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VANGL2 Regulates Membrane Trafficking of MMP14 to Control Cell Polarity and Migration

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VANGL2 Regulates Membrane Trafficking of MMP14 to Control Cell Polarity and Migration Short Report 2141 VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration B. Blairanne Williams1, V. Ashley Cantrell1, Nathan A. Mundell1, Andrea C. Bennett1, Rachel E. Quick1 and Jason R. Jessen1,2,* 1Division of Genetic Medicine/Department of Medicine, and 2Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA *Author for correspondence ([email protected]) Accepted 11 January 2012 Journal of Cell Science 125, 2141–2147 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.097964 Summary Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes. Key words: VANGL2, MMP14 (MT1-MMP), Planar cell polarity, Gastrulation, Fibronectin, Migration Introduction it is unknown whether PCP signaling proteins themselves It has been a decade since homologues of fly planar cell polarity regulate degradation and remodeling of the ECM. Our previous Journal of Cell Science (PCP) genes were shown to be required for convergence and work used HT-1080 cells to probe the relationship between extension movements during frog and fish gastrulation (Djiane human VANGL2, MMP14 and cell–ECM interactions. et al., 2000; Wallingford et al., 2000; Jessen et al., 2002). Knockdown of VANGL2 using siRNA increased secreted Underlying these movements are polarized cell behaviors that levels of active MMP2 and promoted invasion through an ECM contribute to mediolateral narrowing and anterior–posterior substrate (Cantrell and Jessen, 2010). Because MMP2 is activated elongation of the developing embryo. Loss of PCP gene by MMP14 at the cell surface (Sato et al., 1994), we hypothesized function in vivo disrupts polarity, as indicated by changes in that Vangl2 might influence PCP in migrating cells by directly cell elongation, orientation and protrusive activity (Wallingford regulating Mmp14 activity. In this report, we identify VANGL2 et al., 2000; Topczewski et al., 2001; Jessen et al., 2002). As a as a regulator of MMP14 endocytosis and demonstrate that result of altered cell polarity, zebrafish mutants including this membrane-tethered metalloproteinase acts downstream of trilobite/vang-like 2 (vangl2) exhibit a distinct convergence and Vangl2 to control zebrafish convergence and extension cell extension phenotype characterized by shortened and broadened movements. body axes (Sepich et al., 2000; Jessen et al., 2002). Although it is thought that Wnt-mediated PCP signaling impacts the actin Results and Discussion cytoskeleton to regulate polarity and protrusion dynamics in VANGL2 regulates MMP14 endocytosis migrating cells, few downstream effector proteins have been Both endocytosis and recycling of MMP14 provide possible identified. Previously, we showed that zebrafish membrane type- mechanisms to control the level of proteolytic activity in 1 matrix metalloproteinase (Mmp14) is also required for cell polarized cells (Jiang et al., 2001; Remacle et al., 2003; polarity and convergence and extension movements exhibiting a Poincloux et al., 2009). To determine whether VANGL2 genetic interaction with the Wnt co-receptor knypek/glypican4 regulates MMP14 trafficking, we performed an in vitro (Coyle et al., 2008). The temporal requirement for Mmp14 biotinylation assay using a cleavable form of biotin to quantify function during late gastrulation coincides with both that of cell-surface, internalized and recycled MMP14 levels in cells Vangl2 (Sepich et al., 2000) and the appearance of a fibrillar transfected with siRNA against VANGL2. As shown in Fig. 1A, extracellular matrix (ECM) meshwork (Latimer and Jessen, VANGL2-knockdown cells had increased levels of cell-surface 2010), suggesting a functional relationship between cell polarity MMP14 but not total MMP14 protein (supplementary material and proteolysis. However, despite previous connections between Fig. S1). After we stimulated endocytosis, VANGL2-knockdown PCP and ECM assembly (Goto et al., 2005; Dzamba et al., 2009), cells had reduced levels of internalized MMP14 (Fig. 1B,C). To 2142 Journal of Cell Science 125 (9) Fig. 1. VANGL2 regulates