Communication between cells is an important process for survival, especially in multicellular organisms. Cells typically exchange information by releasing small molecules in to their surrounding environment which neighboring cells then receive and respond to. However, there is growing evidence to suggest that cells also pass signals to each other via fatty bubbles called exosomes and tubes connecting their membranes.

Various reports have suggested that these mechanisms can transport larger proteins and nucleic acids which carry the information cells need to make proteins. However, how cells are able to combine their membranes to allow these types of transfer is unclear.

To investigate, Zhang and Schekman studied how human cancer cells and embryonic cells grown in a laboratory pass molecules between each other. This included a string of nucleic acids known as RNA and a protein called Cas9 which can edit the genome of cells to activate an enzyme that has bioluminescence activity. By measuring the level of luminescence, Zhang and Schekman were able to sensitively detect the transfer of Cas9 and RNA to neighboring cells.

The experiments showed that exosomes were not efficient at transporting proteins or RNA. However, cells in near or direct contact transferred both molecules effectively using tube connections, with some cell types being more adept at this mechanism than others. Zhang and Schekman found that the formation of these tubular channels required a protein called syncytin which helps membranes fuse together mainly in the early stages of embryo development.

These findings open a new avenue of investigation on how cells send signals to one another. It is also possible that the protein syncytin has a role in cancer progression, as tumors rely on cell communication to maintain their growth and organize the cells surrounding them. However, further work is needed to investigate this possibility.

Although most forms of intercellular communication are mediated by secreted diffusible proteins and small molecules, considerable interest has developed concerning the possibility that extracellular vesicles (EVs) or exosomes and tubular connections may convey cytoplasmic proteins, RNA molecules, and even organelles between neighboring or distant cells (Aykan, 2013).

Exosomes, one type of EV, are secreted from diverse cells and have been reported to elicit physiologically meaningful effects on target cells (van Niel et al., 2018; Mathieu et al., 2019). Exosomes contain various biological molecules, including lipids, RNA, and proteins, any or all of which may introduce normal or pathological signals to a target cell or tissue (van Niel et al., 2018; Mathieu et al., 2019). Our previous studies reported that two RNA-binding proteins, YBX1 and Lupus La protein, are involved in highly selective microRNA loading into exosomes (Shurtleff et al., 2016; Temoche-Diaz et al., 2019). The use of exosomes as a diagnostic biomarker and as a delivery vehicle for drugs or the CRISPR/Cas9 has generated much interest (Kim et al., 2017; Li et al., 2019; Lin et al., 2018; de Jong et al., 2020). We know little about the means by which exosomes may efficiently deliver cargo to the cytoplasm or nucleus of cells that internalize such vesicles (Kalluri and LeBleu, 2020). Functional delivery will likely require the action of a membrane fusogen to promote fusion of an exosome at the cell surface or after internalization to an endosome.

The tunneling nanotube (TNT) is a membranous structure with the potential for intercellular communication (Rustom et al., 2004). TNTs are thin membrane bridges with open-ended extremities that appear to mediate membrane continuity between cultured mammalian cells (Rustom et al., 2004). TNTs were first described in cultured rat pheochromocytoma PC12 cells, and although considerable evidence has developed for intercellular transfer mediated by TNTs in cell culture, the possibility of a physiological role needs to be further explored (Pinto et al., 2020). Diverse cargo, including small organelles of the endosomal/lysosomal system and mitochondria, calcium, MHC class I proteins, miRNAs, mRNAs, prions, viral and bacterial pathogens, have been found to traverse open-ended TNT connections among a variety of cells (Haimovich et al., 2017; Eugenin et al., 2009; Gerdes, 2009; Gerdes et al., 2007; Gerdes and Carvalho, 2008; Gurke et al., 2008; Wang et al., 2010; Kimura et al., 2012; Kolba et al., 2019). TNTs are usually 50–1000 nm in width; however, thicker tubules of 1–2 μm in width, called tumor microtubes, have been reported in cultures of cancer cells (Roehlecke and Schmidt, 2020). Tumor microtubes may play roles in the tumor microenvironment, especially glioblastoma (Osswald et al., 2015; Venkataramani et al., 2022). This emerging form of intercellular tubular connections may enhance our understanding of cellular community in multicellular organisms and in disease progression (Yamashita et al., 2018). As with exosomes, open-ended tubular connections between cells must involve a membrane fusogen to promote intercellular traffic of proteins, RNA, and organelles.

