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# Tunneling nanotubes enable intercellular transfer in zebrafish embryos

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, Filippo

Del Bene, Chiara Zurzolo

► To cite this version:

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, et al. Tunneling nanotubes enable intercellular transfer in zebrafish embryos. 2024. pasteur-04626630

HAL Id: pasteur-04626630

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Preprint submitted on 27 Jun 2024

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# Tunneling nanotubes enable intercellular transfer in zebrafish embryos

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, Filippo

Del Bene, Chiara Zurzolo

► To cite this version:

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, et al. Tunneling nanotubes enable intercellular transfer in zebrafish embryos. Developmental Cell, In press, 60, 10.1016/j.devcel.2024.10.016. pasteur-04626630v2

HAL Id: pasteur-04626630

https://pasteur.hal.science/pasteur-04626630v2

Submitted on 9 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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Distributed under a Creative Commons Attribution 4.0 International License
# Title: Tunneling nanotubes enable intercellular transfer in zebrafish embryos

**Authors:** Olga Korenkova¹,²,⁴, Shiyu Liu¹,²,⁴, Inès Prlesi¹, Anna Pepe¹, Shahad Albadri³, Filippo Del Bene³ and Chiara Zurzolo*¹.

## Affiliations:

¹ Institut Pasteur, Université Paris Cité, CNRS UMR 3691, Membrane Traffic and Pathogenesis, F-75015 Paris, France.

² Université Paris-Saclay, 91190 Gif-sur-Yvette, France

³ Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 Paris, France

## Author list footnotes:

⁴ these authors contributed equally

* lead contact

correspondence: chiara.zurzolo@pasteur.fr

## Summary

Tunneling nanotubes (TNTs) are thin intercellular connections facilitating the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited similar morphology to TNTs described in cell culture, appeared to have similar formation mechanisms and could be induced by Eps8 overexpression and CK666 treatment. Most notably, to classify them as TNTs, we demonstrated their capability to transfer both soluble cargoes and organelles, which is a defining feature of open-ended TNTs. This study marks the first demonstration of functional TNTs in a living embryo.

## Keywords

Intercellular communication, tunneling nanotubes, TNTs, TNT-like structures, zebrafish embryo, zebrafish gastrula
**Developmental Cell**

# Tunneling nanotubes enable intercellular transfer in zebrafish embryos

**Graphical abstract**

<figure><img src="image_2.png" /></figure>

**Authors**

Olga Korenkova, Shiyu Liu, Inès Prlesi, Anna Pepe, Shahad Albadri, Filippo Del Bene, Chiara Zurzolo

**Correspondence**

chiara.zurzolo@pasteur.fr

**In brief**

Korenkova, Liu, et al. demonstrate the presence of open-ended connections, known as tunneling nanotubes (TNTs), in live zebrafish embryos by showing that they facilitate material transfer between cells. Their findings suggest a functional role for TNTs in living organisms, broadening our understanding of intercellular communication during development.

**Highlights**

* TNT-like structures coexist with other intercellular connections in zebrafish embryos

* Mechanisms of TNT formation are similar in zebrafish embryos and *in vitro*

* Eps8 overexpression in embryos promotes TNTs and causes developmental defects

* TNTs in embryos are open ended and transfer cytosolic proteins and organelles
Introduction

Intercellular communication is a key feature of multicellular organisms. Along with soluble
factors, cellular protrusions play an essential role in communication. Filopodia probe the
environment, participate in cell-cell adhesion and chemoattractant sensing ¹. Cytonemes
are thin and long actin-based signaling protrusions that were first described in Drosophila
wing imaginal disc ², but were further shown to participate in signal transduction in other
models, including tumors ³ and zebrafish embryo development ⁴. In addition to these
mechanisms, more recently discovered structures – Tunneling nanotubes (TNTs),
represent another type of contact-dependent communication.

TNTs are thin (below 1 µm) and long (up to 100 µm) actin-based membranous
connections between cells, that allow for membrane and cytoplasmic continuity. Due to
this feature, TNTs are able to transfer various material between connected cells: ions,
mRNA, proteins, organelles, and, importantly, pathogenic agents, including misfolded
proteins, viruses and bacteria 5,6. TNTs are suggested to participate in progression of
cancer, Parkinson's and Alzheimer's diseases, in the spread of HIV and Sars-CoV2 7-11.
However, the majority of TNT studies are being performed in 2D cell culture, and there is
still a limited number of studies that were performed in more complex 3D environment.
Several studies have shown that TNT-like structures are present in resected tumors,
tumor organoids, and developing embryos 12-14.

The challenge in identifying tunneling nanotubes (TNTs) within 3D models arises due to
the absence of a specific TNT marker. Additionally, the prominent characteristic employed
for TNT identification in vitro, namely the suspension of TNTs above the substrate, is not
present within a 3D environment. In complex experimental settings, TNTs could be
confused with other actin-based protrusions, including filopodia, cytonemes or cytokinetic
bridges. The only reliable criteria to classify a structure as a TNT that is currently available,
is to show that they are able to transfer cellular material. Consequently, structures
observed in 3D models are often referred to as "TNT-like", because their transfer
functionality was not confirmed.

Another cellular structure that is able to transfer cellular materials is the intercellular
bridge (IC). Differently from TNTs, intercellular bridges are cytoplasmic bridges that are
formed by incomplete cytokinesis, keeping the cells interconnected after cell division. ICs
are typically found in germline cells, where they enable communication via transfer of
macromolecules, nutrients and organelles necessary for their further development ¹⁵.
Interestingly, Caneparo and colleagues showed long and thin intercellular connections
connecting cells in the zebrafish gastrula that were able to transfer cytoplasmic material
¹⁶. The formation of these structures was associated with cell division and the authors
suggested that they represent ICs that were formed by dividing cells during blastula stage
and remained stable while cells were migrating apart in the gastrula.

Here we aimed to assess whether TNT-like structures (i.e., communication connections
that do not arise from cell division) exist in a living embryo. We chose the early zebrafish
embryo model for its optical transparency and rapid development that allows to follow in
<figure><img src="image_3.png" /></figure>

Short article

# Tunneling nanotubes enable intercellular transfer in zebrafish embryos

Olga Korenkova,<fnref n="1" />,<fnref n="2" />,<fnref n="4" /> Shiyu Liu,<fnref n="1" />,<fnref n="2" />,<fnref n="4" /> Inès Prlesi,<fnref n="1" /> Anna Pepe,<fnref n="1" /> Shahad Albadri,<fnref n="3" /> Filippo Del Bene,<fnref n="3" /> and Chiara Zurzolo<fnref n="1" />,<fnref n="5" />,*

## SUMMARY

Tunneling nanotubes (TNTs) are thin intercellular connections that facilitate the transport of diverse cargoes, ranging from ions to organelles. While TNT studies have predominantly been conducted in cell cultures, the existence of open-ended TNTs within live organisms remains unverified. Despite the observation of intercellular connections during embryonic development across various species, their functional role in facilitating material transfer between connected cells has not been confirmed. In this study, we performed mosaic labeling of gastrula cells in zebrafish embryos to demonstrate the coexistence of TNT-like structures alongside other cellular protrusions. These embryonic TNT-like connections exhibited a morphology similar to that of TNTs described in cell culture, appeared to have similar formation mechanisms, and could be induced by Eps8 overexpression and CK666 treatment. Most notably, we demonstrated their capability to transfer both soluble cargoes and organelles, thus confirming their open-endedness. This study demonstrates the existence of functional, open-ended TNTs in a living embryo.

## INTRODUCTION

Intercellular communication is a key feature of multicellular organisms. Along with soluble factors, cellular protrusions play an essential role in communication. Filopodia probe the environment and participate in cell-cell adhesion and chemoattractant sensing.<fnref n="1" /> Cytonemes are thin and long actin-based signaling protrusions that were first described in the Drosophila wing imaginal disc,<fnref n="2" /> but have since been shown to participate in signal transduction in other models, including tumors<fnref n="3" /> and zebrafish embryo development.<fnref n="4" /> In addition to these mechanisms, more recently discovered structures called tunneling nanotubes (TNTs) represent another type of contact-dependent communication.

TNTs are thin (below 1 µm) and long (up to 100 µm) actin-based membranous connections between cells that allow for membrane and cytoplasmic continuity. This feature enables TNTs to transfer various materials between connected cells, including ions, mRNA, proteins, organelles, and, importantly, pathogenic agents such as misfolded proteins, viruses, and bacteria.<fnref n="5" />,⁶ TNTs are suggested to participate in the progression of cancer, Parkinson's disease, Alzheimer's disease, and the spread of HIV and SARS-CoV-2.⁷⁻¹¹ However, most TNT studies have been performed in 2D cell culture, and there is still a limited number of studies conducted in more complex 3D environment. Nonetheless, several studies have shown that “TNT-like” structures are present in resected tumors, tumor organoids, and developing embryos.¹²⁻¹⁴

The challenge in identifying bona fide TNTs within 3D models arises from the absence of a specific TNT marker. In complex experimental settings, TNTs could be confused with other actin-based protrusions, including filopodia, cytonemes, or cytokinetic bridges. The only reliable criterion currently available to classify a structure as a TNT is demonstrating its ability to transfer cellular material. Consequently, structures observed in 3D models are often referred to as “TNT-like”, because their transfer functionality was not confirmed.

Another cellular structure capable of transferring materials is the intercellular bridge (IB). Unlike TNTs, IBs are cytoplasmic connections formed by incomplete cytokinesis, maintaining a link between cells after division. These structures are typically found in germline cells, facilitating the exchange of macromolecules, nutrients, and organelles necessary for their development.¹⁵ Caneparo and colleagues observed long, thin intercellular connections in live zebrafish gastrula, which transferred cytoplasmic material.¹⁶ The formation of these connections was associated with cell division, and the authors suggested they represent IBs formed during the blastula stage, remaining stable as cells migrated apart in the gastrula.

In this study, we aimed to assess whether functional TNTs (i.e., communication connections that do not arise from cell division) exist in a living embryo. We chose the early zebrafish embryo model for its optical transparency and rapid development that allows us to follow *in vivo* processes in the living embryo in a non-invasive manner. Using a sparse cell labeling approach, we were

<footnote n="1">¹Institut Pasteur, Université Paris Cité, CNRS UMR 3691, Membrane Traffic and Pathogenesis, 75015 Paris, France</footnote>

<footnote n="2">²Université Paris-Saclay, 91190 Gif-sur-Yvette, France</footnote>

<footnote n="3">³Sorbonne Université, INSERM, CNRS, Institut de la Vision, 75012 Paris, France</footnote>

<footnote n="4">⁴These authors contributed equally</footnote>

<footnote n="5">⁵Lead contact</footnote>

<footnote>*Correspondence: chiara.zurzolo@pasteur.fr</footnote>

<footnote>https://doi.org/10.1016/j.devcel.2024.10.016</footnote>
vivo processes in the living embryo in a non-invasive manner. Using a sparse cell labelling approach, we were able to discriminate and classify by morphology multiple types of protrusions formed by gastrula cells in the whole embryo, including functional TNT-like structures. We demonstrate that the latter not only share characteristics with TNTs observed in cell culture, but also have similar formation mechanisms, namely that they can be induced by overexpression of Eps8 (epidermal growth factor receptor kinase substrate 8) and by treatment with CK666. Most importantly, our results demonstrate that these connections have the ability to transfer intracellular material, such as cytoplasmic proteins and organelles. This study presents the first evidence of functional TNTs in a developing embryo in a whole organism context.

## Results

### Different types of protrusions co-exist in zebrafish gastrula

To visualize intercellular connections, zebrafish embryos were micro-injected with mRNA into one cell at the 16-cell stage, which resulted in mosaic expression of the construct at the gastrula stage (8 hpf) (Fig. 1A). Upon injection of membrane-labeling mRNA, protrusions with various morphologies could be observed within the same field of view (Fig. 1B). These included shorter, non-connecting protrusions, (arrow 1), longer protrusions, with a close-ended tip residing on the neighboring cell (arrow 2), connections with an interruption of membrane labelling often close to the middle of the connection (arrow 3), and uninterrupted connections, that appeared to link distant cells (arrows 4 and 5). The short protrusion (arrow 1) could be generally referred to as filopodia, while long closed-ended protrusions (arrow 2) were clearly distinguishable from filopodia and resembled classical cytonemes, that were previously observed at gastrula stage of zebrafish development ⁴ (Fig. 1C). On the other hand, the interruption in the connection's labelling (arrow 3) hints towards the presence of midbody, thus this third type of connections could represent cytokinetic bridges, which could potentially span longer distances than canonical (1-5 µm) due to the migration of cells during gastrula stage (Fig. 1C). Finally, the uninterrupted connections between distant cells showed substantial variability in length and thickness and presented morphological characteristics of TNTs (Fig. 1C). Therefore, we quantified the connections as TNT-like structures only if they were above 5 µm in length ¹⁷,¹⁸ and appeared uninterrupted. We showed that at the gastrula stage, TNT-like structures connect about 35% of the labelled cells (Fig. 1D). Since TNTs *in vitro* can have different cytoskeletal composition depending on the cell type ¹⁹, in order to further characterize zebrafish TNT-like connections, we injected *lifeAct-mKATE-E2A-EGFP-tubulin* mRNA that labels actin and tubulin of the same cells (Fig. 1E). Quantification showed that the majority of TNT-like connections contained both actin and tubulin, while about 21% contained only actin (Fig. 1F).

### The majority of connections do not derive from cell division

To be able to differentiate the TNT-like connections we observed from cytokinetic bridges, we micro-injected *lifeAct-mKATE-E2A-CEP55-EGFP* mRNA that labels actin and the
<figure><img src="image_4.png" /><figcaption>**Figure 1. Characterization of intercellular connections in zebrafish gastrula**</figcaption></figure>

(A) Schematic representation of the labeling strategy to visualize intercellular connections: zebrafish embryo is injected into one of the central cells at the 16-cell stage, incubated at 28°C until it reaches the gastrula stage (7–9 hpf), and mounted for imaging.

(B) Fluorescent photograph of different protrusions that can be observed at the gastrula stage following membrane labeling by *lyn-mKATE* mRNA injection, identified by yellow arrowheads: (1) short protrusions, (2) long closed-ended protrusions, (3) connections with interrupted membrane labeling, (4) thin TNT-like structures, and (5) thick TNT-like structures.

(C) Cartoon representing the image (B), suggesting the coexistence of (1) filopodia, (2) cytonemes, (3) cytokinetic bridges, (4) TNT-like structures, and (5) bundles of TNT-like structures.

(D) Representative images of CEP55-positive cytokinetic bridges and CEP55-negative TNT-like connections.

(E) Length distribution and average lengths between CEP55-positive ($n = 27$ connections, mean 5.6 µm) and CEP55-negative connections ($n = 114$ connections, mean 7.8 µm), $p = 0.0096$, statistical analysis is t test.

(F) Relative numbers of CEP55-positive connections and CEP55-negative TNT-like connections having lengths below 5 µm, from 5 to 10 µm, and above 10 µm ($n = 141$ connections).

(G) Representative images of Wnt8a-bearing cytonemes and Wnt8a-negative TNT-like connections.

(H) Length distribution and average lengths of Wnt8a-positive ($n = 18$ connections, mean 9.7 µm) and Wnt8a-negative connections ($n = 100$ connections, mean 9.4 µm), $p = 0.7228$, statistical analysis is t test.

(I) Relative numbers of Wnt8a-positive cytonemes and Wnt8a-negative TNT-like connections ($n = 118$ connections).

Scale bars are 10 µm.

See also Figure S1.

able to distinguish and classify multiple types of protrusions formed by gastrula cells within the whole embryo based on morphology, including TNT-like structures. Our results demonstrate that these connections not only share characteristics with TNTs observed in cell culture but also exhibit similar formation mechanisms. Specifically, they can be induced by overexpression of Eps8 (epidermal growth factor receptor kinase substrate 8) and by treatment with CK666, which did not induce the formation of other connections, such as cytokinetic bridges and cytonemes, observed alongside TNTs. Most importantly, we show that these connections can transfer intracellular material, such as cytosolic proteins and organelles. This study provides the evidence of the existence of functional, open-ended TNTs in a developing embryo in the whole organism context.

