The chemotactic defect in wiskott-Aldrich syndrome macrophages is due to the reduced persistence of directional protrusions

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.

Figure 1. WASp is required for the first wave of actin assembly in response to CSF-1.

(A) F-actin content of RAW/LR5 cells in response to CSF-1 stimulation at 37°C or 22°C was quantitatively measured and normalized to the cell number, as described in Material and Methods. (B) F-actin content of RAW/LR5 (LR5), shControl (Ctrl) and shWASp cells in response to CSF-1 stimulation at 22°C was measured as in (A). n = 3–4 experiments per condition. (C) Single cell analysis of F-actin content of shControl, shWASp and Y291F cells in response to CSF-1 stimulation at 22°C. n = 3 experiments, minimum 30 cells were analyzed per condition. (D) Ruffling index of shControl, shWASp and Y291F cells in response to CSF-1 stimulation at 22°C. n = 3 experiments, minimum 30 cells were analyzed per condition. Error bars represent SEM. * p<0.05 compared to unstimulated condition.

Figure 2. WASp and its phosphorylation are not required for directional response to CSF-1.

The role of WASp in directional sensing was examined by monitoring the chemotactic responses of WASp mutant macrophages in a CSF-1 gradient. Representative kymographs from time-lapse images of (A) shControl, (B) shWASp and (C) Y291F cells in response to a micropipette containing CSF-1 are shown (Movies S1, S2, S3). (D) Overlay of the outlines of the membrane protrusions from panels A through C is shown.

Figure 3. WASp and its phosphorylation are required for the persistence of chemotactic protrusion.

Quantification of the protrusive responses of shControl, shWASp and Y291F cells in response to CSF-1 supplying micropipette from time lapse images are shown. (A) Onset of the protrusion (time after CSF-1 stimulation), (B) duration of the protrusion, (C) maximum length of the protrusion and (D) protrusion rate (maximum length/time it takes the protrusion to reach maximum length) in response to CSF-1 were determined. The data represent the mean of at least 30 cells from seven separate videos. Error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001.

Figure 4. WASp is required for the persistence of chemotactic protrusion in primary BMMs.

(A) The role of WASp in directional sensing was examined by monitoring the chemotactic responses of wild-type and WASp-deficient macrophages in a CSF-1 gradient. Representative kymographs from time lapse images of wild-type (WT, top panel) and WASp^−/− (middle panel) BMM in response to CSF-1 containing micropipette (Movies S4 and S5) are shown. The bottom panel shows an overlay of the outlines of the membrane protrusive activities of wild-type and WASp^−/− BMMs. (B) Onset, (C) duration, (D) maximum length and (E) protrusion rate (maximum length/time it takes the protrusion to reach maximum length) after CSF-1 stimulation were determined. The data represent the mean of at least 30 cells from each cell type over a minimum ten separate videos. Error bars represent SEM. * p<0.05, ***p<0.001.

Figure 5. WASp is not required for the recruitment of PI 3-kinase, Rac1, WAVE2 and actin in CSF-1-elicited protrusions from primary BMMs.

Chemotactic protrusions were isolated from wild-type and WASp^−/− BMMs in response to CSF-1 as described in Materials and Methods. (A) Representative western blots of indicated proteins from pseudopod lysates and total cell lysates are shown. (B) Protein amounts from pseudopod lysates were quantified by densitometry and normalized to the corresponding level of a non-specific membrane marker (CD14). Data is shown as relative ratio of each proteins compared to the WT BMM. n = 3 experiments. Error bars represent SEM.