Cell-cell fusion serves vital roles in virtually all organisms from yeast to mammals and plants (Chen and Olson, 2005), Mammals have a limited number of known catalysts of cell fusion that serve specialized cells and tissues. Two examples are myomaker and myomerger for myoblast fusion, and the syncytins for trophoblast cell fusion (reviewed in Brukman et al., 2019). Syncytins are endogenous retroviral envelope glycoproteins: syncytin-1 and syncytin-2 in humans (Blaise et al., 2003; Frendo et al., 2003; Mi et al., 2000) and syncytin-A and -B in mice (Dupressoir et al., 2005; Peng et al., 2007). Syncytin-mediated fusion requires complementary cell surface receptors, including ASCT-2, a widely distributed neutral amino acid transporter, a receptor for syncytin-1 (Blond et al., 2000) and Major Facilitator Superfamily Domain Containing-2 (MFSD2), a sodium-dependent lysophosphatidylcholine transporter, the receptor for syncytin-2 (Esnault et al., 2008). In the mouse, lymphocyte antigen 6E (Ly6e) has been identified as a receptor for syncytin-A (Bacquin et al., 2017). In addition to a role in the formation of a trophoblast syncytium during early embryonic development, syncytins are also involved in the fusion of osteoclast precursors and cancer cells (Bjerregaard et al., 2006; Uygur et al., 2019a).

Cytoplasmic structure-forming proteins are required to organize the plasma membrane as cells adhere in preparation for fusion. For example, invasive protrusions that promote the cell membrane juxtaposition and fusogen engagement are propelled by Arp2/3-mediated branched actin polymerization (Sens et al., 2010; Shilagardi et al., 2013). In this process, dynamin, a large GTPase best known for its role in endocytosis, bundles actin filaments to promote invasive protrusion formation (Zhang et al., 2020).

Here, we report the use of quantitative assays to measure traffic of protein and RNA cargo between cells. A Cas9/gRNA cargo enriched in exosomes was found to be inefficiently delivered for gene editing in a reporter cells. Similar results have been reported elsewhere (de Jong et al., 2020; Haimovich et al., 2017; Somiya and Kuroda, 2021; Albanese et al., 2021). In contrast, we found highly efficient transfer when cells were co-cultured with physical contact. Using live-cell imaging and correlative light and electron microscopy (CLEM), we found that traffic appeared to be mediated by open-ended membrane tubular connections of several microns in diameter. Membrane fusion depended on syncytin in the acceptor cell and a complementary receptor in the donor cell. Optimum transfer was seen with HEK293T cells as a donor and MDA-MB-231 cells and several other tumor cell lines as acceptor. In contrast, HEK293T cells serving as both a donor and an acceptor were not active in intercellular traffic but instead appeared to form close-ended tubular connections.

