Pitstop‐2 and its novel derivative RVD‐127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases

Abstract

Clathrin‐mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop‐2 as a potent CME inhibitor, we and others have reported on substantial clathrin‐independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop‐2, termed RVD‐127, to clarify Pitstop‐2 diverse effects. Using RVD‐127, we were able to trace additional protein targets of Pitstop‐2. Besides inhibiting CME, Pitstop‐2 and RVD‐127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)‐like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop‐2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop‐2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop‐2 and RVD‐127 open up novel avenues.

Pitstop‐2 Upsets The Integrity of Nuclear Pore Complexes (NPCs) by Interaction with β‐Propeller Folds of Npc Scaffold Proteins

“…The binding affinities to Nups are slightly lower or almost equal to its inhibitory binding at the 𝛽propeller fold of the heavy chain of clathrin, and we, therefore, assume that Pitstop-2 may interfere simultaneously with both CME and NPCs, as concluded in our recent studies. [28,29] Substances capable of reducing the stringency of nucleocytoplasmic permeability barriers could facilitate the development of non-viral systems of intranuclear transgene delivery. [35] By causing a massive rearrangement of the NPC scaffold, Pitstop-2 can compromise the barrier's integrity without dissociating the barrier-forming nucleoporins.…”

“…to modulate the barrier and the active nucleocytoplasmic transport functions of the NPCs. [28,29] We reasoned that such similarity might be sufficient for the inhibitor to bind to those Nups and disrupt the structural organization of the NPC scaffold. Our hypothesis was validated by ultrastructural atomic force microscopy (AFM) imaging.…”

“…Based on the substantial similarities in β‐propeller folds in the heavy chain of clathrin and the Y‐complexes and the NPC scaffold, we previously hypothesized that Pitstop‐2 might also be able to modulate the barrier and the active nucleocytoplasmic transport functions of the NPCs. [ 28,29 ]…”

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate

“…Recently, we showed how nanopipettes can deform a cell via intrinsic force between the nanopipette tip and cell surface [14,15], allowing us to estimate the Young's modulus with higher resolution and with less force applied to living cells. We demonstrated that SICM for a Young's modulus estimate enables qualitative and quantitative characterization of the cytoskeleton state during alteration of cytoskeletal proteins such as F-actin and tubulin [16][17][18].…”

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM

“…Furthermore, using correlative confocal imaging of the cytoskeleton, we showed that the estimated Young's modulus is directly related to the state of the cytoskeleton. The developed technique was then successfully used to characterize the cytoskeleton state under different conditions [13][14][15].…”