Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a ) ARHGEF7 (human isoform-a, 646 residues, NM_001113513) domains, structure, and selected residues. The structure is an AlphaFold model (AF-Q14155-1-F1-model_v6_isoform_a) of ARHGEF7 showing the structured SH3, DH, and PH domains with cysteines and nearby residues. ( b ) Accessible surface areas (ASA) of cysteines in ARHGEF7. ( c ) Sequence alignments around C312 in ARHGEF7 orthologs. ( d ) Clickable glutathione approach. Cells expressing a glutathione synthetase mutant (GS M4) synthesize and produce clickable glutathione (N 3 -GSH) using endogenous γGlu-Cys and exogenous azido-Ala. Clickable glutathione forms S-glutathionylation with proteins, which are analyzed after click reactions. ( e - f ) Glutathionylation of ARHGEF7 WT and cysteine mutants in MDA-MB-231 cells in response to high glucose (HG, 25 mM) or low glucose (LG, 5 mM) conditions: cells were incubated for 20 h (n=3). ( g - h ) Glutathionylation of ARHGEF7 constructs in MDA-MB-231 cells upon adding EGF (n=3). EGF was incubated for 20 h. The lysates were used for click reactions with biotin-alkyne and analyzed by western blot with FLAG antibody before (input) and after (eluted) pull-down with streptavidin-agarose. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( e - h ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Sequencing, Expressing, Mutagenesis, Incubation, Construct, Western Blot

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a ) A potential mode of ARHGEF7 activation by S-glutathionylation. In the absence of ROS, the DH-PH active site is relatively closed, and the SH3 domain stays bound to the DH-PH or its binding partners, Rac1 or PAK1, through the C-terminal proline-rich sequence (PRS) in Rac1 and PAK1 (left). Upon ROS generation, ARHGEF7 is glutathionylated at C312 at the PH domain, which opens the active site in the DH-PH domains to which SH3-bound Rac1-GDP binds for GDP-GTP exchange (middle). Rac1 activation follows, resulting in PAK1 phosphorylation and activation (right). ( b ) A model for ARHGEF7 activation in cells. In the absence of ROS, ARHGEF7, in complex with GIT1, may be associated with none or PAK1 in cells, without significant Rac1 activation. In the presence of ROS, glutathionylated ARHGEF7/GIT1/PAK1 complex is recruited to the nascent focal adhesion at the protrusion, where PAK1 is dissociated, and glutathionylated ARHGEF7 increases the binding to Rac1 for Rac1 activation. Active Rac1 increases phosphorylation and activation of PAK1. The active Rac1 and PAK1 activate their downstream signaling, including Arp2/3, LIMK-cofilin, and MEK1/2-ERK1, which increases branching and polymerization of actin filaments, lamellipodia formation, and cell migration.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Activation Assay, Binding Assay, Sequencing, Phospho-proteomics, Migration

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a - b ) In-vitro scratch migration analysis of MDA-MB-231 cells expressing ARHGEF7 construct in high glucose (HG, 25 mM) or low glucose (LG, 5 mM) conditions (n=4) ( a ) or in response to EGF (n=9) ( b ). Yellow indicates areas without cells. ( c - d ) Transwell invasion analysis of MDA-MB-231 cells expressing ARHGEF7 constructs in HL or LG (n=3) ( c ) or upon incubating EGF (n=3) ( d ). Cells were incubated in HG or LG ( a , c ) or with EGF for 20 h ( b , d ). A scale bar = 500 μm. Data represent the mean ± SD. The statistical difference was analyzed by two-way ANOVA and Tukey’s post-hoc test ( a - d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: In Vitro, Migration, Expressing, Construct, Incubation

