Clathrin inhibitor Pitstop-2 disrupts the nuclear pore complex permeability barrier.

Ivan Liashkovich,Gonzalo Rosso,Dzmitry Pasrednik,Valeria Prystopiuk,Hans Oberleithner,Victor Shahin

doi:10.1038/srep09994

Abstract

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.

Highlights

Combining FITC-labeled 250 kDa dextran[14] with TRITC-labeled 65–85 kDa dextran has revealed that only the 65–85 kDa TRITC dextran exhibits accelerated diffusion into the nucleus in presence of Pitstop-2 while 250 kDa FITC dextran remains excluded from the nucleoplasm unless the lipid component of the nuclear envelope is compromised by 1% Triton X100 (Fig. S1)
To test if the NPC permeabilization effect by Pitstop-2 can be attributed to an indirect action via clathrin we examined whether another clathrin inhibitor Pitstop-1, which binds to the β-propeller domain, is able to induce a similar effect
Pitstop-1 was unable to induce the influx of 70 kDa FITC dextran into the nuclei (Fig. S2), strongly indicating that permeabilization of the NPC induced by Pitstop-2 is independent of clathrin

Summary

Introduction

Confocal laser scanning microscopy has revealed that Impβ was able to bind to the NPCs of the digitonin-permeabilized Ea.hy[926] and accumulated in the nucleoplasm in presence of DMSO or the Pitstop-2 negative control compound (Fig. 2A–F). The Pitstop-2 negative control compound had no significant effect on Impβ binding to the NPCs of Ea.Hy926 endothelial cells to its lack of effect on the NPC permeability. To quantify the structural changes we performed cross-sectional analysis of Pitstop-2-treated NPCs and compared it to DMSO control (Fig. 3E) as described previously[5].

Results

Conclusion