Fig 1: Subcellular localisation of EGFP–MyRIP mutants in HUVECs – mutation of the actin-binding region does not perturb endogenous MyoVa recruitment to WPBs. (A) Top, schematic structure of human MyRIP domains and position of the mutated amino acids. Below, confocal immunofluorescence image of a single HUVEC expressing EGFP–MyRIP WT and labelled with phalloidin (red) and specific antibodies against EGFP (green) and VWF (blue). Regions indicated by white boxes are shown as greyscale inserts. (B–D) As for A but for cells expressing EGFP–MyRIP A751P (B), EGFP–MyRIP 4A (C) and EGFP–MyRIP A751P 4A (D). (E,F) Confocal immunofluorescence images of single HUVECs expressing EGFP–MyRIP WT (E) or EGFP–MyRIP 4A (F), and labelled with specific antibodies to EGFP (green) and MyoVa (red). Greyscale images below show, on the same scale, endogenous MyoVa immunoreactivity from regions indicated in the MyRIP-expressing (i) and non-expressing (ii) cells. Scale bars: 10 µm.
Fig 2: Actin-binding properties of EGFP–MyRIP mutants. (Ai–Di) Images from dual colour TIRF movies of live HUVECs co-expressing EGFP–MyRIP constructs (green) and TagRFP–actin (red) showing MyRIP WT (Ai) and MyRIP A751P (Bi), but not MyRIP 4A (Ci) or MyRIP A751P 4A (Di) decorating cortical actin and stress fibres. Images are averages of 134 to 222 consecutive frames acquired at 33 frames/s. Scale bars: 20 µm. Regions indicated by white boxes in Ai–Di are shown in greyscale below the colour merged images. Dual-colour video clips of a small sub-region of each cell are shown in Movies 1–4. (Aii–Dii) Representative examples of single-colour TIRF images of live HUVECs expressing MyRIP WT, MyRIP A751P, MyRIP 4A or MyRIP A751P 4A. Images are averages of 401–900 consecutive frames taken from videos acquired at 50 frames/s. To minimise false detection events during automatic single fluorophore detection and analysis, cells expressing EGFP–MyRIP mutants containing few or no WPBs were selected for recording. (Aiii–Diii) White tracks show the detected trajectories of single EGFP fluorophores for the cells in Aii–Dii (Aiii, 7731 detected objects; Biii, 6448 objects, Ciii, 54 objects; Dii, 89 objects). Scale bars: 20 µm. (E) Lifetime distributions for single fluorophore interaction of EGFP–MyRIP WT (i, n=12,703 objects from 16 cells) or EGFP–MyRIP A751P (ii, n=12,461 objects from 10 cells) and actin derived from experiments illustrated in Aii,iii or Bii,iii, respectively. Red lines show exponential fits (i, R=0.98; ii, R=0.99) to the distributions, and the off-rates determined from the fits are shown on the plots. Events with durations less than 200 ms were excluded to avoid short-lived ‘false’ events (see Materials and Methods).
Fig 3: MyRIP-actin interaction regulates WPB exocytosis. (A) Mean±s.e.m. for the WPB-associated EGFP fluorescence intensity (solid circles) and numbers of fluorescent WPBs (solid squares) in cells expressing MyRIP WT (black, n=7), MyRIP A751P (red, n=28), MyRIP 4A (blue, n=23) and MyRIP A751P 4A (green, n=19). Open symbols show data for cells pre-treated with cytochalasin D (1 µM, 20 min). (B) Cumulative plots of the total number of WPB fusion events detected during ionomycin stimulation (1 µM, applied at t=0 s) for HUVECs expressing VWFpp–EGFP (grey dotted line, n=19 cells, 1074 fusion events, delay to first fusion event, 1.72±0.13 s; mean±s.e.m.), MyRIP WT (no trace, n=7, 0, 0 s), MyRIP A751P (red symbols, n=28, 219, 11.82±4.56 s), MyRIP 4A (blue symbols, n=23, 556, 5.05±0.97 s), MyRIP A751P 4A (green symbols, n=19, 557, 4.03±0.68 s). Dashed lines show data in cells treated with cytochalasin D (+CytoD, 1 µM, 20 min) expressing MyRIP WT (black dashed line, n=14, 91, 8.54±1.78 s) or MyRIP A751P (red dashed line, n=19, 320, 4.44±0.98 s). Delays were compared using ANOVA multiple comparisons with multiplicity adjusted; P-values are given in the main text. Data are scaled to the mean fraction of fluorescent WPBs that underwent fusion in each condition (VWFpp, 65.5±3.2%, n=19 cells; MyRIP WT, 0%, n=7 cells; cytochalasin-D-treated MyRIP WT, 23.2±1.7%, n=14 cells; MyRIP A751P, 17.6±3.4%, n=28 cells; cytochalasin-D-treated MyRIP A751P, 40.89±3.58%, n=19 cells; MyRIP 4A, 47.9±4.1%, n=23 cells; MyRIP A751P 4A, 50.1±4.1%, n=19 cells; mean±s.e.m.).