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: Analysis of ARHGEF7 protein interactions and Rac1-PAK1 downstream signaling. MDA-MB-231 cells expressing ARHGEF7 WT or C312S were incubated in high glucose (HG, 25 mM) or low glucose (LG, 5 mM) for 20 h. ( a ) ARHGEF7 S-glutathionylation increases its binding to Rac1. ARHGEF7 co-immunoprecipitation (co-IP) with Rac1 and PAK1 (n=3). ( b ) Rac1 is activated upon ARHGEF7 S-glutathionylation. Active Rac1 was enriched by purified GST-PBD (p21-binding domain derived from PAK1) and analyzed by western blots (n=3). ( c ) ARHGEF7 S-glutathionylation activates PAK1 and its downstream signaling. Phosphorylation levels of PAK1, LIMK1, and MEK1/2 were analyzed by western blot (n=3). ( d ) ARHGEF7 S-glutathionylation increases Rac1 localization at the cell periphery. The co-localization images of ARHGEF7 and Rac1 (left). Cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n=10 images). White arrows indicate the intense co-localization of ARHGEF7 and Rac1. Pearson’s correlation coefficients (right) were calculated after Intermodes thresholding to select for colocalization of ARHGEF7 and Rac1 at the cell periphery (see also Figure S6). A scale bar = 10 μm. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a-d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Expressing, Incubation, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Purification, Derivative Assay, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a - c ) In vitro binding analysis of ARHGEF7 constructs (DP or SDP) with GST-Rac1. Purified ARHGEF7 constructs (DP and SDP) were incubated with oxidized glutathione (GSSG) to induce S-glutathionylation. Following dialysis, ARHGEF7 constructs were incubated with GST-Rac1 immobilized on glutathione-Sepharose. GST-Rac1 and ARHGEF7 constructs were analyzed by Western blot before and after GST pull-down. ( a ) Comparison of Rac1 binding to ARHGEF7 DP WT and ARHGEF7 SDP WT (n=3). ( b ) Comparison of Rac1 binding to ARHGEF7 SDP WT and ARHGEF7 SDP C312S (n=3). ( c ). Comparison of ARHGEF7 SDP WT binding to Rac1 with different active site states (none, GDP, GTPγS) (n=3). ( d ) In vitro binding analysis of ARHGEF7 with PI(3,5)P 2 . Purified ARHGEF7 SDP treated without and with GSSG were incubated with biotinylated PI(3,5)P 2 on micelle. Bound ARHGEF7 SDP constructs were analyzed by Coomassie stain (CM) (n=3). ( e - f ) ARHGEF7 increases its GDP-GTP exchange rate upon S-glutathionylation. Different purified ARHGEF7 constructs were incubated with GSSG and used for enzyme kinetics. Rates were determined by a loss of fluorescence when Bodipy-GDP bound to Rac1 dissociates from Rac1. ( e ) Rate measurement by purified ARHGEF7 DP or SDP constructs without or with incubation of GSSG. Rates were determined by increasing enzyme concentration (n=3) (left) and substrate concentration (Michaelis-Menten) (n=3) (right). ( f ) Rate measurement by a full-length ARHGEF7 construct without or with glutathionylation. FLAG-ARHGEF7 purified by affinity purification was incubated with GSSG and used to measure rates (n=4). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a-d ) or unpaired t-test ( f ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: In Vitro, Binding Assay, Construct, Purification, Incubation, Western Blot, Comparison, Staining, Fluorescence, Concentration Assay, Affinity Purification

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: (A) Purified Lsm12 protein used throughout this study. Coomassie blue staining of an SDS-PAGE gel shows a single band near 25 kDa, consistent with the predicted molecular mass of 23.8 kDa. (B) Cytosolic application of purified Lsm12 reduced lysosomal TPC2 currents activated by 0.3 µM PI(3,5)P 2 in a concentration-dependent manner. For most Lsm12 concentrations, n = 10; n = 7 for 0.1 µM Lsm12 and n = 8 for the washout condition (“0 again”). (C) Purified Lsm12 (2 µM) markedly reduced endolysosomal TPC1 currents activated by 0.3 µM PI(3,5)P 2 ( n = 7). (D) Purified Lsm12 (2 µM) had little effect on endolysosomal TRPML1 currents activated by 0.3 µM PI(3,5)P 2 ( n = 6). Left panels in B–D: representative current-voltage (I-V) relationships obtained using a voltage ramp from −120 mV to +120 mV, together with schematic illustrations of whole-endolysosome recordings showing the major cations on either side of the membrane, from vacuolin-1-enlarged endolysosomes acutely isolated from HEK293 cells. Right panels in B–D: summary of current amplitudes measured at −120 mV (B, D) or +120 mV (C), showing individual data points and mean ± SEM, normalized to the corresponding current recorded in the absence of Lsm12. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Purification, Staining, SDS Page, Concentration Assay, Membrane, Isolation

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: (A) Representative time course of TPC2 PM currents at −120 mV during perfusion with 0.3 µM PI(3,5)P 2 . After current activation under control conditions, 0.5 µM Lsm12 was applied to inhibit the current, and subsequent washout of Lsm12 led to recovery. (B, C) Summary of the normalized current amplitudes derived from the maximal (activation) and minimal current (inhibition) levels ( B ) and time constants (τ) ( C ) during the control, Lsm12 wash-on, and wash-off phases from recordings as shown in A ( n = 5). (D) Effects of heat-treated Lsm12 on PI(3,5)P 2 -activated TPC2 PM currents, normalized to currents recorded in the absence of heat-treated Lsm12. (E) Fraction of unbound BODIPY FL PI(3,5)P 2 remaining after incubation of 0.3 µM lipid with 0.05, 0.3, or 1 µM 6×His-tagged Lsm12 ( n = 4), determined from the fluorescence remaining in solution after precipitation of bead-bound Lsm12 with IMAC beads. (F) Representative I–V relationships of constitutively active TPC2 PM carrying the L690A/L694A mutation, recorded under bath conditions alone or after addition of the indicated concentrations of Lsm12 or PI(3,5)P 2 . (G, H) Representative I–V relationships of TPC2 PM currents activated by 0.3 µM ( G ) or 1 µM ( H ) PI(3,5)P 2 in the absence and presence of the indicated concentrations of Lsm12. (I) Concentration-response relationships for inhibition by Lsm12 of TPC2 PM currents activated by 0.3 µM ( n = 10) or 1 µM ( n = 9) PI(3,5)P 2 . (J) PI(3,5)P 2 concentration-response relationships for TPC2 PM currents in the absence and presence of 2 µM Lsm12. (K) Lsm12 concentration-response relationships for TPC2 PM pseudo-WT ( n = 10) and Δ45–50 mutant ( n = 8) channels activated by 0.3 µM PI(3,5)P 2 . Left, modeled structure of Lsm12 highlighting residues 45–50 in stick representation. Summarized data are presented as plots showing mean ± SEM, with or without individual data points, as indicated. ns, not significant ( P > 0.05).