MMP14 endocytosis in vitro. (A) Quantification of cell-surface MMP14 in cells transfected with non-targeting control (NT) siRNA or siRNA to knock down VANGL2. MESNA treatment removes cell-surface biotin. Graph of representative experimental data normalized to GAPDH expression from total protein extracts. (B) Endocytosis was quantified after shifting the temperature to 37˚C and MESNA treatment to remove non-internalized proteins. MMP14 recycling was quantified after incubating the cells an additional hour followed by a second MESNA treatment. Graph shows representative data from one experiment. (C) Quantification of all internalization and recycling data from three independent experiments. Graph depicts the mean percentage of internalized MMP14 (total intracellular MMP14 divided by total cell-surface MMP14; NT siRNA594.6%, VANGL2 siRNA548.6%) and recycled MMP14 (total intracellular after endocytosis divided by total intracellular after 1 hour of recycling; NT siRNA, 25%; VANGL2 siRNA, 33%) with s.d. (D) Endocytosis assay performed Journal of Cell Science using an antibody to label cell-surface MMP14 at 4˚C followed by a temperature shift to 37˚C. Although more MMP14 is observed at the plasma membrane in VANGL2-knockdown cells (arrows), less is internalized compared with controls. (E) Transferrin endocytosis following VANGL2 knockdown. HT-1080 cells transfected with NT or VANGL2 siRNA were labeled at 4˚C with Alexa-Fluor-594-labeled transferrin followed and shifted to 37˚C. Similar numbers of transferrin-positive intracellular vesicles were observed between control and VANGL2-knockdown cells (mean number of vesicles counted per cell with s.d. from three independent experiments; NT538614, n5186 cells; VANGL2543611, n5221 cells). test whether MMP14 recycling is impaired, after endocytosis Fig. S2). These data indicate that VANGL2 specifically regulates cells were treated with MESNA and incubated at 37˚C for an endocytosis of MMP14; however, it is possible that VANGL2 additional hour followed by a second MESNA treatment. Here, influences the trafficking of additional membrane proteins in western blot of biotin-labeled proteins gives a quantitative polarized cells. For example, studies from fly and fish suggest measurement of the remaining non-recycled pool of MMP14. that PCP proteins regulate endocytosis of cadherins to modulate Our data show that a similar ratio of intracellular MMP14 was cell adhesion (Classen et al., 2005; Ulrich et al., 2005). recycled to the cell surface in both control and VANGL2- knockdown cells (Fig. 1B,C). To confirm these findings, we VANGL2 and MMP14 colocalization in HT-1080 cells performed an antibody-uptake assay to visualize MMP14- MMP14 expression in vitro reflects its regulation by endocytosis positive endocytic vesicles. Cells were labeled with antibody and recycling in that little protein is present on the plasma against MMP14 and either fixed or shifted to 37˚C to permit membrane with the majority localized to intracellular vesicles endocytosis. VANGL2-knockdown cells were confirmed to have (Steffen et al., 2008). Recruitment of fly Van Gogh to the plasma a decreased ability to internalize cell-surface MMP14 (Fig. 1D). membrane is thought to be a crucial event for PCP signaling in To determine whether loss of VANGL2 globally disrupts epithelial cells (Strutt and Strutt, 2009). However, recent in vitro endocytosis, we performed endocytosis assays using and in vivo studies have identified VANGL2 expression within fluorescently labeled transferrin (to ascertain clathrin-mediated vesicular compartments (Merte et al., 2010; Cha et al., 2011). endocytosis) or EEA1 antibody (to detect early endosomes). In VANGL2 and MMP14 colocalization in HT-1080 cells was neither case did we observe a difference between control and detected at the plasma membrane (Fig. 2A and supplementary VANGL2-knockdown cells (Fig. 1E and supplementary material material Fig. S3A). VANGL2 was also expressed in vesicle VANGL2 regulates MMP14 trafficking 2143 Fig. 2. VANGL2 and MMP14 colocalization in HT-1080 cells. (A) GFP–VANGL2 and MMP14 expression at membrane protrusions (arrows). (B–D) GFP–VANGL2 and MMP14 expression shown in relation to different vesicular markers (EEA1, RAB11 and
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