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. 2016 Jun 3;6:27085.
doi: 10.1038/srep27085.

Drosophila Cells Use Nanotube-Like Structures to Transfer dsRNA and RNAi Machinery Between Cells

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Free PMC article

Drosophila Cells Use Nanotube-Like Structures to Transfer dsRNA and RNAi Machinery Between Cells

Margot Karlikow et al. Sci Rep. .
Free PMC article

Abstract

Tunnelling nanotubes and cytonemes function as highways for the transport of organelles, cytosolic and membrane-bound molecules, and pathogens between cells. During viral infection in the model organism Drosophila melanogaster, a systemic RNAi antiviral response is established presumably through the transport of a silencing signal from one cell to another via an unknown mechanism. Because of their role in cell-cell communication, we investigated whether nanotube-like structures could be a mediator of the silencing signal. Here, we describe for the first time in the context of a viral infection the presence of nanotube-like structures in different Drosophila cell types. These tubules, made of actin and tubulin, were associated with components of the RNAi machinery, including Argonaute 2, double-stranded RNA, and CG4572. Moreover, they were more abundant during viral, but not bacterial, infection. Super resolution structured illumination microscopy showed that Argonaute 2 and tubulin reside inside the tubules. We propose that nanotube-like structures are one of the mechanisms by which Argonaute 2, as part of the antiviral RNAi machinery, is transported between infected and non-infected cells to trigger systemic antiviral immunity in Drosophila.

Figures

Figure 1
Figure 1. Nanotube-like structures are present in Drosophila cells.
Stable cell lines expressing eGFP or dsRed under the control of an actin promoter were mixed at a 1:1 ratio, grown overnight and examined by confocal microscopy (a–g). Note that images have been voluntarily saturated to better visualize the nanotube-like structures. (a) Merged image of eGFP and dsRed cells stained for tubulin and F-actin. Zoom of (a) is depicted in (b) to better visualize the structures indicated by arrows 1 and 2. (c) dsRed positive cells. (d) eGFP positive cells. Cells were stained for tubulin in blue (f) and F-actin using Phalloidin 647 Alexa-Fluor (g). The inset in (a) depicts the corresponding (x–z) section through the marked nanotube-like structures (arrow). Arrows indicate projections between cells and bars represent 10 μm (a) and 1 μm (hi). Scanning electron microscopy of S2 cells showing projections between cells (h,i).
Figure 2
Figure 2. Nanotube-like structures are more abundant during viral infection.
(a,b) Non-infected S2 cells (S2n), S2 cells during acute DCV infection, S2 cells persistently infected with either DCV (S2pDCV), or with FHV (S2pFHV), and S2 cells infected with bacteria (S2n Erwinia-GFP) were plated on glass coverslips overnight as described in Materials and Methods. Cells were stained for DAPI and Phalloidin, and nanotube-like structures were counted in at least 1,000 cells per treatment group. Error bars indicate standard deviation of the mean of three independent experiments and three biological replicates per experiment. **p ≤ 0.0079. *p = 0.0159. ****p < 0.0001. ns: non-significant. All statistical analyses were performed with Prism 6 software, using a non-parametric Mann-Whitney test. (cf) Immunofluorescence during acute infection of S2 cells with DCV (c,d) or FHV (e,f). Cells were stained for CG4572 and viral capsid. DAPI and Phalloidin 647 Alexa-Fluor were used to mark nuclei and F-actin, respectively. The insets in (c,e) depict the corresponding (x–z) section through the marked nanotube-like structure (arrow). Higher magnification images of nanotube-like structure (arrow), viral proteins and CG4572 are shown in (d,f).
Figure 3
Figure 3. The RNAi machinery localizes along nanotube-like structures.
Immunofluorescence and confocal microscopy in S2 cells. Cells were stained for F-actin using Phalloidin 647 Alexa-Fluor (a,d,g). Rab7 and Ago2 (b,c), dsRNA and Ago2 (e,f) and CG4572 protein and Ago2 (h,i) were detected along nanotube-like structures. DAPI is used to mark nuclei. The insets in (b,e,h) depict the corresponding (x-z) section through the marked nanotube-like structure (arrow). Higher magnification images of nanotube-like structures (arrow) and RNAi proteins are shown in (c,f,i).
Figure 4
Figure 4. Ago2 localises inside nanotube-like structures and is transferred between cells.
Drosophila S2 cells were stained for F-actin using Phalloidin 647 Alexa-Fluor, Ago2 in green and Tubulin in red. DAPI was used to stain the nucleus. SR-SIM acquisition in a plan view (a) or seen represented as 3D view with an image rotation (b). A surface (in grey) was defined following the Phalloidin staining (c) and a zoom was performed to only visualize the proteins that localize beneath the surface (proteins outside the surface are not visible in this representation) (d). (e) S2n cells were mixed 1:1 with S2R+ dsRed cells and gates were set. P1 quadrant shows double negative cells dsRed−GFP−, P2 quadrant shows dsRed+ GFP− cells, P3 quadrant shows double positive cells dsRed+ GFP+, P4 quadrant shows dsRed-GFP+ cells. (f) Analysis of S2n cells plated with supernatant of S2R+ dsRed transfected with Ago2-GFP. (g) Analysis of S2n cells mixed 1:1 with S2R+ dsRed cells transfected with Ago2-GFP. (h) Statistical analysis of the percentage of S2n cells that became GFP+ (P4 quadrant) when plated with the supernatant of transfected cells (0.42%) (f) or mixed with the transfected cells (1.184%) (g). Error bars indicate standard deviation of the mean of three independent experiments and two biological replicates per experiment. p = 0.0179. All statistical analyses were performed with Prism 6 software, using a non-parametric Mann-Whitney test.

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