Proteomic Analysis of Microtubule-associated Proteins during Macrophage Activation

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-γ 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-γ/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 HSP90β in stabilization of the MT cytoskeleton during macrophage activation.Microtubules (MTs)¹ are major structural components of the cytoskeleton that are intricately involved in cell morphology, motility, division, and intracellular organization and transport. The diverse roles of MTs are dependent on the polymer having the capacity to be both dynamic and static in nature. Individual MTs alternate between growing and shrinking by the rapid attachment and detachment of tubulin subunits at their ends (1, 2). Thus, MTs can continually reorganize and undergo cycles of growing, pausing, and shortening. A number of mechanisms exist to regulate this dynamic equilibrium and involve association of proteins with the MT lattice. MT-associated proteins (MAPs), such as MAP4 and tau, stabilize MTs by binding to the wall thus inhibiting MT disassembly (3, 4). Recently MT plus (+) end-binding proteins have been implicated in stabilizing MTs by associating with cortical proteins to tether the MT end to peripheral target sites (5–7). Stabilized MT subsets are biochemically distinct and acquire posttranslational modifications that can be used to differentiate them from dynamic subsets. For example, posttranslational modifications such as glutamylation (8), detyrosination (8, 9), and acetylation (10) occur on MTs that exhibit increased stability. Stabilized MTs have been implicated in MT transport by allowing increased binding of MT motors (11, 12). Numerous other MAPs have been shown to regulate MT form and function including control of MT nucleation and elongation, MT linkage to and movement of organelles, and modulation of MT growth to allow scaffolding of signal transduction events (13).The extensive MT network provides a large surface area to serve as a platform for the binding of a large number of proteins that is likely heavily influenced by local cellular events and cell type. Traditionally the term MAP referred to proteins that bind directly to tubulin within the MT polymer, and a lot of recent debate and controversy have surrounded the definition of a MAP (14, 15). In this and other reports the definition of MAPs is considered to also include proteins that indirectly or transiently interact with MTs, co-localize with MTs, or influence MT growth dynamics in some way (16). The advent of proteomics has allowed cytoskeleton researchers to resolve the spectrum of MAPs. To date, the MT proteome has been resolved by MS analysis in developmentally important animal and plant models including Xenopus laevis egg extracts (17), Drosophila melanogaster embryos (18), Artemia franciscana embryos (19), Arabidopsis suspension cells (20), and complex mammalian tissues such as rat brain (21). The MT proteome has also been described for specialized MT structures including mitotic spindles (22–24), centrosomes (25, 26), and cilia (27, 28).Macrophages are key regulators of the immune system connecting innate and specific immune responses. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, is a potent activator of monocytes and macrophages. LPS triggers the abundant secretion of many cytokines from macrophages including IL-1 (29), IL-6, (30), and tumor necrosis factor-α (31), which together contributes to the pathophysiology of septic shock. IFN-γ is a proinflammatory cytokine produced by the host in response to intracellular pathogens. IFN-γ binds to IFN-γ receptors on macrophages, and IFN-γ signaling induces the production and/or release of cytokines, like IL-1 or tumor necrosis factor-α, which enhance LPS-mediated effects (32). Thus, the synergy between LPS and inflammatory cytokines such as IFN-γ represents an important regulatory mechanism by which the host tackles a significant, ongoing infection before it activates potent effector responses (33). It has been demonstrated that LPS may cause changes in monocyte cytoskeleton and directly influence assembly of isolated MTs (34). Recently we observed that classical activation of murine resident peritoneal or RAW264.7 macrophages with a combination of IFN-γ and LPS induces an increase in stabilized cytoplasmic MTs (5). A significant effort has been made to unravel the importance of stable MTs in cellular processes over the past few years. With respect to macrophage function, stable MTs could potentially function as tracks for vesicle secretion of cytokines and matrix metalloproteinases necessary to effect the enhanced inflammatory response observed in classically activated macrophages. We recently demonstrated that stable MTs are important for cell spreading as well as the binding of large particles in activated macrophages (5). The stabilization of macrophage interphase MTs is uniquely rapid, thus serving as an ideal model for studying MAPs involved in MT modulation in mammalian cells.The focus of the present study was to identify the MT-associated proteins involved in altering and stabilizing MT structures and also to resolve the spectrum of proteins within the MT proteome of a mammalian cell. To achieve this goal, we used a proteomics approach involving a MAP purification technique based on MT co-sedimentation (35) followed by off-line fractionation and identification of MAPs using LC-MS/MS. Information provided by mass spectrometry analysis allowed us to analyze the changes in MAP abundance during activation of macrophages by IFN-γ/LPS. These studies also provided candidate proteins for selective molecular intervention for chronic inflammatory disorders.