In this study, we employed two reporter approaches designed to detect and quantify intercellular transfer of cargo proteins mediated by exosomes and cell contact-dependent connections. A Cas9-based dual-luciferase system and a trifluorescence split-GFP assay were used to measure Cas9-mediated genome editing and the formation of active GFP from fragments shared between cells in contact. We found that exosomes enriched in Cas9 were internalized by acceptor cells but largely failed to release Cas9 for editing purposes. The same was true for exosomes enriched in a fragment of GFP that failed to form active GFP in acceptor cells. In contrast, quite efficient transfer was seen in co-cultures of cells grown to near confluence. Transfer among cells in co-culture was dependent on actin but was not blocked under conditions that arrested endocytosis. Imaging experiments revealed occasional intercellular tubular connections of a range of diameter that in certain donor-acceptor cell pairs appeared to be open-ended to allow cytoplasmic continuity (Figures 4 and 9G). As a donor cell line, HEK293T was quite efficient in cargo transfer and formed open-ended tubular connections to MDA-MB-231 acceptor cells. In contrast, the tubular connections between HEK293T cells were not open-ended and transferred little if any cargo. The transfer process appeared to consist of at least three stages: an initial tubular protrusion, possibly driven by F-actin, emanating from a donor or acceptor cell; contact by the tubule with an opposing cell surface; and membrane fusion in favorable circumstances, particularly when the acceptor cell was MDA-MB-231 or one of several other tumor cell lines. Tubules formed and made contact with opposing cells but did not fuse unless the endogenous membrane fusogen, syncytin (particularly syncytin-2), was expressed on the acceptor cell. The syncytin-2 receptor protein, MFSD2A, appeared to be required to promote fusion for the intercellular transfer.

Many reports have described the intercellular traffic of proteins, RNA, and organelles such as mitochondria and lysosomes mediated by tubular connections; however, the physiological function and molecular mechanism of this pathway have not been reported (Pinto et al., 2020; Roehlecke and Schmidt, 2020; Yamashita et al., 2018; Dagar et al., 2021). In contrast, exosomes are widely believed to mediate intercellular traffic of proteins and RNA, yet no reports have probed the mechanism of the membrane fusion event that must accompany the discharge of exosome content to the cytoplasm or nucleus of targeted cells.

We considered other pathways of traffic mediated by cell-cell contact that could explain our results. Trogocytosis is a means by which one cell nibbles another cell (Joly and Hudrisier, 2003). In this process, lymphocytes (B, T, and NK cells) extract surface molecules from antigen-presenting cells and express them on their own surface (Joly and Hudrisier, 2003; Gutiérrez-Vázquez et al., 2013). Previous studies reported that trogocytosis requires PI3K activation, which is blocked by such inhibitors as wortmannin (Lis et al., 2010; Martínez-Martín et al., 2011). We found that the PI3K inhibitors wortmannin and LY294002 did not block the transfer of Cas9 (Figure 3A). Alternatively, some cells in contact form an ‘immunological synapse’ that could promote the local traffic of exosomes possibly leading to selected delivery of cargo proteins and RNA (Dustin, 2014; Grakoui et al., 1999). However, we found intercellular traffic relatively unaffected by inhibitors of endocytosis, which would be required for the uptake of vesicles produced and consumed at an immunologic-like synapse.

A variety of other cell-cell adhesions, some of which involve tubular connections, have been reported. Many such connections remained close-ended such as in gap junctions, synaptic junctions, and cytonemes (Yamashita et al., 2018; Kornberg and Roy, 2014; Ramírez-Weber and Kornberg, 1999). We found large lateral surfaces form and remain stable at the junction of tubules and the cell surface under conditions where membrane fusion does not occur. These adhesions may require molecules employed for other stable cell-cell junctions, and open-ended membrane tubes such as TNTs and tumor microtubes (Roehlecke and Schmidt, 2020; Yamashita et al., 2018). The role of such stable junctions as a prelude to membrane fusion remains to be considered.