## RESULTS

### Different types of protrusions coexist in zebrafish gastrula

To visualize intercellular connections, zebrafish embryos were micro-injected with synthetized mRNAs of choice into one cell at the 16-cell stage, which resulted in mosaic expression of the construct at the gastrula stage (8 hours post fertilization [hpf]).

(Figure 1A). Upon injection of membrane-labeling mRNA, protrusions with various morphologies could be observed within the same field of view (Figure 1B). These included shorter, non-connecting protrusions (arrow 1), longer protrusions with a close-ended tip residing on the neighboring cell (arrow 2), connections with an interruption of membrane labeling, often near the middle (arrow 3), and uninterrupted connections that appeared to link distant cells (arrows 4 and 5). The short protrusions (arrow 1) were generally referred to as filopodia, which typically do not exceed 5 µm in length,¹,¹⁷ while long closed-ended protrusions (arrow 2) were distinguishable from filopodia and resembled classical cytonemes, previously observed at gastrula stage of zebrafish development⁴ (Figure 1C). The interruption in the connection's labeling (arrow 3) suggests the presence of a midbody, indicating that this third type of connection could represent cytokinetic bridges, which could potentially span longer distances than the canonical range (1–5 µm) due to cell migration during gastrula stage (Figure 1C). The uninterrupted connections between distant cells showed substantial variability in length and thickness and presented morphological characteristics of TNTs (Figure 1C, arrows 4 and 5). Therefore, based on these morphological criteria, we identified the connections as “TNT-like structures” only if they were above 5 µm in length¹⁸,¹⁹ and
midbody marker CEP55 in the same cells (Fig. 1G). CEP55 was previously shown to label
cytokinetic bridges in the zebrafish embryo throughout their lifespan, from the beginning
of bridge formation until the midbody remnant release <sup>20</sup>. In our timelapse recordings
CEP55 was present from cytokinetic bridge formation until abscission, while CEP55-
positive midbody remnants could also be observed (data not shown). We show that 19%
of all identified connections were CEP55-positive and hence represented cytokinetic
bridges (Fig. 1I), while the majority of connections were of a distinct nature. CEP55-
positive cytokinetic bridges were significantly shorter than CEP55-negative connections,
and their length never exceeded 10 µm (Fig. 1H), as expected for canonical cytokinetic
bridges. Of interest, a portion of CEP55-negative connections had the length above 10
µm, and they could reach up to 30 µm in length (Fig. 1H, I).

A substantial amount of CEP55-negative connections had the length below 5 µm (Fig. 1I), which could correspond to filopodia that were reported to fall in this length range <sup>17,18</sup>; thus, for further analysis of TNT-like structures we only included connections with a length above 5 µm.

Interestingly, in rare embryos we could observe unexpectedly long and stable cytokinetic
bridges (Supplementary Fig.1). These structures exceeded 40 µm in length and remained
stable for at least 30 minutes of the timelapse recording (Movie 1). While embryos
containing these structures were rarely observed (around ten embryos from hundreds of
embryos imaged in total), within these embryos several bridges could be seen in the same
field of view. This observation suggests that these particular embryos experienced
dysregulation during development, and the observed structures could possibly be DNA
bridges formed as consequence of failed abscission <sup>21</sup>. Overall, our approach allowed
the discrimination of TNT-like structures from other intercellular connections in the
embryo.

*Connections can be formed via two mechanisms similarly to TNTs in cell culture*

Two mechanisms of TNT formation have been described *in vitro*: formation from filopodia-like structures that connect and fuse; and formation by cell dislodgement, when cells migrate apart and leave membrane tether behind <sup>5</sup>. To be able to track formation of the TNT-like connections in the living embryo, we performed time-lapse experiments at 8 hpf. We were able to show that connections in the embryo can be formed from two filopodia-like structures, similarly to TNTs *in vitro* (Fig. 2A, Movie 2). Connections appeared very dynamic (example on Fig 2A was fully formed within 4 minutes) and remained stable for at least 5 minutes after formation (Movie 2). Moreover, we observed that connections could also be formed by cell dislodgement. Interestingly, in some instances TNT-like structures formed between cells remained connected while one of them underwent cell division (Fig. 2B, Movie 3). This is highlighted in Fig. 2B and Movie 3, which show the top cell (white asterisk) that migrates away from two bottom cells and leaves a membrane tether behind (yellow arrowhead). At the same time, top cells divides and both daughter cells maintain the connections with bottom cells. A cytokinetic bridge of typical morphology could also be observed between the two daughter cells (white arrowhead). This data further confirms that TNT-like structures can coexist with cytokinetic bridges, which are formed by distinct mechanism and hence represent a different type of connections.
**Developmental Cell**
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<figure><img src="image_5.png" /></figure>

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appeared uninterrupted. Our quantification showed that at the gastrula stage, TNT-like structures connect about 35% of the labeled cells (Figure S1A).

TNTs were initially described in neuronal cells as actin-based protrusions that lack microtubules.²⁰ However, further studies in other cell types showed that TNTs can also contain tubulin.²¹⁻²³ To further characterize zebrafish TNT-like connections, we injected *lifeAct-mKATE-E2A-EGFP-tubulin* mRNA that labels actin and tubulin of the same cells (Figure S1B). Quantification revealed that the majority of TNT-like connections contained both actin and tubulin, while in about 21% of connections, tubulin was not detected (Figure S1C).

To assess the significance of the presence of tubulin in the TNT-like connections in the embryo, we treated the embryos with 1 µM nocodazole for 1 h (Figure S1D). We found that neither the frequency nor the length of TNT-like connections was affected by the treatment (Figures S1E and S1F), suggesting that tubulin is not required for the formation or stability of connections in the embryo. This result is consistent with the previous studies of tubulin-containing TNTs *in vitro*.²²

**The majority of connections do not derive from cell division and do not bear the cytoneme marker Wnt8a**

To differentiate the observed TNT-like connections from cytokinetic bridges, zebrafish embryos were micro-injected with *lifeAct-mKATE-E2A-EGFP-CEP55* mRNA, which labels actin and the midbody marker CEP55 (Figure 1D). CEP55 was previously shown to be present in cytokinetic bridges in the zebrafish embryos throughout their lifespan (from the beginning of formation to midbody remnant release)²⁴ and to be expressed in mouse ring canals (IBs between germ cells formed by incomplete cytokinesis following failed abscission).²⁵ Therefore, CEP55 is expected to label cytokinetic bridges at all stages of their formation, as well as IBs.¹⁵ We found that 19% of all identified connections were CEP55-positive cytokinetic bridges (Figure 1F), which were significantly shorter than CEP55-negative connections, and did not exceed 10 µm in length (Figure 1E). The majority of connections were CEP55 negative, indicating that they were distinct from cytokinetic bridges. Interestingly, some of these CEP55-negative connections exceeded 10 µm in length, reaching up to 30 µm in length (Figures 1E and 1F), while a substantial number were shorter than 5 µm (Figure 1F), likely representing filopodia, which typically fall within this length range.¹⁸,¹⁹ Therefore, for further analysis of TNT-like structures, only connections longer than 5 µm were included.

Unlike cytokinetic bridges, there is no specific marker for cytonemes, making it challenging to distinguish TNTs from cytonemes (reviewed in Korenkova et al.¹⁴ and Yamashita et al.²⁶). However, they have been differentiated from filopodia using the signaling proteins that are present at their tip.⁴ For example, Wnt8a-positive cytonemes have been observed in zebrafish gastrula, at the same stage when we observe TNT-like structures.²⁷ To assess the proportion of Wnt8a-cytonemes within the group of TNT-like connections, we co-injected *lyn-GFP* mRNA with *wnt8a-mCherry* mRNA (Figure 1G) and quantified the connections longer than 5 µm (Figure 1H). We found that Wnt8a-cytonemes constituted 15% of TNT-like connections, while the majority of connections were of a different nature (Figure 1I). Importantly, the length of Wnt8a-positive cytonemes was

not significantly different from Wnt8a-negative connections (Figure 1H), as expected.

Therefore, by using CEP55 and Wnt8a labeling, we estimated the proportion of cytokinetic bridges and Wnt8a-cytonemes among all intercellular connections in the embryo. Our findings suggest that the majority of connections in the embryo are neither cytokinetic bridges nor Wnt8a-cytonemes, indicating that they could potentially be TNTs.

Overall, while TNT-like structures coexist with other intercellular connections in the zebrafish embryo, our approach enables us to enrich the population of TNT-like structures in our quantification of embryonic connections.

**In vivo TNT-like connections can be formed via two mechanisms, similar to *in vitro***

Two mechanisms of TNT formation have been described *in vitro*: the formation from filopodia-like structures that connect and fuse, and the formation by cell dislodgement, when cells migrate apart and leave membrane tethers behind.⁵

To track the formation of TNT-like connections in the living embryo, we performed time-lapse experiments at 8 hpf. This approach allowed us to demonstrate that connections in the embryo can form from two filopodia-like structures, similar to TNTs *in vitro* (Figure 2A; Video S1). These connections were highly dynamic (example in Figure 2A was fully formed within 4 min) and remained stable for at least 5 min after formation (Video S1). Additionally, we observed that connections could also form through cell dislodgement. Interestingly, in some instances TNT-like connections formed between cells remained stable while one of the connected cells underwent cell division. Figure 2B and Video S2 show the top cell migrating away from two bottom cells, leaving a membrane tether behind. Concurrently, the top cell undergoes division, with both daughter cells maintaining the connections with the bottom cells, while a cytokinetic bridge of typical morphology is observed between the two daughter cells. These data confirm that TNT-like structures can coexist with cytokinetic bridges, further supporting the idea that distinct types of protrusions can form simultaneously within the same cells.

To reinforce these findings and compare the formation mechanisms of TNT-like structures *in vivo* and *in vitro*, specifically in zebrafish-derived cells, we utilized PAC2 zebrafish embryonic cell culture. First, we confirmed that PAC2 cells form TNTs at a similar frequency to cells in the zebrafish gastrula (50% of connected cells on average; Figure S2A). Next, we demonstrated that PAC2 cells can form TNT-like connections through both mechanisms: filopodia driven (Figure S2C) and cell dislodgement (Figure S2D). PAC2 cells did not show a significant preference for either formation mechanism (out of 34 formation events, 59% were filopodia driven and 41% resulted from cell dislodgement). The average length of connections formed by the two mechanisms was not significantly different, although we observed several examples of longer connections formed by filopodia-driven mechanism (Figure S2B).

As a complementary approach to confirm the formation of TNT-like structures through a mechanism distinct from cytokinesis, we conducted clonal labeling of zebrafish embryos by injecting *lyn-Cerulean* mRNA into one cell at the 8-cell stage, followed by the injection of *lyn-mKATE* mRNA at the 16-cell
As a complementary approach to confirm the formation of TNT-like structures through a mechanism distinct from cytokinesis, we conducted clonal labeling of zebrafish embryos. This involved injecting *lyn-Cerulean* mRNA into one of the cells at the 8-cell stage, followed by the injection of *lyn-mKATE* mRNA at the 16-cell stage (Fig. 2C). Using this method, two distinctly labeled cell populations were created and demonstrated that the connections could be labeled with a single color or both colors. Consequently, these connections could originate from either one or both of the connected cells (Fig. 2D). Interestingly, time-lapse recordings at 8 hpf showed that connections could be formed by elongation on top of each other, possibly serving each other as a support (Fig. 2E, Movie 4). This could lead to the formation of bundles of connections, which was previously shown to be one of the unique features of TNTs in cell culture<sup>22,23</sup>.

## *Eps8 and CK666 induce TNT-like structures in the embryo*

Besides morphological criteria that allowed us to classify some of the connections observed in the embryo as TNT-like structures, we wanted to assess whether the latter were regulated by similar mechanisms as reported for TNTs *in vitro*. Our group has previously identified several ways to induce TNT formation in cell culture<sup>17,24</sup>. One of the potent inducers of TNTs between cells in culture is *Eps8*, an actin binding protein with two distinct activities: actin-bundling and actin-capping. Upon overexpression of *Eps8*, the number of TNT-connected neuronal cells was increased, which was due to the bundling activity of the protein<sup>24</sup>.

To overexpress *Eps8* in the embryo, *eps8-HaloTag* mRNA was generated and micro-injected together with membrane-labeling *lyn-mKate* mRNA. Overexpression of the wildtype form of *Eps8* (*Eps8-WT*) did not affect the number of TNT-connected cells but decreased average length of the connections (Fig. 3A, B, Supplementary Fig. 2A). This is probably due to dual activity of *Eps8*, namely, via its capping activity it could reduce the overall length of connections and hence the number of connections that we quantified following the length criteria. This hypothesis is supported by the fact that the number of shorter connections (5-10 µm) was increased (Fig. 3C).

Interestingly, when we overexpressed the mutant form of *Eps8* lacking its capping activity (*Eps8-dCAP*), we observed an increase in the number of connected cells (Fig. 3A, D). Overexpression of *Eps8-dCAP* did not affect the length of connections (Fig. 3E, Supplementary Fig. 2B). These results are consistent with previous data where *Eps8* was up-regulating TNTs in cell culture through its bundling activity<sup>24</sup>.

To complement our characterization of TNT-like structures in the embryo in comparison with data obtained *in vitro*, we used a drug which differentially affected TNTs and other cell protrusions in cultured cells<sup>17,22</sup>. Specifically, treatment of cells with the drug *CK666*, which inhibits Arp2/3 protein and hence branched actin network<sup>25</sup>, was shown to induce TNT formation in neuronal cells while inhibiting attached filopodia and lamellipodia<sup>17,22</sup>. To check whether *CK666* would have a similar effect on the connections in the embryo, we incubated embryos with two different concentrations of the drug that were previously used in cell culture, for 1 hour prior to imaging (Fig. 3F). At 200 µM, *CK666* appeared to be toxic to the embryos, that were either dying at early stages or, if survived, had
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stage (Figure 2C), which resulted in two distinctly labeled cell populations. Observed connections were labeled with one color or both colors, indicating that these connections could originate from either one or both of the connected cells (Figure 2D). Interestingly, time-lapse recordings at 8 hpf showed that connections could be formed by elongation on top of each other (Figure 2E; Video S3), which could lead to the formation of bundles of connections. Similar observations have been reported in living cells in culture,²⁸ while bundle organization has been described as one of the unique features of TNTs in neuronal cell culture.²⁹

## Eps8 and CK666 induce TNT-like structures in the embryo

Next, we wanted to determine whether these connections were regulated by similar mechanisms to those reported for TNTs *in vitro*. One of the potent inducers of TNTs between cells in cul-

<caption>**Figure 2.** Formation mechanisms of TNT-like connections</caption>

(A) Time-lapse recording of the zebrafish embryo at 8 hpf shows two neighboring cells extending filopodia (yellow arrowheads) that subsequently merge into a single membranous connection that remains stable for at least 5 min.

(B) Time-lapse recording of the zebrafish embryo at 8 hpf shows three neighboring cells forming TNT-like connections through cell dislodgement (yellow arrowhead) while the top cell is dividing (white asterisks). The daughter cells maintain connections with the lower cells for at least 15 min (yellow arrowhead), while a cytokinetic bridge between the daughter cells is also visible (white arrowhead).

(C) Schematic representation of the strategy for labeling two cell populations: zebrafish embryo is injected with *lyn-Cerulean* mRNA into one of the central cells at the 8-cell stage, followed by injection at the 16-cell stage with *lyn-mKATE* mRNA, and then imaged at the gastrula stage (7–9 hpf). (C')—Expected labeling outcomes: TNT-like structures formed by either one or both cell populations.

(D) Representative image showing connections formed by either one (green and magenta arrowheads) or both (yellow arrowhead) cell populations.

