Fig. S1. Expression of ssEGFP leads to incorporation into the lumen of the ER and in WPBs in HUVECs. (A) Top, shows a representative fluorescence image of HUVECs 24 hours after nucleofection with ssEGFP (right panel and green in color merge). The cell was fixed and counterstained with a specific antibody to the ER resident marker PDI (middle panel and red in color merge). The region indicated by the white box in the colour merge image in the top panel of A is shown on an expanded scale in the lower panels. (B) An example of colocalisation of expressed ssEGFP fluorescence (right image and green in color merge) with specific antibodies to the WPB proteins VWF (left image, red in color merge) and Proregion (middle image, cyan in color merge). The region indicated by the white box in the color merge image is shown on an expanded scale in the lower panels,showing the characteristic rod-like morphology of the WPBs.

Fig. S2. Additional histograms of protein mobilities in the cytoplasm and plasma membrane of HUVECs. (A) Comparison of mobility histograms of EGFP in the HUVEC cytoplasm (expression of EGFP−N1, bin size 2 m²/second, shown in cyan) and in the HUVEC ER (as in Fig. 1D, expression of ssEGFP, bin size 1 m²/second, shown in grey), both obtained by point-FRAP. (B) Histograms of EGFP−hRab27a mobilities in WPBs obtained in confocal FRAP (black bars, data for control mature WPBs; see Fig. 4D) and of EGFP−Rab35 mobilities in plasma membrane of HUVECs obtained by point-FRAP (red bars). Bin sizes 0.2 m²/second.

Fig. S3. All WPBs located at the cell periphery acquire endogenous Rab27a or expressed EGFP−hRab27a, while immature WPBs lacking Rab27a are specifically located in a perinuclear region. (A) Representative color merge fluorescence image of a HUVEC stained with specific antibodies to endogenous Rab27a (green) and VWF (red). Grayscale images from perinuclear (i) and peripheral (ii) regions of the cell, indicated by the white boxes, are shown on expanded scales on the right in both panels. (B) Representative image of a HUVEC expressing EGFP−hRab27a (green) and counterstained with a specific antibody to VWF (red). (C) Representative color merge fluorescence image of a HUVEC expressing P-selectin−EGFP (8 hours post-nucleofection) and stained with specific antibodies to endogenous Rab27a (red) and VWF (blue). Grayscale images from perinuclear (i) and peripheral (ii) regions of the cell, indicated by the white boxes, are shown on expanded scales on the right. Panel (i) shows that P-selectin−EGFP is enriched within the immature Rab27a-negative WPBs.

Fig. S4. ‘Corona’ bleaching and FRAP analysis of protein mobility in WPBs. (A) The ‘corona’ effect revealed by repeated bleaching (20−30 times) of a dried layer of FITC deposited on a glass coverslip. Sample was scanned bidirectionally at high zoom using a 100× objective. The same laser power profile set for all bleachings (during left to right scan) is shown as a solid black line, with a region bleached at 100% laser power indicated by the yellow ROI. Note a decrease in average fluorescence of unbleached region, ‘corona’. (B) The average normalised fluorescence profile of five repeated bleaching experiments (same horizontal scale, solid black line; blue lines indicate ±s.d. levels). Red line, linear fit to the extended bleaching profile, slope is 0.79 m⁻¹. Identical results were obtained with 63× objective. (C) Schematic representation of the post-bleaching fluorescence profile, incorporating the linear extension due to the ‘corona’ effect, which was used in the diffusion model. (D) Horizontally aligned confocal images of a mature WPB expressing EGFP−CD63 in the membrane in control (left) and during the ‘fly’ mode bleach (right), the horizontal image size is 2.2 m. Two ROIs used for quantification in F are shown in white. Graph symbols corresponding to the unbleached (circles) and bleached (triangles) ROIs are shown under the images. (E) Left, spatial profiles of fluorescence in images in D are shown superimposed; the red trace corresponds to the control image. (E) Right, the difference of these profiles illustrating the amount of bleached fluorescence. The lengths of horizontal lines indicate L=1.79 m, αL=0.76 m (nominal bleaching, see A), βL=1.30 m (extended bleaching, the profile in C). (F) The average ROI fluorescences F1 (black circles) and F2 (red triangles) were scaled according to Eqn 2 and plotted against time t. The smooth solid lines represent the diffusion fit to the recovery time courses using the model (Eqn 3) with the parameters defined above. The best fit D value was 0.044 m²/second. An alternative model fit relying solely on α and L values without accounting for the extended bleaching (see profile in A) underestimated diffusion by 32% (D=0.030 m²/second).

Fig. S5. Complete bleaching of mature EGFP−hRab27a-positive WPB demonstrates absence of EGFP−hRab27a recovery from a cytoplasmic pool. (A) Left, control images of WPB in HUVEC coexpressing EGFP−hRab27a (top, green) and the core protein Proregion−mRFP (middle panel, red). Right, two-color images obtained at t=246 seconds post bleaching. (B) The time course for EGFP−hRab27a fluorescence during the experiment in A. Top, the fluorescent images of mature WPB expressing EGFP−hRab27a obtained at times indicated below the images. Note the absence of recovery. The residual fluorescence is due to incomplete EGFP bleaching (see residual Proregion−mRFP fluorescence in A, top right). Bottom, the graph of total EGFP−hRab27a fluorescence in the WPB plotted on time.

Fig. S6. Collapse of WPB structure following prolonged exposure to NH₄Cl. Fluorescence images showing VWF immunoreactivity in individual HUVEC in control (left) and after a 3 hour incubation with 40 mM NH₄Cl (right).