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

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.

Facilitating plasmid nuclear delivery by interfering with the selective nuclear pore barrier

“…However, they also pointed out the significant cytotoxicity of 1,2‐TCHD for Vero cells at concentrations starting from 1%. Our previous works reveal cytotoxicity of 1,2‐TCHD with increasing concentrations in different cell types . Here, we present 1,6‐HD as promising lead substance for the design of pharmacologically potent NBBs facilitating nuclear delivery of nanoparticles.…”

“…Our previous works reveal cytotoxicity of 1,2-TCHD with increasing concentrations in different cell types. 16,17 Here, we present highly disordered brushes of FG-Nups which form the entropic barrier as described in the "polymer brush" model. 21 Larger molecules are generally unable to diffuse in this model as they cause a strain on the system by restricting the conformational freedom of the highly disordered FG-Nups anchored to the scaffold due to their large molecular mass or volume.…”

“…Cells were permeabilized with digitonin, which permeabilizes the plasma membrane but leaves the nuclear envelope intact. 20 They were then treated with the different NBBs to find out, using western blot analysis, whether or not the addition of the NBBs would lead to FG-Nups dissociation from NPCs 16,17 (Figure 3). Western blots were performed with cell nuclei.…”

“…10,11 Several amphiphilic alcohols disrupt NPC selectivity and therefore break its barrier function by acting on FG-Nups, for example, trans-1,2-cyclohexanediol (1,2-TCHD). [12][13][14][15] Upon the addition of this NPC barrier breaker (NBB), NPCs become permeable to 70 kDa dextran 16,17 and single pDNA particles. 13 1,2-TCHD, however, exhibits significant cytotoxic effects.…”

A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression

“…Graphs represent an average of at least three biological replicates specifically due to CME inhibition, we examined the effect of another CME inhibitor, dynasore. 18,25,26 Treatment with dynasore, a cell-permeable inhibitor of dynamin GTPases that disrupts CME, also resulted in a significant increase in Calcein Blue fluorescence relative to untreated cells ( Figure 4I-L). Together, these observations support the idea that alkalization of endosomal lumen by NHE9 leads to elevated clathrin-mediated endocytic uptake of GNPs.…”