Fusion has been seen at other tubular junctions, particularly for the formation of open-ended contact with thin tubules called TNTs. TNTs range in diameter from 50 to 1000 nm and in length from a few to 100 μm (Roehlecke and Schmidt, 2020). Previous studies have reported the transfer of proteins, mRNA, and organelles such as mitochondria and lysosomes mediated by TNTs (Pinto et al., 2020). Tumor microtubes produced by cancer cells generate tubular connections of 1–2 μm in diameter, perhaps to allow the passage of larger organelles (Roehlecke and Schmidt, 2020; Osswald et al., 2015; Latario et al., 2020). Such membrane tubes were detected in tumors (Osswald et al., 2015; Lou et al., 2012), possibly serving a role in tumor maintenance and progression. MDA-MB-231 cells transferred cytoplasmic cargo to other cancer cell lines, including U2OS, A549, and MCF5, as well as to other MDA-MB-231 cells (Figure 2G). The tubular connections we found range from 2 to 4 μm in diameter (Figures 4—6) and were not confined to tumor cells but included our standard donor cell line, HEK293T (Figures 4—6 and Figure 8). We observed membrane tubules projecting from HEK293T cells connecting to MDA-MB-231 cells (Figures 4 and 5) and vice versa (Figure 8). Such structures from nontransformed cells could indicate a more normal role in cell and tissue physiology and at the border of a tumor as in the hijacking of mitochondria from immune cells (Saha et al., 2022).

At present, it is not possible to offer distinct roles for TNTs and thick tubules in the transfer of cytoplasmic cargo between cells. Our observations are consistent with either or both such structures engaged in the transfer of Cas9 and our other reporter proteins. Aside from the example of cancer cells and their interface in tumors, the role of TNTs and larger tubular connections in normal physiological functions remains to be explored.

Among the possible catalysts of open-ended connections, we considered the role of the one known mammalian viral-like fusogen, syncytin (syncytin-1 and syncytin-2). Syncytins are mammalian endogenous fusogens that facilitate trophoblast cell fusion in the early embryo (Blaise et al., 2003; Mi et al., 2000; Grandi and Tramontano, 2018). In this study, we used immunoblot to detect syncytins corresponding in SDS-PAGE mobility to precursor and/or furin-processed mature proteins expressed in several cell lines (Figure 7—figure supplement 1). Using CRISPRi, we found that knockdown of syncytins in MDA-MB-231 cells, but not in HEK293T cells, blocked transmission of Cas9 and the formation of open-ended tubular connections (Figures 7 and 8). Previous studies reported a number of other proteins involved in TNT formation, including M-Sec and the exocyst complex (Hase et al., 2009; Kimura et al., 2016), LST1 and RalA (Schiller et al., 2013), ERp29 (Pergu et al., 2019), S100A4 and RAGE (Sun et al., 2012), Myosin10 (Gousset et al., 2013), Rab8-Rab11-Rab35 (Bhat et al., 2020; Zhu et al., 2018), MICAL2PV (Wang et al., 2021), IRSp53 (Prévost et al., 2015; Delage et al., 2016), and others (Dagar et al., 2021). However, it is not clear in any of these cases if the protein is involved in tube formation, adhesion, or fusion. Nonetheless, it is likely that proteins, in addition to syncytin, such as the receptor proteins MFSD2A (for syncytin-2), ASCT2 (for syncytin-1), and Ly6e (for mouse syncytin-A), are involved specifically in fusion. The failure of endogenous syncytins to support fusion in HEK293T cells serving as an acceptor target could relate to unexplored aspects of intracellular traffic or regulation of fusogen activity. Although we found some increase in Cas9 traffic to HEK293T cells ectopically expressing mouse syncytin-A (Figure 7E), full fusion activity may depend on unknown variables.

Inefficient sorting of functional syncytins or their receptors may explain the failure of exosomes isolated from HEK293T and MDA-MB-231 cells to deliver Cas9 for editing in the other target cell (Figure 1). Indeed, the requirement for an active fusogen incorporated into exosomes may explain other examples of failed delivery (de Jong et al., 2020; Haimovich et al., 2017; Somiya and Kuroda, 2021; Albanese et al., 2021). The question remains to explain the many observations that suggest functional delivery by exosomes. A closer look by quantitative measures may show that apparent success in exosome-mediated transfer is quite inefficient. A contrasting example may be in the activity of trophoblasts that may express active syncytins and their receptors at the cell surface and may likewise traffic extracellular vesicles within the developing placenta (Tolosa et al., 2012; Uygur et al., 2019b; Vargas et al., 2014). Lessons learned from trophoblasts may inform the rational design of functional exosomes useful in the delivery of heterologous protein, such as Cas9 protein, between cells.

