Sequential binding of ezrin and moesin to L-selectin regulates monocyte protrusive behaviour during transendothelial migration

ABSTRACT Leukocyte transendothelial migration (TEM) is absolutely fundamental to the inflammatory response, and involves initial pseudopod protrusion and subsequent polarised migration across inflamed endothelium. Ezrin/radixin/moesin (ERM) proteins are expressed in leukocytes and mediate cell shape changes and polarity. The spatio-temporal organisation of ERM proteins with their targets, and their individual contribution to protrusion during TEM, has never been explored. Here, we show that blocking binding of moesin to phosphatidylinositol 4,5-bisphosphate (PIP2) reduces its C-terminal phosphorylation during monocyte TEM, and that on–off cycling of ERM activity is essential for pseudopod protrusion into the subendothelial space. Reactivation of ERM proteins within transmigrated pseudopods re-establishes their binding to targets, such as L-selectin. Knockdown of ezrin, but not moesin, severely impaired the recruitment of monocytes to activated endothelial monolayers under flow, suggesting that this protein plays a unique role in the early recruitment process. Ezrin binds preferentially to L-selectin in resting cells and during early TEM. The moesin–L-selectin interaction increases within transmigrated pseudopods as TEM proceeds, facilitating localised L-selectin ectodomain shedding. In contrast, a non-cleavable L-selectin mutant binds selectively to ezrin, driving multi-pseudopodial extensions. Taken together, these results show that ezrin and moesin play mutually exclusive roles in modulating L-selectin signalling and shedding to control protrusion dynamics and polarity during monocyte TEM.


Fig. S2 Reconstitution of ezrin-GFP into knock-down cells.
(A) THP-1 cells stably expressing lentiviral shRNA targetting ezrin (clone 5) were reconstituted with shRNA-immune WT ezrin-GFP. Immunoblot shows knockdown of endogenous ezrin and reconstitution of ezrin-GFP to similar expression levels. Actin and ezrin expression were probed simultaneously with anti-ezrin (rabbit, 3145S, Cell Signalling) and anti-actin (mouse monoclonal antiβ-actin, clone AC-74, Sigma Aldrich) antibody. Green and red signals correspond to LI-COR secondary antibody, IRDye® 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse (Odyssey). Merged signals to the right rendered in black and white. Expression levels are compared against THP-1 cells expressing nontargetting (NT) shRNA.
(B) Anti-phospho-ERM Western blots reveal that ezrin-GFP can be phosphorylated in response to 25 nM calyculin A (Cal A). THP-1 cells expressing non-targetting shRNA (NT) show the relative phosphorylation levels of ezrin and moesin, which both increase in response to Cal A stimulation. Mouse Antivinculin monoclonal antibody (Sigma-Aldrich V9131) was used as loading control in this immunoblot. (A) THP-1 cells expressing WT or mutant versions of moesin tagged to GFP were harvested and resuspended to a density of 2 x 10 6 cells per mL. Approximately 50 µL of each cell suspension was spotted onto 13 mm diameter poly-L-lysinecoated coverslips and allowed to adhere for 10 min at room temperature. Cells were subsequently fixed in excess (~ 300 µL) 4% paraformaldehyde for 10 minutes, washed in PBS, permeabilised in PBS containing 0.1% (v/v) NP-40 (Fluka) and then blocked in 15% bovine serum albumin for 20 min at room temperature, and stained with TRITC-phalloidin. Panel of images reveal the subcellular localisation of moesin-GFP with TRITC-phalloidin staining. Scale bar for all images = 5.3 µm. TA and TD are respectively constitutively inactive and active forms of moesin (where T558 is mutated to either an alanine (A) or aspartate (D)). The 4N mutant represents the PIP2-binding mutant of moesin, and 4NTD is where the PIP2 binding mutant has been rendered constitutively active. (B) MOC between cells lines reveals significant reduction in membrane localisation of 4N moesin-GFP. Interestingly, the 4NTD mutant associates strongly with phalloidin. This is likely due to the C-terminal domain can interact strongly with the actin cytoskeleton. Statistical analysis: One-way ANOVA followed by Tukey's post test. ***=p<0.001.

