Figure 3

Subcellular localization of fluorescently-tagged PCP proteins in polarized MEC1 cells. (A) Photomicrograph of migrating polarized MEC1 cell with precisely defined leading and trailing edge. VANGL2-EGFP is significantly enriched in the trailing edge of the cell. To visualize the cytoskeleton, actin-RFP was co-transfected with the VANGL2 construct. Arrow indicates the direction of migration. Size bar = 10 μm. (B) Co-localization of EGFP-VANGL2 with endogenous CD44, a marker of the trailing edge. (C,D,E) Polarized structure of migrating MEC1 cells expressing ROR2-mCherry, DVL3-EYFP and β-arrestin 2-EGFP. Arrows indicate the direction of cell migration. Size bars = 10 μm. (F) To quantify the enrichment of VANGL2-EGFP in the trailing edge, the ratio of the EGFP signal intensity from leading and trailing edge was calculated using ImageJ software. Size bar = 10 μm. (G) Ratios of the signal intensity in the trailing edge divided by the signal intensity in the leading edge for all PCP proteins shown in A-E. One dot represents one analyzed cell. Red line indicates ideally symmetric distribution. (H) The effect of Cytochalasin D treatment on VANGL2-EGFP transfected MEC1 cell is shown after 0, 15 and 30 min. The localization of VANGL2-EGFP does not change even after the disruption of the actin cytoskeleton. (I) Quantification of individual cells recorded in E. The polarization of VANGL2-EGFP is expressed as the ratio between the EGFP signal in the trailing edge compared to the leading edge. Cytochalasin B and Cytochalasin D treatment does not affect the localization of VANGL2-EGFP in the MEC1 cell even though the typical migration morphology has been disrupted by the applied treatment. (n.s. – not significant).