(E) Time-lapse recording of the zebrafish embryo at 8 hpf shows two cells belonging to different cell populations extending protrusions (yellow arrowhead) that elongate along each other.

Scale bars are 15 µm for (A) and (B), 10 µm for (D) and (E), and time is indicated as h:mm:ss.

See also Videos S1, S2, and S3 and Figure S2.

ture is Eps8, an actin-binding protein with two actin-regulating activities: capping actin barbed ends and cross-linking actin filaments.³⁰ Overexpression of Eps8 has been shown to increase the number of TNT-connected neuronal cells due to the protein's bundling activity.³¹

To overexpress Eps8 in the embryo, we micro-injected *eps8-HaloTag* mRNA together with membrane-labeling *lyn-*

*mKate* mRNA. Overexpression of the wild-type form of mouse Eps8 (mEps8-WT) did not affect the number of TNT-connected cells but decreased the average length of the connections (Figures 3A–3C). This is probably due to the dual activity of Eps8; under WT conditions, the preferred activity of Eps8 might be capping actin filaments. Additionally, by not considering structures less than 5 µm in length, we may have overlooked changes in the total amount of TNTs, as the overall length of connections decreased. This hypothesis is supported by the increased number of shorter connections (5–10 µm) (Figure 3D).

Interestingly, when we overexpressed the mutant form of Eps8 lacking its capping activity (Eps8-dCAP), we observed an increase in the number of connected cells (Figures 3A and 3E), while the length of connections was not affected (Figures 3F and 3G). These results are consistent with studies on the effect of Eps8 on TNTs in cell culture and might be due to an
observable tail twist at 48 hpf (Supplementary Fig. 3). This deleterious treatment resulted in the decrease of the number of connected cells and the average length of connections (Fig. 3G, H, I, Supplementary Fig. 2C). At 50 µM, CK666 was not altering the gross morphology of the embryo at the same developmental stage and the overall survival. However, at this dose the treatment led to an increase of the average length of connections as well as the number of longer connections (above 10 µm), while the total number of connected cells was not affected (Fig. 3G, H, I, Supplementary Fig. 2C). This data is consistent with the role of Arp2/3 as a TNT inhibitor in cell culture ¹⁷. However, as Arp2/3 participates in the cytokinetic bridge abscission ²⁶, we next questioned whether the length of cytokinetic bridges would also be affected by CK666 treatment. Incubation of *LifeAct-mKATE-E2A-CEP55-EGFP*-injected embryos with 50 µM CK666 resulted in a significant increase in the average length of cytokinetic bridges (from an average of 4.7 µm in DMSO-treated embryos to an average of 5.7 µm in CK666-treated embryos). Nevertheless, CEP55-positive connections still did not exceed 10 µm in length (Supplementary Fig. 2D). These observations indicate that Arp2/3 inhibition had a prominent effect on TNT-like connections in zebrafish similar to what has been shown in *in vitro* cell culture studies ¹⁷,²².

## TNT-like structures are able to transfer material

The most important criteria to define TNTs is to assess their transfer function. Thus, we next investigated whether the characterized TNT-like connections *in vivo* could transfer material and hence represent functional TNTs. To this purpose we set up several approaches. First, to be able to observe the transfer of cytoplasmic proteins, we made use of the *Dendra2* photoconvertible protein. The molecular weight of *Dendra2* protein (26 kDa) gives a possibility to rule out gap junction-mediated transfer that has a cutoff of 1 kDa. Embryos were injected with *dendra2* mRNA along with *lyn-HaloTag* mRNA, which allowed us to visualize membranous connections (Fig. 4A, indicated by a yellow arrowhead) and subsequently photoconvert one of the connected cells (marked with a white asterisk) from green-*Dendra2* to red-*Dendra2*. Time-lapse recording revealed that within 20 minutes, the connected cell (indicated by a white arrowhead) received red labeling from the photoconverted cell, while the fluorescent intensity of not connected neighboring cells (1, 2, 3) remained unchanged (Fig. 4A, B, Movie 5). Moreover, linear regression curve fitting showed that the photoconverted donor cell was losing red fluorescent signal with similar speed as the acceptor cell was receiving the red signal (slope = -0,46; R² = 0,86 for donor cell, slope = 0,49; R² = 0,89 for acceptor cell) (Fig. 4B). This first approach therefore reveals active transfer of *Dendra2* photoconverted protein between the two cells through the *Lyn-HaloTag*-labeled open-ended TNT connection.

Next, to test whether TNT-like structures are able to transfer organelles, we injected *Lyn-mKATE-E2A-phb-EGFP* mRNA that would label membrane and mitochondria of the same cells (Fig. 4C) and therefore enable assessment of the ability of TNTs to transfer mitochondria. Quantification showed that approximately 7% of the TNT-like structures contained mitochondria, indicating the possibility of their transfer between cells (Fig. 4D). Time-lapse recording indeed confirmed that mitochondria could move along membranous TNT-like structures and be transferred between connected cells (Fig. 4E, Movie 6).
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bryos, median 36.21%, *p* value is >0.9999, analyzed using the Mann-Whitney test. (J) Average length of TNT-like connections. DMSO control group: *n* = 541 connections, mean 11.29 µm; CK666 50 µM group: *n* = 328 connections, mean 12.90 µm, *p* value is <0.0001, analyzed using a t test. (K) TNT length distribution. DMSO control group: *n* = 541 connections; CK666 50 µM group: *n* = 328 connections, *p* = 0.0008, analyzed using the chi-squared test. See also Figure S3.

increase in TNT stability and/or an increase in TNT formation induction.<sup>18,31</sup>

To determine whether the effect of Eps8 overexpression is specific to TNT-like structures, we assessed its impact on IBs and Wnt8a-cytonemes. We overexpressed Eps8-dCAP in the embryos labeled with cytokinetic bridge marker CEP55 or cyto-neme marker Wnt8a. We found that the number of CEP55-pos-itive cytokinetic bridges was not changed, while their average length decreased (Figures S3A and S3B). Neither number nor length of Wnt8a-cytonemes was affected by Eps8-dCAP over-expression (Figures S3E and S3F). However, the number of Wnt8a-negative connections increased (Figure S3E). The data confirm that Eps8-dCAP overexpression specifically upregu-lates TNT-like structures and not cytokinetic bridges or Wnt8a-cytonemes in the embryo.

Treatment of cells with the drug CK666, which inhibits Arp2/3 protein and hence the branched actin network,<sup>32,33</sup> was shown to induce TNT formation in neuronal cells while inhibiting

attached filopodia and lamellipodia.<sup>18,29</sup> To investigate whether CK666 would have a similar effect on the connections in the em-bryo, we incubated embryos with 50 µM CK666 for 1 h prior to imaging (Figure 3H). The treatment resulted in an increase in the average length of connections and the number of longer con-nections (above 10 µm), while the total number of connected cells was not affected (Figures 3H–3K). These data are in line with cell culture findings, where CK666 releases the pool of actin from the branched actin network machinery, facilitating linear actin polymerization and enabling the formation of longer connections.<sup>18,29</sup>

To assess specificity, we investigated whether CK666 treat-ment also affects cytokinetic bridges and cytonemes in em-bryos. Incubating *lifeAct-mKATE-E2A-EGFP-CEP55*-injected embryos with 50 µM CK666 significantly increased the average length of cytokinetic bridges, although it did not alter their num-ber (Figures S3C and S3D). The elongation of cytokinetic bridges may be due to Arp2/3's role in cytokinetic bridge abscission.<sup>34</sup>

<caption>**Figure 3.** Effect of TNT inducers on zebra-fish intercellular connections</caption>

(A–G) Effect of overexpression of Eps8-WT (A)–(D) and its mutant Eps8-dCAP (A) and (E)–(G). (A) Representative images of the zebrafish embryo cells connected by TNT-like structures (yellow ar-rowheads), following Eps8-WT and Eps8-dCAP overexpression. Graphs (B) and (E) represent rela-tive numbers of connected cells, graphs (C) and (F) show violin plots of lengths of TNT-like con-nections, and graphs (D) and (G) represent length distributions of TNT-like connections.

(B) Control group: *n* = 15 embryos, median 35.38%; Eps8-WT overexpression group: *n* = 22 embryos, median 33.77%. *p* = 0.6521, analyzed using the Mann-Whitney test.

(C) Control group: *n* = 220 connections, mean 15.30 µm; Eps8-WT overexpression group: *n* = 247 connections, mean 12.99 µm. *p* = 0.0011, analyzed using a t test.

(D) Control group: *n* = 220 connections; Eps8-WT overexpression group: *n* = 247 connections, *p* = 0.0030, analyzed using the chi-squared test.

(E) Control group: *n* = 23 embryos, median 36.71%; Eps8-dCAP overexpression group: *n* = 28 em-bryos, median 40.19%. *p* = 0.0436, analyzed using the Mann-Whitney test.

(F) Control group: *n* = 343 connections, mean 11.77 µm; Eps8-dCAP overexpression group: *n* = 489 connections, mean 11.20 µm. *p* = 0.1717, analyzed using a t test.

(G) Control group: *n* = 343 connections; Eps8-dCAP overexpression group: *n* = 489 connec-tions, *p* = 0.0516, analyzed using the chi-squared test.

(H–K) Effect of CK666 treatment of the embryo on TNT-like connections. (H) Representative images of the zebrafish embryo cells connected by TNT-like structures (yellow arrowheads), following CK666 treatment. (I) Relative numbers of con-nccted cells. DMSO control group: *n* = 57 embryos, median 34.88%; CK666 50 µM group: *n* = 44 em-
Finally, we investigated whether TNT-like structures could facilitate the transfer of vesicular structures. To track early endosomes, we labeled them using the Early Endosome Antigen 1 (EEA1) marker. For this experiment, we injected either *lyn-HaloTag* (control) or *eps8-dCAP-HaloTag* mRNA into the embryos at the 1-cell stage, followed by the injection of *lifeAct-mKATE-E2A-EGFP-EEA1* mRNA at the 16-cell stage. Subsequently, injected embryos were treated with the HaloTag 647 ligand prior to imaging. As result, two distinct cell populations were observed: donor cells with membrane (or cytoplasm), actin, and endosome labeling, and acceptor cells with only membrane (or cytoplasm) labeling (Fig. 4F). Timelapse experiments at 8 hpf showed that early endosomes can indeed be transferred via TNTs between labelled cells (Movie 7). Steady images of these injected embryos revealed LifeAct-mKate-negative, EGFP-EEA1-positive acceptor cells, indicating that they received early endosomes from LifeAct-mKate/EGFP-EEA1-positive donor cells (Fig. 4F, yellow arrowheads). Furthermore, quantification showed that the relative number of acceptor cells that acquired early endosomes is significantly increased upon overexpression of Eps8-dCAP (Fig. 4G). Since overexpression of Eps8-dCAP was also increasing the number of TNT-connected cells (Fig. 3D), we conclude that the observed early endosome transfer is TNT-related.

Discussion

TNT studies *in vivo* are very challenging due to the absence of specific markers, which makes it necessary to rely majorly on the functional characterization. The main morphological criterion to classify a connection as a TNT in cell culture studies is that TNT should be hovering above and not attach to the substrate. This positional criterion cannot be used in 3D models because the cells are embedded in extracellular matrix, which represents substrate of 2D models. Another complication of TNT visualization *in vivo* is that cells are very densely packed, which makes it impossible to observe intercellular structures if all of the cells are labelled. In our study we took advantage of the zebrafish embryo model which allows mosaic labeling of the embryonic cells following micro-injection of mRNA into one of the cells at 8-16 cell stage. We have chosen micro-injection into one of the central cells at 16-cell stage to achieve optimal labeling density.

Following micro-injections of different labeling mRNAs, we concluded that different types of protrusions can co-exist during zebrafish embryonic development. Analysis of the protrusions' morphology suggested co-existence of filopodia, cytokinetic bridges, cytonemes and TNT-like structures within the same cell population of zebrafish gastrula cells. We then used additional criteria to the solely membrane labelling approach in order to clearly discriminate the different observed protrusions from TNT-like structures.

Gastrula cells are expected to form multiple filopodia as they are actively migrating during gastrulation. However, filopodia generally does not exceed 5 µm in length¹,¹⁷, so we used the criteria of length threshold to discriminate TNT-like connections from filopodia. In parallel to active migration, gastrula cells are continuously dividing, so other frequently observed structures are cytokinetic bridges. Although conventional cytokinetic bridges should not exceed 5 µm in length, similarly to filopodia, there is a possibility that they get
CK666 increased the length of both TNT-like structures and cytokinetic bridges; however, the length of CEP55-positive connections remained below 10 µm (Figure S3D), while CK666 only affected TNT-like structures longer than 10 µm (Figure 3K). Therefore, the elongation of cytokinetic bridges did not interfere with the quantification of TNT-like structures or the results obtained.

Furthermore, treating *lyn*-GFP/wnt8a-mCherry-injected embryos with CK666 did not affect the number or length of Wnt8a-positive cytonemes but did increase the average length of Wnt8a-negative connections (Figures S3G and S3H), suggesting a differential effect of CK666 on cytonemes and TNTs.

Overall, these observations indicate that Arp2/3 inhibition predominantly affects TNT-like connections in zebrafish, consistent with *in vitro* studies.<sup>18,29</sup>

We next investigated whether Eps8-dCAP overexpression and CK666 treatment could affect embryo development. Following the respective treatments, we examined the embryos at 30 hpf. No significant developmental differences were observed in the control and the CK666-treated group (Figures S3I and S3J). This may be because CK666 treatment increased the length but not the number of TNT-like connections (Figures 3I-3K), which might not impact their biological function. However, in the group of embryos injected with *eps8-dCAP* mRNA, we observed embryos with developmental abnormalities, including twisted tails, smaller eye sizes, and shorter yolk extensions, while a significant portion of embryos was failing to develop normally (Figures S3J and S3K). While only 12% of the control group exhibited mild abnormalities, the Eps8-dCAP-injected group showed 26% with mild abnormalities and 54% with severe abnormalities (Figure S3K). Since Eps8-dCAP overexpression also led to the ectopic formation of TNTs in the embryo (Figure 3E), these results suggest that dysregulation in TNT-mediated communication could impair normal embryonic development.

## TNT-like structures are able to transfer material

To investigate whether the characterized TNT-like connections *in vivo* could transfer material and hence represent functional TNTs, we set up several approaches. First, to observe the transfer of cytosolic proteins, we used the Dendra2 photoconvertible protein. The molecular weight of Dendra2 protein (26 kDa) rules out gap junction-mediated transfer, which has a cutoff of 1 kDa. Embryos were injected with *dendra2* mRNA along with *lyn*-HaloTag mRNA, which allowed us to visualize membranous connections (Figure 4A, yellow arrowhead) and subsequently photoconvert one of the connected cells (white asterisk) from green-Dendra2 to red-Dendra2. Time-lapse recording revealed that within 20 min, the connected cell (white arrowhead) received red labeling from the photoconverted cell, while the fluorescence intensity of not-connected neighboring cells (1, 2, and 3) remained unchanged (Figures 4A and 4B; Video S4). Linear regression curve fitting showed that the photoconverted donor cell lost the red fluorescent signal at a rate similar to the gain of the red signal in the acceptor cell (slope = -0.46; R² = 0.86 for donor cell, slope = 0.49; R² = 0.89 for acceptor cell, Figure 4B). This result suggests direct cytoplasm transfer, which can occur only through an open-ended connection. This approach, therefore, demonstrates the transfer of

Dendra2 photoconverted protein between two cells through an open-ended connection.

