An extracellular vesicle targeting ligand that binds to Arc proteins and facilitates Arc transport in vivo
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eLife assessment
Extracellular vesicles (EVs) are emerging as important mediators of cell-to-cell signaling. Lee et al show convincingly that Stranded at second (Sas), a Drosophila cell surface protein, is trafficked by and localized to Drosophila EVs. However, the data supporting interaction with dArc and whether Sas facilitates the intercellular transfer of dArc protein or mRNA is incomplete. Moreover, almost all experiments rely on gain-of-function and over-expression of Sas, thus the relevance to normal physiological signaling is unclear.
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Abstract
Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. Here, we identify the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2) cells. Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. We used co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1 and mammalian Arc. dArc1 and Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the APP ICD also binds to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo.
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eLife assessment
Extracellular vesicles (EVs) are emerging as important mediators of cell-to-cell signaling. Lee et al show convincingly that Stranded at second (Sas), a Drosophila cell surface protein, is trafficked by and localized to Drosophila EVs. However, the data supporting interaction with dArc and whether Sas facilitates the intercellular transfer of dArc protein or mRNA is incomplete. Moreover, almost all experiments rely on gain-of-function and over-expression of Sas, thus the relevance to normal physiological signaling is unclear.
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Reviewer #1 (Public Review):
Extracellular vesicles (EVs) are emerging as important mediators of cell-to-cell signaling. In this paper the authors aim to demonstrate that Stranded at second (Sas), a Drosophila cell surface protein, binds to dArc1 and Ptp10D to mediate intercellular transport of dArc1 via EVs. dArc1 protein has been shown to form virus-like capsids that carry dArc1 mRNA from neurons to muscle, but little is known about this new intercellular communication pathway. Similarly, not much is known generally about how EVs are targeted to specific cell types, or how specific EV cargo can be delivered. Thus, this work is of interest to cell biologists and neuroscientists. However, the jumbled description of the results and general lack of rigor of experiments diminish the impact and interpretability of the conclusions. Moreover, …
Reviewer #1 (Public Review):
Extracellular vesicles (EVs) are emerging as important mediators of cell-to-cell signaling. In this paper the authors aim to demonstrate that Stranded at second (Sas), a Drosophila cell surface protein, binds to dArc1 and Ptp10D to mediate intercellular transport of dArc1 via EVs. dArc1 protein has been shown to form virus-like capsids that carry dArc1 mRNA from neurons to muscle, but little is known about this new intercellular communication pathway. Similarly, not much is known generally about how EVs are targeted to specific cell types, or how specific EV cargo can be delivered. Thus, this work is of interest to cell biologists and neuroscientists. However, the jumbled description of the results and general lack of rigor of experiments diminish the impact and interpretability of the conclusions. Moreover, almost all experiments rely on gain-of-function and over-expression of Sas, thus the relevance to normal physiological signaling is unclear.
Major strengths:
1. The data showing that Sas is released into EVs and delivered to cells is strong.
2. The EM data showing Sas localization to EVs is clear.Major weaknesses:
The description of the results omits some data in the figures and is not in a logical order. This made it hard to read and follow. There is also a lack of rigor and quantification in some experiments. Specifically:1. Figure 2: Description of dArc1 putative capsids is absent from the results section (2f,g) until describing fig 4 data (line 362). Given that there is no immuno-EM labeling of dArc1 protein, it is not clear if Sas and dArc1 are localized to the same EVs. Nor is it clear if the double membrane EVs are actually EVs that contain capsids. Overall, the EM data lacks quantification. How many EVs on average show Sas labeling? How many EVs have double membranes? The dense protein staining surrounding EVs seems unusual, is this due to artifact of the purification? EV kits are generally non-specific and isolate non-EV membranes, corroboration using ultracentrifugation or size exclusion chromatography methods would be beneficial. SAS-FL overexpression results in more EVs, which confounds subsequent experiments suggesting that Sas targets EVs to specific cell types/regions.
