Journal: bioRxiv

Article Title: Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

doi: 10.1101/2023.03.03.529094

Figure Lengend Snippet: (A) GINIP binds to active but not inactive Gαi3. Left , representative Coomassie-stained gel showing binding of His-Gαi3 loaded with the indicated nucleotides (GDP, GDPꞏAlF 4 − , GTPγS) to immobilized GST-GINIP. Right , quantification of His-Gαi3 binding to GST-GINIP based on band densitometry. K D ’s were determined based on fitting the data to a one-site binding nonlinear regression curve. Mean ± S.E.M., n=3-4. (B) GINIP binds to Gαi3, but not to other Gα’s of the same family (Gαo, Gαz), or other families (Gαs, Gαq, Gα12). Lysates of HEK293T cells expressing the indicated G proteins were incubated with GST or GST-Gαi3 immobilized on glutathione-agarose beads in the presence of GDP or GDPꞏAlF 4 − , as indicated. Bead-bound proteins were detected by Ponceau S staining or by immunoblotting (IB). (C) GINIP does not affect the enzymatic activity of Gαi. Nucleotide exchange on Gαi3 was determined by GTPγS binding, whereas nucleotide hydrolysis by Gαi1 RM/AS was determined by the production of free phosphate (Pi) from GTP. GINIP, 2 μM, DAPLE, 1 μM, RGS4, 0.2 μM. Mean ± S.E.M., n=3. (D) Gαi3 region 205-262 is required for GINIP binding. Left , diagram of Gαi3 (orange) /Gαo (green) chimeras. Sequence alignment of the Gαi3 205-262 region with Gαi1, Gαi2 and Gαo, indicating mutations tested in panel (E). Right , representative experiment showing binding of purified His-GINIP to the indicated G proteins in the presence of GDPꞏAlF 4 − . Bead-bound proteins were detected by Ponceau S staining or by immunoblotting (IB). (E) Mutation of residues in the α3 helix and Switch II of Gαi ablate GINIP binding. Left, Structural model of Gαi1-(GDPꞏAlF 4 − ) (PDB: 2G83). Red indicates residues in the α3/Switch II region that result in loss of GINIP when mutated, whereas blue indicates a residue in the same region that does not affect GINIP binding when mutated. Center, Mutation of some residues in the α3 helix from Gαi3 to cognate amino acids from Gαo ablate binding to GINIP. Right, representative experiment showing binding of purified His-GINIP to the indicated G proteins in the presence of GDPꞏAlF 4 − . Bead-bound proteins were detected by Ponceau S staining or by immunoblotting (IB). (F) Mutation of residues within, but not adjacent to, the effector binding region (α3/Switch II groove) of Gαi impair GINIP binding. Left , Structural model of Gαi1-(GDPꞏAlF 4 − ) (PDB: 2G83) displaying the residues investigated by site-directed mutagenesis. Right , representative Coomassie-stained gel showing binding of the indicated Gαi3 proteins loaded with GTPγS to immobilized GST-GINIP. All protein electrophoresis results are representative of n ≥ 3 experiments.

Article Snippet: HEK293T cells (Lenti-X 293T, Cat# 632180, Takara Bio) were plated on 150 mm diameter dishes (~2.5 million cells / dish) and cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.

Techniques: Staining, Binding Assay, Expressing, Incubation, Western Blot, Activity Assay, Sequencing, Purification, Mutagenesis, Residue, Protein Electrophoresis

Journal: bioRxiv

Article Title: Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

doi: 10.1101/2023.03.03.529094

Figure Lengend Snippet: (A) GINIP prevents Gαi-mediated inhibition of adenylyl cyclase (AC) upon stimulation of 3 different G i -coupled GPCRs. Top row, kinetic traces of BRET measurement of cAMP in HEK293T cells expressing the GABA B R in the presence or absence of GINIP treated with forskolin (FSK) and GABA as indicated. Bottom row, quantified inhibition of FSK-stimulated cAMP upon stimulation of GABA B R, α2-AR, or D2R with GABA (1 μM), Brimonidine (5 μM), or dopamine (0.2 μM). Immunoblot (IB) validates GINIP expression. Mean ± S.E.M., n=3-5. **p<0.01, ***p<0.001, paired t-test. (B) GINIP prevents the association of active Gαi3 with AC5 in cells. Left, changes in BRET (ΔBRET) were determined in HEK293T cells expressing Gαi3-Nluc WT or Gαi3-Nluc Q204L upon transfection of increasing amounts of GINIP. Mean ± S.E.M., n=6. Right , validation of GINIP expression by IB. (C) GINIP prevents the association of Gαi3 with AC5 upon GPCR stimulation. BRET was measured in HEK293T cells expressing the GABA B R or the α2 A -AR upon transfection of different amounts of GINIP DNA. Kinetic traces correspond to cells expressing no GINIP (‘CTRL’ blue) or transfected with 2 μg of GINIP plasmid (red). Cells were treated with the indicated GPCR agonists/antagonists, and BRET changes (ΔBRET) one minute after agonist stimulation for each amount of GINIP transfection. Mean ± S.E.M., n=3. (D) GINIP blocks the regulation of AC by Gαi in vitro . Coomassie-stained gel shows the purified proteins used to reconstitute and modulate AC activity in vitro . Bar graph shows that FSK (5 μM) or Gαs-GTPγS (0.1 μM), but not myr-Gαi1 (2 μM) or GINIP (2 μM), promote the activation of reconstituted AC (AC5 (c1) + AC2 (C2)). Right, Gαs-stimulated AC activity was determined in the presence of increasing concentrations of myr-Gαi1-GTPγS in the absence (blue) or presence of GINIP (2 μM, red). Mean ± S.E.M., n=5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA for presence/ absence of GINIP x myr-Gαi1 concentration, with multiple comparisons at each concentration using Fisher’s LSD test. (E) Diagram summarizing the proposed mechanism action of GINIP on Gαi-mediated modulation of AC activity (competitive binding).

