Figure 1.
VEC molecules are internalized by adjacent cells via trans-endocytosis.
(A) Co-culture of primary microvascular endothelial cells from mice lungs. The primary microvascular endothelial cells were isolated separately from VEC-EGFP knock-in mice and Rosa26-mTmG mice expressing mTomato fluorescent protein in all cells and fixed after 24 hours of the co-culture. The trans-endocytosed VEC-EGFP molecules in mTomato fluorescent protein expressing cells were stained by anti-GFP antibody without (upper images) and with permeabilization (lower images). (B) Higher magnification of images of the indicated area in A. Arrows in upper images show VEC-EGFP molecule could not be stained by anti-GFP antibody in non-permeabilized cells. Arrowheads in lower images show VEC-EGFP molecule stained by anti-GFP antibody in permeabilized cells. (C) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT. Trans-endocytosis of VEC occurred in HUVECs (scale bar = 20 µm). (D) Higher magnification of the indicated area in C. Arrow shows VEC-EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. Asterisks show the extended filopodia or adherens junctions from the neighboring cell. Arrow heads show VEC-TagRFPT molecules were trans-endocytosed by VEC-EGFP expressing cells. Scale bar = 5 µm.
Figure 2.
VEC trans-endocytosis occurs between HUVECs expressing endogenous VEC and COS7 cells expressing VEC fusion proteins.
(A) Co-culture of COS7 cells expressing VEC-EGFP (COS7-VEC-EGFP) and HUVECs expressing TagRFP657 (HUVEC-TagRFP657). Lower images are higher magnification of the indicated area in upper images. VEC trans-endocytosis occurred between COS7-VEC-EGFP and HUVEC-TagRFP657, due to endogenous VEC in HUVECs. Exogenously expressed VEC molecules in COS7 cells form cell-cell junctions. Arrows show trans-endocytosed VEC-EGFP molecules from COS7 cells by adjacent HUVECs labeled with TagRFP657. Scale bars = 20 µm. (B) Time-course images of co-culture of COS7-VEC-EGFP and HUVECs-VEC-TagRFP657. The interaction between endogenous VEC in HUVECs and over-expressed VEC-EGFP in COS7 cells can induce trans-endocytosis of VEC. Arrows show the trans-endocytosed VEC-EGFP molecules from a COS7 cell that appeared to bud off from cell-cell junctions and be pulled into a HUVEC. Scale bar = 10 µm. See also Movie S1. (C) Co-culture of COS7 cells expressing VEC-EGFP (COS7-VEC-EGFP) and COS7 cells expressing TagRFP657 (COS7-TagRFP657). Lower images are higher magnification of the indicated area in upper images. No trans-endocytosis occurred between these cells. Scale bars = 20 µm.
Figure 3.
VEC trans-endocytosis mediates transport of junctional proteins.
(A) Co-culture of HUVECs expressing VEC-Y658E-EGFP and HUVECs expressing VEC-Y658F-TagRFPT. Arrow heads show VEC-Y658F-TagRFPT molecules were trans-endocytosed by VEC-Y658E-EGFP expressing cells. (B) Co-culture of HUVECs expressing p120-EGFP and HUVECs expressing VEC-TagRFPT. Arrow shows p120-EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. (C) Co-culture of HUVECs expressing β-catenin-EGFP and HUVECs expressing VEC-TagRFPT. Arrows show β-catenin -EGFP molecules were trans-endocytosed by VEC-TagRFPT expressing cells. (D) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC/α-catenin-FLAG. Arrowheads show VEC/α-catenin-FLAG molecules were trans-endocytosed by VEC-EGFP expressing cells. (E) Co-culture of HUVECs expressing EGFP and HUVECs expressing VEC-TagRFPT. Arrow shows EGFP molecules were trans-endocytosed and co-localized with VEC-TagRFPT in the recipient cell. (F) Co-culture of HUVECs labeled with iRFP and VEC-EGFP expressing HUVECs transfected with Cy3-labeled scramble siRNA. Arrowhead shows siRNA molecules were trans-endocytosed and co-localized with VEC-EGFP in the recipient cell. (A–F) Lower images are higher magnification of the indicated area in upper images. Scale bars = 20 µm, upper images; 5 µm, lower images.