Interestingly, IBs were previously shown to transfer Dendra2 protein between connected cells in zebrafish embryo.<sup>16</sup> To assess whether it is possible to distinguish Dendra2 transfer mediated by TNTs from that by cytokinetic bridges, we performed Dendra2 transfer experiment in the embryos injected with *dendra2-E2A-EGFP-CEP55* mRNA. Our results showed that both CEP55-positive cytokinetic bridges and CEP55-negative connections can transfer cytosolic Dendra2 (Figures S4A and S4B). Quantification of the proportion of transferring versus non-transferring connections showed that the majority of cytokinetic bridges (81%) enable transfer, compared to 28% of CEP55-negative connections. These results suggest that 72% of CEP55-negative connections represent closed-ended structures (filopodia, cytonemes, or closed-ended TNTs), while 28% correspond to open-ended TNTs. Given that most connections in the embryo are CEP55-negative (81%, Figures 1F and 4C), the overall occurrence of cytosolic transfer mediated by TNTs and cytokinetic bridges in the embryo is comparable (23% versus 15%, respectively, Figure 4C).

Comparison of the kinetics of Dendra2 transfer showed that during the first 10 min, the transfer occurs faster through cytokinetic bridges than through TNTs. However, after 12 min, the fluorescent signal of the acceptor cell reaches a plateau and is not significantly different between the groups (Figure S4C). The faster transfer in cytokinetic bridges is likely due to their shorter and thicker structure compared to TNTs (Figure 1E), suggesting that cytosolic transfer through open-ended connections occurs through passive diffusion (reviewed in Korenkova et al.<sup>14</sup>). Overall, these data demonstrate that CEP55-negative connections, which do not derive from cell division, can be open ended and enable the transfer of material between cells in the embryo.

Next, to test whether TNT-like structures are able to transfer organelles, we labeled early endosomes using the early endosome antigen 1 (EEA1) marker. For this experiment, we injected either *lyn*-HaloTag (control) or *eps8-dCAP-HaloTag* mRNA into the embryos at the 1-cell stage, followed by the injection of *lifeAct-mKATE-E2A-EGFP-EEA1* mRNA at the 16-cell stage, and followed by treatment with the HaloTag 647 ligand. This allowed us to observe two distinct cell populations: donor cells with membrane (or cytoplasm), actin, and endosome labeling, and acceptor cells with only membrane (or cytoplasm) labeling (Figure 4E). Time-lapse experiments at 8 hpf showed that early endosomes can indeed be transferred via TNTs between labeled cells (Figure 4D; Video S5). Steady images of these injected embryos revealed LifeAct-mKATE-negative, EGFP-EEA1-positive acceptor cells, indicating that they received early endosomes from LifeAct-mKATE/EGFP-EEA1-positive donor cells (Figure 4E, yellow arrowheads). Furthermore, quantification showed that the relative number of acceptor cells that acquired early endosomes is significantly increased upon overexpression of Eps8-dCAP (Figure 4F). Since overexpression of Eps8-dCAP increased the number of TNT-like connections (Figure 3E) without affecting the number of cytokinetic bridges (Figure S3A) or Wnt8a-cytonemes (Figure S3E), we conclude that the observed early endosome transfer is TNT related.
elongated when daughter cells migrate apart from each other, as previously hypothesized ¹⁶. Therefore, cytokinetic bridges might be difficult to discriminate from TNT-like structures only based on length. Here, we have used the CEP55 midbody marker to distinguish TNT-like structures from cytokinetic bridges and have shown that the majority of intercellular connections (81%) did not derive from cell division. Based on our observations, CEP55-positive cytokinetic bridges never exceeded 10 µm in length, while TNT-like connections could span several tens of micrometers. However, out of hundreds of embryos imaged overall, in some embryos we were able to observe unexpectedly long and stable cytokinetic bridges (Supplementary Fig.1). Given that these structures could be observed between multiple daughter cells within the same embryo, we hypothesize that these particular embryos might have had alterations in development and the observed bridges could represent DNA bridges that are formed between daughter cells due to chromosome mis-segregation, and failed abscission ²¹.

Cytonemes, closed-ended structures that morphologically resemble TNTs ²,²⁷, were shown to exist during zebrafish gastrulation, where they participate in Wnt signaling ²⁸. In our study, we also observed cytoneme-like protrusions that appeared to be closed-ended. Consequently, we did not include them in the quantification of TNT-like protrusions, and only considered protrusions connecting two labeled cells and displaying continuous membrane labeling. However, solely relying on this morphological criterion, we cannot rule out the possibility that some structures counted as TNT-like might actually be cytonemes, as we lacked specific exclusion markers. In fact, similarly to TNTs, specific marker for cytonemes has not yet been identified.

Thus, we pursued our analysis to address whether the TNT-like connections, that in the embryo appeared morphologically distinct from filopodia, cytokinetic bridges and cytonemes, could represent actual TNTs that were well characterized in cell culture. TNTs were initially described as actin-based protrusions that lack microtubules ²⁹. However, further studies in other cell types showed that TNTs can also contain tubulin ³⁰,³¹. We found that the majority of zebrafish TNT-like connections contained both actin and tubulin, while a proportion of connections contained only actin.

Next, we assessed the mechanism of TNT-like structures formation in the embryo and investigated whether they could be regulated by some of the factors identified for TNTs in cell culture. By timelapse microscopy, we found that both mechanisms previously described in cell culture, namely filopodia extension and cell dislodgement ⁵,¹⁴, lead to the formation of TNT-like structures between gastrula cells. Moreover, we could show that upon formation TNT-like connections tend to serve as a support for each other extension which might lead to the formation of bundles of connections. Similar observation has been reported in living cells in culture ²³, while bundle organization has been described for TNTs in neuronal cells ²². Interestingly, we observed that TNT-like structures can form in parallel with cell division and cytokinetic bridge formation, which further supports the idea that distinct types of protrusions can coexist simultaneously within the same cells.

To assess whether intercellular connections that we observed in zebrafish embryos could be influenced by the effectors that were shown to induce TNT-formation in cell culture, we decided to investigate the role of Eps8, an actin-binding protein that is expressed during
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Figure360▷

For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.devcel.2024.10.016.

(A) Time-lapse recording of the zebrafish embryo at 8 hpf shows two cells connected by a membranous TNT-like connection (yellow arrowhead). Cytoplasmic Dendra2 was photoconverted to red in the cell on the left (white asterisk), the cell on the right (white arrowhead) receives red labeling within 20 min, while the fluorescence intensity of other neighboring cells (1, 2, and 3) is not changed.

(B) Relative mean fluorescent intensity (MFI) quantification for time-lapse (A). Linear regression fitting for donor and acceptor cells performed in GraphPad Prism software, with equations and goodness of fit (R²) visualized on the graph.

(C) Relative numbers of transferring and non-transferring connections in accordance with the proportions of CEP55-positive cytokinetic bridges and CEP55-negative TNT-like connections (upper chart: *n* = 141 connections; lower charts: CEP55-negative *n* = 25 connections, CEP55-positive *n* = 21 connections).

(D) Time-lapse recording of the zebrafish embryo at 8 hpf shows two cells connected by an actin-positive TNT-like connection, which enables the transfer of early endosomes. The white arrowhead points the vesicles that were already present in the connection at the beginning of the recording, while the magenta arrowhead points the vesicles that entered the connection from the connected cell below.

(E) Representative images showing violet Lyn-HaloTag 647 or Eps8-dCAP-HaloTag 647 acceptor cells containing green dots of EEA1-positive vesicles (yellow arrowheads). The green signal in the left panels was enhanced compared to the right panels to better visualize vesicles in the acceptor cells.

(F) Quantification of the relative amount of acceptor cells (normalized to the amount of donor cells) that received EEA1-positive vesicles. Control group: *n* = 17 embryos, median 0.02344, Eps8-dCAP overexpression group: *n* = 18 embryos, median 0.09467. *p* = 0.0184, analyzed using the Mann-Whitney test. Box lines show medians, box edges represent quartiles, and whiskers - minimal to maximal values.

Scale bars are 10 µm; time is indicated as: h:mm:ss.

See also Videos S4, S5, and S6 and Figure S4.

One of the distinctive functions of TNTs is their ability to transfer mitochondria.⁵,⁶,¹² To assess whether TNT-like connections in the embryo can transfer mitochondria, we injected *lyn-mKATE-E2A-phb-EGFP* mRNA, which labels the membrane and mitochondria of the same cells (Figure S4D). Quantification showed that approximately 7% of the TNT-like structures contained mitochondria, indicating the possibility of their transfer between cells (Figure S4E). Time-lapse recording confirmed that mitochondria indeed can move along membranous TNT-like structures in the embryo (Figure S4F; Video S6). However, unlike the EEA1 transfer, mitochondria transfer events were rare, and our setup did not allow us to quantify the percentage of transfer or the effect of Eps8-dCAP overex-

pression. This might be due to several factors, including the developmental stage of the embryo or technical limitations.

DISCUSSION

*In vivo* studies of TNTs are very challenging due to the absence of specific markers, necessitating a major reliance on functional characterization. In cell culture studies, TNTs are typically identified by their morphology, particularly their hovering position above the substrate. This criterion is not applicable in 3D models, where cells are embedded in extracellular matrix. Another complication in visualizing TNTs *in vivo* is that cells are very densely packed, which makes it impossible to observe
gastrulation<sup>32</sup> and was previously shown to induce TNT-formation in neuronal cells through its bundling activity<sup>24</sup>. Eps8 displays two actin-regulating activities: capping actin barbed ends and cross-linking actin filaments<sup>33</sup>. Overexpression of Eps8-WT, which possesses both activities, led to an increase in the number of shorter connections, probably because under WT conditions its preferred activity is capping actin filaments. In addition, by not considering structures less than 5 µm in length, we may have overlooked changes in the total amount of TNTs because the overall length of connections decreased. On the other hand, when Eps8-dCAP mutant (lacking capping activity) was overexpressed, the number of cells connected by TNT-like structures was increased. This is consistent with studies on the effect of Eps8 on TNTs in cell culture and might be due to an increase of the TNT stability and/or to an increase of TNT formation induction as previously reported<sup>17,24</sup>.

As an alternative approach to affect TNT formation in the whole embryo, we used CK666, a drug that inhibits Arp2/3 branched actin network<sup>25</sup> and robustly induces TNTs in culture<sup>17</sup>. Interestingly, we found that it significantly increased the proportion of longer TNT-like connections. This is in line with cell culture data where CK666 releases the pool of actin from branched actin network and hereby enables formation of longer connections<sup>17,22</sup>. Arp2/3 has also been demonstrated to participate in cytokinetic bridge abscission<sup>26</sup>. Therefore, inhibiting Arp2/3 could potentially influence the stability of cytokinetic bridges. We hypothesized that if cytokinetic bridges persist longer, their length might increase due to continuous cell migration during gastrulation. As expected, we observed that CK666 treatment increased the average length of CEP55-positive cytokinetic bridges, although they still did not exceed 10 µm in length. Interestingly, CK666 only affected TNT-like connections that were above 10 µm in length, indicating that elongated cytokinetic bridges did not interfere with the results obtained for TNT-like connections.

Taken together, our data suggests that TNT-like intercellular connections in the zebrafish embryo share multiple characteristics with TNTs observed in cell culture, namely: i) a similar morphology, ii) similar formation mechanisms, and iii) similar response to known TNT inducers. However, the only unambiguous way to confirm that observed structures are veritable TNTs is to show that they are able to transfer material. By timelapse experiments we could demonstrate that TNT-like intercellular connections enable cytoplasmic continuity and transfer of cytoplasmic Dendra2 protein, suggesting that they should be open-ended and hence represent canonical TNTs. Indeed, Dendra2 protein molecular weight is 26 kDa which makes it impossible to pass through the gap junctions that allow transport of molecules smaller than 1 kDa. The fact that the speed of signal acquisition by the acceptor cell is almost equal to the speed of signal intensity loss from the donor cell, suggests direct cytoplasm transfer that can happen only in the case of open-ended connection. Moreover, we successfully demonstrated that TNTs in zebrafish facilitate the transfer of organelles, including mitochondria and early endosomes, which represents one of the most distinctive functions of TNTs.

Overall, our study represents the first demonstration of functional TNTs in the living zebrafish embryo. The identified structures share multiple morphological and dynamic characteristics with TNTs described in cell culture and enable the transfer of cellular
intercellular structures if all cells are uniformly labeled. In our study, we took advantage of the zebrafish embryo model, which allows mosaic labeling of the embryonic cells following the micro-injection of mRNA into one of the cells at the 16-cell stage.

Following micro-injections of various labeling mRNAs and observations at the gastrula stage, we concluded that different types of protrusions can coexist during zebrafish embryonic development. Analysis of the protrusions' morphology suggested the coexistence of filopodia, cytokinetic bridges, cytonemes, and TNT-like structures within the same cell population of zebrafish gastrula cells. Using morphological criteria, we quantified "TNT-like structures"—connections that morphologically resemble TNTs.

Our data suggest that TNT-like intercellular connections in the zebrafish embryo share multiple characteristics with TNTs observed in cell culture, including (1) similar morphology, (2) similar formation mechanisms, and (3) similar responses to known TNT inducers. Through time-lapse experiments using spinning disk microscopy, we showed that TNT-like intercellular connections enable cytoplasmic continuity and transfer of cytosolic proteins and organelles, including mitochondria and early endosomes, demonstrating the existence of functional TNTs in the living embryo.

TNTs may play a crucial role in cell-to-cell communication during development, complementing the functions of other connections that exist concurrently.

For example, cytokinetic bridges were shown to participate in the coordination of transcription onset during midblastula transition in the early zebrafish embryo.²⁴ In various species, stable IBs between germline or embryonic cells are suggested to be implicated in the synchronization of cell division, differentiation, and directional transfer of nutrients.¹⁵ Furthermore, our study provides evidence that cytokinetic bridges with midbodies can persist for 10–20 min in zebrafish embryos at the gastrula stage, a phenomenon previously only observed in blastula stages. These cytokinetic bridges can transfer cytosolic proteins, unveiling an alternative mechanism of intercellular communication between cells during zebrafish gastrula development. Cytonemes were shown to enable morphogen transfer in various species, including Drosophila, zebrafish, chicken, and mice, and could be crucial for the gradient formation of membrane-associated morphogens, such as Wnt, Sonic hedgehog, or Delta-Notch.³⁵⁻³⁸ It is suggested that membrane-bound proteins can be delivered via the release of exosomes from the tip of the cytoneme.³⁸,³⁹ Interestingly, in addition to facilitating long-distance ligand-receptor communication through the transfer of ligands or receptors, cytonemes have been recently shown to transfer active ligand-receptor complexes, thereby enabling the recipient cell to become responsive to the ligand.⁴⁰

While cytonemes transfer proteins and protein complexes, and cytokinetic bridges facilitate communication between dividing cells, TNTs are currently the only structure shown to allow the direct exchange of cytoplasmic content between cells from distinct populations. This TNT-mediated exchange ranges from electrical coupling to organelle transfer and can significantly influence the fate of connected cells. It has been suggested that TNTs may contribute to the coordination of cell migration through electrical coupling, thereby orchestrating morphogenic movements.⁴¹ TNTs have also been shown to

transfer the death signal Fas ligand and active caspases between T cells *in vitro*, suggesting their potential role in inducing apoptosis in recipient cells.⁴² Additionally, TNTs are involved in osteoclastogenesis, facilitating the transfer of the regulatory protein DC-STAMP (dendritic cell-specific transmembrane protein) between osteoclast precursors.⁴³

TNT-mediated mitochondrial transfer has been observed in various physiological contexts. For instance, mitochondria transfer from mesenchymal stromal cells to T regulatory (Treg) cells via TNTs can help maintain the Treg naive phenotype and stabilize the population.⁴⁴ This type of mitochondrial transfer has also been shown to drive cellular reprogramming, such as endothelial-to-myocyte transdifferentiation and cardiomyocyte dedifferentiation.⁴⁵,⁴⁶ In cancer, tumor cells can form TNTs with cells in the tumor microenvironment, acquiring healthy mitochondria through TNT-mediated transfer. This process enhances cancer cell proliferation, increases invasiveness, and contributes to therapy resistance.⁴⁷⁻⁵⁰

Although the role of TNT-mediated intercellular communication in development is not fully understood, studies suggest that TNTs may be involved in developmental processes such as coordinated cell movement, differentiation, proliferation, and survival. Our observation of TNTs in a living organism suggests that cells can communicate directly and selectively over considerable distances within tissues. This TNT-mediated communication could involve molecular, electrical, and mechanical signals, potentially complementing morphogen gradient signaling to ensure proper tissue and organ formation during embryonic development.¹⁴ This hypothesis is supported by the developmental defects we observed following ectopic TNT formation due to Eps8-dCAP overexpression, which may lead to dysregulated intercellular communication. Depending on the cargo transferred, this dysregulation could result in uncoordinated migration, increased apoptosis, or differentiation defects during early development.

