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First published online 29 May 2007
doi: 10.1242/jcs.007781

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High-pressure freezing provides insights into Weibel-Palade body biogenesis

Helen L. Zenner^1,*, Lucy M. Collinson^2,*,, Grégoire Michaux^1, and Daniel F. Cutler^1,¶

¹ MRC Laboratory of Molecular Cell Biology, Cell Biology Unit, and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK
² Electron Microscopy Facility, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (184K): [in this window] [in a new window]   Fig. 1. Weibel-Palade bodies forming in HUVECs show some connections to the Golgi and are often clathrin-coated. Cultures of HUVECs prepared for conventional transmission EM after chemical fixation give hints of the early biogenesis of WPBs. (A) Swollen Golgi cisternae (arrows) may be the earliest stage of WPB biogenesis, although no striations are apparent. Bar, 200 nm. (B) Weibel-Palade bodies juxta-Golgi have striations (arrows) and a clathrin coat (arrowheads) but the relationship to the Golgi is unclear. Bar, 200 nm. (C) Candidate WPBs (arrows) contain striations and have varying degrees of clathrin coat, but direct connections to the Golgi are unclear. Bar, 200 nm. (D) A single image showing the connections of a WPB that is connected (arrow) to the Golgi. Bar, 200 nm.

View larger version (155K): [in this window] [in a new window]   Fig. 2. Images of HUVECs prepared using HPF for transmission EM show a huge improvement in structural preservation. (A) A low magnification image showing immature WPBs (arrows) in the Golgi region. Bar, 500 nm. (B) A VWF tubule is seen within the TGN (arrowheads). Bar, 200 nm. (C,D) Immature WPBs with stalk-like attachments to the Golgi (arrows), Bars, 200 nm (C) and 500 nm (D). G, Golgi; M, mitochondrion; RER, rough endoplasmic reticulum; TGN, trans-Golgi network.

View larger version (88K): [in this window] [in a new window]   Fig. 3. Tubule organisation in immature WPBs. After HPF, immature WPBs found in the juxta-Golgi region of HUVECs were examined to assess their organisation. (A) A clathrin-coated immature WPB containing a single tubule of VWF. Bar, 200 nm. (B) A WPB containing two tubules that are disordered. The tubules touch the membrane only at the tip of the WPB. Bar, 200 nm. (C,D) More elongated WPBs containing three and seven tubules, respectively, which, while disorganised, together extend over the full length of the WPB. Bars, 500 nm (C) and 200 nm (D). (E) WPBs were grouped using the number of tubules apparent in the section and the width was measured. n=4-20 per bar; error bars represent standard error.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Clathrin has a dual role in the maturation of WPBs. (A,B) Clathrin in a supportive role. This is shown by its presence at the tip (A) and in a region where the tubules seem to be organising themselves relative to one another (B) (arrowheads). Bars, 200 nm. (C) Clathrin has a potential retrieval role shown by the classical clathrin-coated vesicle budding from the WPB (arrow). Bar, 500 nm.

View larger version (91K): [in this window] [in a new window]   Fig. 5. An increase in electron density is coupled to a decrease in width as WPBs mature. (A) A low magnification view shows both immature (arrow) and mature (arrowhead) WPBs within one section. Bar, 5 µm. (B-D) Mature WPBs found in the periphery of the cells show a dramatic increase in electron density. Bars, 500 nm (B), 200 nm (C) and 500 nm (D). (E) Percentage of WPBs in each width size bin for immature (white, n=51) and mature (black, n=34). G, Golgi; M, mitochondrion; N, nucleus; PM, plasma membrane; TGN, trans-Golgi network; RER, rough endoplasmic reticulum.

View larger version (94K): [in this window] [in a new window]   Fig. 6. Membrane-bound structures containing subunits of VWF in cells fixed by HPF. (A-C) Subunits in membrane-bound structures (arrowheads), adjacent to the Golgi in B and C, and with arrows indicating possible connections between subunits in C. Bars, 200 nm. (D) Combination of subunits (closed arrowheads) and cross-sections (open arrowheads). Bar, 200 nm. (E) Cross-sections only (open arrowheads). Bar, 200 nm. G, Golgi.

View larger version (113K): [in this window] [in a new window]   Fig. 7. WPBs manoeuvre within cells by folding at hinges. (A) HUVECs were fixed, permeabilised and labelled for VWF by indirect immunofluorescence (see Materials and Methods). The gallery shows a selection of the possible shapes of WPB, as seen by confocal microscopy. Bar, 2 µm. (B) HUVECs were transfected with VWF-GFP. A time-lapse movie (see Materials and Methods) was made 2 days after transfection. The sequence shows the movement of a single WPB over a 24-minute time period. Bar, 4 µm. See also Movie 1. (C,D) Images of WPBs after HPF and freeze substitution confirm that WPBs can bend as well as twist and branch. Bars, 200 nm (C) and 500 nm (D).

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