Figure 4.
VEC trans-endocytosis is not dependent on known endocytic pathway.
(A) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT, then stained with anti-Rab5 antibody. The trans-endocytosed VEC molecules by the adjacent cell co-localized with Rab5 in the recipient cells to a low extent. Lower images are higher magnification of the indicated area in upper images. Scale bars = 20 µm, upper images; 5 µm, lower images. (B) Quantitative analysis of the number of trans-endocytosed VEC molecules co-localized with Rab5 in the recipient cells. About 15–20% of trans-endocytosed VEC molecules co-localized with Rab5 in the recipient cells. The percentage of Rab5 co-localization with VEC-TagRFPT was counted over 6-9 different fields of view for each time point; n = 6 (3 h), n = 8 (5 h) and n = 10 (7 h). Data were expressed as mean ± SD. (C) Co-culture of COS7 cells expressing VEC-EGFP and COS7 cells expressing VEC-TagRFPT with or without various inhibitors. Arrows show the trans-endocytosed VEC-EGFP molecules by VEC-TagRFPT expressing cells. Arrowheads show trans-endocytosed VEC-TagRFPT molecules by VEC-EGFP expressing cells. The trans-endocytosis occurred even with several inhibitors for clathrin-dependent endocytosis, macropinocytosis or phagocytosis. Dynasore (20 µM), an inhibitor for clathrin/dynamin dependent endocytosis, 5-(N-ethyl-N-isopropyl) amiloride (25 µM), the macropinocytosis inhibitor, Y27632 (10 µM), ROCK inhibitor, and bafilomycin A1 (200 nM), a specific inhibitor of the vacuolar type H(+)-ATPase for phagocytosis were used. Scale bars = 20 µm. (D) Quantification of the number of trans-endocytosis positive cells with indicated inhibitors. The percentage of trans-endocytosis positive cells was counted over 10 different fields of view for each inhibitor; n = 88 (DMSO), n = 65 (dynasore), n = 57 (amiloride), n = 63 (Y27632) and n = 57 (bafilomycin A1). Data were expressed as mean ± SD.
Figure 5.
VEC trans-endocytosis is dependent on actin/myosin force and Vinculin.
(A) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing LifeAct-TagRFPT. The trans-endocytosed VEC-EGFP structure clearly associated with F-actin visualized by LifeAct-TagRFPT. Lower images are time-course images of higher magnification of the indicated area in upper images (only for merged image). Arrow shows the trans-endocytosed VEC-EGFP structure and arrowheads show F-actin associated with VEC-EGFP during the trans-endocytosis process. Scale bars = 10 µm, upper images; 5 µm, lower images. See also Movie S2. (B) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT with (−)-blebbistatin. (−)-Blebbistatin, a selective inhibitor of non-muscle myosin II, inhibited trans-endocytosis of VEC vesicles. Arrows show trans-endocytosed VEC-EGFP vesicles by VEC-TagRFPT expressing cells. Scale bars = 10 µm, upper images; 5 µm, lower images. See also Movie S3. (C) Quantitative analysis of the number of trans-endocytosed VEC-EGFP structures from VEC-EGFP expressing cells to VEC-TagRFPT expressing cells in B. The number of trans-endocytosed vesicles after 5 minutes was counted and plotted. 100 µM of (−)-blebbistatin were added at 20 minutes after starting the time-lapse acquisition to inhibit the trans-endocytosis of VEC. (D–F) Co-culture of HUVECs expressing VEC-EGFP or α-catenin constructs and HUVECs expressing VEC-TagRFPT. When HUVECs expressing VEC-TagRFPT were co-cultured with HUVECs expressing VEC-EGFP (D) or α-catenin-EGFP (E), trans-endocytosis of VEC occurred as shown by arrowheads. When HUVECs expressing VEC-TagRFPT were co-cultured with HUVECs expressing α-catenin-ΔVBS-EGFP (F), trans-endocytosis of VEC did not occur. Lower images are higher magnification of the indicated area in upper images. Scale bars = 10 µm, upper images; 5 µm, lower images.
