Abstract
Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp−/− bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 – elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1.
Highlights
Monocytes and macrophages circulate throughout the organism and their ability to be recruited to various sites is crucial for their function as important mediators of innate and adaptive immunity
It has long been known that Wiskott-Aldrich syndrome protein (WASp) is an important regulator of the actin cytoskeleton that is required for the proper function of immune cells, including migration of B and T cells, dendritic cells and macrophage
We demonstrate that colony stimulating factor-1 (CSF-1) stimulation of macrophages results in two transient waves of actin assembly, where WASp is required for the initial peak, while WAVE2 controls the second [14,15]
Summary
Monocytes and macrophages circulate throughout the organism and their ability to be recruited to various sites is crucial for their function as important mediators of innate and adaptive immunity. Cellular migration can be mechanistically described as a sequence of events that repeats until the cell reaches its destination. The initial event in cell migration is polarization and extension of protrusions in the direction of migration [1]. These protrusions persist and are stabilized through the formation of new adhesions to the underlying substratum. The cell body moves forward, propelled by actin-myosin mediated contraction. A popular model of chemotaxis hypothesizes that, while chemotactic receptors are evenly distributed over the cell surface, the cells utilize receptor-mediated signaling network as a molecular ‘‘compass’’ to sense small differences of chemoattractant outside of the cell leading to directional migration [3]
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