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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 33

Transfer of signals and nucleic acids
In addition to Ca mediated signaling, TNTs are capable of transferring several components of different
2+
signaling pathways. Initial discoveries of such transfer were made in immune cells, where it was observed
that activation of Fas receptors on T-cells promoted TNT formation with neighboring T-cells, leading to
[80]
movement of membrane-bound FasL and active caspase-3 . Natural killer (NK) cells can form functional
nanotubes upon their activation and eventually cause cytotoxicity of target cells. Accumulation of DAP10,
and Major Histocompatibility Complex (MHC) class-I chain-related protein A (MICA) at the tip of
[81]
nanotubes between NK cells and target cells was sufficient for such immune interaction . The cytoplasmic
stain Calcein-AM was also observed to be transferred between mesenchymal multipotent stromal cells
[73]
(MMSCs) and rat renal tubular cells (RTCs) in a co-culture system . Similarly, other cytoplasmic stains
and dyes like CFSE and Lucifer Yellow have also been shown to move between cells via TNTs [75,80] . Notably,
cytosolic EGFP has recently been reported to move from layer I-III cortical astrocytes to layer V neurons via
[82]
tunneling nanotubes .
Finally, movement of nucleic acids between cells via TNTs provides a mechanism of regulation of gene
expression at a global level of connected cells. Several forms of nucleic acids have been reported to reach
target cells via the route of nanotubes, such as mitochondrial DNA [72,83] , messenger RNA [84,85] , viral
RNAs [35,46,86] , and regulatory miRNAs [87-89] . This potentially allows for the donor cell to regulate the
transcriptomic and metabolomic profiles of the target cells, with critical implications in neurological
pathologies, and other conditions such as cancers, involving regulatory nucleic acids.

Plasma membrane component, intracellular vesicles and organelles
Besides exchanging cytosolic materials, TNTs can allow the transfer of membrane components such as cell
surface receptors and membrane-anchored proteins. In their first description of TNTs, Rustom et al.
[41]
showed the transfer of membrane-bound (farnesylated) Ras to a connected PC12 neuronal cell . Cell
surface MHC-I can be present on nanotubes and be transferred between immune cells [75,90] . Additionally,
surface-expressed CD59 and CD81 have also been reported to be exchanged bidirectionally between Jurkat
[80]
T-cells .

Labeling of intracellular vesicles with lipophilic dyes and immunostaining with vesicle/organelle-specific
markers have proven to be reliable approaches in assessing vesicular transfer between donor and acceptor
cells via TNTs. Endosomes, lysosomes, mitochondria, Golgi, and endoplasmic reticulum have been reported
[91]
to utilize TNTs for transfer between cells (reviewed in ). However, the intercellular exchange of
mitochondria has been of particular interest because of their potential involvement in regulating the
metabolism of acceptor cells and alleviating the health of diseased cells [83,92,93] . Mitochondrial dysfunction in
dopaminergic neurons has been shown to be attenuated by transfer of functional mitochondria from
astrocytes . In the presence of pathogenic load of α-Syn in astrocytes, healthy mitochondria are transferred
[94]
[95]
via TNTs from neighboring astrocytes as a way of restoring homeostasis . Recent findings also suggest that
mitochondria move in TNTs from microglia to neuronal cells in co-culture, preferably to α-Syn loaded cells,
[96]
compared to healthy cells . Besides mitochondria, lysosomes actively move between cells via TNTs [41,97] .
Such transfer provides a route for the movement of α-Syn aggregates that hitchhike functionally
[70]
compromised lysosomes to spread and propagate α-Syn pathology .
Pathogens
Several pathogens such as bacteria and viruses utilize TNTs as a route of propagation. In the earliest
description, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been reported to move between
human monocyte-derived macrophages . Human Immunodeficiency virus (HIV) facilitates its spreading
[40]
by inducing TNT formation, eventually associating with endosomes and MVBs for transfer between