Existence of a selective nucleocytoplasmic permeability barrier is attributed to Phenylalanine-Glycine rich proteins (FG-nups) within the central channel of the nuclear pore complex (NPC). Limited understanding of the FG-nup structural arrangement hinders development of strategies directed at disrupting the NPC permeability barrier. In this report we explore an alternative approach to enhancing the NPC permeability for exogenous macromolecules. We demonstrate that the recently discovered inhibitor of clathrin coat assembly Pitstop-2 compromises the NPC permeability barrier in a rapid and effective manner. Treatment with Pitstop-2 causes a collapse of the NPC permeability barrier and a reduction of Importin β binding accompanied by alteration of the NPC ultrastructure. Interestingly, the effects are induced by the same chemical agent that is capable of inhibiting clathrin-mediated endocytosis. To our knowledge, this is the first functional indication of the previously postulated evolutionary relation between clathrin and NPC scaffold proteins.

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To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.