利用空間蛋白質體學揭示KIF5A–RAC1運輸模組驅動神經母細胞瘤中粒線體經隧道奈米管之輸送

Abstract

隧道奈米管（Tunneling nanotubes, TNTs）為F-actin構成的膜性突起結構，使細胞間長距離的物質傳遞更有效率。在高度異質性的幼兒實體腫瘤，像是神經母細胞瘤（neuroblastoma, NB）中，缺氧微環境會使細胞的代謝適應與細胞間合作更緊密。癌細胞可透過TNT介導的粒線體轉移，以維持代謝穩定並在壓力環境下提升存活能力。然而，目前與此過程相關的機制仍不清晰。 此研究建立一套針對TNT的空間蛋白質體學策略，以分析神經母細胞瘤SK-N-BE(2)C細胞株在常氧與缺氧共培養條件下所形成的TNT。透過光標記（photolabeling）方法選擇性富集TNT相關蛋白。質譜分析共鑑定出270,367個質譜訊號與56,207條胜肽。在篩選出具有兩條以上唯一胜肽支持的蛋白質後，相較於未進行光標記的對照組，建立了一組高可信度的TNT蛋白質體，共包含1,520個僅在光標記樣本中偵測到的蛋白質。 功能富集分析顯示，TNT蛋白質體與纖毛及微管調控過程具有顯著關聯，顯示 TNT 內的長距離物質運輸可能依賴微管導引機制。進一步聚焦於粒線體運輸，蛋白質間交互作用分析顯示 kinesin 馬達蛋白KIF5A為粒線體運輸的重要核心節點之一。在TNT富集的粒線體相關蛋白中，RAC1與MIRO1為唯二被預測可與KIF5A交互作用的蛋白。先前研究已證實MIRO1為TNT中粒線體運輸的重要接合蛋白，支持本研究的結果。 免疫細胞化學染色與鄰位連接技術進一步證實KIF5A與RAC1及粒線體在TNT中具有交互作用。利用 shRNA抑制KIF5A或RAC1表現，觀察到不影響TNT形態的前提下，TNT內的線粒體含量明顯減少，並損害了細胞間的線粒體轉移。此外，，利用流式細胞儀（flow cytometry）分析的結果與ICC一致，shRNA介導的KIF5A或RAC1基因敲低，明顯減少了線粒體從常氧細胞向缺氧細胞的轉移也降低了細胞整體的代謝能力與存活率。 整體來說，使研究以空間蛋白質體學對神經母細胞瘤TNT進行高解析度的分子特徵分析，並鑑定出一個參與TNT介導粒線體運輸的核心調控模組。結果顯示了KIF5A–RAC1運輸模組，或許能應用於未來腫瘤治療發展的潛在標的。

Tunneling nanotubes (TNTs) are a type of membrane protrusion that is mainly composed of F-actin. TNTs enable long-distance intercellular transfer of cellular cargoes. In highly heterogeneous solid tumors such as neuroblastoma (NB), hypoxic microenvironments promote metabolic adaptation and intercellular cooperation. The cancer cells engage in TNT-mediated mitochondrial transfer to support metabolic homeostasis and survival under stress. However, the molecular machinery involving this process remains poorly defined. Here, we provided a TNT-specific spatial proteomics strategy to characterize TNTs formed in a co-culture model of the neuroblastoma cell line SK-N-BE(2)C under normoxic and hypoxic conditions. TNT-associated proteins were selectively enriched using a photolabeling-based approach. Across analyses, we identified 270,367 mass spectrometry spectra and 56,207 peptides. After filtering for proteins supported by more than two unique peptides, we defined a high-confidence TNT proteome consisting of 1,520 proteins uniquely detected in photo-labeled samples after comparison with non-photo-labeled controls. Functional enrichment analysis revealed a significant relation of cilia-associated transport and microtubule regulatory processes, suggesting that long-range cargo transport within TNTs relies on microtubule-guided mechanisms. Focusing on mitochondrial trafficking, protein–protein interaction network analysis identified the kinesin motor protein KIF5A as a central hub within mitochondria-related transport. RAC1 and MIRO1 were the only mitochondria-associated proteins enriched in TNTs that were predicted to interact with KIF5A. MIRO1 has been previously established as a key adaptor for mitochondrial transport within TNTs, supporting the biological relevance of our findings. Immunocytochemistry and proximity ligation assays confirmed the co-localization of KIF5A with RAC1 and mitochondria within TNTs. shRNA-mediated knockdown of KIF5A or RAC1 significantly reduced mitochondrial presence within TNTs and impaired intercellular mitochondrial transfer without affecting TNT morphology. Moreover, consistent with the results of ICC, analysis by flow cytometry showed that shRNA-mediated knockdown of KIF5A or RAC1 significantly reduced mitochondrial transfer from normoxic to hypoxic cells, while also compromising cellular metabolic capacity and viability. In conclusion, this study applies spatial proteomics to achieve high-resolution molecular characterization of neuroblastoma TNTs and identifies a core regulatory module specifically involved in TNT-mediated mitochondrial transport. These findings suggest that the KIF5A–RAC1 transport module may represent a potential target for tumor therapies.