Fig. S4 Monitoring TEM of THP-1 cells expressing WT, TA or TD moesin-GFP.
Endogenous moesin was depleted from THP-1 cells using the clone 2 lentiviral shRNA (Sigma-Aldrich Mission). Cells were subsequently reconstituted with shRNA-resistant WT, TA or TD moesin-GFP to determine to contribution of the C-terminal threonine 558 phosphorylation in regulating TEM (see corresponding Western blots in Fig.1A of main manuscript). Cells were subjected to continuous perfusion for 30 minutes over TNF-activated HUVEC. Timelapse video microscopy allowed THP-1 cells to be scored for forming protrusions during TEM, expressed as a percentage of the total recruited cells (TRC) from flow. (A) Three representative images were taken for each cell line: 10 min, 20 min and 30 min. In this example, at 10 min perfusion, cells were found exclusively on top of the endothelium. At 20 and 30 min, only the WT and TA cell lines are forming subendothelial protrusions. Scale bar = 16 µm. (B) Data are represented more comprehensively in Figure 2 C-G in the main manuscript. One-way ANOVA followed by Bonferroni's post-test. * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig S7. Working model: the biological significance underlying sequential interaction of L-selectin with ezrin and then moesin, and its impact on protrusive behaviour during TEM and beyond
(A) Stage 1: During TEM, the pool of WT L-selectin (green bars) within transmigrating pseudopods interacts preferentially with ezrin (E). Given that ezrin has the potential to interact with the regulatory subunit of PI3K (Gautreau et al., 1999), the biological significance of this interaction could be to promote pseudopod extension during TEM and facilitate cell invasion-like behaviour. Stage 2: As time progresses, ezrin is exchanged for moesin (M). In this setting, moesin is likely to contribute to clustering of L-selectin prior to shedding by its protease (ADAM17). Stage 3&4: Clustering of L-selectin can drive its own ectodomain shedding in neutrophil suspensions (Palecanda et al., 1992), but whether this can occur specifically in monocytes during TEM is currently not understood. Numerous reports have shown that ectodomain shedding of Lselectin can be triggered by p38 MAPK or PKC (Killock and Ivetic, 2010;Preece et al., 1996;Smolen et al., 2000). Moreover, activation of p38 MAPK lies upstream of ADAM17 mobilisation to the plasma membrane and its proteolytic activity, as judged by threonine phosphorylation of the ADAM17 cytoplasmic tail (Killock and Ivetic, 2010;Xu and Derynck, 2010). L-selectin shedding, specifically during TEM, is essential for the establishment of front-back polarity of transmigrated monocytes. (B) Monocytes that express a non-cleavable mutant of L-selectin (ΔM-N) interact preferentially with ezrin, but not moesin. It is believed that the sustained interaction between ΔM-N and ezrin underlies the excessive multi-pseudopodial phenotype, which could be due to uncontrolled signalling to PI3K. Ultimately, cells will fail to establish front-back polarity, which is essential for interstitial migration towards target sites of inflammation.

Movie 1
Primary human monocytes were perfused over TNF activated HUVEC for a 4 min and 40 sec at a density of 1.0 x 10 6 per mL. Cells are initially captured from flow, after which they spread, before contracting the spread area just prior to TEM. White arrow shows on the rare occasion a CD14-positive monocyte undergoing full-TEM, highlighting the fact that this flow assay had been optimised for capturing cells in mid-TEM. Scale bar = 100 µm.

Movie 2
A THP-1 cell stably expressing WT L-selectin-GFP, recruited from flow and subsequently undergoing TEM during the course of the flow assay. Note in this example, the cell is producing a single pseudopodial extension beneath the TNFactivated endothelial monolayer.

Movie 3
A THP-1 cell stably expressing WT L-selectin-GFP, recruited from flow and subsequently undergoing TEM during the course of the flow assay. The screen is split into phase (left hand side) and GFP (right hand side) channels. Note in this example, the cell is producing two pseudopodial extension beneath the TNFactivated endothelial monolayer.

Movie 4
A THP-1 cell stably expressing ΔM-N L-selectin-GFP, recruited from flow and subsequently undergoing TEM during a 25 min period of flow. The screen is split into phase (left hand side) and GFP (right hand side) channels. Note in this example, the cell is producing multi-pseudopodial extensions beneath the TNFactivated endothelial monolayer.