2. Figure 3: There are no data showing the expression of Sas in SG cells using the GAL4 lines. Is this expression restricted to just SG cells? The results jump from a-b to f-g. c-e are out of order. The quantification in g should be broken into two and paired with the actual data (c-e, and f). It is not clear how the quantification in g was performed. How many WBs were analyzed? There seems to be a bubble in the first lane of f, which would preclude quantification. Why is d not quantified and there seems to be an overall increase in background staining in e that is not specific to discs. The source data files are not labelled and these data should be incorporated into annotated supplemental figures. Is transfer in a-b due to Ptp10D? How many WBs were quantified in g?
3. Figure 4: C and d, IP data has no inputs for IPs, no sizing markers, and no IgG controls for antibody specificity. These data would also be more convincing if done with FL Sas and included co-Ips from cell lysates.
4. In general, the WBs in the figures show very white backgrounds with high contrast, which suggests the images may have been manipulated. Total protein controls are also missing.
5. Figure 5: Ashley et al (Cell 2018) showed that dArc1 mRNA transfer required the 3'UTR so it is puzzling that the authors used heterologous UTRs. The results using FISH on endogenous dArc1 mRNA are dramatic. The authors should show definitively that their probe does not pick up over-expressed dArc1.
6. Many of the conclusions would be strengthened by the loss of function experiments, especially showing a requirement for Sas in dArc1 transfer.
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Reviewer #2 (Public Review):
The manuscript addresses the important question of how EVs are targeted to their recipient cells once they are produced and released.
The present manuscript contains 4 messages:
First, it shows that the transmembrane protein Sas gets incorporated into EVs and that this protein binds to its receptor Ptp10D on target cells, thus targeting the EVs. Second, the manuscript shows that the Sas cytoplasmic domain ICD binds to dARC1 protein (and perhaps darc1 RNAs), which are incorporated into EVs where they form capsids, before being targeted to recipient cells. dARC1 is important for neuron development in flies! Interestingly the motif in the Sas ICD is conserved in mammalian APP that also binds ARC1, suggesting a conserved mechanism of targeting EVs in mammalian neural development. Third, exposure of target cells …Reviewer #2 (Public Review):
The manuscript addresses the important question of how EVs are targeted to their recipient cells once they are produced and released.
The present manuscript contains 4 messages:
First, it shows that the transmembrane protein Sas gets incorporated into EVs and that this protein binds to its receptor Ptp10D on target cells, thus targeting the EVs. Second, the manuscript shows that the Sas cytoplasmic domain ICD binds to dARC1 protein (and perhaps darc1 RNAs), which are incorporated into EVs where they form capsids, before being targeted to recipient cells. dARC1 is important for neuron development in flies! Interestingly the motif in the Sas ICD is conserved in mammalian APP that also binds ARC1, suggesting a conserved mechanism of targeting EVs in mammalian neural development. Third, exposure of target cells (ex vivo wing discs) to EVs positive to FL Sas leads to its increased targetting when the target cells also expressed Ptp10D and Numb, which are acting as Sas receptors in a synergetic manner. Fourth, dARC1 ORF expression in the EV-producing cells (SG) leads to the increased expression of dARC1 protein and mRNAs in the recipient cells in vivo (Trachea). Many techniques are used, including IEM, fly genetics, S2 cells, and Ips. It is broad, and well executed, and the questions are interesting.However, the manuscript should be strengthened. It is a lot of data and techniques but because there are so many messages in the paper, each needs more substances and controls.
1: Use of more extensive fly genetics using specific Ptp10D LOF in wing discs and trachea (to show the converse of the GOF).
Does Ptp10D acts as the MAIN receptor to FL Sas? Numb LOF, a combination of LOF and GOF?
does Ptp10D GOF compensate for Numb and vice versa?2: What is the specificity for FL Sas? The expression of short Sas should not lead to its incorporation in EVs and their overnight addition should not lead to the same effect (Figure 3). This should be better investigated as short Sas is a good control for FL Sas.
3: A better quantitative analysis should be provided. For instance, there is no quantitative data for Figure 5.
