Deciphering the molecular mechanisms of TNT formation and function using a multi-omic approach

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Our research is focused on the role of tunneling nanotubes (TNTs)—a novel mechanism of functional connectivity between cells—in the spreading of viruses, misfolded protein aggregates (leading to neurodegenerative diseases), as well as the part they may play in the proliferation and persistence of cancer. TNTs have been found in numerous cell types, allowing the transport of cytosolic and membrane-bound molecules, organelles, calcium flux, and the spreading of pathogens. In vitro, these structures are very heterogeneous and numerous disparities have emerged both in their structure and functions. Similar filopodia- like structures also exist in vivo and in tissue explants. Unfortunately, little is currently known about the basic mechanism of TNT formation, their structural components, or the signaling pathways involved. Recent studies have revealed that TNTs do play an important physiological role in both health and disease. Indeed, TNTs are significant mediators of electrical, antigen, and genomic signaling, while also promoting cellular recovery after ischemic, inflamatory, and hypoxic injury. What's more, retroviruses, such as the HIV-1, HSV-1, HTLV-1, and influenza exploit these subcellular structures to facilitate infection by evading immune surveillance. Moreover, pathogenic particles and proteins, such as Aβ, prions, and HIV-1 Nef, are found to induce, and then usurp TNT-like structures to spread between cells. Spreading through TNTs is highly efficient, since it avoids diffusive transfer and evades immune detection. Finally, TNTs can mediate the direct transfer of metabolic and genetic material between tumor cells and their stroma enhancing tumor cell chemoresistance, tumor progression, and metastasis. With a previous NIH SCORE SC2 Pilot Project Award, we successfully developed a novel method to specifically isolate distinct protrusion subtypes—based on their morphology or fluorescent markers—using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrated that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Finally, our method confirmed that different subtypes of protrusions have distinct proteomes. Therefore, our method created a unique opportunity to characterize TNTs shedding light on their role in health and disease. In this SCORE SC1 grant, we propose a three-step strategy to utilize our LCM/MS method to study TNT formation and function. This entails: 1) Expanding the TNT proteome by incorporating different cell types, induction methods, and TNT substructures using our LCM/MS method; 2) Collecting the TNT transcriptome to limit the detection bias of the individual platforms while at the same time cross-validating TNT protein/pathway identifications; and, 3) Identifying conserved TNT proteins and pathways, as well as potentially druggable proteins and biomarkers.