All data generated or analysed during this study are included in the manuscript and supporting file. Source data files have been provided for Figure 1B; Figure 1- figure supplement 1A and 1B; Figure 3- figure supplement 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, and 2J; Figure 7- figure supplement 1A, 1B, 1D, 1F, 1G, and 1H; Figure 8- figure supplement 1D; Figure 9B and 9D.

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Decision letter after peer review:

Thank you for submitting your article "Syncytin-mediated open-ended membrane tubular connections facilitate the intercellular transfer of cargos including Cas9 protein" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editor, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Gal Haimovich (Reviewer #2).

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

Essential revisions:

One reviewer is more extensive in the comments which need to be addressed as best as is possible. Most of these are clarifications but the reviewer indicates that more convincing evidence is needed.

1) Clarify text to address questions raised by reviewer 2.

2) An experiment suggested by reviewers 2 and 3 is to use immunofluorescence to localize the presence of the fusion determinants on the membrane. Perhaps this will clarify the differences in receptivity in the cell line. There is some confusion here as to the level of the protein and the corresponding ability to form open connections.

3) A clarification of the differences between the role of these observed membrane tubules and the TNTs reported in other works in transfer of molecules (eg mRNA).

Is it just a thickness difference?

Reviewer #1 (Recommendations for the authors):

It would be more generalizable to living systems to look at primary cells rather than cell lines.

Reviewer #2 (Recommendations for the authors):

1. Related to public comment 1: If str-CD63 is absent from the transiently transfected cells, then an additional comparison should be to transiently transfect SBP-Cas9-GFP to cells expressing myc-str-CD63 and the Nluc reporter, in the presence or absence of biotin. This will show whether the str-SBP interaction is inhibiting Cas9 nuclear import and if biotin actually releases SBP-Cas9-GFP for nuclear import (as an additional control to the co-IP in Figure 1 – supp figure 1A).

2. Related to public comment 2:

c. Assuming Syncytin-2 is not required for TNTs transfer, a simple experiment to distinguish TNT and membrane tubules will be to track TNT formation, or transfer of known TNTs cargos (e.g. vesicles, mitochondria, mRNA) in WT vs Syncytin K/O.

d. To rule out the possibility that the observed phenotype is due to mRNA transfer through TNTs, the authors could transfect the acceptor cells with siRNA against the SBP-Cas9-GFP mRNA. The phenotype should not change if mRNA transfer does not play a role here.

e. At the very least, the authors should discuss these possibilities and why they think TNTs do not play a role in the Cas9/split-GFP transfer since both the protein and mRNA can transfer through TNTs, yielding the observed results.

3. Related to public comment #3: Split GFP assay

a. A FACS analysis of mixed (not co-cultured) cells similar to the one presented in Figure 2C, F should be shown.

b. Knocking-down Syncytin for the split-GFP assay will strengthen the claim that both assays measure the same process.

4. Related to public comment #4c: The authors should show live imaging of single-culture acceptor cells to see the background in the mCherry and GFP channel.

5. Related to public comment #5: I think a relatively simple experiment to examine the function of Syncytin in HEK293T will be to determine the syncytin localization in MDA vs HEK cells (e.g. at cell surface, contact sites, membrane tubules). I think it can greatly improve the authors' claim on the role of Syncytin and resolve the HEK problem.

