Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

• Published in: Journal of leukocyte biology Vol. 92; no. 3; pp. 633 - 639
• Main Authors: Green, Teresa D., Park, Joungjoa, Yin, Qi, Fang, Shijing, Crews, Anne L., Jones, Samuel L., Adler, Kenneth B.
• Format: Journal Article
• Language: English
• Published: United States Society for Leukocyte Biology 01.09.2012
• Subjects: Actin Cytoskeleton - immunology; Actin Cytoskeleton - metabolism; Animals; Blotting, Western; Cell Development, Differentiation, & Trafficking; chemotaxis; Chemotaxis, Leukocyte - physiology; cytoplasmic translocation; Intracellular Signaling Peptides and Proteins - immunology; Intracellular Signaling Peptides and Proteins - metabolism; J774A.1 cells; Macrophages - immunology; Macrophages - metabolism; Membrane Proteins - immunology; Membrane Proteins - metabolism; Mice; Microscopy, Confocal; monocyte chemotactic protein 1; Myristoylated Alanine-Rich C Kinase Substrate; Protein Transport - physiology
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1189/jlb.1211604

MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin. A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N‐terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS‐related peptide could affect migration of macrophages, using the mouse macrophage‐like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP‐1 (25–100 ng/ml) or C5a (5–20 ng/ml). Cells were preincubated (15 min) with MANS or a mis‐sense control peptide (RNS), both at 50 μM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP‐1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP‐1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.