Cholesterol oxidase treatment impairs CXCR4-mediated T cell migration.

IF 8.2 2区 生物学 Q1 CELL BIOLOGY
Sofía R Gardeta, Eva M García-Cuesta, Blanca Soler Palacios, Rosa Ayala Bueno, Adriana Quijada-Freire, Noelia Santander Acerete, José Miguel Rodríguez-Frade, Mario Mellado
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引用次数: 0

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: This study identifies cholesterol as a crucial regulator of CXCR4 lateral mobility and spatial organization, enabling cells to effectively sense chemoattractant gradients.

胆固醇氧化酶处理损害cxcr4介导的T细胞迁移。
背景:胆固醇是哺乳动物细胞膜的关键成分,可调节脂质双分子层的性质,并影响包括G蛋白偶联受体(gpcr)在内的膜受体的构象状态。这些作用是通过与跨膜区域内特定残基的直接相互作用和周围脂质双分子层的调节来介导的。趋化因子受体,一个GPCR亚家族,采用不同的构象与特定的细胞功能相关。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尚不清楚。本研究探讨了胆固醇在调节趋化因子受体CXCR4中的作用。方法:我们使用脂质组学分析测量细胞胆固醇水平,光栅图像相关光谱评估胆固醇消耗对膜流动性的影响。在TIRF模式下,利用单粒子跟踪技术研究了CXCR4的纳米聚类和动力学特性。通过FRET和FLIM分析评估CXCR4二聚体的形成,并使用微流控趋化室测量定向细胞迁移。用特异性抗体和CXCL12-ATTO700流式细胞术检测受体表达和配体结合。其他检测包括钙通量和信号分子的western blotting。统计分析采用非配对t检验、单因素方差分析和双尾Mann-Whitney检验。结果:我们的研究结果表明,使用胆固醇氧化酶进行适度的胆固醇消耗可增加膜流动性,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。该处理还诱导了CXCR4构象和空间分布的改变,但没有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,对脂质富集微域没有显著影响。结论:本研究确定胆固醇是CXCR4横向移动和空间组织的重要调节因子,使细胞能够有效地感知化学引诱剂梯度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
11.00
自引率
0.00%
发文量
180
期刊介绍: Cell Communication and Signaling (CCS) is a peer-reviewed, open-access scientific journal that focuses on cellular signaling pathways in both normal and pathological conditions. It publishes original research, reviews, and commentaries, welcoming studies that utilize molecular, morphological, biochemical, structural, and cell biology approaches. CCS also encourages interdisciplinary work and innovative models, including in silico, in vitro, and in vivo approaches, to facilitate investigations of cell signaling pathways, networks, and behavior. Starting from January 2019, CCS is proud to announce its affiliation with the International Cell Death Society. The journal now encourages submissions covering all aspects of cell death, including apoptotic and non-apoptotic mechanisms, cell death in model systems, autophagy, clearance of dying cells, and the immunological and pathological consequences of dying cells in the tissue microenvironment.
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