Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

Abstract

Tunneling nanotubes (TNTs) are open actin- and membrane-based channels, connecting remote cells and allowing direct transfer of cellular material (e.g. vesicles, mRNAs, protein aggregates) from cytoplasm to cytoplasm. Although they are important especially in pathological conditions (e.g., cancers, neurodegenerative diseases), their precise composition and their regulation were still poorly described. Here, using a biochemical approach allowing to separate TNTs from cell bodies and from extracellular vesicles and particles (EVPs), we obtained the full composition of TNTs compared to EVPs. We then focused to two major components of our proteomic data, the CD9 and CD81 tetraspanins, and further investigated their specific roles in TNT formation and function. We show that these two tetraspanins have distinct functions: CD9 participates in the initiation of TNTs, whereas CD81 expression is required to allow the functional transfer of vesicle in the newly formed TNTs, possibly by regulating fusion with the opposing cell.

Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking

“…If your cells are transfected with actin-chromobody-GFP (Manzano et al, 2022), no additional fluorescent dye is required to visualize TNTs. In contrast, if the cells do not express fluorescent protein, they are labeled with WGA-Alexa Fluor (488 /546/647) in a 1:1000 dilution in 1 ml of complete cell growth medium.…”

“…A good compromise to reconcile sufficient resolution and reduced phototoxicity is the use of several high-resolution microscopes with fast image acquisitions, such as spinning disk confocal microscopy, Airyscan confocal microscopy, or light sheet microscopy. Live imaging of TNTs can then be achieved with the further use of protein markers, e.g., fluorescent fusion proteins targeted to the plasma membrane or the actin cytoskeleton, such as actin-chromobody-GFP (Manzano et al, 2022), (see also Alternate Protocol 2). Furthermore, the entire plasma membrane of TNTs can be labeled with fluorescently conjugated proteins that bind glycolipids and glycoproteins, such as WGA (Pepe et al, 2023).…”

“…Alternatively, we can use SH-SY5Y that have been stably transfected with actinchromobody-GFP (Manzano et al, 2022).…”

“…Since it is very difficult to detect newly formed TNTs, we capture pre-existing TNTs and calculate their lifetime using Fiji software (Pepe et al, 2023). Importantly, the lifetime of TNTs may change depending on the use of a particular dye or fluorescent protein (e.g., WGA, actin-chromobody-GFP) as shown in Manzano et al, 2022and Pepe et al, 2023(Manzano et al, 2022Pepe et al, 2023).…”