AbstractMigratory macrophages (Mφ) play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. We have employed 3D-SIM and 3D-STORM super-resolution techniques to map the subcellular localisation of adhesion and motility related proteins in bone marrow derived mouse Mφ, and to study how their distribution changes under different conditions. Super-resolution imaging shows that Mφ form adhesion and actin cytoskeletal structures that are quite different from those seen in less motile cells or other immune cells, consisting of countless tiny focal complexes and point contacts. Mφ also form podosomes, which are actin-rich adhesion structures that degrade extracellular matrix. Matrix degradation capabilities were assessed by plating Mφ on a fluorescently labelled layer of matrix and studying the protein distribution around degraded areas. 3D-SIM and 3D-STORM are able to determine the exact subcellular location of fluorescently labelled motility- and adhesion-related proteins, and unravel the differences between different conditions on a scale never seen before. We have found that Mφ refine their adhesion structures as they differentiate from immature poorly spread Mφ to mature well spread Mφ. Functionally this translates into striking increases in motility and matrix degradation. These results will help in deciphering the roles of up- and downregulated proteins in macrophage function. In further work, we plan to perturb the expression (by siRNA technology) or function (by the use of inhibitors) of these proteins, and to characterise their roles in Mφ adhesion and migration by live-cell super-resolution microscopy.
University of Western Australia
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