## Limitations of the study

Despite our findings, several critical questions remain unanswered due to the limitations of our study. We demonstrated the coexistence of multiple types of cellular protrusions within the same cells, underscoring the complexity of their regulation *in vivo* and the need for further investigation into the mechanisms governing TNT formation. While we showed that TNT-mediated transfer of cellular components occurs in the embryo, a deeper exploration of the specific molecular signals and mechanisms involved is required to fully understand their biological function in development. Additionally, although Eps8-dCAP overexpression specifically upregulates TNT-like structures without significantly affecting cytokinetic bridges or Wnt8a-cytonemes, the broader consequences of this upregulation are unclear. Furthermore, while developmental abnormalities were observed in the embryos overexpressing Eps8-dCAP, we cannot definitively attribute these phenotypes solely to altered TNT-mediated communication, as Eps8-dCAP's role in enhancing actin filament bundling may also impact other actin-based structures, complicating interpretation.

Further research is needed to disentangle the specific role of TNTs in development and to determine whether the observed defects are directly linked to changes in TNT-mediated
material, which is to date the only reliable criteria to classify a given protrusion as a tunneling nanotube. Our results open the way to further investigations on the role of TNTs in the developing embryo. Of interest, several studies, including from our group ³⁴⁻³⁷, support potential implication of TNTs in various developmental processes, such as coordinating cell movement, cell differentiation, and cell proliferation. The current demonstration of the presence of these structures in living organisms strongly indicates that cells have the ability to communicate directly and selectively over considerable distances within tissues. TNT-mediated communication may encompass molecular, electrical, and mechanical signals and could potentially work alongside morphogen gradient signaling during embryonic development ¹⁴. Employing a targeted disruption of TNT formation in living organisms would be an effective strategy to assess the role of TNT-based communication. However, these studies will require better and more specific tools as well as further improved 3D imaging systems.

## Author contributions

Conceptualization, O.K., A.P., S.A., F.D.B. and C.Z.; Methodology, O.K., S.L., A.P. and S.A.; Investigation, O.K., S.L. and I.P.; Formal Analysis, O.K. and S.L.; Writing – Original Draft, O.K.; Writing – Review & Editing, O.K., S.L., A.P., S.A., F.D.B. and C.Z.; Supervision, C.Z.; Funding Acquisition, C.Z.

## Acknowledgements

Authors would like to acknowledge the help of the following Institut Pasteur facilities: Zebrafish Projects Hub, Photonic BioImaging platform, Biomaterials and Microfluidics platform, Image Analysis Hub, Bioinformatics and Biostatistics Hub. Authors thank Frédéric Rosa for his kind advice and help with the assembly of *pXT7-lyn-mKATE-E2A-phb-EGFP* plasmid. Authors would like to thank all members of the Zurzolo group for fruitful discussions and Reine Bouyssie, a member of the administrative staff of the Membrane Traffic and Pathogenesis Unit, for her continuous support. Authors would like to thank Janelia Research Campus Advanced Imaging Center members Teng-Leong Chew and Chad Hobson for valuable insights on the project.

This work was supported by grants Equipe FRM #EQU202103012692 and ANR-20-CE13-0032-01 to Chiara Zurzolo. Olga Korenkova was supported by the Pasteur-Paris University (PPU) International PhD Program and by FRM (Fondation Recherche Médicale) End of thesis grant #FDT202204014868. Shiyu Liu is supported by the "Programme Explore Cancer" de l'Institut Pasteur.

## Declaration of interests

The authors declare no competing interests.
# Developmental Cell
## Short article

<figure><img src="image_27.png" /></figure>

communication. This includes a more detailed analysis of the materials transported via TNTs and their impact on receiving cells. Spinning disk confocal imaging used in this study, while advanced, has inherent limitations in the field of view and acquisition duration. To better study TNT formation, material transfer, and their functional consequences in vivo, advancements in 4D imaging techniques will be essential. Most importantly, the identification of specific TNT markers and the development of tools to selectively disrupt TNT formation in vivo will be crucial for assessing their role during various stages of development.

In summary, our study provides compelling evidence of functional TNTs in zebrafish embryos sharing multiple characteristics with TNTs in cell culture. Addressing the challenges and open questions highlighted here will be essential for fully understanding the significance of TNT-mediated communication in embryonic development and beyond.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chiara Zurzolo (chiara.zurzolo@pasteur.fr).

### Materials availability

All plasmids generated in this study are available from the lead contact without restriction.

### Data and code availability

* All data reported in this paper will be shared by the lead contact upon request.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

Authors would like to acknowledge the help of the following Institut Pasteur facilities: Zebrafish Projects Hub, Photonic Biolmaging platform, Biomaterials and Microfluidics platform, Image Analysis Hub, and the Bioinformatics and Biostatistics Hub. The authors would like to thank the following: Frédéric Rosa for his kind advice and help with the assembly of pXT7-lyn-mKATE-E2A-phb-EGFP plasmid; Steffen Scholpp for kindly providing the pCS2+-zfWnt8a-mCherry plasmid and PAC2 cells; all members of the Zurzolo group for fruitful discussions; and Reine Bouyssie, a member of the administrative staff of the Membrane Traffic and Pathogenesis Unit, for her continuous support.

This work was supported by grants Equipe FRM # EQU202103012692 and ANR-20-CE13-0032-01 to C.Z. O.K. was supported by the Pasteur-Paris University (PPU) International PhD Program and by FRM (Fondation Recherche Médicale) End of thesis grant # FDT202204014868. S.L. is supported by the "Programme Explore Cancer" de l'Institut Pasteur.

## AUTHOR CONTRIBUTIONS

Conceptualization, O.K., A.P., and C.Z.; methodology, O.K., S.L., A.P., and S.A.; investigation, O.K., S.L., and I.P.; formal analysis, O.K., and S.L.; writing—original draft, O.K.; writing—review and editing, O.K., S.L., A.P., S.A., F.D.B., and C.Z.; supervision, C.Z.; funding acquisition, C.Z.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* PAC2 cells

* Zebrafish

* METHOD DETAILS

* Plasmids and mRNA

* Embryo micro-injections

* Embryo mounting and image acquisition

* Dendra2 photoconversion and time-lapse imaging

* PAC2 experiments

* Image analysis

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2024.10.016.

Received: September 12, 2023

Revised: June 25, 2024

Accepted: October 18, 2024

Published: November 13, 2024

## REFERENCES

1. Mattila, P.K., and Lappalainen, P. (2008). Filopodia: molecular architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 9, 446–454. https://doi.org/10.1038/nrm2406.

2. Ramírez-Weber, F.A., and Kornberg, T.B. (1999). Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599–607. https://doi.org/10.1016/s0092-8674(00)80771-0.

3. Fereres, S., Hatori, R., Hatori, M., and Kornberg, T.B. (2019). Cytoneme-mediated signaling essential for tumorigenesis. PLoS Genet. 15, e1008415. https://doi.org/10.1371/journal.pgen.1008415.

4. Stanganello, E., Hagemann, A.I., Mattes, B., Sinner, C., Meyen, D., Weber, S., Schug, A., Raz, E., and Scholpp, S. (2015). Filopodia-based Wnt transport during vertebrate tissue patterning. Nat. Commun. 6, 5846. https://doi.org/10.1038/ncomms6846.

5. Abounit, S., and Zurzolo, C. (2012). Wiring through tunneling nanotubes—from electrical signals to organelle transfer. J. Cell Sci. 125, 1089–1098. https://doi.org/10.1242/jcs.083279.

6. Sisakhtnezhad, S., and Khosravi, L. (2015). Emerging physiological and pathological implications of tunneling nanotubes formation between cells. Eur. J. Cell Biol. 94, 429–443. https://doi.org/10.1016/j.ejcb.2015.06.010.

7. Chastagner, P., Loria, F., Vargas, J.Y., Tois, J., I Diamond, M., Okafo, G., Brou, C., and Zurzolo, C. (2020). Fate and propagation of endogenously formed Tau aggregates in neuronal cells. EMBO Mol. Med. 12, e12025. https://doi.org/10.15252/emmm.202012025.

8. Dilsizoglu Senol, A., Samarani, M., Syan, S., Guardia, C.M., Nonaka, T., Liv, N., Latour-Lambert, P., Hasegawa, M., Klumperman, J., Bonifacino, J.S., and Zurzolo, C. (2021). α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes. PLoS Biol. 19, e3001287. https://doi.org/10.1371/journal.pbio.3001287.

9. Pepe, A., Pietropaoli, S., Vos, M., Barba-Spaeth, G., and Zurzolo, C. (2022). Tunneling nanotubes provide a route for SARS-CoV-2 spreading. Sci. Adv. 8, eab00171. https://doi.org/10.1126/sciadv.abo0171.

10. Hashimoto, M., Bhuyan, F., Hiyoshi, M., Noyori, O., Nasser, H., Miyazaki, M., Saito, T., Kondoh, Y., Osada, H., Kimura, S., et al. (2016). Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in
## Figure legends

**Figure 1. Characterization of intercellular connections in zebrafish gastrula.**

(A) Schematic representation of the labeling strategy to visualize intercellular connections: zebrafish embryo is injected into one of the central cells at 16-cell stage, incubated at 28°C until it reaches gastrula stage (7-9 hpf) and mounted for imaging.

(B) Fluorescent photograph of different protrusions that can be observed at gastrula stage following membrane labeling by *lyn-mKATE* mRNA injection, pointed by yellow arrowheads: (1) short protrusions, (2) long closed-ended protrusions, (3) connections with interrupted membrane labeling, (4) thin TNT-like structures, (5) thick TNT-like structures.

(C) Cartoon representing the image (B), suggesting the co-existence of: (1) filopodia, (2) cytonemes, (3) cytokinetic bridges, (4) TNT-like structures, (5) bundles of TNT-like structures.

(D) Graph representing relative number of labelled cells, connected by TNT-like structures, each point representing separate embryo (n=38, median 36,6%).

(E, F) Cytoskeletal composition of TNT-like structures. Embryos were injected as described in (A), using *lifeAct-mKATE-E2A-EGFP-tubulin* mRNA. All connections could be divided into 3 groups depending on the presence of tubulin. (E) – representative images, (F) – pie chart representing relative numbers of the 3 types of connections (n=72).

(G, H) Presence of midbody in the intercellular connections. Embryos were injected as described in (A), using *lifeAct-mKATE-E2A-CEP55-EGFP* mRNA. (G) Representative images of CEP55-positive cytokinetic bridges and CEP55-negative TNT-like connections.

(H) Graph representing difference in length distribution and average lengths between CEP55-positive (n=27, mean 5,6 µm) and CEP55-negative connections (n=114, mean 7,8 µm), p=0.0096, statistical analysis is t-test.

(I) – pie chart representing relative numbers of CEP55-positive connections and CEP55-negative TNT-like connections having lengths below 5 µm, from 5 µm to 10 µm, and above 10µm (n=141).

Scale bars are 10 µm.

**Figure 2. Formation mechanisms of TNT-like connections.**

(A) Time-lapse recording of the zebrafish embryo at 8hpf shows two neighboring cells extending filopodia (yellow arrowheads) that subsequently merge into single membranous connection that remains stable for at least 5 minutes.

(B) Time-lapse recording of the zebrafish embryo at 8hpf shows three neighboring cells forming TNT-like connections by cell dislodgement (yellow arrowhead), while the top cell is dividing (white asterisks). Daughter cells maintain connections with lower cells for at least 15 minutes (yellow arrowhead), while cytokinetic bridge between daughter cells is also visible (white arrowhead).

(C) Schematic representation of the strategy for labeling two cell populations: zebrafish embryo is injected into one of the central cells at 8-cell stage with *lyn-Cerulean* mRNA, followed by injection at 16-cell stage with *lyn-mKATE* mRNA, incubated at 28°C until it reaches gastrula stage (7-9 hpf) and mounted for imaging. (C') – expected labeling outcomes: TNT-like structures formed by either one or both cell populations.
Macrophages. J. Immunol. **196**, 1832-1841. https://doi.org/10.4049/jimmunol.1500845.

11. Taiarol, L., Formicola, B., Fagioli, S., Sierrri, G., D'Aloia, A., Kravicz, M., Renda, A., Viale, F., Dal Magro, R., Ceriani, M., and Re, F. (2021). The 3.0 Cell Communication: New Insights in the Usefulness of Tunneling Nanotubes for Glioblastoma Treatment. Cancers (Basel) **13**, 4001. https://doi.org/10.3390/cancers13164001.

12. Pinto, G., Saenz-de-Santa-Maria, I., Chastagner, P., Perthame, E., Delmas, C., Toulas, C., Moyal-Jonathan-Cohen, E., Brou, C., and Zurzolo, C. (2021). Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids. Biochem. J. **478**, 21-39. https://doi.org/10.1042/bcj20200710.

13. Pinto, G., Brou, C., and Zurzolo, C. (2020). Tunneling Nanotubes: The Fuel of Tumor Progression? Trends Cancer **6**, 874-888. https://doi.org/10.1016/j.trecan.2020.04.012.

14. Korenkova, O., Pepe, A., and Zurzolo, C. (2020). Fine intercellular connections in development: TNTs, cytonemes, or intercellular bridges? Cell Stress **4**, 30-43. https://doi.org/10.15698/cst2020.02.212.

15. Haglund, K., Nezis, I.P., and Stenmark, H. (2011). Structure and functions of stable intercellular bridges formed by incomplete cytokinesis during development. Commun. Integr. Biol. **4**, 1-9. https://doi.org/10.4161/cib.4.1.13550.

16. Caneparo, L., Pantazis, P., Dempsey, W., and Fraser, S.E. (2011). Intercellular bridges in vertebrate gastrulation. PLoS One **6**, e20230. https://doi.org/10.1371/journal.pone.0020230.

17. Henderson, J.M., Ljubojevic, N., Belian, S., Chaze, T., Castaneda, D., Battistella, A., Giai Gianetto, Q., Matondo, M., Descroix, S., Bassereau, P., and Zurzolo, C. (2023). Tunnelling nanotubes formation is driven by Eps8/IRSp53-dependent linear actin polymerization. EMBO J. **42**, e113761. https://doi.org/10.15252/embj.2023113761.

18. Henderson, J.M., Ljubojevic, N., Chaze, T., Castaneda, D., Battistella, A., Gianetto, Q.G., Matondo, M., Descroix, S., Bassereau, P., and Zurzolo, C. (2022). Arp2/3 inhibition switches Eps8's network associations to favour longer actin filament formation necessary for tunneling nanotubes. Preprint at bioRxiv. https://doi.org/10.1101/2022.08.24.504515.

19. Ljubojevic, N., Henderson, J.M., and Zurzolo, C. (2021). The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions. Trends Cell Biol. **31**, 130-142. https://doi.org/10.1016/j.tcb.2020.11.008.

20. Rustom, A., Saffrich, R., Markovic, I., Walther, P., and Gerdes, H.H. (2004). Nanotubular highways for intercellular organelle transport. Science **303**, 1007-1010. https://doi.org/10.1126/science.1093133.

21. Önfelt, B., Nedvetzki, S., Benninger, R.K., Purbhoo, M.A., Sowinski, S., Hume, A.N., Seabra, M.C., Neil, M.A., French, P.M., and Davis, D.M. (2006). Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J. Immunol. **177**, 8476-8483. https://doi.org/10.4049/jimmunol.177.12.8476.

