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. 2022 Oct 19;8(4):e10425.
doi: 10.1002/btm2.10425. eCollection 2023 Jul.

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

Ivan Liashkovich  1 , Sílvio Terra Stefanello  1 , Reshma Vidyadharan  2 , Günter Haufe  2 , Alexander Erofeev  3   4 , Peter V Gorelkin  3 , Vasilii Kolmogorov  3 , Caren Rigon Mizdal  1 , Alexander Dulebo  5 , Etmar Bulk  1 , Ian U Kouzel  6 , Victor Shahin  1
Affiliations

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

Ivan Liashkovich et al. Bioeng Transl Med. .

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.

Keywords: atomic force microscopy; cellular physiology; clathrin; nanomedicine; nuclear pores; pharmacology; small GTPases.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

FIGURE 1
FIGURE 1
Experimental validation of the biological activities of the fluorescent derivative of Pitstop‐2, RVD‐127 with confocal fluorescence microscopy. (a) 100 μM RVD‐127 retains the ability to inhibit uptake of the classical substrate for clathrin‐mediated endocytosis, transferrin (red), to a similar extent as 30 μM Pitstop‐2. The medium of cells contains 70 kDa FITC‐dextran (green), which remains excluded from cellular uptake thereby delivering a negative image of the cells. Control cells are treated with the solvent of Pitstop‐2 and RVD‐127 (dimethyl sulfoxide; N = 5, and 60 or more cells each). (b) 30 μM Pitstop‐2 and 100 μM RVD‐127 are able to prevent association of importin‐β (Alexa‐488 labeled, green) with the nuclear pore complexes, and its intranuclear accumulation (N = 5, ≥60 cells each). Scale bars = 10 μm.
FIGURE 2
FIGURE 2
RVD‐127 disrupts selective nucleocytoplasmic transport across nuclear pore complexes (NPCs) by direct binding to the small GTPase Ran. (a) Subcellular distribution of the RVD‐127 (cyan) targets (N = 3, ≥60 cells each). Scale bar = 10 μm. (b) NPCs staining with mAb414 antibody (magenta), which stains phenylalanine‐glycine‐nucleoporins (FG‐Nups). (c) Merged image of (a) and (b). (d) Fluorescent staining with 100 μM RVD‐127 reveals a gradient of the distribution of its targets along the entire cytoplasmic (Cyt.) and nucleoplasmic (Nuc.) length of the NPC peaking at the nuclear basket 200 nm away from the FG‐nucleoporins labeled with mAb414. Three separate experiments were performed and the fluorescence intensity section profiles of 100 NPCs from 20 different cells were averaged and plotted (a.U., arbitrary units). (e) The gradient of the RVD‐127 staining within the NPC is clarified by its direct interaction with Ran GTPase. Heterologously expressed and purified recombinant Ran (Hexahistidin‐Tag) immobilized on Ni‐NTA agarose beads displays a characteristic staining of the bead edge when exposed to RVD‐127. Beads functionalized with other proteins (streptavidin, negative control) lack rim staining, which is indicative of a very limited interaction (N = 3). Scale bar = 100 μm. (f) Dynamics of intranuclear accumulation of RVD‐127 indicative of GDP‐like conformation of Ran induced by RVD‐127 (N = 3). Scale bar = 10 μm.
FIGURE 3
FIGURE 3
Pitstop‐2 and RVD‐127 disrupt actin dynamics and associated cell migration and mechanics. (a) High‐speed AFM imaging (frame rate = two images per minute) reveals that 7.5 μM Pitstop‐2 inhibits lamellipodial dynamics and causes gradual dismantling of cortical actin network within minutes of its application to living endothelial cells (bottom row, Video S1). (b,c) 50 μM RVD‐127 and 7.5 μM Pitstop‐2 induce an arrest of the endothelial cell motility. Quantification of the short‐term cell dynamics was performed by outlining the cell borders at time‐points 0 and 30 min for each condition (initial motility, inhibitor treatment, and inhibitor washout) and quantifying the cell area at time‐point 