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Activation Assay, Control, Derivative Assay, Inhibition, Incubation, Fluorescence, Mutagenesis, Concentration Assay

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: ( A ) NAADP elicits TPC2 PM currents in the presence of PI(3,5)P 2 and Lsm12. Left : representative time course of TPC2 PM currents recorded at −120 mV during continuous application of 0.3 µM PI(3,5)P 2 , followed by sequential addition of 50 nM Lsm12 alone and then 50 nM Lsm12 together with increasing concentrations of NAADP (10, 100, and 1000 nM), and subsequent withdrawal of NAADP and Lsm12. Middle : summary of current amplitudes measured at −120 mV and normalized to the initial current activated by 0.3 µM PI(3,5)P 2 alone. Right : representative I–V relationships obtained under the corresponding conditions. ( B ) NADP does not elicit TPC2 PM currents in the presence of PI(3,5)P 2 and Lsm12. Current amplitudes were measured at −120 mV and normalized to the initial current activated by 0.3 µM PI(3,5)P 2 alone. ( C ) NAADP fails to induce currents of PI(3,5)P 2 -insensitive mutant TPC2 PM channels. Current amplitudes were measured at −120 mV under the indicated conditions. Path-clamp recordings in (A-C) were performed under the same configuration and ionic conditions. (D) NAADP does not alter the relative Na + /Ca 2+ permeability of PI(3,5)P 2 -activated TPC2 PM currents. Middle: representative I–V relationships under the indicated conditions. Left and right: summaries of normalized current amplitudes at +120 mV and reversal potentials, respectively. For reversal potential analysis, only I–V plots with maximal current amplitudes (+120 mV) between 0.5 and 1 nA were included to minimize error. Throughout this figure, the numbered steps and time points correspond to the color-coded conditions. Unless otherwise specified, the patch-clamp configurations and ionic conditions are illustrated schematically on the left. Bar graphs show individual data points and mean ± SEM. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, p > 0.05.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Mutagenesis, Permeability, Patch Clamp

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: (A) Summary of current amplitudes and representative I-V relationships showing that NAADP at 0.1 or 1 µM elicits currents of endolysosomal TPC2 in the presence of 0.3 µM PI(3,5)P 2 and 1 µM Lsm12. ( B ) Representative I–V relationships comparing Lsm12 alone and Lsm12 plus NAADP, showing that NAADP produces minimal activation of TPC2 currents from Lsm12-KO cells in the absence of PI(3,5)P 2 . ( C ) Summary of 1 µM NAADP-induced currents in Lsm12-KO cells supplemented with purified Lsm12 (2 µM), in the presence or absence of 0.3 µM PI(3,5)P 2 . ( D ) Summary of current amplitudes and representative I-V relationships showing that NAADP (1 µM) elicits currents of endolysosomal TPC1 in the presence of 0.3 µM PI(3,5)P 2 and 1 µM Lsm12. Current amplitudes for summary plots were measured at −120 mV in (A) and (C), and at +120 mV in (D). Where indicated, currents were normalized to those recorded in the absence of NAADP and Lsm12 within the same recording. Patch-clamp configurations and ionic conditions were identical for (A–C) and are illustrated schematically to the left of (A) and (D). Bar graphs show individual data points and mean ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Activation Assay, Purification, Patch Clamp

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: (A) Endogenous PI(3,5)P 2 contributes to NAADP-evoked Ca²⁺ release. Antibodies or lipid-binding “grip” proteins targeting PI(3,5)P 2 or PI(4,5)P2 were co-injected with NAADP into cells. Left: Representative Ca²⁺ indicator traces. Right: Summary of peak NAADP-evoked Ca²⁺ responses. Bar graphs show individual data points and mean ± SEM. ***, p < 0.001. ( B ) Schematic summary of the interplay among PI(3,5)P 2 , Lsm12, and NAADP in TPC regulation. PI(3,5)P 2 activates TPCs, whereas Lsm12 binding to TPCs competitively suppresses PI(3,5)P 2 -activated channels. NAADP binding to Lsm12 relieves this suppression, restoring channel opening in the presence of PI(3,5)P 2 . The interacting sites of the ligand and proteins, as well as the number of Lsm12 molecules depicted in the cartoon, are arbitrary.

Article Snippet: 500 μl 0.3 μM BODIPY FL PI(3,5)P 2 (Echelon Biosciences #C-35F6) was incubated with designated concentrations (0, 0.05, 0.3, and 1 μM) of 6×His tagged Lsm12 for 10 min in the K + -based solution (145mM KMeSO3, 5mM KCl, and 10mM HEPES (pH 7.4)) that was used for inside-out patch-clamp recordings.

Techniques: Binding Assay, Injection