22. Sáenz-de-Santa-Maria, I., Bernardo-Castiñeira, C., Enciso, E., García-Moreno, I., Chiara, J.L., Suarez, C., and Chiara, M.D. (2017). Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes. Oncotarget **8**, 20939-20960. https://doi.org/10.18632/oncotarget.15467.

23. Resnik, N., Erman, A., Veranič, P., and Kreft, M.E. (2019). Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes. Histochem. Cell Biol. **152**, 311-317. https://doi.org/10.1007/s00418-019-01806-3.

24. Adar-Levor, S., Nachmias, D., Gal-Oz, S.T., Jahn, Y.M., Peyrieras, N., Zaritsky, A., Birnbaum, R.Y., and Elia, N. (2021). Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis. Proc. Natl. Acad. Sci. USA **118**, e2021210118. https://doi.org/10.1073/pnas.2021210118.

25. Iwamori, T., Iwamori, N., Ma, L., Edson, M.A., Greenbaum, M.P., and Matzuk, M.M. (2010). TEX14 interacts with CEP55 to block cell abscission. Mol. Cell. Biol. **30**, 2280-2292. https://doi.org/10.1128/MCB.01392-09.

26. Yamashita, Y.M., Inaba, M., and Buszczak, M. (2018). Specialized Intercellular Communications via Cytonemes and Nanotubes. Annu. Rev. Cell Dev. Biol. **34**, 59-84. https://doi.org/10.1146/annurev-cellbio-100617-062932.

27. Mattes, B., Dang, Y., Greicius, G., Kaufmann, L.T., Prunsche, B., Rosenbauer, J., Stegmaier, J., Mikut, R., Özbek, S., Nienhaus, G.U., et al. (2018). Wnt/PCP controls spreading of Wnt/beta-catenin signals by cytonemes in vertebrates. eLife **7**, e36953. https://doi.org/10.7554/eLife.36953.

28. Chang, M., Lee, O.C., Bu, G., Oh, J., Yunn, N.O., Ryu, S.H., Kwon, H.B., Kolomeisky, A.B., Shim, S.H., Doh, J., et al. (2022). Formation of cellular close-ended tunneling nanotubes through mechanical deformation. Sci. Adv. **8**, eabj3995. https://doi.org/10.1126/sciadv.abj3995.

29. Sartori-Rupp, A., Cordero Cervantes, D., Pepe, A., Gousset, K., Delage, E., Corroyer-Dulmont, S., Schmitt, C., Krijnse-Locker, J., and Zurzolo, C. (2019). Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat. Commun. **10**, 342. https://doi.org/10.1038/s41467-018-08178-7.

30. Vaggi, F., Disanza, A., Milanesi, F., Di Fiore, P.P., Menna, E., Matteoli, M., Gov, N.S., Scita, G., and Ciliberto, A. (2011). The Eps8/IRSp53/VASP network differentially controls actin capping and bundling in filopodia formation. PLoS Comput. Biol. **7**, e1002088. https://doi.org/10.1371/journal.pcbi.1002088.

31. Delage, E., Cervantes, D.C., Pénard, E., Schmitt, C., Syan, S., Disanza, A., Scita, G., and Zurzolo, C. (2016). Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes. Sci. Rep. **6**, 39632. https://doi.org/10.1038/srep39632.

32. Hetrick, B., Han, M.S., Helgeson, L.A., and Nolen, B.J. (2013). Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem. Biol. **20**, 701-712. https://doi.org/10.1016/j.chembiol.2013.03.019.

33. Dutta, A., and Kumar Sinha, D. (2015). Turnover of the actomyosin complex in zebrafish embryos directs geometric remodelling and the recruitment of lipid droplets. Sci. Rep. **5**, 13915. https://doi.org/10.1038/srep13915.

34. Advedissian, T., Frémont, S., and Echard, A. (2024). Cytokinetic abscission requires actin-dependent microtubule severing. Nat. Commun. **15**, 1949. https://doi.org/10.1038/s41467-024-46062-9.

35. Roy, S., and Kornberg, T.B. (2015). Paracrine signaling mediated at cell-cell contacts. BioEssays **37**, 25-33. https://doi.org/10.1002/bies.201400122.

36. Sanders, T.A., Llagostera, E., and Barna, M. (2013). Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature **497**, 628-632. https://doi.org/10.1038/nature12157.

37. Stanganello, E., and Scholpp, S. (2016). Role of cytonemes in Wnt transport. J. Cell Sci. **129**, 665-672. https://doi.org/10.1242/jcs.182469.

38. Hall, E.T., Dillard, M.E., Cleverdon, E.R., Zhang, Y., Daly, C.A., Ansari, S.S., Wakefield, R., Stewart, D.P., Pruett-Miller, S.M., Lavado, A., et al. (2024). Cytoneme signaling provides essential contributions to mammalian tissue patterning. Cell **187**, 276-293. e23. https://doi.org/10.1016/j.cell.2023.12.003.

39. Hall, E.T., Dillard, M.E., Stewart, D.P., Zhang, Y., Wagner, B., Levine, R.M., Pruett-Miller, S.M., Sykes, A., Temirov, J., Cheney, R.E., et al. (2021). Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex. eLife **10**, e61432. https://doi.org/10.7554/eLife.61432.

40. Zhang, C., Brunt, L., Ono, Y., Rogers, S., and Scholpp, S. (2024). Cytoneme-mediated transport of active Wnt5b-Ror2 complexes in zebrafish. Nature **625**, 126-133. https://doi.org/10.1038/s41586-023-06850-7.

41. Gerdes, H.H., Rustom, A., and Wang, X. (2013). Tunneling nanotubes, an emerging intercellular communication route in development. Mech. Dev. **130**, 381-387. https://doi.org/10.1016/j.mod.2012.11.006.

42. Arkwright, P.D., Luchetti, F., Tour, J., Roberts, C., Ayub, R., Morales, A.P., Rodriguez, J.J., Gilmore, A., Canonico, B., Papa, S., and Esposti, M.D.
(D) Representative image showing connections formed by either one (green and red arrowheads) or both (yellow arrowhead) cell populations.

(E) Time-lapse recording of the zebrafish embryo at 8hpf shows two cells belonging to different cell populations, extending protrusions (yellow arrowhead) that elongate along each other.

Scale bars are 15 µm for (A) and (B), 10 µm for (D) and (E), time is indicated as h:mm:ss.

**Figure 3.** Effect of TNT-inducers on zebrafish intercellular connections.

(A-E) Effect of overexpression of Eps8-WT (A-C) and its mutant Eps8-dCAP (A, D, E).

(A) Representative images of the zebrafish embryo cells connected by TNT-like structures (yellow arrowheads), following Eps8-WT and Eps8-dCAP overexpression. Graphs (B) and (D) represent relative numbers of connected cells, graphs (C) and (E) represent length distributions of TNT-like connections.

(B) control group: n=15, median 35.38%, Eps8-WT overexpression group: n=22, median 33.77%. p= 0.6521, analysed by Mann Whitney test.

(C) control group: n=220, Eps8-WT overexpression group: n=247. p= 0.0030, analysed by chi-square test.

(D) control group: n=23, median 36.71%, Eps8-dCAP overexpression group: n=28, median 40.19. p= 0.0436, analysed by Mann Whitney test.

(E) control group: n=343, Eps8-dCAP overexpression group: n=489. p= 0.0516, analysed by chi-square test.

(F-I) Effect of CK666 treatment of the embryo on TNT-like connections.

(F) Schematic representation of experiment set-up: embryo was injected into one of the central cells at 16-cell stage, incubated at 28°C until it reaches 6hpf, treated with CK666 for 1 hour and mounted for imaging at gastrula stage (7-9 hpf).

(G) Representative images of the zebrafish embryo cells connected by TNT-like structures (yellow arrowheads), following CK666 treatment. Graph (H) shows relative numbers of connected cells, graph (I) represents length distributions of TNT-like connections.

(H) DMSO control group: n=57, median 34.88%, CK666 50 µM group: n=44, median 36.21%, CK666 200 µM group: n=24, median 26.81%. p-values are >0.9999 for DMSO vs. CK666 50µM, 0.0347 for DMSO vs. CK666 200µM and 0.0054 for CK666 50µM vs. CK666 200µM, analysed by Kruskal Wallis test.

(I) DMSO control group: n= 541, CK666 50 µM group: n=328, CK666 200 µM group: n=298. p-values are 0.0008 for DMSO vs. CK666 50µM, 0.0297 for DMSO vs. CK666 200µM and <0.0001 for CK666 50µM vs. CK666 200µM, analysed by chi-square test.

**Figure 4.** TNT-like structures are able to transfer material.

(A) Time-lapse recording of the zebrafish embryo at 8hpf shows two cells connected by a membranous TNT-like connection (yellow arrowhead). Cytoplasmic Dendra2 was photoconverted to red in the cell on the left (white asterisk), recording shows that cell on the right (white arrowhead) receives red labeling within 20 minutes, while the fluorescence intensity of other neighboring cells (1, 2, 3) is not changed.
**Developmental Cell**
Short article

<figure><img src="image_28.png" /></figure>

CellPress

OPEN ACCESS

(2010). Fas stimulation of T lymphocytes promotes rapid intercellular ex-
change of death signals via membrane nanotubes. Cell Res. 20, 72-88.
https://doi.org/10.1038/cr.2009.112.

43. Takahashi, A., Kukita, A., Li, Y.J., Zhang, J.Q., Nomiyama, H., Yamaza, T., Ayukawa, Y., Koyano, K., and Kukita, T. (2013). Tunneling nanotube formation is essential for the regulation of osteoclastogenesis. J. Cell. Biochem. 114, 1238-1247. https://doi.org/10.1002/jcb.24433.

44. Do, J.S., Zwick, D., Kenyon, J.D., Zhong, F., Askew, D., Huang, A.Y., Van't Hof, W., Finney, M., and Laughlin, M.J. (2021). Mesenchymal stromal cell mitochondrial transfer to human induced T-regulatory cells mediates FOXP3 stability. Sci. Rep. 11, 10676. https://doi.org/10.1038/s41598-021-90115-8.

45. Acquistapace, A., Bru, T., Lesault, P.F., Figeac, F., Coudert, A.E., le Coz, O., Christov, C., Baudin, X., Auber, F., Yiou, R., et al. (2011). Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer. Stem Cells 29, 812-824. https://doi.org/10.1002/stem.632.

46. Koyanagi, M., Brandes, R.P., Haendeler, J., Zeiher, A.M., and Dimmeler, S. (2005). Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novel mechanism for cell fate changes? Circ. Res. 96, 1039-1041. https://doi.org/10.1161/01.RES.0000168650.23479.0c.

47. Burt, R., Dey, A., Aref, S., Aguiar, M., Akarca, A., Bailey, K., Day, W., Hooper, S., Kirkwood, A., Kirschner, K., et al. (2019). Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells

from oxidative stress. Blood 134, 1415-1429. https://doi.org/10.1182/blood.2019001398.

48. Hekmatshoar, Y., Nakhle, J., Galloni, M., and Vignais, M.L. (2018). The role of metabolism and tunneling nanotube-mediated intercellular mitochon-dria exchange in cancer drug resistance. Biochem. J. 475, 2305-2328. https://doi.org/10.1042/bcj20170712.

49. Goliwas, K.F., Libring, S., Berestesky, E., Gholizadeh, S., Schwager, S.C., Frost, A.R., Gaborski, T.R., Zhang, J., and Reinhart-King, C.A. (2023). Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer. J. Cell Sci. 136, jcs260419. https://doi.org/10.1242/jcs.260419.

50. Halász, H., Tárnai, V., Matkó, J., Nyitrai, M., and Szabó-Meleg, E. (2024). Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes. Cells 13, 607. https://doi.org/10.3390/cells13070607.

51. Wühr, M., Obholzer, N.D., Megason, S.G., Detrich, H.W., and Mitchison, T.J. (2011). Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos. Methods Cell Biol. 101, 1-18. https://doi.org/10.1016/B978-0-12-387036-0-00001-3.

52. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682. https://doi.org/10.1038/nmeth.2019.
(B) Relative mean fluorescent intensity (MFI) quantification for timelapse (A). Linear regression fitting for donor and acceptor cells performed in GraphPad Prism software, equations and goodness of fit (R²) visualized on the graph.

(C) Representative image of the zebrafish embryo cells connected by TNT-like structures, where some connections contain mitochondria (green arrowhead). Red arrowhead points at TNT-like structure quantified as not containing mitochondria.

(D) Quantification of the relative number of TNT-like connections containing mitochondria (red part – no mitochondria, green part – containing mitochondria), n=564.

(E) Time-lapse recording of the zebrafish embryo at 8hpf shows two cells connected by a membranous TNT-like connection which enable the transfer of mitochondria (yellow arrowhead).

(F) Representative images showing magenta Lyn-HaloTag 647 or Eps8-dCAP-HaloTag 647 acceptor cells containing green dots of EEA1-positive vesicles (yellow arrowheads). Green signal on the left panels was enhanced compared to the right panels, in order to better visualize vesicles in acceptor cells.

(G) Quantification of the relative amount of acceptor cells (normalized to the amount of donor cells) that received EEA1-positive vesicles. Control group: n=17, median 0.02344, Eps8-dCAP overexpression group: n=18, median 0.09467. p= 0.0184, analysed by Mann Whitney test.

Scale bars are 10µm, time is indicated as: h:mm:ss.

# Methods

## Zebrafish husbandry

AB wild-type zebrafish (Danio rerio) were maintained in 3,5L tanks at a maximal density of seven per liter, in 28.5°C and pH 7.4 water, on a 14 h light/10 h dark cycle following to European Union guidelines for handling of laboratory animals. All procedures of animal caring were carried out in accordance with the official regulatory standards of the department of Paris and conformed to French and European ethical and animal welfare directives.

## Plasmids

The following plasmids were used to make mRNA for micro-injections in zebrafish embryos:

* pXT7-lyn-mKATE-E2A-phb-EGFP, pDEST-LifeAct-mKATE-E2A-CEP55-EGFP, pDEST-LifeAct-mKATE-E2A-EGFP-tubulin, pDEST-lyn-Cerulean, pHTC-lyn-HaloTag, pDEST-LifeAct-mKATE-E2A-EGFP-EEA1, pDEST-Dendra2, pDEST-zEps8-HaloTag, pDEST-mEps8dCAP-HaloTag.

