drosophila Stock Center	BDSC:6292; FLYB:FBst0006292; RRID:BDSC_6292	y1 w*; P{UAS-Rac1.N17}1
Genetic reagent (D. melanogaster)	UAS-Rho1DN	Bloomington Drosophila Stock Center	BDSC:7328; FLYB:FBst0007328; RRID:BDSC_7328	w*; P{UAS-Rho1.N19}2.1
Genetic reagent (D. melanogaster)	UAS-GFPNLS	Bloomington Drosophila Stock Center	BDSC:4776; FLYB:FBst0004776; RRID:BDSC_4776	w1118; P{UAS-GFP.nls}8
Genetic reagent (D. melanogaster)	UAS-GFP-Myc-2x-FYVE	Bloomington Drosophila Stock Center	BDSC:42712; FLYB:FBst0042712; RRID:BDSC_42712	w*; P{UAS-GFP-myc-2xFYVE}2
Genetic reagent (D. melanogaster)	UAS-YFP-Rab5	Bloomington Drosophila Stock Center	BDSC:9775; FLYB:FBst0009775; RRID:BDSC_9775	y1 w*; P{UASp-YFP.Rab5}Pde808b
Genetic reagent (D. melanogaster)	60H12-Gal4	Bloomington Drosophila Stock Center	BDSC:39268; FLYB:FBst0039268; RRID:BDSC_39268	w1118; P{GMR60H12-GAL4}attP2
Genetic reagent (D. melanogaster)	UAS-shiDN	Bloomington Drosophila Stock Center	BDSC:5822; FLYB:FBst0005822; RRID:BDSC_5822	w*; TM3, P{UAS-shi.K44A}3-10/TM6B, Tb1
Genetic reagent (D. melanogaster)	NrxIV-GFP	Bloomington Drosophila Stock Center	BDSC:50798; FLYB:FBst0050798; RRID:BDSC_50798	y1 w*; P{PTT-GA}Nrx-IVCA06597
Genetic reagent (D. melanogaster)	Df(1)BSC867	Bloomington Drosophila Stock Center	BDSC:29990; FLYB:FBst0029990; RRID:BDSC_29990	Df(1)BSC867, w1118/Binsinscy
Genetic reagent (D. melanogaster)	UAS-ogre RNAi	Vienna Drosophila Resource Center	VDRC:7136; FLYB:FBst0470569	w1118; P{GD3264}v7136
Genetic reagent (D. melanogaster)	byn-Gal4	Singer et al., 1996	FLYB:FBal0137290	P{GawB}bynGal4
Genetic reagent (D. melanogaster)	UAS-GFPPA	Lynn Cooley; McLean and Cooley, 2013	FLYB:FBti0148163	P{20XUAS-IVS-Syn21-mC3PA-GFP-p10}
Genetic reagent (D. melanogaster)	UAS-NDN	Rebay et al., 1993	NA	NA
Genetic reagent (D. melanogaster)	UAS-shi-Venus	Stefano De Renzis; Fabrowski et al., 2013	NA	NA
Genetic reagent (D. melanogaster)	UAS-GFP-ogre	Andrea Brand; Spéder and Brand, 2014	FLYB:FBtp0127574	ogreUAS.N.GFP
Genetic reagent (D. melanogaster)	UAS-Gapdh2-GFPPA	This paper	NA	Transgenic line created through gene synthesis and embryo injection. Codon-optimized D. melanogaster Gapdh2 fused to GFPPAunder UAS control.
Antibody	anti-GFP(Rabbit polyclonal)	Thermo Fisher Scientific	Cat# A11122; RRID:AB_221569	IF (1:1000)
Antibody	anti-HA (Rat monoclonal)	Roche	Cat# 11867423001; RRID:AB_390918	IF (1:100)
Antibody	anti-Inx3(Rabbit polyclonal)	Reinhard Bauer; Lehmann et al., 2006	RRID:AB_2568555	IF (1:75)
Antibody	Anti-Rabbit Alexa Fluor 488 (Goat)	Thermo Fisher Scientific	Cat# A32731; RRID:AB_2633280	IF (1:2000)
Antibody	Anti-Rabbit Alexa Fluor 568 (Goat)	Thermo Fisher Scientific	Cat# A-11011; RRID:AB_143157	IF (1:2000)
Antibody	Anti-Rat Alexa Fluor 633 (Goat)	Thermo Fisher Scientific	Cat# A-21094; RRID:AB_2535749	IF (1:2000)
Other	DAPI stain	Sigma-Aldrich	Cat# D9542	(1:5000)