MRC National Institute for Medical Research, London
Science for Health
Tom Carter group project:
Movie gallery
Resulting from inter-Divisional collaborations (Peter Rosenthal, Physical Biochemistry and Matthew Hannah, Molecular Neuroendocrinology) high resolution cryo-EM tomographic images of individual WPBs in intact vitrified ECs have led to the elucidation of the three dimensional structure (Movie 1) and packaging of the coagulation factor VWF, and have begun to reveal the structural transformations (Movie 2) that occur in VWF during WPB exocytosis (Berriman et al 2009).
Movie 1 - Tomogram of WPB in a frozen hydrated HUVEC
Flash player must be installed in order for you to view this video
A structural model for VWF tubules of a single WPB derived from a tomogram of a frozen hydrated HUVEC. The WPB membrane is shown in red. Those tubules running the full-length of the granule are shown in yellow, shorter tubules are shown in blue and one, showing bending at the upper end of the granule is shown in orange (see Berriman et al 2009).
Movie 2 - Exocytosis of a EGFP-CD63 containing WPB
Flash player must be installed in order for you to view this video
Marrow shaped WPB showing disordered tubules at the neck of a thin membrane projection to the plasma membrane. Similar membrane projections are observed during WPB exocytosis in living cells elicited by physiological stimuli (see movie WPB exocytosis and detection of secreted VWF), (see Berriman et al 2009).
Movie 3 - Intracellular trafficking of WPBs
Flash player must be installed in order for you to view this video
Single HUVEC (37oC) expressing EGFP-Rab3D. Rab3D is recruited specifically to the membranes of WPBs. Image series are projections of 5 optical z-sections acquired at ~2.5s intervals using a Nipkow Spinning-disc confocal microscope.
Movie 4 - Hormone evoked WPB exocytosis
Flash player must be installed in order for you to view this video
Movie showing a single HUVEC expressing the propolypeptide of VWF fused to EGFP (proregion-EGFP) that labels WPBs specifically. Cell was stimulated (time indicated by green box in top left corner) with histamine. Examples of WPB collapse with retention of proregion-EGFP are indicated by red circles.
Movie 5 - Complete and partial exocytosis of WPBs
Dual colour movie shows a small sub region of a HUVEC co-expressing the propolypeptide of VWF fused to EGFP that labels WPBs specifically (green) and mito-DsRed that labels mitochondria (red). Cell was stimulated with histamine.
Movie 6 - WPB exocytosis and detection of secreted VWF
Flash player must be installed in order for you to view this video
Dual colour movie showing an example of a single VWF-EGFP containing WPB (green) undergoing stimulated exocytosis (top panel and green in colour merge). Secreted extracellular VWF is detected by an extracellular fluorescent anti-VWF antibody (middle panel and red in colour merge).
Movie 7 - Multi-colour FRAP of individual secretory granules
Movie showing an example of a FRAP experiment in a single WPB. The WPB is co-expressing the VWF- propolypeptide fused to mRFP (proregion-mRFP; red) and the membrane protein EGFP-CD63 (green). Approximately half of the WPB was exposed to a brief period of intense illumination to bleach mRFP and EGFP fluorescence. The graphs on the left show the mean fluorescence intensity in the nominally unbleached (red circles) and bleached (black triangles) parts of the WPB with time. Proregion is immobile but CD63 recovers slowly. Data curves for EGFP-CD63 fluorescence are fitted with a diffusion equation to yield the apparent diffusion coefficient for this molecule in the WPB membrane.
Selected publications
- Berriman JA, Li S, Hewlett LJ, Wasilewski S, Kiskin FN, Carter T, Hannah MJ, Rosenthal PB. (2009)
Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells.
Proceedings of the National Academy of Sciences of the United States of America 106, 17407-17412 PubMed abstract
Internal links
© MRC National Institute for Medical Research
The Ridgeway, Mill Hill, London NW7 1AA
Top of page