Figure 6.
VEC trans-endocytosis is Rac1 dependent.
(A) Co-culture of HUVECs expressing VEC-EGFP and HUVECs expressing iRFP with 100 µM of NSC23766. NSC23766, a specific inhibitor for Rac1 activation, inhibited trans-endocytosis of VEC-EGFP, though cell-cell junctions remained intact. Arrow shows intact cell-cell junction. Cells were first pre-treated for an hour with 100 µM of NSC23766 before co-culture, then were mixed and incubated for 4 hours with 100 µM of NSC23766. Scale bar = 10 µm. (B) Quantitative analysis of the number of trans-endocytosed structures with Rac1 or Cdc42 inhibitors. ML141, a specific inhibitor of Cdc42, and NSC23766 were used at concentrations around their IC50 values. The IC50 values of ML141 and NSC23766 are 2.6 µM and 50 µM, respectively. NSC23766 inhibited trans-endocytosis of VEC in a dose-dependent manner. The number of trans-endocytosed structures was counted for over 10-13 different fields of view per time point; n = 24-35 (ML141) and n = 19-38 (NSC23766). *, p<0.01 vs DMSO. Data were expressed as mean ± SD. (C) Co-culture of HUVECs expressing PA-Rac1-CA and HUVECs expressing VEC-EGFP. PA-Rac1-CA was accumulated at cell-cell junctions and co-localized with VEC-EGFP after its activation at 0 time point. Arrowheads show co-localization of PA-Rac1-CA with VEC-EGFP at cell-cell junctions. Scale bar = 10 µm. See also Movie S4. (D) Co-culture of HUVECs expressing PA-Rac1-DN and HUVECs expressing VEC-EGFP. PA-Rac1-DN was not accumulated at cell-cell junctions, nor co-localized with VEC-EGFP after its activation at 0 time point. Scale bar = 10 µm. See also Movie S4.
Figure 7.
VEC trans-endocytosis occurs in sprouting HUVECs and endothelial cells in zebrafish embryos.
(A) A single z-plane, x-z and y-z cross-sectional images of the tube-like structure of sprouting HUVECs. HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT were co-cultured in three-dimension fibrin gels. The Z-stack images were taken at the connection between HUVECs expressing VEC-EGFP and HUVECs expressing VEC-TagRFPT. Images were collected at 0.3 µm intervals with the 488 nm and 561 nm lasers to create a stack in the Z axis with a 60x objective. Scale bar = 20 µm. See also Movie S5. (B) Higher magnification of a 3-dimensional projection image of the indicated area in A. Arrows show VEC-EGFP molecules were trans-endocytosed by HUVECs expressing VEC-TagRFPT. Scale bar = 10 µm. (C) A three dimensional projection image, x-z and y-z cross-sectional images of the connection between the dorsal longitudinal anastomotic vessel (DLAV) and an intersegmental vessel (ISV) of a zebrafish embryo. Zebrafish VEC-EGFP (zVEC-EGFP) plasmids were injected into Tg(flkl:myr-mCherry) zebrafish using Tol2 system for transient mosaic expression of zVEC-EGFP. Images were collected at 1 µm intervals using the 488 nm and 561 nm lasers to create a stack in the Z axis with a 60x objective. Scale bar = 5 µm. See also Movie S6. (D) Sequential 3-dimensional projection image of the zebrafish vessel in C. Arrows show a zVEC-EGFP positive structure budding to inside of the endothelial cell. Scale bars = 5 µm.
Figure 8.
Model of trans-endocytosis of VEC.
VEC complex, VEC, p120, β-catenin, α-catenin and vinculin are pulled into the cell by actin/myosin force in a Rac1-dependent manner. The trans-endocytosed structure also contains some volume of adjacent cytosolic components that may include proteins or RNA molecules.