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.

The ubiquity and importance of cytoplasm sharing led us to seek out novel examples in the tractable animal model Drosophila melanogaster. Here, we report an animal-wide screen for tissues that share cytoplasm. We identify a novel mechanism of cytoplasm sharing in the rectal papilla, a resorptive intestinal epithelium (10) and known site of pathogen localization (11). Unlike We next examined whether cytoplasm sharing requires the distinctive papillar cell cycle program, which completes prior to sharing onset (FigS1D). Larval papillar cells first undergo endocycles, which increase cellular ploidy, and then pupal papillar cells undergo polyploid mitotic cycles, which increase cell number (14). Knockdown of the endocycle regulator fizzy-related (15) significantly disrupts cytoplasm sharing (FigS1E,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 (14), does not prevent sharing (FigS1E,G,H). Thus, papillar cytoplasm sharing requires developmentally programmed endocycles but not mitotic cycles.
As our dBrainbow approach only identifies cytoplasm sharing events that do not involve failed 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 (16,17). We conducted a candidate dBrainbow-based RNAi screen (77 genes, Fig2A, Table S1) of myoblast fusion regulators and other plasma membrane components. Remarkably, 0/15 myoblast fusion genes from our initial screen regulate papillar cytoplasm sharing (Fig2A, FigS2A, Table S1). Furthermore, dominant-negative forms of Rho family GTPases have no impact on Brainbow labeling (FigS2B), 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) (Fig2A) 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 (18) (FigS2A). 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.
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, Fig2B, Table S2) including 3 more vacuolar H+ ATPase subunits, 5 more exocyst components, and the Dynamin GTPase shibire 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 (FigS2D-F). Together, our screens reveal that membrane trafficking, particularly Dynamin-mediated endocytosis and early/recycling endosome trafficking, regulates papillar cytoplasmic 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) and a Venus-tagged shi before and after sharing. Endosomes are evenly distributed shortly before sharing, but become highly polarized at 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 (19) and related insects (20). 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 (Fig3F-F''). Just basal to the microvilli, apical cell-cell junctions compress from a straight to a more tortuous morphology around the time of cytoplasm sharing onset (FigS3D-D''). One of the most striking changes, coincident with Shi re-localization, is formation of pan-cellular endomembrane stacks surrounding mitochondria. These stacks are likely ion transport sites (Fig3G-G''). 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 (Fig3H-J). Additionally, apical junctions do not compress as in controls (FigS3E-G). Notably, membrane stacks are greatly reduced (Fig3K-M). shi RNAi animals exhibit numerous trapped vesicles, consistent with a known role for Dynamin in membrane vesicle severing (21,22) (Fig3L, inset). Together, we find that Shi and endosomes extensively remodel membranes during cytoplasm 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 (FigS4A) (19,20). Instead, through our GTPase knockdown studies, we identified a striking alteration in the apical cell-cell interface that strongly correlates with cytoplasm sharing.

FigS3H-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 (Fig4A-B, FigS5A-A''). Given our electron micrograph results, we determined which innexins, the protein family associated with gap junctions in invertebrates (23,24), are expressed in rectal papillae. From RNA-seq data (Methods), we determined that ogre (Inx1), Inx2, and Inx3 are most highly expressed (Fig4C). This combination of innexins is not unique; the non-sharing brain and optic lobe (FigS1A) also express high levels of all three (25). We examined localization of Inx3 (a gap junction component), and compared it to a septate junction component, NeurexinIV (NrxIV). NrxIV localizes similarly both pre and post-sharing onset (Fig4D-D'), indicative of persistent septate junctions remaining between papillar cells. In contrast, Inx3 organizes apically only after cytoplasm sharing (Fig4E-E', FigS5B-B'). We tested whether innexins are required for cytoplasm sharing. Knocking down these three genes individually causes mild yet significant cytoplasm sharing defects (Fig4F). However, we see larger defects when we express dominant-negative ogre DN (Fig4F-G), which contains a C-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 (Fig4F, 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. One test of papillar function is viability following the challenge of a high-salt diet (15,26). Using either pan-hindgut or papillae-specific (FigS5C-D, Methods) knockdown of cytoplasm sharing regulators, we find both shi DN and ogre DN animals are extremely sensitive to the high-salt diet (mean survival <1 day, Fig4H). These results underscore an important function for gap junction proteins, as well as membrane remodeling by Shibire, in cytoplasm sharing.
Our findings identify Drosophila rectal papillae as a new and distinctive example of cytoplasm sharing in a simple, genetically tractable system. Papillar cytoplasm sharing is developmentally regulated, occurring over a brief 6-hour window, and requires membrane remodeling by trafficking GTPases, which apically position gap junction proteins (Fig4I, FigS5H). These membrane and junctional changes are required for normal rectum function. 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. Given the absence of other clear canals, channels, or breaks in lateral membrane, our data suggest a specialized function of gap junction proteins facilitates cytoplasm sharing between neighboring cells in an otherwise intact epithelium (Fig4I). Although gap junctions typically transfer molecules of <1kDa, elongated proteins up to 18 kDa are observed to pass through certain vertebrate gap junctions (27). Our results have several implications for functions and regulation of multinucleation. Given that cytoplasm sharing facilitates pathogen spread (4), and that papillae are an avenue of entry for mosquito viruses (11), our findings may impact insect vector control strategies. Our prior work (15) 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, a finding relevant to syncytial cancers. In the future, our Brainbow-based approach could be applied to other contexts to identify other tissues with gap junction-dependent but membrane breach-independent cytoplasm sharing. Collectively, our findings highlight the expanding diversity of multicellular tissue organization strategies.