30 min which does not overlap with the initial cell position. The white, dark gray and light gray box shading in the plot corresponds to initial motility, inhibitor treatment, and inhibitor washout, respectively (N = 4, n = 20 cells for treatment with each type of inhibitor). Details about exact statistical analysis are given in the corresponding part in Experimental section. (d) Alexa‐488 phalloidin staining of EA.hy926 cells treated with 7.5 μM Pitstop‐2 or 50 μM RVD‐127 reveals ultrastructural alterations of actin cytoskeleton induced by the inhibitors. White arrowheads point to punctate foci of actin polymerization in dimethyl sulfoxide (DMSO)‐treated cells which are largely substituted by ring‐like structures in cells treated with Pitstop‐2 and RVD‐127 (N = 3, n ≥ 60 each). Scale bar = 10 μm.
FIGURE 4
FIGURE 4
Pitstop‐2‐induced disruptive effects on the Actin cytoskeleton and global cell motility are paralleled by substantial increase in cell volume and decrease in cell stiffness: Simultaneous investigation of topography and mechanics of living EA.hy926 cells before and after treatment with Pitstop‐2, using Scanning Ion Conductance Microscopy (noncontact imaging and low‐stress mechanical properties measurements). (a) Continuous mapping of the topography of EA.hy926 cells before and after the addition of Pitstop‐2. At 12 min was added Jasplakinolide (Jasp), a commonly used Actin filament polymerizing and stabilizing compound. (b) Simultaneously to topography mapping, continuous mapping of the mechanics (stiffness: Elastic Young's modulus, E) of EA.hy926 cells before and after the addition of Pitstop‐2. (c) Young's modulus distribution of EA.hy926 before and after treatment with Pitstop‐2; (d) Dynamic of mean value of cell Young's modulus during treatment with Pitstop‐2. (e) Cell volume change during treatment with Pitstop‐2 (N = 3, 10 cells each, and in each cell, five different areas were analyzed for Young's modulus measurements). Asterisks indicate a statistically significant difference, p < 0.05, performed by Analysis of variance (ANOVA) test.
FIGURE 5
FIGURE 5
Pitstop‐2 and RVD‐127 directly interact with the small GTPase Rac1 and inhibit downstream signaling. (a) Direct interaction of RVD‐127 with heterologously expressed and purified recombinant small GTPases Rac1 (Hexahistidin‐Tag, 6His), immobilized on Ni‐NTA agarose beads, demonstrated by confocal imaging. Characteristic staining of the bead periphery is detectable for beads functionalized with small GTPases but not for the empty beads or unrelated proteins (calmodulin) used as controls. Scale bar = 100 μm. (b) Biochemical assay based on quantification of Rac1 interaction with its cognate effector PAK demonstrates that Pitstop‐2 abolishes this interaction even in presence of nonhydrolyzable GTP analog (GTPγS, right lane) which otherwise strongly promotes this interaction (left lane) compared with a much weaker binding in presence of GDP (middle lane; N = 3, 20 beads each). DMSO, dimethyl sulfoxide
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References

    1. McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin‐mediated endocytosis. Nat Rev Mol Cell Biol. 2011;12:517‐533. - PubMed
    1. Mitsunari T, Nakatsu F, Shioda N, et al. Clathrin adaptor AP‐2 is essential for early embryonal development. Mol Cell Biol. 2005;25:9318‐9323. - PMC - PubMed
    1. Schmid EM, McMahon HT. Integrating molecular and network biology to decode endocytosis. Nature. 2007;448:883‐888. - PubMed
    1. Humphries AC, Way M. The non‐canonical roles of clathrin and Actin in pathogen internalization, egress and spread. Nat Rev Microbiol. 2013;11:551‐560. - PubMed
    1. Wu F, Yao PJ. Clathrin‐mediated endocytosis and Alzheimer's disease: an update. Ageing Res Rev. 2009;8:147‐149. - PubMed