6. The authors show that transferred Cas9, as detected by immunofluorescence (Figure 1H), is maintained in vesicular formations in the acceptor cells – probably endosomes or lysosomes as previously demonstrated for EV uptake (e.g. PMID: 27114500), but the authors do not provide more details on these structures. Is the IF signal for CD63 represents the endogenous CD63 in acceptor cells or myc-str-CD63 coming from the donor cells? The figure legend reads "anti-CD63" antibody suggesting endogenous, but no such antibody appears in the list in Materials and methods section, so maybe the authors meant anti-myc? Furthermore, co-IF with antibodies against early/late endosome/lysosome markers could clarify at which stage Cas9 gets "stuck".

7. The authors perform the co-culture experiments on a time scale of days. From my experience with TNT-mediated mRNA transfer, this is most likely because the initial culture was at 10% confluence – with low chance of cell-cell contact. Have the authors tried looking at transfer in a 1:1 or 10:1 culture plated already at 60-70% confluence? This could yield more interactions that could be detected and studied.

8. Latrunculin – my experience suggests it is very toxic to cells (I used 100-200nM) and typically the cells die within several hours. Yet here the authors treated the cells for 3-6 days. Furthermore, such a treatment will likely affect cell motility and proliferation – thus limiting not only formation of tubules but also decrease the probability for cell-contacts. The authors should state if they checked for cell viability and if cell proliferation was affected – thus reducing confluence. Experiments with high-confluence at initiation and therefor reduced co-culture duration (item #2 above) could help with the toxicity by reducing time of exposure to the drug.

9. The authors spend two paragraphs in the discussion to suggest a role in cancer (transformed) cells vs non-transformed cells. However, the only example of non-transformed cells they have are the HEK293T cells – and the lack of transfer can be a unique issue of this particular cell line. I suggest to limit the discussion of this subject or provide data from at least one more non-transformed cell line (which also expresses Syncytin).

10. The section on the split GFP is very confusing and hard to follow – for example I needed to read several times until I understood what "Q2-4/Q4-4+Q2-4". And what are the black-labeled cell populations and how are they different from the green or purple populations in the dot-plots? It will also be helpful to label the axes with the relevant fluorescent protein measured, rather the filter name. I suggest using simpler labeling and more explanations for better clarity.

11. The authors suggest in their model that Syncytin and MFSD2A on opposite cells "meet" at the tip of membrane tubules. I believe imaging of Syncytin and MFSD2A proteins will help support this model and may resolve the inconsistent result with HEK293T cells (see public comment #5).

Reviewer #3 (Recommendations for the authors):

1. A formin inhibitor could be used to test their potential involvement.

2. Statistical analyses may be used to assess the occurrence of cargo transfer along a broad cell-cell contact zone.

3. Antibody staining of syncytins and/or MFSD2A could be performed (if antibodies are available) to detect the presence of these proteins on the cell surface in difference conditions.

Essential revisions:

1) Clarify text to address questions raised by reviewer 2.

Response to each question as below.

2) An experiment suggested by reviewers 2 and 3 is to use immunofluorescence to localize the presence of the fusion determinants on the membrane. Perhaps this will clarify the differences in receptivity in the cell line. There is some confusion here as to the level of the protein and the corresponding ability to form open connections.

Since the commercial antibodies for syncytins are not good for immunofluorescence, we relied on cells transfected with fluorescent protein-tagged syncytins. The results show that syncytins partially localized to the cell surface (Figure 8—figure supplement 1A-C), including at the junction between a tubule and the target cell surface (Figure 8—figure supplement 1E, F). We found that overexpressed syncytin-1 and -2 induced cell fusion in HEK293T cells, consistent with the cell surface localization of at least a fraction of these proteins. Nonetheless, the basal level of syncytins at the surface of HEK293 cells may be inadequate to promote open-ended tubular connections in cultures of this cell line. Answering this question will require more sensitive probes to detect the endogenous syncytins.

As an independent test of cell surface localization, we used surface biotinylation to show that a fraction of the syncytins can be labeled externally (Figure 8—figure supplement 1D). This fraction shows evidence of proteolytic processing consistent with furin cleavage whereas the overwhelming majority of transfected syncytins detected in a blot of lysates suggests that most remains in the unprocessed precursor form, consistent with the punctate and reticular fluorescence images (Figure 8—figure supplement 1A-C).