All plasmids were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs). pXT7 and pDEST vectors were a kind gift from the group of Laure Bally-Cuif, pG1-flk1-eGFP-tubulinα2 was a kind gift from Anne Schmidt, pENTR_Mm_CEP55(WT) was a gift from Guillermo de Cárcer & Marcos Malumbres (Addgene plasmid # 136333; http://n2t.net/addgene:136333; RRID: Addgene_136333),
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>HaloTag 646 Janelia Fluor ligand</td><td>Promega</td><td>Cat#GA1120</td></tr><tr><td>CK-666 Arp2/3 inhibitor</td><td>Sigma-Aldrich</td><td>Cat#SML0006; CAS 442633-00-3</td></tr><tr><td>Pronase</td><td>Roche</td><td>Cat# PRON-RO</td></tr><tr><td>Rhodamine-phalloidin</td><td>Invitrogen</td><td>Cat#R415</td></tr><tr><td>CellMask Deep Red Actin Tracking Stain</td><td>Invitrogen</td><td>Cat#A57245</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>Cat#M1404; CAS 31430-18-9</td></tr><tr><td colspan="3">Critical commercial assays</td></tr><tr><td>NEBuilder® HiFi DNA Assembly Master Mix</td><td>New England Biolabs</td><td>Cat#E2621</td></tr><tr><td>mMessage mMachine SP6 Transcription Kit</td><td>Invitrogen</td><td>Cat#AM1340</td></tr><tr><td>mMessage mMachine T7 Transcription Kit</td><td>Invitrogen</td><td>Cat#AM1344</td></tr><tr><td colspan="3">Experimental models: Cell lines</td></tr><tr><td>Zebrafish: PAC2 cells line</td><td>Laboratory of Steffen Scholpp, University of Exeter (Stanganello et al.*)</td><td>RRID: CVCL_5853</td></tr><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>Zebrafish: AB wild-type</td><td>Zebrafish Projects Hub, Institut Pasteur</td><td>RRID: ZFIN_ZDB-GENO-960809-7</td></tr><tr><td colspan="3">Recombinant DNA</td></tr><tr><td>Plasmid: pXT7-lyn-mKATE-E2A-phb-EGFP</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-LifeAct-mKATE-E2A-EGFP-CEP55</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-LifeAct-mKATE-E2A-EGFP-tubulin</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-lyn-mKATE-E2A-EGFP-tubulin</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-lyn-Cerulean</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-lyn-GFP</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pHTC-lyn-HaloTag</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-LifeAct-mKATE-E2A-EGFP-EEA1</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-Dendra2</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-Dendra2-E2A-EGFP-CEP55</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-mEps8-HaloTag</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pDEST-mEps8dCAP-HaloTag</td><td>This paper</td><td>N/A</td></tr><tr><td>Plasmid: pCS2++-zfWnt8a-mCherry</td><td>Laboratory of Steffen Scholpp, University of Exeter (Stanganello et al.*)</td><td>N/A</td></tr><tr><td colspan="3">Software and algorithms</td></tr><tr><td>Fiji ImageJ</td><td>Schindelin et al.⁵²</td><td>https://imagej.net/software/fiji/downloads</td></tr><tr><td>Imaris v9</td><td>Oxford Instruments</td><td>https://imaris.oxinst.com</td></tr><tr><td>SHADE Shiny application</td><td>Bioinformatics and Biostatistics Hub, Institut Pasteur</td><td>https://shade.pasteur.fr</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>https://www.graphpad.com</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>N/A</td></tr><tr><td colspan="3">Other</td></tr><tr><td>Nikon Ti2E Spinning disk microscope</td><td>Nikon</td><td>N/A</td></tr><tr><td>Ti2 FRAP module</td><td>Nikon</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
actc1b-mKate2-EEA1 was a gift from Rob Parton (Addgene plasmid # 109508 ; http://n2t.net/addgene:109508 ; RRID: Addgene_109508), PhOTO-M (pMTB:memb-Dendra2-2A-H2B-Cerulean) was a gift from Periklis Pantazis (Addgene plasmid # 92401 ; http://n2t.net/addgene:92401 ; RRID: Addgene_92401), pHTC HaloTag® CMV-neo vector was purchased from Promega, pXT7-lyn-mKATE-E2A-phb-EGFP was kindly assembled by Frédéric Rosa.

Capped mRNA was prepared from purified linearized plasmids, using mMessage mMachine SP6 or T7 Transcription Kits (Invitrogen), following manufacturer's protocol. Use of E2A sequence in the plasmids gives a possibility to inject single mRNA that will be transcribed into two separate proteins.

## Embryo micro-injections

For visualization of TNT-like connections, zebrafish embryos were micro-injected with mRNA at 16-cell stage at concentration of 100 ng/µl, into one of the central cells. For clonal expression, injection of *lyn*-Cerulean mRNA (100 ng/µl) at 8-cell stage was followed by injection of *lyn*-mKATE-E2A-phb-EGFP mRNA (100 ng/µl) at 16-cell stage. For Eps8 overexpression, embryos were injected at 1-cell stage with *Eps8-HaloTag* mRNA (100 ng/µl) and further injected with *lyn*-mKATE-E2A-phb-EGFP mRNA (50 ng/µl) at 16-cell stage. Embryos injected with only *lyn*-mKATE-E2A-phb-EGFP mRNA (100 ng/µl) at 16-cell stage were used as control.

## Embryo mounting and image acquisition

Imaging was performed live at gastrula stage of development (7-9 hpf) using Nikon Ti2E Spinning disk microscope with 40x NA 1.15 WD 0.6 water immersion objective. Prior to imaging, embryos were dechorionated at 5-6 hpf, by treatment with 0,5 mg/ml pronase at 25°C for 2 minutes, followed by manual removal of chorions in agarose-coated dishes using Dupont N5 forceps. Dechorionated embryos were mounted into custom-made agar chambers in a 35 mm glass bottom µ-dish (Ibidi) and oriented for the animal pole to face the coverslip.

Preparation of agar chambers: agar wells were prepared using a custom made PDMS (Polydimethylsiloxane) mold, designed according to Wühr et al. ³⁸, with slight modifications. In short, a mold with 16 wells, with sides from 550 µm to 700 µm, was placed in a 35 mm glass bottom µ-dish. Wells were created by pouring 1,5 ml of 2% melted agarose into the µ-dish with the mold inside. After agar solidifying, the mold was carefully removed.

To visualize HaloTag expression, dechorionated embryos were incubated with HaloTag 647 ligand (Promega) at 28°C for 1 hour, followed by washing in E3 medium and mounting. For CK666 experiments, embryos were mounted into E3 medium containing either DMSO (control), or 50 µM CK666, or 200 µM CK666 and incubated for 1 hour at 28°C prior to imaging.
<figure><img src="image_29.png" /></figure>

**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Stereo microscope Zeiss Stemi 2000</td><td>Zeiss</td><td>N/A</td></tr><tr><td>Sensor Technics Picospritzer III</td><td>Sensor Technics | Parker Automation</td><td>N/A</td></tr><tr><td>Olympus SZX16 stereo microscope</td><td>Olympus</td><td>N/A</td></tr></tbody></table>

**EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

**PAC2 cells**

PAC2 zebrafish fibroblast cells (RRID:CVCL_5853), derived from 24h old embryos, were kindly provided by Steffen Scholpp (University of Exeter). Cells were maintained in Lebowitz-15 media supplemented with 10% fetal bovine serum (Gibco) and 1x Penicillin-Streptomycin (Gibco), at 28°C and without additional CO₂ supply.

**Zebrafish**

Zebrafish embryos were obtained from AB wild-type zebrafish (Danio rerio). Zebrafish were maintained in 3,5L tanks at a maximal density of seven per liter, in 28.5°C and pH 7.4 water, on a 14 h light/10 h dark cycle following to European Union guidelines for handling of laboratory animals. All procedures of animal caring were carried out in accordance with the official regulatory standards of the department of Paris and conformed to French and European ethical and animal welfare directives.

The offspring of the same cross was selected for all experimental groups, based on anatomical/developmental good health. Injections were performed at 1-cell, 8-cell, or 16-cell stage. Embryos were maintained at 28°C in E3 medium until gastrula stage (7hpf), when they mounted for live imaging. The imaging was performed between 7 hpf and 9 hpf.

**METHOD DETAILS**

**Plasmids and mRNA**

The plasmids that were used to prepare mRNA for micro-injections in zebrafish embryos are listed in the key resources table.

pXT7-lyn-mKATE-E2A-phb-EGFP was a kind gift from Frédéric Rosa (Bally-Cuif group, Institut Pasteur), pCS2+-zfWnt8a-mCherry was kindly provided by Steffen Scholpp (University of Exeter). Other plasmids used in this study were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs), according to the manufacturer's instructions, using the following fragments as templates: pXT7 and pDEST vectors were a kind gift from the group of Laure Bally-Cuif (Institut Pasteur), pG1-flk1-eGFP-tubulinα2 was a kind gift from Anne Schmidt (Institut Pasteur), pENTR_Mm_CEP55(WT) was a gift from Guillermo de Cárcer & Marcos Malumbres (Addgene plasmid # 136333; http://n2t.net/addgene:136333; RRID:Addgene_136333), actc1b-mKate2-EEA1 was a gift from Rob Parton (Addgene plasmid # 109508; http://n2t.net/addgene:109508; RRID:Addgene_109508), PhOTO-M (pMTB:memb-Dendra2-2A-H2B-Cerulean) was a gift from Periklis Pantazis (Addgene plasmid # 92401; http://n2t.net/addgene:92401; RRID:Addgene_92401), pHTC HaloTag® CMV-neo vector was purchased from Promega.

Capped mRNA was prepared from purified linearized plasmids, using mMessage mMachine SP6 or T7 Transcription Kits (Invitrogen), following manufacturer's protocol. Use of E2A sequence in the plasmids allows to inject single mRNA that will be transcribed into two separate proteins.

**Embryo micro-injections**

For visualization of TNT-like connections, zebrafish embryos were micro-injected with mRNA of choice at 16-cell stage at concentration of 100 ng/µl, into one of the central cells (Figure 1A) using Zeiss Stemi 2000 stereo microscope and Sensor Technics Picospritzer III pump. For clonal expression, injection of *lyn-Cerulean* mRNA (100 ng/µl) at 8-cell stage was followed by injection of *lyn-mKATE-E2A-phb-EGFP* mRNA (100 ng/µl) at 16-cell stage (Figure 2C). For Eps8 overexpression, embryos were injected at 1-cell stage with *mEps8-HaloTag* mRNA (100 ng/µl) or *mEps8dCAP-HaloTag* mRNA (100 ng/µl) and further injected with *lyn-mKATE-E2A-phb-EGFP* mRNA (50 ng/µl), or *LifeAct-mKATE-E2A-EGFP-CEP55* mRNA (50 ng/µl), or a mixture *lyn-EGFP +zfWnt8a-mCherry* mRNAs (100 ng/µl each) at 16-cell stage. Embryos injected with only *lyn-mKATE-E2A-phb-EGFP* mRNA (100 ng/µl) at 16-cell stage were used as control.

**Embryo mounting and image acquisition**

Imaging was performed live at gastrula stage of development (7-9 hpf) using Nikon Ti2E Yokagawa CSU-W1 Spinning disk microscope with 40x NA 1.15 WD 0.6 water immersion objective and Photometrics Prime 95B camera (pixel size 11 µm, 1200×1200 pixels). Dechorionated embryos were mounted into custom-made agar chambers in a 35 mm glass bottom µ-dish (Ibidi), oriented for the animal pole to face the coverslip and imaged up to 50 µm from the animal pole.

**Preparation of agar chambers**

Agar wells were prepared using a custom made PDMS (Polydimethylsiloxane) mold, designed according to Wühr et al.,⁵¹ with a modification in the number of wells. The custom mold had 16 square wells, with sides 550, 600, 650 or 700 µm. To create wells,
## *Dendra2* *photoconversion* and *time-lapse imaging*

Cytoplasmic *dendra2* mRNA was co-injected together with *lyn-HaloTag* mRNA at 16-cell stage. Dechorionated embryos were incubated with HaloTag 647 ligand (Promega) at 28°C for 1 hour, followed by washing in E3 medium and mounting. Photoconversion of Dendra2 was performed using the FRAP module of Nikon Ti2E Spinning disk microscope, by 0,5 second exposure to 405 nm laser (at 25% laser power). ROI was imaged for 30 minutes with the time interval of 40 seconds between the stacks.

For all presented time-lapse recordings, time interval was kept in the range of 20-45 seconds between the stacks, representing the best time resolution available on the system.

## *Image analysis and statistics*

3D renderings of acquired Z-stacks were generated using the Imaris® software (version 9, Bitplane). The total number of labelled cells in the field of view was counted manually using Imaris Spots function. The length of every TNT-like connection was measured using Imaris Measurement points tool. The protrusion was annotated as a TNT-like connection if it was connecting two labelled cells, seemed continuous and had length above 5 µm. Every image represented separate embryo and had 50-100 labelled cells in the field of view, 20-40 images per group were analyzed.

For Dendra2 photoconversion experiment analysis, SUM intensity projection of the movie was used to measure mean fluorescent intensity (MFI) of donor cell, acceptor cell and 3 neighboring cells using Fiji software. Measured MFI was corrected for bleaching, using the bleaching speed calculated from another group of cells converted in the same embryo. For the visualization on the graph, corrected MFIs were normalized to the first time point MFI of the donor cell.

Statistical analysis was performed using Microsoft Excel and GraphPad Prism. All the statistical tests performed were two-tailed, and their significance level was set at 5%. To compare relative numbers of connected cells Mann Whitney test and Kruskal Wallis test were used, to compare average lengths of connections t-test and one-way ANOVA were used, to compare length distributions chi-square test was used.
the mold was placed in a 35 mm glass bottom µ-dish (Ibibi) and 1,5 ml of 2% melted agarose was poured into the µ-dish with the mold inside. After agar solidifying, the mold was carefully removed.

### Embryo treatment

Prior to imaging, embryos were dechorionated at 5-6 hpf by treatment with 0,5 mg/ml pronase (Roche) solution in E3 medium, at 25°C for 2 minutes, followed by manual removal of chorions in agarose-coated dishes using Dupont N5 forceps.

To visualize HaloTag expression, dechorionated embryos were incubated with HaloTag 646 Janelia Fluor ligand (Promega) at 28°C for 1 hour, followed by washing in E3 medium and mounting. For CK666 experiments, embryos were mounted into E3 medium containing either DMSO (control) or 50 µM CK666 (Sigma-Aldrich) and incubated for 1 hour at 28°C prior to imaging. For nocodazole treatment experiment, embryos were mounted into E3 medium containing either DMSO (control) or 1 µM nocodazole (Sigma) and incubated for 1 hour at 28°C prior to imaging.

### Imaging the effect of the treatments on the development of the embryos

Embryos injected with mEps8dCAP-HaloTag mRNA or treated with 50 µM CK666, as well as respective controls, were incubated in E3 medium at 28°C until 30 hpf. At 30 hpf, embryos were imaged with Olympus stereo microscope SZX16 supplied with DP73 camera. Based on visual observation, embryos were classified as normally developing, with mild or severe abnormalities.

### Dendra2 photoconversion and time-lapse imaging

Cytoplasmic *dendra2* or *dendra2-E2A-EGFP-CEP55* mRNA was co-injected together with *lyn-HaloTag* mRNA (100 ng/µl each) at 16-cell stage. Dechorionated embryos were incubated with HaloTag Janelia Fluor 646 ligand (Promega) at 28°C for 1 hour, followed by washing in E3 medium and mounting. Photoconversion of Dendra2 was performed using the FRAP module of Nikon Ti2E Spinning disk microscope, by 0,5 second exposure to 405 nm laser (at 25% laser power). ROI was imaged for 30 minutes with the time interval of 40 seconds between the stacks.

For all presented time-lapse recordings, time interval was kept in the range of 20-45 seconds between the stacks, representing the best time resolution available on the system.

### PAC2 experiments

PAC2 zebrafish cells were seeded for imaging at 70% density in 35 mm glass bottom µ-dishes (Ibidi). For TNT-counting, cells were fixed with 4% PFA and 0,05% glutaraldehyde for 20 minutes at 28°C and further labelled with rhodamine-phalloidin (Invitrogen) diluted 1/250 in PBS, for 20 minutes at room temperature. For live timelapse imaging, cells were labelled using CellMask Deep Red Actin Tracking Stain (Invitrogen), according to the manufacturer's protocol. Imaging was performed in phenol-free L15 media (Sigma) using Nikon Ti2E Spinning disk microscope with 40x NA 1.15 WD 0.6 water immersion objective, ROI was imaged for 60 minutes with the time interval of 40 seconds between the stacks.

### Image analysis

3D renderings of acquired Z-stacks were generated using the Imaris® software (version 9, Oxford Instruments). The total number of labelled cells in the field of view was counted manually using Imaris Spots function. The length of every TNT-like connection was measured using Imaris Measurement points tool. The protrusion was annotated as a TNT-like connection if it was connecting two labelled cells, seemed continuous and had length above 5 µm. Every image represented separate embryo and had 50-100 labelled cells in the field of view.

For Dendra2 photoconversion experiment analysis, SUM intensity projection of the movie was used to measure mean fluorescent intensity (MFI) of donor cell, acceptor cell and 3 neighboring cells using Fiji software.⁵² Measured MFI was corrected for bleaching, using the bleaching speed calculated from another group of cells converted in the same embryo. For the visualization on the Graph 4B, corrected MFIs were normalized to the first time point MFI of the donor cell; for Graph S4C corrected MFIs of acceptor cells were normalized to the first time point MFI of each acceptor cell.

### QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed using Microsoft Excel and GraphPad Prism. To compare relative numbers of connected cells Mann Whitney test was used. Illustrations of these statistical analyses are displayed as dot plots, violin plots or box plot, showing the median and 25%-75% quartiles. To compare average lengths of connections Student unpaired two-tailed t-test was used, the Shapiro-Wilk normality test was used to confirm the normality of the data. Illustrations of these statistical analyses are displayed as violin plots showing the mean +/- standard deviation (SD). To compare connection length distributions and developmental abnormalities ratios the chi-squared test was used. p-values smaller than 0.05 were considered as significant. *, p<0.05, **, p < 0.01, ***, p < 0.001.

Each experiment was performed in at least 10 biological replicates. The necessary number of replicates for each experiment was estimated depending on the effect size using SHADE application developed by the Bioinformatics and Biostatistics Hub of Institut Pasteur. N numbers, mean or median numbers and exact p-values for all experiments are indicated in the respective figure legends.
**Supplementary files' legends**

**Supplementary figure 1.** Stable cytokinetic bridges during zebrafish gastrulation.
In several embryos we could observe unexpectedly long cytokinetic bridges. Timelapse recording at 8 hpf shows two pairs of daughter cells (white and black asterisks point corresponding pairs) connected by mitochondria-containing cytokinetic bridge that remains stable for at least 30 minutes of recording.

**Supplementary figure 2.**
(A, B, C) Violin plots of length of TNT-like connections following treatments: Eps8-WT overexpression (A), Eps8-dCAP overexpression (B), CK666 treatment (C).
(A) control group: n=220, mean 15.30 µm, Eps8-WT overexpression group: n=247, mean 12.99 µm. p= 0.0011, analysed by t-test.
(B) control group: n=343, mean 11.77 µm, Eps8-dCAP overexpression group: n=489, mean 11.20 µm. p= 0.1717, analysed by t-test.
(C) DMSO control group: n= 541, mean 11.29 µm, CK666 50 µM group: n=328, mean 12.90 µm, CK666 200 µM group: n=298, mean 10.34 µm. p-values are <0.0001 for DMSO vs. CK666 50µM, 0.0190 for DMSO vs. CK666 200µM and <0.0001 for CK666 50µM vs. CK666 200µM, analysed by one-way ANOVA.
(D) Violin plot of length of CEP55-positive connections following CK666 treatment. DMSO control group: n= 82, mean 4.639 µm, CK666 50 µM group: n=51, mean 5.572 µm. p= 0.0192, analysed by t-test.

**Supplementary figure 3.** Toxicity of CK666 treatment on zebrafish embryos.
Zebrafish embryos were treated with CK666 for 1 hour at the 6 hpf stage of development and left at 28°C for 24h and for 48h. Treatment with 50 µM CK666 did not alter the gross morphology of the embryos, while treatment with 200 µM CK666 resulted in embryonic death at early stages and twisting of the tail of the survived embryos at 48 hpf.

**Movie 1.** Stable cytokinetic bridges during zebrafish gastrulation.
Time-lapse recording of the zebrafish embryo at 8 hpf shows two pairs of daughter cells connected by mitochondria-containing cytokinetic bridges (yellow arrowheads) that remain stable for at least 30 minutes of recording.

**Movie 2.** TNT-like structures formed from two filopodia.
Time-lapse recording of the zebrafish embryo at 8 hpf shows two neighboring cells extending filopodia (white arrowheads) that subsequently merge into single membranous connection that remains stable for at least 5 minutes.

**Movie 3.** TNT-like structures formed by cell dislodgement in parallel with cell division.
Time-lapse recording of the zebrafish embryo at 8 hpf shows three neighboring cells forming TNT-like connections by cell dislodgement (yellow arrowheads), while the top cell is dividing (white asterisks). Daughter cells maintain connections with lower cells for at least 15 minutes (yellow arrowheads), while cytokinetic bridge between daughter cells is also visible (white arrowhead).
**Movie 4.** TNT-like connections elongate along each other.

Time-lapse recording of the zebrafish embryo at 8hpf shows two cells belonging to different cell populations (top red cell and bottom green cell), extending protrusions of corresponding colors (red and green arrowheads) that elongate along each other.

**Movie 5.** Zebrafish TNTs allow transfer of cytoplasmic Dendra2 protein.

Time-lapse recording of the zebrafish embryo at 8hpf shows two cells connected by a membranous TNT-like connection (yellow arrowhead). Cytoplasmic Dendra2 was photoconverted to red in the cell on the left (donor), recording shows that cell on the right (acceptor) receives red labeling within 20 minutes. In the end of the recording connection breaks (white arrowhead).

**Movie 6.** Zebrafish TNTs allow transfer of mitochondria.

Time-lapse recording of the zebrafish embryo at 8hpf shows two cells connected by a membranous TNT-like connection, which enables the transfer of mitochondria (white arrowhead).

**Movie 7.** Zebrafish TNTs allow transfer of early endosomes.

Timelapse recording of the zebrafish embryo at 8hpf shows actin-rich connection between two cells (white arrowhead) that allows the transfer of early endosomes (yellow arrowhead).
References

1. Mattila, P.K., and Lappalainen, P. (2008). Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9, 446-454. 10.1038/nrm2406.

2. Ramirez-Weber, F.A., and Kornberg, T.B. (1999). Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599-607. 10.1016/s0092-8674(00)80771-0.

3. Fereres, S., Hatori, R., Hatori, M., and Kornberg, T.B. (2019). Cytoneme-mediated signaling essential for tumorigenesis. PLoS Genet 15, e1008415. 10.1371/journal.pgen.1008415.

4. Stanganello, E., Hagemann, A.I., Mattes, B., Sinner, C., Meyen, D., Weber, S., Schug, A., Raz, E., and Scholpp, S. (2015). Filopodia-based Wnt transport during vertebrate tissue patterning. Nat Commun 6, 5846. 10.1038/ncomms6846.

5. Abounit, S., and Zurzolo, C. (2012). Wiring through tunneling nanotubes--from electrical signals to organelle transfer. J Cell Sci 125, 1089-1098. 10.1242/jcs.083279.

6. Sisakhtnezhad, S., and Khosravi, L. (2015). Emerging physiological and pathological implications of tunneling nanotubes formation between cells. Eur J Cell Biol 94, 429-443. 10.1016/j.ejcb.2015.06.010.

7. Chastagner, P., Loria, F., Vargas, J.Y., Tois, J., I Diamond, M., Okafo, G., Brou, C., and Zurzolo, C. (2020). Fate and propagation of endogenously formed Tau aggregates in neuronal cells. EMBO Mol Med 12, e12025. 10.15252/emmm.202012025.

8. Dilsizoglu Senol, A., Samarani, M., Syan, S., Guardia, C.M., Nonaka, T., Liv, N., Latour-Lambert, P., Hasegawa, M., Klumperman, J., Bonifacino, J.S., and Zurzolo, C. (2021). α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes. PLoS Biol 19, e3001287. 10.1371/journal.pbio.3001287.

9. Pepe, A., Pietropaoli, S., Vos, M., Barba-Spaeth, G., and Zurzolo, C. (2022). Tunneling nanotubes provide a route for SARS-CoV-2 spreading. Sci Adv 8, eabo0171. 10.1126/sciadv.abo0171.

10. Hashimoto, M., Bhuyan, F., Hiyoshi, M., Noyori, O., Nasser, H., Miyazaki, M., Saito, T., Kondoh, Y., Osada, H., Kimura, S., et al. (2016). Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages. J Immunol 196, 1832-1841. 10.4049/jimmunol.1500845.

11. Taiarol, L., Formicola, B., Fagioli, S., Sierrri, G., D'Aloia, A., Kravicz, M., Renda, A., Viale, F., Dal Magro, R., Ceriani, M., and Re, F. (2021). The 3.0 Cell Communication: New Insights in the Usefulness of Tunneling Nanotubes for Glioblastoma Treatment. Cancers (Basel) 13. 10.3390/cancers13164001.

12. Pinto, G., Saenz-de-Santa-Maria, I., Chastagner, P., Perthame, E., Delmas, C., Toulas, C., Moyal-Jonathan-Cohen, E., Brou, C., and Zurzolo, C. (2021). Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids. Biochem J 478, 21-39. 10.1042/bcj20200710.

13. Pinto, G., Brou, C., and Zurzolo, C. (2020). Tunneling Nanotubes: The Fuel of Tumor Progression? Trends Cancer 6, 874-888. 10.1016/j.trecan.2020.04.012.
14. Korenkova, O., Pepe, A., and Zurzolo, C. (2020). Fine intercellular connections in development: TNTs, cytonemes, or intercellular bridges? *Cell Stress* 4, 30-43. 10.15698/cst2020.02.212.

15. Haglund, K., Nezis, I.P., and Stenmark, H. (2011). Structure and functions of stable intercellular bridges formed by incomplete cytokinesis during development. *Commun Integr Biol* 4, 1-9. 10.4161/cib.4.1.13550.

16. Caneparo, L., Pantazis, P., Dempsey, W., and Fraser, S.E. (2011). Intercellular bridges in vertebrate gastrulation. *PLoS One* 6, e20230. 10.1371/journal.pone.0020230.

17. Henderson, J.M., Ljubojevic, N., Chaze, T., Castaneda, D., Battistella, A., Gianetto, Q.G., Matondo, M., Descroix, S., Bassereau, P., and Zurzolo, C. (2022). Arp2/3 inhibition switches Eps8's network associations to favour longer actin filament formation necessary for tunneling nanotubes. *bioRxiv*, 2022.008.2024.504515. 10.1101/2022.08.24.504515.

18. Ljubojevic, N., Henderson, J.M., and Zurzolo, C. (2021). The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions. *Trends Cell Biol* 31, 130-142. 10.1016/j.tcb.2020.11.008.

19. Resnik, N., Erman, A., Veranič, P., and Kreft, M.E. (2019). Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes. *Histochem Cell Biol* 152, 311-317. 10.1007/s00418-019-01806-3.

20. Adar-Levor, S., Nachmias, D., Gal-Oz, S.T., Jahn, Y.M., Peyrieras, N., Zaritsky, A., Birnbaum, R.Y., and Elia, N. (2021). Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis. *Proc Natl Acad Sci U S A* 118. 10.1073/pnas.2021210118.

21. Fernández-Casañas, M., and Chan, K.L. (2018). The Unresolved Problem of DNA Bridging. *Genes* (Basel) 9. 10.3390/genes9120623.

22. Sartori-Rupp, A., Cordero Cervantes, D., Pepe, A., Gousset, K., Delage, E., Corroyer-Dulmont, S., Schmitt, C., Krijnse-Locker, J., and Zurzolo, C. (2019). Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. *Nature Communications* 10, 342. 10.1038/s41467-018-08178-7.

23. Chang, M., Lee, O.C., Bu, G., Oh, J., Yunn, N.O., Ryu, S.H., Kwon, H.B., Kolomeisky, A.B., Shim, S.H., Doh, J., et al. (2022). Formation of cellular close-ended tunneling nanotubes through mechanical deformation. *Sci Adv* 8, eabj3995. 10.1126/sciadv.abj3995.

24. Delage, E., Cervantes, D.C., Penard, E., Schmitt, C., Syan, S., Disanza, A., Scita, G., and Zurzolo, C. (2016). Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes. *Sci Rep* 6, 39632. 10.1038/srep39632.

25. Hetrick, B., Han, M.S., Helgeson, L.A., and Nolen, B.J. (2013). Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. *Chem Biol* 20, 701-712. 10.1016/j.chembiol.2013.03.019.

26. Bompard, G., Rabeharivelo, G., and Morin, N. (2008). Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway. *BMC Cell Biology* 9, 42. 10.1186/1471-2121-9-42.

27. Yamashita, Y.M., Inaba, M., and Buszczak, M. (2018). Specialized Intercellular Communications via Cytonemes and Nanotubes. *Annu Rev Cell Dev Biol* 34, 59-84. 10.1146/annurev-cellbio-100617-062932.
28. Mattes, B., Dang, Y., Greicius, G., Kaufmann, L.T., Prunsche, B., Rosenbauer, J., Stegmaier, J., Mikut, R., Ozbek, S., Nienhaus, G.U., et al. (2018). Wnt/PCP controls spreading of Wnt/beta-catenin signals by cytonemes in vertebrates. Elife 7. 10.7554/eLife.36953.

29. Rustom, A., Saffrich, R., Markovic, I., Walther, P., and Gerdes, H.H. (2004). Nanotubular highways for intercellular organelle transport. *Science* **303**, 1007-1010. 10.1126/science.1093133.

30. Onfelt, B., Nedvetzki, S., Benninger, R.K., Purbhoo, M.A., Sowinski, S., Hume, A.N., Seabra, M.C., Neil, M.A., French, P.M., and Davis, D.M. (2006). Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. *J Immunol* **177**, 8476-8483. 10.4049/jimmunol.177.12.8476.

31. Sáenz-de-Santa-María, I., Bernardo-Castiñeira, C., Enciso, E., García-Moreno, I., Chiara, J.L., Suarez, C., and Chiara, M.D. (2017). Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes. *Oncotarget* **8**, 20939-20960. 10.18632/oncotarget.15467.

32. Thisse, B., and Thisse, C. (2004). Fast Release Clones: A High Throughput Expression Analysis. *ZFIN Direct Data Submission*.

33. Vaggi, F., Disanza, A., Milanesi, F., Di Fiore, P.P., Menna, E., Matteoli, M., Gov, N.S., Scita, G., and Ciliberto, A. (2011). The Eps8/IRSp53/VASP network differentially controls actin capping and bundling in filopodia formation. *PLoS Comput Biol* **7**, e1002088. 10.1371/journal.pcbi.1002088.

34. Ariazi, J., Benowitz, A., De Biasi, V., Den Boer, M.L., Cherqui, S., Cui, H., Douillet, N., Eugenin, E.A., Favre, D., Goodman, S., et al. (2017). Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions. *Front Mol Neurosci* **10**, 333. 10.3389/fnmol.2017.00333.

35. Zurzolo, C. (2021). Tunneling nanotubes: Reshaping connectivity. *Curr Opin Cell Biol* **71**, 139-147. 10.1016/j.jeb.2021.03.003.

36. Cordero Cervantes, D., Khare, H., Wilson, A.M., Mendoza, N.D., Coulon-Mahdi, O., Lichtman, J.W., and Zurzolo, C. (2023). 3D reconstruction of the cerebellar germinal layer reveals tunneling connections between developing granule cells. *Sci Adv* **9**, eadf3471. 10.1126/sciadv.adf3471.

37. Gerdes, H.H., Rustom, A., and Wang, X. (2013). Tunneling nanotubes, an emerging intercellular communication route in development. *Mech Dev* **130**, 381-387. 10.1016/j.mod.2012.11.006.

38. Wühr, M., Obholzer, N.D., Megason, S.G., Detrich, H.W., and Mitchison, T.J. (2011). Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos. *Methods Cell Biol* **101**, 1-18. 10.1016/B978-0-12-387036-0.00001-3.
<figure><img src="image_2.png" /><figcaption>FIGURE 1</figcaption></figure>

LifeAct-mKATE-E2A-CEP55-GFP

CEP55+ connection

CEP55- connection

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /><figcaption>FIGURE 2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIGURE 3</figcaption></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2023.10.12.562005; this version posted October 12, 2023. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
<figure><img src="image_7.png" /><figcaption>FIGURE 4</figcaption></figure>

This version of the article is licensed under a Creative Commons Attribution 4.0 International license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as the work is properly cited.

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>SUPPLEMENTARY FIGURE 1</figcaption></figure>
SUPPLEMENTARY FIGURE 2

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<table><caption>SUPPLEMENTARY FIGURE 3</caption><thead><tr><th></th><th>control</th><th>50μM CK666</th><th>200μM CK666</th></tr></thead><tbody><tr><td>24 hpf</td><td></td><td></td><td></td></tr><tr><td>48 hpf</td><td></td><td></td><td></td></tr></tbody></table>