Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies

Abstract

Weibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings.This article has an associated First Person interview with the first authors of the paper.

Exocytosis of Weibel–Palade bodies: how to unpack a vascular emergency kit

“…Finally, by monitoring of the redistribution of EGFP–CD63 through the membranes of fusing granules, a related but mechanistically distinct mode of homotypic WPB fusion, termed sequential or cumulative exocytosis, was observed . In this mode, a postfusion WPB is used as a membrane fusion site for subsequent cumulative fusion of additional WPBs, culminating in the ginger root‐like fusion structures that were observed in ultrastructural studies .…”

“…Homotypic fusion modes can serve to concentrate cargo release at hotspots when access to fusion sites on the membrane becomes limiting. It may also augment the secretory response by providing indirect access to the plasma membrane to a pool of WPBs situated deeper into the cytoplasm . Lingering‐kiss fusion not only saves vesicle reformation and retrieval of vesicle membrane proteins, but may also help in preserving a delicate balance between endocytosis and exocytosis by limiting membrane expenditure.…”

“…Lingering‐kiss fusion not only saves vesicle reformation and retrieval of vesicle membrane proteins, but may also help in preserving a delicate balance between endocytosis and exocytosis by limiting membrane expenditure. Exocytosis–endocytosis coupling is pertinent to the choice of secretory mode, as pharmacological blocking of compensatory endocytosis resulted in a shift from full fusion towards cumulative exocytosis, possibly because of the inability to retrieve membrane components from the postfusion WPBs . Distinct fusion modes also allow the endothelium to fine‐tune its secretory response to specific vascular conditions: lingering‐kiss fusion releases only a subset of molecules, such as chemokines, whereas prothrombotic cargo is not released .…”

“…Distinct fusion modes also allow the endothelium to fine‐tune its secretory response to specific vascular conditions: lingering‐kiss fusion releases only a subset of molecules, such as chemokines, whereas prothrombotic cargo is not released . Postfusion entanglement of VWF tubules in both compound and cumulative fusion structures does not preclude the eventual formation of VWF strings, but results in shorter strings when cumulative fusion is promoted .…”

Weibel−Palade bodies at a glance

“…where multiple WPBs can be present at a single fusion site (Kiskin et al, 2014;Stevenson et al, 2017;Valentijn et al, 2010). Such cumulative exocytic events occur when WPBs fuse with a pre-fused WPB, rather than with the plasma membrane directly.…”