3) A clarification of the differences between the role of these observed membrane tubules and the TNTs reported in other works in transfer of molecules (eg mRNA).

Is it just a thickness difference?

The results in this study cannot distinguish between roles for TNTs or thick membrane tubules. We are not yet aware of distinct requirements for the formation of these two tubular connections. It may be that TNTs contain actin only, whereas thick tubules contain both actin and microtubules (ref: PMID: 17142745), which may result in the transfer of different cargos, for example, the transfer of microtubule-mediated cargos through thick tubular connections. Such functional distinctions bear further exploration thus we have been careful avoid implying a role for one or the other, except insofar as our visual detection has focused on the more obvious role of thick tubules in intercellular transfer.

Reviewer #2 (Recommendations for the authors):

1. Related to public comment 1: If str-CD63 is absent from the transiently transfected cells, then an additional comparison should be to transiently transfect SBP-Cas9-GFP to cells expressing myc-str-CD63 and the Nluc reporter, in the presence or absence of biotin. This will show whether the str-SBP interaction is inhibiting Cas9 nuclear import and if biotin actually releases SBP-Cas9-GFP for nuclear import (as an additional control to the co-IP in Figure 1 – supp figure 1A).

We added new data in Figure 1—figure supplement 1D which suggests that CD63-tethering itself does not affect Cas9 function.

2. Related to public comment 2:

c. Assuming Syncytin-2 is not required for TNTs transfer, a simple experiment to distinguish TNT and membrane tubules will be to track TNT formation, or transfer of known TNTs cargos (e.g. vesicles, mitochondria, mRNA) in WT vs Syncytin K/O.

d. To rule out the possibility that the observed phenotype is due to mRNA transfer through TNTs, the authors could transfect the acceptor cells with siRNA against the SBP-Cas9-GFP mRNA. The phenotype should not change if mRNA transfer does not play a role here.

e. At the very least, the authors should discuss these possibilities and why they think TNTs do not play a role in the Cas9/split-GFP transfer since both the protein and mRNA can transfer through TNTs, yielding the observed results.

The results in this study do not rule out a role for TNTs in intercellular transfer nor do we suggest that syncytin-2 is not required for TNT-mediated transfer. This point is clarified in the discussion.

3. Related to public comment #3: Split GFP assay

a. A FACS analysis of mixed (not co-cultured) cells similar to the one presented in Figure 2C, F should be shown.

b. Knocking-down Syncytin for the split-GFP assay will strengthen the claim that both assays measure the same process.

A FACS analysis of mixed (not co-cultured) cells was included. The results in this study do not rule out a role for TNTs in the transfer of both Cas9 and split-GFP.

4. Related to public comment #4c: The authors should show live imaging of single-culture acceptor cells to see the background in the mCherry and GFP channel.

Images of single-culture acceptor cells have been added in Figure 4—figure supplement 1.

5. Related to public comment #5: I think a relatively simple experiment to examine the function of Syncytin in HEK293T will be to determine the syncytin localization in MDA vs HEK cells (e.g. at cell surface, contact sites, membrane tubules). I think it can greatly improve the authors' claim on the role of Syncytin and resolve the HEK problem.

We have performed new visualization and cell surface labeling experiments to show that syncytins appear to be partially localized at the cell surface of MDA-MB-231 cells, and even at sites of cell-cell contact (Figure 8—figure supplement 1). We also find that overexpressed syncytin-2 localizes to the plasma membrane of HEK293T cells to induce cell fusion. As stated above, the problem remains to explain why basal levels of expression of syncytins are inadequate to promote the formation of open-ended tubular connections.

