Proteomic Analysis Reveals CD9 and CD81 Tetraspanins as Key Regulators of Tunneling Nanotubes

Core Concepts

CD9 and CD81 tetraspanins play distinct and complementary roles in the formation and function of tunneling nanotubes, with CD9 stabilizing the nanotubes and CD81 facilitating the completion and functionality of these intercellular connections.

Abstract

The content describes a study that aimed to identify the structural components and regulators of tunneling nanotubes (TNTs), which are thin membranous channels that allow direct transfer of cellular material between remote cells. Using a biochemical approach to isolate TNTs from cell bodies and extracellular vesicles, the authors obtained the full proteomic composition of TNTs (TNTome) in U2OS cells.
The TNTome analysis revealed that it is enriched in membrane-associated proteins, cytoskeletal components, and factors involved in RNA processing and metabolism. Two major components identified were the tetraspanins CD9 and CD81, which are known markers of extracellular vesicles.
The authors further investigated the specific roles of CD9 and CD81 in TNT formation and function using the SH-SY5Y neuronal cell model. They found that CD9 plays a key role in stabilizing TNTs, as knocking out CD9 reduced the number of TNT-connected cells, while overexpressing CD9 increased TNT formation. In contrast, CD81 knockout did not affect TNT number but impaired the transfer of vesicles through the TNTs, suggesting a role in the completion and functionality of these structures.
Mechanistically, the authors showed that CD9 clustering on the cell surface, induced by an anti-CD9 antibody, led to an increase in TNT stability and duration. This stabilizing effect of CD9 did not require CD81. On the other hand, CD81 appeared necessary for the full completion of TNTs, as its knockout resulted in the accumulation of vesicles within the TNTs, which were unable to reach the opposing cell.
In summary, the study demonstrates that CD9 and CD81 have distinct, non-redundant functions in the regulation of TNTs - CD9 stabilizes the nanotubes, while CD81 facilitates the docking or fusion of the nanotubes with the opposing cell membrane, enabling the transfer of cellular material.

Stats

The mean diameter of extracellular vesicles and particles (EVPs) was 60 nm, while the mean diameter and length of TNTs were 69 nm and 372 nm, respectively.
The percentage of TNT-connected cells was reduced from 31.6% in wild-type to 25.2% in CD9 knockout, 30.4% in CD81 knockout, and 16.3% in CD9 & CD81 double knockout SH-SY5Y cells.
Overexpression of CD9 increased the percentage of TNT-connected cells from 29.8% in wild-type to 45.3%, while overexpression of CD81 had no significant effect (29.5%).
The percentage of acceptor cells receiving vesicle transfer from donor cells was reduced from 32.7% in wild-type to 18.4% in CD9 knockout, 19.1% in CD81 knockout, and 10.5% in CD9 & CD81 double knockout.
Overexpression of CD9 or CD81 in donor cells increased the percentage of acceptor cells receiving vesicle transfer to 61.3% and 55.6%, respectively.

Quotes

"CD9 participates in stabilizing TNTs, whereas CD81 expression is required to allow the functional transfer of vesicle in the newly formed TNTs, possibly by regulating docking to or fusion with the opposing cell."
"CD9 clustering on the cell surface, induced by an anti-CD9 antibody, led to an increase in TNT stability and duration."
"CD81 knockout resulted in the accumulation of vesicles within the TNTs, which were unable to reach the opposing cell."

Key Insights Distilled From

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

by Notario Manz... at www.biorxiv.org 12-22-2022

https://www.biorxiv.org/content/10.1101/2022.12.21.521537v2

Deeper Inquiries

How might the distinct roles of CD9 and CD81 in TNT regulation be exploited for therapeutic applications, such as in the context of neurodegenerative diseases or cancer?

