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Published on: March 24, 2015
Cholesterol oxidase treatment impairs CXCR4-mediated T cell migration
Sofía R Gardeta1, Eva M García-Cuesta1, Blanca Soler Palacios1
1Chemokine Signaling group, Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, Madrid, E-28049, Spain.
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.
Insights
Cholesterol regulates chemokine receptor CXCR4 organization and movement, which is vital for T cells to sense chemical signals and migrate effectively. This finding highlights cholesterol's role in cell signaling and immune responses.
Area of Science:
- Cellular Biology
- Membrane Biophysics
- Immunology
Background:
- Cholesterol influences cell membrane properties and receptor conformation, including G protein-coupled receptors (GPCRs).
- Chemokine receptors, like CXCR4, adopt specific conformations affecting cell functions such as directional migration.
- The exact mechanisms of cholesterol's control over chemokine receptor spatial organization are not fully understood.
Purpose of the Study:
- To investigate the role of cholesterol in modulating the chemokine receptor CXCR4.
- To understand how cholesterol affects CXCR4's spatial organization and dynamics.
- To elucidate cholesterol's impact on CXCR4-mediated cell migration and signaling.
Main Methods:
- Lipidomic analysis and raster image correlation spectroscopy to assess cholesterol levels and membrane fluidity.
- Single-particle tracking, FRET, and FLIM to analyze CXCR4 nanoclustering, dynamics, and dimerization.
- Microfluidic chemotaxis, flow cytometry, calcium flux, and western blotting to evaluate cell migration, receptor expression, ligand binding, and signaling.
Main Results:
- Moderate cholesterol depletion increased membrane fluidity and impaired T cell migration towards CXCL12.
- Cholesterol depletion altered CXCR4 conformation and spatial distribution, affecting larger clusters.
- Ligand binding and other signaling pathways were not significantly affected by cholesterol depletion.
Conclusions:
- Cholesterol is a critical regulator of CXCR4 lateral mobility and spatial organization.
- Effective sensing of chemoattractant gradients by cells relies on cholesterol's modulation of CXCR4.
- This study provides insights into cholesterol's role in immune cell navigation and signaling.
