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Proteomic Profiling of Macrophages by 2D Electrophoresis
Published on: November 4, 2014
Proteomic analysis of microtubule-associated proteins during macrophage activation
Prerna C Patel1, Katherine H Fisher, Eric C C Yang
1Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario M1C 1A4, Canada.
Abstract:
Classical activation of macrophages induces a wide range of signaling and vesicle trafficking events to produce a more aggressive cellular phenotype. The microtubule (MT) cytoskeleton is crucial for the regulation of immune responses. In the current study, we used a large scale proteomics approach to analyze the change in protein composition of the MT-associated protein (MAP) network by macrophage stimulation with the inflammatory cytokine interferon-gamma and the endotoxin lipopolysaccharide. Overall the analysis identified 409 proteins that bound directly or indirectly to MTs. Of these, 52 were up-regulated 2-fold or greater and 42 were down-regulated 2-fold or greater after interferon-gamma/lipopolysaccharide stimulation. Bioinformatics analysis based on publicly available binary protein interaction data produced a putative interaction network of MAPs in activated macrophages. We confirmed the up-regulation of several MAPs by immunoblotting and immunofluorescence analysis. More detailed analysis of one up-regulated protein revealed a role for HSP90beta in stabilization of the MT cytoskeleton during macrophage activation.
Insights
Macrophage activation alters microtubule-associated proteins (MAPs). Proteomics identified changes in MAPs, revealing HSP90beta
Area of Science:
- Immunology and Cell Biology
- Proteomics and Cytoskeletal Dynamics
Background:
- Macrophage activation is critical for immune responses, involving significant cellular changes.
- The microtubule (MT) cytoskeleton plays a vital role in regulating immune cell function.
- Understanding changes in microtubule-associated proteins (MAPs) during activation is key.
Purpose of the Study:
- To analyze the proteomic changes in the MAP network of macrophages upon activation.
- To identify specific MAPs that are differentially regulated by inflammatory stimuli.
- To investigate the functional role of specific MAPs in activated macrophages.
Main Methods:
- Large-scale proteomics to identify proteins associated with microtubules.
- Macrophage stimulation using interferon-gamma and lipopolysaccharide.
- Bioinformatics analysis of protein interaction networks and validation via immunoblotting and immunofluorescence.
Main Results:
- Identified 409 proteins associated with microtubules in macrophages.
- Found 52 up-regulated and 42 down-regulated MAPs after stimulation.
- Revealed a role for HSP90beta in stabilizing the microtubule cytoskeleton in activated macrophages.
Conclusions:
- Macrophage activation significantly remodels the microtubule-associated protein network.
- Specific MAPs, including HSP90beta, are dynamically regulated and functionally important.
- These findings provide insights into cytoskeletal regulation during immune cell activation.