The distinct roles of CD9 and CD81 in regulating TNT formation and function offer promising therapeutic opportunities in various disease contexts. In neurodegenerative diseases, where intercellular communication and transfer of cellular material play crucial roles, targeting CD9 and CD81 could potentially modulate TNT formation and enhance the transfer of essential molecules like mRNAs, proteins, or organelles between cells. For example, in conditions like Alzheimer's or Parkinson's disease, where impaired intercellular communication contributes to disease progression, enhancing TNT functionality through CD9 and CD81 modulation could facilitate the transfer of neuroprotective factors or clearance of toxic aggregates, potentially slowing down disease progression.
In cancer, where TNTs have been implicated in promoting tumor growth, metastasis, and resistance to therapy, targeting CD9 and CD81 could offer therapeutic strategies. By understanding the specific roles of these tetraspanins in TNT formation and function, interventions could be designed to disrupt TNT formation in cancer cells, thereby inhibiting the transfer of oncogenic signals, drug resistance factors, or metastasis-promoting molecules. Additionally, modulating CD9 and CD81 levels could potentially be used to enhance the transfer of anti-cancer agents or immune-stimulating molecules between cells, improving the efficacy of cancer treatments.
Overall, leveraging the distinct functions of CD9 and CD81 in TNT regulation could open up new avenues for therapeutic interventions in neurodegenerative diseases and cancer by targeting intercellular communication and material transfer mechanisms mediated by TNTs.

How might the distinct roles of CD9 and CD81 in TNT regulation be exploited for therapeutic applications, such as in the context of neurodegenerative diseases or cancer?

The distinct roles of CD9 and CD81 in regulating TNT formation and function offer promising therapeutic opportunities in various disease contexts. In neurodegenerative diseases, where intercellular communication and transfer of cellular material play crucial roles, targeting CD9 and CD81 could potentially modulate TNT formation and enhance the transfer of essential molecules like mRNAs, proteins, or organelles between cells. For example, in conditions like Alzheimer's or Parkinson's disease, where impaired intercellular communication contributes to disease progression, enhancing TNT functionality through CD9 and CD81 modulation could facilitate the transfer of neuroprotective factors or clearance of toxic aggregates, potentially slowing down disease progression.
In cancer, where TNTs have been implicated in promoting tumor growth, metastasis, and resistance to therapy, targeting CD9 and CD81 could offer therapeutic strategies. By understanding the specific roles of these tetraspanins in TNT formation and function, interventions could be designed to disrupt TNT formation in cancer cells, thereby inhibiting the transfer of oncogenic signals, drug resistance factors, or metastasis-promoting molecules. Additionally, modulating CD9 and CD81 levels could potentially be used to enhance the transfer of anti-cancer agents or immune-stimulating molecules between cells, improving the efficacy of cancer treatments.
Overall, leveraging the distinct functions of CD9 and CD81 in TNT regulation could open up new avenues for therapeutic interventions in neurodegenerative diseases and cancer by targeting intercellular communication and material transfer mechanisms mediated by TNTs.

How might the distinct roles of CD9 and CD81 in TNT regulation be exploited for therapeutic applications, such as in the context of neurodegenerative diseases or cancer?

The distinct roles of CD9 and CD81 in regulating TNT formation and function offer promising therapeutic opportunities in various disease contexts. In neurodegenerative diseases, where intercellular communication and transfer of cellular material play crucial roles, targeting CD9 and CD81 could potentially modulate TNT formation and enhance the transfer of essential molecules like mRNAs, proteins, or organelles between cells. For example, in conditions like Alzheimer's or Parkinson's disease, where impaired intercellular communication contributes to disease progression, enhancing TNT functionality through CD9 and CD81 modulation could facilitate the transfer of neuroprotective factors or clearance of toxic aggregates, potentially slowing down disease progression.
In cancer, where TNTs have been implicated in promoting tumor growth, metastasis, and resistance to therapy, targeting CD9 and CD81 could offer therapeutic strategies. By understanding the specific roles of these tetraspanins in TNT formation and function, interventions could be designed to disrupt TNT formation in cancer cells, thereby inhibiting the transfer of oncogenic signals, drug resistance factors, or metastasis-promoting molecules. Additionally, modulating CD9 and CD81 levels could potentially be used to enhance the transfer of anti-cancer agents or immune-stimulating molecules between cells, improving the efficacy of cancer treatments.
Overall, leveraging the distinct functions of CD9 and CD81 in TNT regulation could open up new avenues for therapeutic interventions in neurodegenerative diseases and cancer by targeting intercellular communication and material transfer mechanisms mediated by TNTs.