4: All experiments are done with flies. There is no data on mammalian neurons in culture. This is missing. Exposure of neurons with SAS-positive EVs (or APP)
5: Are the capsid reconstitution with purified dARC1 and 2 performed in the presence of darc1 rRNA? Any RNA (figure 2).
6: The dAC1 increased expression in the target cells upon dARC1 increased production in SG(Figure 5) becomes an important part of the paper (and the model) but is not investigated!
How does it work? Does the delivery of darc1 mRNAs packaged in capsids simply lead to more dARC1 translation? Is it proportional?
OR is there also stimulation of darc1 transcription? Is there also an increase in the mRNA level (I cannot see the SG control of 5o (sage>+) supporting the authors' claim on line 562!).7: Most (all) experiments are performed with overexpression of FL Sas or ICD. Does endogenous Sas bind endogenous Ptp10D and dARC1? ICDs? Also full-length APP?
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Reviewer #3 (Public Review):
Lee et al. identify the Stranded at second (Sas) cell surface protein as an extracellular vesicle (EV) component in Drosophila. They first show that different isoforms of Sas exhibit differential tissue distribution in vivo, with the EV-enriched full-length Sas isoform exhibiting distribution at distant sites away from its cells of origin. They show that Sas is present in EVs purified from Drosophila S2 cells, as assessed using exosome isolation kits and via immuno-electron microscopy. Their data suggest that Sas-bearing EVs preferentially target cells expressing Ptp10D, a receptor tyrosine phosphatase that is a known binding partner of Sas, both in the context of S2 cells and imaginal discs engineered to overexpress Ptp10D and the endocytosis regulatory protein Numb. Through immunoprecipitation (IP) of Sas …
Reviewer #3 (Public Review):
Lee et al. identify the Stranded at second (Sas) cell surface protein as an extracellular vesicle (EV) component in Drosophila. They first show that different isoforms of Sas exhibit differential tissue distribution in vivo, with the EV-enriched full-length Sas isoform exhibiting distribution at distant sites away from its cells of origin. They show that Sas is present in EVs purified from Drosophila S2 cells, as assessed using exosome isolation kits and via immuno-electron microscopy. Their data suggest that Sas-bearing EVs preferentially target cells expressing Ptp10D, a receptor tyrosine phosphatase that is a known binding partner of Sas, both in the context of S2 cells and imaginal discs engineered to overexpress Ptp10D and the endocytosis regulatory protein Numb. Through immunoprecipitation (IP) of Sas from S2 cell EVs, as well as validation co-IPs and peptide binding assays, the authors found that Sas can interact with the dArc1 protein (i.e. the orthologue of mammalian Arc, which has the ability to form capsid-like structures) via a conserved protein motif of Sas. Finally, they show that Sas increases the transfer of dArc1 protein and mRNA from Sas-expressing cells to Ptp10D-enriched tissues in vivo. The authors conclude that Sas facilitates the delivery of dArc1 capsids that carry dArc1 mRNA to recipient cells that express Ptp10D.
General Strengths: The in vivo and in vitro data conveying the selective targeting of the full-length Sas isoform to EVs, and the impact on the delivery of dArc1 to distant Ptp10D-expressing cells, are generally strong and supportive of the proposed model. The authors also show convincing data confirming the interaction of Sas with dArc1 by IP-MS and binding assays.
General Weakness: It is not clear if the major biological function of the endogenous Sas-Ptp10D interaction is mediated via EVs. The inclusion of additional data evaluating dArc1 mRNA EV-mediated transfer to the trachea in Sas and/or Ptp10D null mutant flies would strongly enhance the paper and support the role of these proteins in tissue-specific EV targeting in vivo. Moreover, throughout the paper, there are several controls and quantifications missing that would be required in order to strengthen the general conclusions and proposed regulatory model. For instance, it is not clear to what extent Sas and dArc1 proteins are co-enriched within purified EV specimens. Immuno-EM studies or nanoparticle analysis strategies should be implemented to address this aspect. Several of the IF- and FISH-based labeling experiments lacked controls. Also, there are few if any quantifications provided as to the number of tissue specimens that were examined in the various assays as a basis for making specific conclusions.
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