Zenner Helen L, Collinson Lucy M, Michaux Grégoire, Cutler Daniel F
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.
J Cell Sci. 2007 Jun 15;120(Pt 12):2117-25. doi: 10.1242/jcs.007781. Epub 2007 May 29.
The Weibel-Palade bodies (WPBs) of endothelial cells play an important role in haemostasis and the initiation of inflammation, yet their biogenesis is poorly understood. Tubulation of their major content protein, von Willebrand factor (VWF), is crucial to WPB function, and so we investigated further the relationship between VWF tubule formation and WPB formation in human umbilical vein endothelial cells (HUVECs). By using high-pressure freezing and freeze substitution before electron microscopy, we visualised VWF tubules in the trans-Golgi network (TGN), as well as VWF subunits in vesicular structures. Tubules were also seen in WPBs that were connected to the TGN by membranous stalks. Tubules are disorganised in the immature WPBs but during maturation we found a dramatic increase in the spatial organisation of the tubules and in organelle electron density. We also found coated budding profiles suggestive of the removal of missorted material after initial formation of these granules. Finally, we discovered that these large, seemingly rigid, organelles flex at hinge points and that the VWF tubules are interrupted at these hinges, facilitating organelle movement around the cell. The use of high-pressure freezing was vital in this study and it suggests that this technique might prove essential to any detailed characterisation of organelle biogenesis.
内皮细胞的魏贝尔-帕拉德小体（WPB）在止血和炎症起始过程中发挥着重要作用，但其生物发生过程却鲜为人知。其主要内容蛋白血管性血友病因子（VWF）的管状化对WPB功能至关重要，因此我们进一步研究了人脐静脉内皮细胞（HUVEC）中VWF小管形成与WPB形成之间的关系。通过在电子显微镜检查前使用高压冷冻和冷冻置换技术，我们观察到了反式高尔基体网络（TGN）中的VWF小管以及囊泡结构中的VWF亚基。在通过膜性柄与TGN相连的WPB中也发现了小管。在未成熟的WPB中小管排列紊乱，但在成熟过程中，我们发现小管的空间组织和细胞器电子密度显著增加。我们还发现了有被出芽轮廓，提示在这些颗粒初步形成后对错误分选物质的清除。最后，我们发现这些大的、看似刚性的细胞器在铰链点处弯曲，并且VWF小管在这些铰链处中断，这有利于细胞器在细胞周围移动。高压冷冻技术在本研究中至关重要，这表明该技术可能对细胞器生物发生的任何详细表征都至关重要。
