Gardeta Sofía R, García-Cuesta Eva M, Palacios Blanca Soler, Bueno Rosa Ayala, Quijada-Freire Adriana, Acerete Noelia Santander, Rodríguez-Frade José Miguel, Mellado Mario
Chemokine Signaling group, Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, Madrid, E-28049, Spain.
Cell Commun Signal. 2025 Oct 17;23(1):444. doi: 10.1186/s12964-025-02392-9.
Cholesterol, a key component of mammalian cell membranes, modulates the properties of the lipid bilayer and influences the conformational states of membrane receptors, including G protein-coupled receptors (GPCRs). These effects are mediated through direct interactions with specific residues within the transmembrane regions and modulation of the surrounding lipid bilayer. Chemokine receptors, a GPCR sub-family, adopt distinct conformations associated with specific cell functions. For example, CXCL12 triggers receptor clustering, essential for directional cell migration. However, the precise mechanisms by which cholesterol controls the spatial organization of these receptors remain unclear. This study investigated the role of cholesterol in modulating the chemokine receptor CXCR4.
We used lipidomic analysis to measure cellular cholesterol levels, and raster image correlation spectroscopy to assess the impact of cholesterol depletion on membrane fluidity. CXCR4 nanoclustering and dynamics were examined using single-particle tracking in TIRF mode. CXCR4 dimer formation was evaluated by FRET and FLIM analyses, and directed cell migration was measured using microfluidic chemotaxis chambers. Receptor expression and ligand binding were determined by flow cytometry with specific antibodies and CXCL12-ATTO700. Additional assays included calcium flux, and western blotting for signaling molecules. Statistical analysis used unpaired t-tests, one-way ANOVA, and two-tailed Mann-Whitney tests.
Our findings demonstrate that moderate cholesterol depletion using cholesterol oxidase increases membrane fluidity, impairs T cell migration towards CXCL12 gradients, and enhances CXCL12-mediated β1-integrin activation. This treatment also induced alterations in CXCR4 conformation and spatial distribution, without significantly affecting ligand binding or other chemokine-mediated signaling pathways. Immunocytochemical analysis indicated that cholesterol oxidase primarily affected the largest CXCR4 clusters, with no significant impact on lipid-enriched microdomains.
This study identifies cholesterol as a crucial regulator of CXCR4 lateral mobility and spatial organization, enabling cells to effectively sense chemoattractant gradients.
胆固醇是哺乳动物细胞膜的关键成分，可调节脂质双层的特性，并影响膜受体的构象状态，包括G蛋白偶联受体（GPCRs）。这些作用是通过与跨膜区域内的特定残基直接相互作用以及对周围脂质双层的调节来介导的。趋化因子受体是GPCR的一个亚家族，具有与特定细胞功能相关的独特构象。例如，CXCL12触发受体聚集，这对细胞定向迁移至关重要。然而，胆固醇控制这些受体空间组织的确切机制仍不清楚。本研究调查了胆固醇在调节趋化因子受体CXCR4中的作用。
我们使用脂质组学分析来测量细胞胆固醇水平，并使用光栅图像相关光谱法评估胆固醇消耗对膜流动性的影响。使用TIRF模式下的单粒子追踪来检查CXCR4纳米簇集和动力学。通过FRET和FLIM分析评估CXCR4二聚体形成，并使用微流控趋化室测量定向细胞迁移。使用特异性抗体和CXCL12-ATTO700通过流式细胞术测定受体表达和配体结合。其他检测包括钙流和信号分子的蛋白质印迹分析。统计分析使用未配对t检验、单因素方差分析和双尾曼-惠特尼检验。
我们的研究结果表明，使用胆固醇氧化酶适度消耗胆固醇会增加膜流动性，损害T细胞向CXCL12梯度的迁移，并增强CXCL12介导的β1整合素激活。这种处理还诱导了CXCR4构象和空间分布的改变，而不会显著影响配体结合或其他趋化因子介导的信号通路。免疫细胞化学分析表明，胆固醇氧化酶主要影响最大的CXCR4簇，对富含脂质的微结构域没有显著影响。
本研究确定胆固醇是CXCR4横向流动性和空间组织的关键调节因子，使细胞能够有效地感知趋化因子梯度。