6. The authors show that transferred Cas9, as detected by immunofluorescence (Figure 1H), is maintained in vesicular formations in the acceptor cells – probably endosomes or lysosomes as previously demonstrated for EV uptake (e.g. PMID: 27114500), but the authors do not provide more details on these structures. Is the IF signal for CD63 represents the endogenous CD63 in acceptor cells or myc-str-CD63 coming from the donor cells? The figure legend reads "anti-CD63" antibody suggesting endogenous, but no such antibody appears in the list in Materials and methods section, so maybe the authors meant anti-myc? Furthermore, co-IF with antibodies against early/late endosome/lysosome markers could clarify at which stage Cas9 gets "stuck".

Here we used anti-CD63 and have added that in the list in Materials and methods section. We observed endogenous CD63 in the PBS control images, suggesting that Cas9 is at least partially localized in endosome related vesicles.

7. The authors perform the co-culture experiments on a time scale of days. From my experience with TNT-mediated mRNA transfer, this is most likely because the initial culture was at 10% confluence – with low chance of cell-cell contact. Have the authors tried looking at transfer in a 1:1 or 10:1 culture plated already at 60-70% confluence? This could yield more interactions that could be detected and studied.

We tried 10:1 culture plated at 60-70% confluence, and cultured for short times but found less transfer (1d vs 3d in Figure 2—figure supplement 1A).

8. Latrunculin – my experience suggests it is very toxic to cells (I used 100-200nM) and typically the cells die within several hours. Yet here the authors treated the cells for 3-6 days. Furthermore, such a treatment will likely affect cell motility and proliferation – thus limiting not only formation of tubules but also decrease the probability for cell-contacts. The authors should state if they checked for cell viability and if cell proliferation was affected – thus reducing confluence. Experiments with high-confluence at initiation and therefor reduced co-culture duration (item #2 above) could help with the toxicity by reducing time of exposure to the drug.

For latrunculin treatment, we used latrunculin A at 40, 80, 200 nM and latrunculin B at 1, 2.5, 5 µM. We checked the cell viability, and found that high concentrations of latrunculin (such as latrunculin A at 200 nM, and latrunculin B at 5 µM) affected cell growth. Thus for the high concentrations, at initiation we used a higher cell density (a little more than DMSO) and cultured for longer times until near confluence to diminish the toxic effect. The Nluc/Fluc signal detected after co-culture suggested that more than half of the cells remained viable during drug treatment.

9. The authors spend two paragraphs in the discussion to suggest a role in cancer (transformed) cells vs non-transformed cells. However, the only example of non-transformed cells they have are the HEK293T cells – and the lack of transfer can be a unique issue of this particular cell line. I suggest to limit the discussion of this subject or provide data from at least one more non-transformed cell line (which also expresses Syncytin).

We reduced this part of the Discussion to one paragraph.

10. The section on the split GFP is very confusing and hard to follow – for example I needed to read several times until I understood what "Q2-4/Q4-4+Q2-4". And what are the black-labeled cell populations and how are they different from the green or purple populations in the dot-plots? It will also be helpful to label the axes with the relevant fluorescent protein measured, rather the filter name. I suggest using simpler labeling and more explanations for better clarity.

This is a valuable suggestion to help for clarity. We now label the axes with the relevant fluorescent protein measured. The black-labeled cell populations should be the doublet cells, the plot in the Figure 2C and F were displayed with an all-cell mode.

11. The authors suggest in their model that Syncytin and MFSD2A on opposite cells "meet" at the tip of membrane tubules. I believe imaging of Syncytin and MFSD2A proteins will help support this model and may resolve the inconsistent result with HEK293T cells (see public comment #5).

Imaging data for syncytin and MFSD2A proteins are now included. We find that syncytins partially localize at cell surface of MDA-MB-231 cells, including at cell-cell contact sites (Figure 8—figure supplement 1). MFSD2A partially is localized to the plasma membrane of HEK293T cells.

Author details

1. Congyan Zhang

   Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, United States

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0737-3876

2. Randy Schekman

   Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   schekman@berkeley.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-8615-6409

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