Identification and characterization of TNTs in vitro and in vivo – LiveTuneL

WP1 employed micropatterns, biophysical assays, and live imaging to show that inhibition of Arp2/3 shifts actin dynamics to favor TNT formation through Eps8 and IRSp53 interaction.

WP2 developed a novel biochemical TNT purification strategy, followed by proteomic profiling, identifying CD9 and CD81 tetraspanins as functional regulators of TNT formation and vesicle transfer.

WP3 used live-cell imaging and fluorescent cargo transfer assays to characterize neuron-microglia TNTs in vitro. These were shown to mediate selective mitochondrial transfer and α-Syn aggregate spread, modulating neurodegenerative burden.

WP4 applied cryo-EM and functional assays to define TNT ultrastructure, revealing N-Cadherin as central for structural integrity and cargo flow, regulated via p120-catenin and NMIIA.

WP5 utilized zebrafish embryos with mosaic labeling and pharmacological perturbation to demonstrate functional, open-ended TNT-like connections in vivo, capable of transporting cargo during development.

Key discoveries include:

WP1: Demonstrated that linear actin polymerization, rather than branched networks, drives TNT elongation. Identified Eps8/IRSp53 cooperation as key to TNT architecture.

WP2: Replaced an inconsistent imaging screen with a proteomic strategy. Identified CD9 as essential for TNT stability and CD81 for functional cargo transfer—marking some molecular signatures of TNT functions.

WP3: Showed α-Synuclein promotes TNT connectivity. Neurons transfer α-Syn to microglia, while microglia send mitochondria to affected neurons—highlighting TNTs as mediators of neuroimmune interaction.

WP4: Uncovered a mechanosensitive TNT regulation mechanism driven by N-Cadherin and cytoskeletal tension. Described how actomyosin dynamics govern TNT cargo transport efficiency.

WP5: Provided first in vivo evidence for functional TNTs in vertebrate development. Demonstrated cargo and organelle transfer in zebrafish embryos, influenced by actin regulators and mirroring in vitro findings.

This is the first project to combine molecular, biophysical, and ultrastructural analysis of TNTs with in vivo functional demonstration. The discovery of functional, cargo-transferring TNTs in developing zebrafish provides a new paradigm for studying intercellular communication in embryogenesis.

The project opens new avenues for targeting TNTs in cancer and neurodegenerative diseases, where they may mediate both pathology and repair. The identification of some TNT-specific markers may offer opportunities for diagnostic and therapeutic innovation. Mechanistic insights into actin dynamics and N-Cadherin signaling further expand the field of membrane biology.

Future work will refine TNT classification, explore their immunological roles, and test therapeutic modulation. The zebrafish model provides a high-throughput platform to screen for TNT modulators in vivo, accelerating translational research on cell-cell communication.

Novel actin-based membrane protrusions named tunneling nanotubes (TNTs) have been found to connect remote cells allowing communication during development, and disease transmission, suggesting that TNTs are ubiquitous in both physiological and pathological contexts. However, their existence in vivo has not been demonstrated and the mechanisms of TNT formation and fusion with a target cell are unknown. This project seeks to understand how protein-protein/protein lipid specific interactions are involved in forming TNTs, and what are the mechanical and dynamical differences distinguishing TNTs from filopodia. Here the involvement of actin, actin-regulating proteins, membrane connectors, and novel candidates identified by a screening approach, will be ascertained in real time in vitro and correlated to mechanical measurements and then tested in vivo in zebrafish embryos. Complementary correlative cryoCLEM will elucidate native TNT structure in different cellular contexts and in the animal.