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

我们的研究集中在隧道纳米管(TNTs)的作用上，这是一种新的功能机制 细胞之间的连通性-在病毒的传播中，错误折叠的蛋白质聚集体(导致 神经退行性疾病)，以及它们可能在癌症的扩散和持久性中所起的作用。 TNTs在多种细胞类型中被发现，允许胞浆和膜结合的运输 分子、细胞器、钙flUX和病原体的传播。在体外，这些结构非常 它们在结构和功能上都出现了异质性和许多差异。类似的filpodia- 类似的结构也存在于体内和组织外植体中。不幸的是，目前人们对基本的 TNT的形成机制、其结构成分或所涉及的信号通路。 最近的研究表明，TNTs确实在健康和健康方面发挥着重要的生理作用 疾病。事实上，TNTs是电、抗原和基因组信号的重要媒介，同时也 促进细胞在缺血、炎症和缺氧损伤后的恢复。更重要的是，逆转录病毒，如 HIV-1、HSV-1、HTLV-1和流感病毒利用这些亚细胞结构通过逃避 免疫监视。此外，致病颗粒和蛋白质，如Aβ，Pron，和HIV-1Nef，是 被发现诱导，然后篡夺TNT样结构在细胞之间传播。通过TNTs传播的几率很高 高效，因为它避免了扩散转移和逃避免疫检测。最后，TNTs可以调解直接的 代谢和遗传物质在肿瘤细胞及其基质间的转移增强肿瘤细胞 化疗耐药、肿瘤进展和转移。 在之前的NIH SC2试点项目奖下，我们成功地开发了一种新的方法来 根据其形态或荧光标记，专门分离不同的突起亚型 激光捕获显微解剖(LCM)。结合独特的固定和蛋白质提取方案，我们 突破了微蛋白质组学的极限，并证明了来自LCM分离突起的蛋白质可以 使用超高场轨道搭载技术成功地、可重复性地通过质谱学进行鉴定。 最后，我们的方法证实了不同类型的突起具有不同的蛋白质组。因此，我们的 方法创造了一个独特的机会来描述TNTs的特征，揭示了它们在健康和疾病中的作用。 在这个SC1分数授予中，我们提出了一个三步策略，利用我们的LCM/MS方法来研究 TNT的形成和功能。这需要：1)通过结合不同类型的细胞来扩展TNT蛋白质组， 2)收集TnT转录组，以 限制单个平台的检测偏差，同时交叉验证TNT蛋白/途径 识别fi阳离子；以及，3)鉴定保守的TnT蛋白和途径，以及潜在的可药物 蛋白质和生物标志物。

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.