Fig 4: MyRIP–actin interaction regulates WPB trafficking. (A) Representative x-y trajectories of individual WPBs in single HUVECs expressing VWFpp–EGFP (i), EGFP–MyRIP WT (ii), EGFP–MyRIP A751P (iii), EGFP–MyRIP 4A (iv) or EGFP–MyRIP A751P 4A (v). Trajectories were determined here and elsewhere from TIRFM videos using the ASPT function of GMimPro software (see Materials and Methods). The number of cells imaged and trajectories detected were: VWFpp, n=15 cells, 3890 trajectories; MyRIP WT, n=11 cells, 2978 trajectories; MyRIP A751P, n=13 cells, 1278 trajectories; MyRIP 4A, n=8 cells, 1862 trajectories; MyRIP A751P 4A, n=11 cells, 1310 trajectories. (B,C) Mean±s.e.m. parameters determined from detected trajectories of long-range (Bi–iii) or short-range (C) movements of WPBs in HUVECs expressing EGFP fusion proteins of VWFpp, MyRIP WT and MyRIP mutants as indicated. Number of WPBs analysed for short-range movements were: VWFpp, n=277; MyRIP WT, n=86; MyRIP A751P, n=242; MyRIP 4A, n=250; MyRIP A751P 4A, n=387. P values are with respect to VWFpp–EGFP (one-way ANOVA using Tukey multiple comparisons test).
Fig 5: Microtubule disruption abolishes long-range movements of WPBs carrying actin-binding-defective EGFP–MyRIP mutants. (Ai–Ci) Representative confocal immunofluorescence images of individual HUVECs expressing EGFP fusion proteins of MyRIP WT (Ai), MyRIP 4A (Bi) or MyRIP A751P 4A (Ci) exposed to vehicle (0.1% DMSO, 20 min: −Noco) and labelled with specific antibodies to EGFP (green), α-tubulin (red) and actin (phalloidin; blue). Scale bars: 20 µm. (Aii–Cii) Representative examples of the x-y trajectories of individual WPBs in control (−Noco) HUVECs expressing EGFP fusion proteins of MyRIP WT (Aii), MyRIP 4A (Bii) or MyRIP A751P 4A (Cii). Number of cells imaged and trajectories detected were: MyRIP WT, n=7 cells, 1007 trajectories; MyRIP 4A, n=3 cells, 448 trajectories; MyRIP A751P 4A, n=8 cells, 1667 trajectories. Scale bar: 10 µm for Aii-Cii (shown in Cii). (Aiii–Ciii) Representative confocal immunofluorescence images of individual HUVECs expressing the same EGFP fusion proteins and stained as described in Ai–Ci, but after treatment with nocodozole (1 µM, 20 min: +Noco). Scale bars: 20 µm. (Aiv–Civ) Representative examples of the x-y trajectories of individual WPBs in +Noco treated HUVECs expressing EGFP fusion proteins of MyRIP WT (Aiv), MyRIP 4A (Biv) or MyRIP A751P 4A (Civ). Number of cells imaged and trajectories detected were: MyRIP WT, n=3 cells, 497 trajectories; MyRIP 4A, n=7 cells, 125 trajectories; MyRIP A751P 4A, n=10 cells, 392 trajectories. Scale bar: 10 µm for Aiv-Civ (shown in Civ). (Di–iii) Mean±s.e.m. values for the fraction of WPBs with movements greater than 3 µm in length (Di), maximum displacement from start of recording (Dii) and maximum velocity (Diii) determined from the detected trajectories of the WPBs in control (−Noco) or nocodozole (+Noco) treated cells expressing the MyRIP fusion proteins as indicated. Number of cells imaged and trajectories detected in −Noco and +Noco treated cells are as described above. P values were calculated with a one-way ANOVA using Tukey multiple comparisons test.
Supplier Page from Abcam for Anti-MYRIP antibody