Tunneling nanotubes enable intercellular transfer in zebrafish embryos
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Abstract
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.
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e most important criteria to define TNTs is to assess their transfer function
I really loved this section!
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Tunneling nanotubes (TNTs)
A very cool paper, with some very cool images showing tunneling nanotubes in zebrafish embryos!
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At 200 µM, CK666 appeared to be toxic to the embryos, that were either dying at early stages or, if survived, had observable tail twist at 48 hpf
Is there a third intermediate concentration you could try to see if you get an intermediate effect?
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Figure 3.
Panels C and E and the significance labels are a bit confusing. I'm wondering if there's a different a way to display this data?
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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.
I really love this approach, and I think this data is very cool! I don't know much about these specific labels but I'm curious if you could see transfer of colors between cells at all?
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5 minutes after formation
Could you quantify the lifetime of different types of connections? Just wondering about different ways to classify TNTs vs other types of connections.
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We were able to show that connections in the embryo can be formed from two filopodia-like structures, similarly to TNTs in vitro
I love this figure and video of the two filopodia reaching out and connecting!
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Of interest, a portion of CEP55-negative connections had the length above 10 µm, and they could reach up to 30 µm in length
I'm interested in the thickness of the connections - the bridges seem quite thick.
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o 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 midbody marker CEP55 in the same cells
This is a clever way to differentiate between cytokinetic bridges and the TNT-like connections, but I don't know that it fully rules out that the TNT-like connections could be related to division. Is there a way to block division and see if you still get TNT-like connections?
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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
Did you only do this staining and analysis with TNT-like protrusions? It's clear that there's some diversity of the cytoskeletal composition of these structures, but I wonder if compared to other types of protrusions, some pattern might emerge. For example, comparatively, filopodia are likely only actin the majority of the time, making them quite different than these structures.
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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
Might be nice to add a citation here since you have specific number values.
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developing embryos
Have these been seen in living embryos before or is your study the first report of this?
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