ACKNOWLEDGMENTS
We thank members of the Fox laboratory and Drs. Dong Yan and Tony Harris for valuable feedback. Ying Hao (Duke Eye Center) provided assistance with electron microscopy. Funding: This work was supported by NIH grants GM118447 to D.F. and HL140811 to N.P. Author contributions: experimental design, execution, and data analysis-all authors, manuscript writing and editing-N.P. and D.F. Competing interestsauthors declare no competing interests. Data and materials availabilityall data is available in the main text or the supplementary materials.

Materials and Methods
Table S1-S6

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P r e -s h a r i n g W T

Fly Stocks and Genetics
Flies were raised at 25C on standard media (Archon Scientific, Durham, NC) unless specified otherwise. See Table S4 for a list of fly stocks used. See Table S3 for a full list of fly lines screened in primary and secondary screens. See Table S5 for panel-specific genotypes.
brachyenteron (byn)-Gal4 was the driver for all UAS transgenes with the exception of the screen in FigS1A, which used Tubulin-Gal4, and the shi knockdown in Fig4H, 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 DN (FigS5C-G). For all Gal4 experiments, UAS expression was at 29C, except in Fig1D-F, where it was at 25C. If byn-Gal4 expression of a given UAS-transgene was lethal, the experiment was repeated with a temperature-sensitive Gal80 ts repressor transgene and animals were kept at 18C until shifting to 29C at an experimentallydetermined time point that would both result in viable animals and permit time to express the transgene prior to syncytium formation.
For salt feeding assays, age-and sex-matched siblings were transferred into vials containing 2% NaCl food made with Nutri-Fly MFâ food base (Genesee Scientific) or control food (15). Flies were monitored for survival each day for 10 days.

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 x 1024 EMCCD (pixel size 13um) and the Andor Zyla For live imaging, hindguts were dissected and cultured based on previous protocols (14). 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 PA-GFP movement in three dimensions. PA-GFP was activated by either point activation or region of interest activation with the 405nm laser set to between 5-20%, depending on the microscope and sample of interest. For each imaging session, test activations on nearby tissues were performed prior to quantified experiments to ensure that only single cells were being activated. After activation, the wizard software was used to acquire time lapses of 15s-2min of a single activation plane in order to capture protein movement. Extremely low 488nm and 405nm laser power was used in acquisition of the time lapse images of GFP and Hoechst respectively. Low level 405nm scanning did not significantly activate PA-GFP, and control experiments were performed without the use of 405nm 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 65C overnight. The blocks were cut into semithin (0.5µm) sections using Leica Reichert Ultracuts and the sections were stained with 1% methylene blue. After inspection, ultra-thin sections (65nm -75nm) 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 35mm port camera.

Image Analysis
All image analysis was performed using ImageJ and FIJI (28,29).
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.

Genotype and experiment-specific method notes
Some additional methodological details, including animal genotype, applied to only a specific figure panel. Please see Table S5 for this information.

1G-H
byn-Gal4 / UAS-PA-GFP, live-imaged during adulthood. Single secondary and primary cells were photoactivated and imaged every 3s. 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 25C and shifted to 29C at L3. If a given RNAi or DN line was lethal when expressed with the byn-Gal4 driver, a Gal80 ts was crossed in and the animals raised at 18C with a shift to 29C 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". 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 25C and shifted to 29C at L3. If a given RNAi line was lethal when expressed with the byn-Gal4 driver, a Gal80 ts was crossed in and the animals raised at 18C with a shift to 29C at pupation. Given the robustness of cytoplasmic sharing in WT animals, gene knockdowns with even single cell defects in sharing were considered "hits". 2C Secondary screen (Hsp70>cre ; UAS-dBrainbow ; byn-Gal4) of dominant-negative and constitutively-active variants of the Drosophila Rab GTPases. UAS-Rab11 DN and UAS-Rab14 DN required a Gal80 ts repressor and temperature shifts from 18C to 29C at pupation. UAS-Rab1 DN and UAS-Rab5 DN required papillar-specific expression using an alternative Gal4 driver (60H12-Gal4), Gal80 ts repressor, and temperature shifts from 18C to 29C at pupation.