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Chakraborty et al. Extracell Vesicles Circ Nucleic Acids 2023;4:27-43 https://dx.doi.org/10.20517/evcna.2023.05 Page 31

Mechanism of filopodia versus TNT formation
From a structural point of view, TNTs present striking homologies with filopodia . Consistent with that
[55]
observation, many of the molecular actors that have been identified so far to promote TNT formation also
play central roles in filopodial formation, such as Rab35, the unconventional Myosin X, Eps8 as well as
IRSp53 [53,54,56] . Therefore, a central question of the field lies in understanding whether TNTs originate from
[63]
the differentiation of a subset of filopodia as it is believed to be the case for cytonemes , or if they are
distinct structures from the beginning. Recent evidence obtained using cryo-correlative light and electron
microscopy (cryo-CLEM) of TNTs and VASP-induced filopodia in murine neuronal-like (CAD) cells leads
us to hypothesize that TNTs are unique structures from the beginning of their formation .
[30]

Cryo-CLEM showed that the Actin bundle within filopodia and TNTs arrange in hexagonal arrays with a
comparable average distance separating the filaments (~4.7 nm for filopodia, ~5.5 nm for TNTs). This
suggests redundancy in the Actin bundlers present in both structures. However, filopodia in CAD cells are
individual close-ended protrusions, while TNTs imaged at high resolution consist of a bundle of small
open-ended tubes called iTNTs (individual TNTs) running parallel to each other . Additionally, TNTs in
[30]
vitro are always non-adherent to the substrate, suggesting a different protein and lipid composition of the
membrane of the protrusions, or a different activation pattern of adherent proteins. These differences per se
are not sufficient to allow us to exclude the possibility that TNTs differentiate from preformed filopodia.
However, Actin filaments within each iTNT in the bundle run uninterrupted all along the imaged areas
(1.2-1.5 µm), and F-Actin continuity within filopodia is interrupted every 0.3 to 1.1 μm. These results,
associated with the fact that TNTs reach far greater distances compared to filopodia, suggest that different
Actin polymerizers with different processivities are responsible for the growth of protrusions. If so, it would
[57]
mean TNTs and filopodia arise from different molecular actors early during their formation .
Mechanism of ectosomes versus TNT formation
Surprisingly, TNTs also share interesting similarities with ectosomes. During early formation, they both rely
on the formation of microdomains at the plasma membrane regulating the recruitment of protein
complexes leading to negative membrane curvature. In fact, knock-down of IRSp53 was shown to decrease
ectosome shedding [64,65] . As such, the involvement of IRSp53 in TNT formation suggests the potential
[53]
convergence of signaling pathways in regulating ectosomes and TNTs [Figure 1]. Additionally, they have in
common the presence of tetraspanins such as CD9 and CD81, known to interact with integrins and to play
[66]
a major role in sperm-egg fusion during fertilization [Figure 1]. As both ectosomes and TNTs share a
similar fate (fusing with a neighbor cell), these membrane proteins could be involved in the same process.
Recent investigation on the proteome of TNTs versus EVs has shown the presence of specific but also
[60]
common components . Thus, it is possible that from an evolutionary point of view, TNTs emerge from
molecular pathways involved in filopodial formation and ectosome shedding. Further investigation will be
necessary to test this intriguing hypothesis.

FUNCTIONAL ROLES OF TUNNELING NANOTUBES
The major characteristic point that distinguishes TNTs from any other kind of cellular protrusions is their
ability to transfer cargoes between connected cells. The different cytosolic and/or engulfed materials that
have been reported to be transferred (ions, vesicles, nucleic acids, organelles, pathogens, proteins and
proteinaceous aggregates) suggest critical roles of TNTs in maintenance of homeostasis within the cellular
network, as well as in spreading of pathologies [28,59,67,68] [Figure 2]. Movement across a nanotube can occur
either uni-directionally or bi-directionally, depending on the context. As a plausible mechanism for diluting
the effects of stress, an unhealthy cell can transfer materials such as damaged organelles or protein
aggregates to the connected cell in a unidirectional manner . However, this unidirectional transfer can also
[69]
lead to spread of neurodegenerative pathologies such as PD and AD, wherein movement of α-Synuclein (α-