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First published online 19 August 2003
doi: 10.1242/jcs.00711

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Weibel-Palade bodies recruit Rab27 by a content-driven, maturation-dependent mechanism that is independent of cell type

Matthew J. Hannah1,*, Alistair N. Hume2, Monica Arribas1,{ddagger}, Ross Williams1, Lindsay J. Hewlett1, Miguel C. Seabra2 and Daniel F. Cutler1,§

1 MRC Laboratory of Molecular Cell Biology, Cell Biology Unit, University College London, Gower St, London WC1E 6BT, UK
2 Cell and Molecular Biology Section, Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK



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  		Fig. 1. Localization of Rab27 on WPBs in HUVECs by immunofluorescence microscopy. HUVECs grown on gelatine-coated glass coverslips were fixed, permeabilized and double immunolabelled for VWF (A,D and red in C,F) and endogenous Rab27 (B,E and green in C,F). Arrows in C and F point to WPBs that are positive for both VWF and Rab27, and the curly brackets indicate the population of VWF-positive organelles that do not stain for Rab27. Scale bar, 20 µm (F).

 


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  		Fig. 2. Rab27 purifies with VWF on density gradients. A post-nuclear supernatant was prepared from HUVECs that had been grown to confluence and the organelles contained therein were separated according to density on two sequential self-forming Percoll gradients (aliquots of fractions 7-9 from gradient 1 were pooled and run on gradient 2). (A) The membrane proteins present in each fraction were enriched by TX-114 partitioning, separated according to size by SDS-PAGE and transferred onto a PVDF membrane. The membrane was probed with an antibody to Rab27 and detected with an enzymatically coupled secondary antibody and ECL. (B) The quantification of the Rab27 immunoblot data in (A) (filled black circles) plotted together with the VWF-ELISA values of the same fractions (open squares). Both these data are plotted as percentages of the sum of immunoreactivities across the whole gradient.

 


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  		Fig. 3. Exogenously expressed EGFP-Rab27a localizes with WPB in HUVECs. HUVECs were transfected with expression vectors for either EGFP-Rab27a (A-C) or MYC-Rab1a (D-F) and, 24 hours later, fixed, permeabilized and immunolabelled for VWF (A-C) or VWF and the MYC epitope (D-F). VWF immunoreactivity is shown in (A,D) and is red in (C,F). EGFP fluorescence and MYC immunoreactivity are shown in (B) and (E), respectively, and are green in (C) and (F), respectively. A single EGFP-Rab27a-expressing cell can be seen in (B) and, in green, in (C). The arrows in (C) point to WPBs that are positive for both VWF and EGFP-Rab27a, and the curly bracket in (A) delineates a group of juxtanuclear WPBs that are EGFP-Rab27a negative. Scale bars, 20 µm (C,F).

 


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  		Fig. 4. Expression of a VWF-EGFP fusion protein in HUVECs reveals that newly formed WPBs are Rab27-negative. HUVECs were nucleofected with pre-pro-VWF-EGFP, replated onto gelatine-coated glass coverslips and allowed to recover for either 5 hours (A-C) or 24 hours (D-F), after which they were fixed and processed for immunocytochemistry as described in Materials and Methods. The images shown are single confocal sections of endogenous Rab27 immunoreactivity (A,D and red in C,F) and EGFP fluorescence/immunoreactivity (B,E and green in C,F). Arrowheads indicate examples of green-fluorescent WPBs that are not immunoreactive for Rab27, asterisks indicate examples of Rab27-immunoreactive WPBs that do not contain EGFP and arrows indicate examples of WPBs that are positive for both EGFP and Rab27 immunoreactivity. Scale bars, 10 µm.

 


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  		Fig. 5. In HEK-293 cells endogenous Rab27 immunoreactivity is not enriched on lysosomes, but it is recruited to the WPB-like organelles induced by expression of VWF. HEK-293 cells were either mock transfected (A-D) or transfected with VWF (E-L); after 48 hours, they were fixed and processed for immunocytochemistry as described in Materials and Methods. The images shown are composite confocal images of Rab27 (A,E,I and green in D,H,L), LAMP1 (B,F,J and red in D,H,L) and VWF (C,G,K and blue in D,H,L) immunoreactivities. The same confocal parameters were used to collect the data for both groups (transfected and untransfected). (I-L) An enlarged area from (H) (outlined by the white box). Asterisk in (A) indicates immunoreactivity associated with the microtubule organizing centre. Arrows in (A,B) indicate the slight association of Rab27 immunoreactivity with a minority of the LAMP1-positive structures. Arrows in (E,G,I,K) indicate examples of co-localization between the VWF-positive structures and Rab27, and the arrowheads point to examples of VWF-positive structures that are negative for Rab27 immunoreactivity. Scale bars, 10 µm (D,H), 2 µm (L).

 


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  		Fig. 6. Exogenously expressed EGFP-Rab27a localizes with WPB-like organelles and not lysosomes in HEK-293 cells. HEK-293 cells were transfected with either EGFP-Rab27a (A-D) or EGFP-Rab27a together with VWF-WT (E-L) and, after 48 hours, they were fixed and processed for immunocytochemistry as described in Materials and Methods. The images shown are composite confocal images of EGFP (Rab27a) (A,E,I and green in D,H,L), LAMP1 (B,F,J and red in D,H,L) and VWF (C,G,K and blue in D,H,L). The same confocal parameters were used to collect the data for both groups (plus and minus VWF-WT). (I-L) An enlarged area from (H) (outlined by the white box). Arrows in (I,K) indicate examples of co-localization between the VWF-positive structures and EGFP, and the arrowheads indicate examples of VWF-positive structures that are negative for EGFP. Scale bars, 10 µm (D,H), 2 µm (L).

 

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© The Company of Biologists Ltd 2003