Article Snippet: HEK293T cells (Lenti-X 293T, Cat# 632180, Takara Bio) were plated on 150 mm diameter dishes (~2.5 million cells / dish) and cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.

Techniques: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, In Vitro, Staining, Purification, Activity Assay, Activation Assay, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

doi: 10.1101/2023.03.03.529094

Figure Lengend Snippet: (A) GINIP enhances Gβγ-mediated signaling triggered by GABA B R. Left, diagram of G protein activation/deactivation cycle and BRET-based detection of free Gβγ. Center, BRET was measured in HEK293T cells expressing the GABA B R in the absence (black) or presence (red) of GINIP. Kinetic traces correspond to cells expressing no GINIP (black) or transfected with 2 μg of GINIP plasmid (red). Cells were treated with GABA and CGP54626 as indicated and the amplitude of the BRET responses quantified 1 min after agonist stimulation. Right, G protein deactivation rates were determined by normalizing the BRET data to maximum response and fitting the post-antagonist data to an exponential decay curve to extract rate constant values ( k ). Mean ± S.E.M., n=4-7. ns = not significant, **p<0.01, ***p<0.001, paired t-test. (B) GINIP antagonizes GAIP-mediated acceleration of Gβγ deactivation for Gi but not Go proteins. BRET experiments were carried out and analyzed as in (A) with cells expressing Gαi3 or Gαo in the absence (grey) or presence of GAIP (blue) or GAIP plus GINIP (orange). Expression of GAIP and GINIP was validated by immunoblotting IB. Mean ± S.E.M., n=5. ns = not significant, **p<0.01, ***p<0.001, one-way ANOVA corrected for multiple comparisons (Tukey). (C) GINIP antagonizes the acceleration of Gβγ deactivation mediated by representative members of all RGS families. BRET experiments were carried out and analyzed as in (B), expect that RGS8 (R4), RGS7 (R7), or RGS12 (R12) were used instead of GAIP (RZ). For experiments with RGS7, cells were also co-transfected with plasmids encoding Gβ 5 and R7BP. Mean ± S.E.M., n=3-6. *p<0.05, **p<0.01, one-way ANOVA corrected for multiple comparisons (Tukey). (D) GINIP antagonizes the GAP activity of RGS4 on Gαi in vitro . Nucleotide hydrolysis by Gαi1 RM/AS (WT or W258F) was determined in the presence of RGS4 and/or GINIP as indicated. Mean ± S.E.M., n=3. ns = not significant, *p<0.05, **p<0.01, one-way ANOVA corrected for multiple comparisons (Tukey). (E) GINIP competes with RGS4 for binding to Gαi3. Left, Structural model of Gαi1-(GDPꞏAlF 4 − ) bound to RGS4 (PDB: 1AGR). Right, increasing concentrations of purified His-GINIP and a fixed amount of His-RGS4 (20 nM) were incubated with GST or GST-Gαi3 (WT or W258F) immobilized on glutathione-agarose beads in the presence of GDPꞏAlF 4 − . Bead-bound proteins were detected by Ponceau S staining or by immunoblotting (IB). One representative result of three independent experiments is shown. (F) Diagram summarizing the proposed mechanism by which GINIP biases G protein responses by favoring Gβγ-dependent signaling in detriment of Gαi-dependent signaling. Binding of GINIP to G proteins simultaneously impairs Gαi binding to its effector (AC) and to negative regulators (RGS GAPs).

Article Snippet: HEK293T cells (Lenti-X 293T, Cat# 632180, Takara Bio) were plated on 150 mm diameter dishes (~2.5 million cells / dish) and cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Activity Assay, In Vitro, Binding Assay, Purification, Incubation, Staining

Journal: bioRxiv

Article Title: Spatio-temporal analysis of the innate immune response to cytoplasmic dsDNA using a novel cGAMP biosensor

doi: 10.1101/2024.06.10.598238

Figure Lengend Snippet: A , B , Live cell imaging analyses of HEK293T and HFF-1 cGAMP-biosensor cells treated with 2 μM diABZI. C , IFNb-Luc reporter assays in HEK293T cells transfected with empty plasmid or wt STING, or HEK293T cGAMP-biosensor cells, and treated with the indicated concentrations of the STING agonist diABZI. Note that HEK293T do not express endogenous cGAS or STING.

Article Snippet: To generate the HeLa and HEK293T cGAMP biosensor cells, the cGAMP biosensor was cloned into the PiggyBAC backbone plasmid (Biocat PB510B-1-SBI).

Techniques: Live Cell Imaging, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Immunostaining of the EGFR on Hek 293T cells (left) and the MDA MD 468 cells (right). Images were taken via confocal laser scanning microscopy.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Immunostaining, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Schematic representation of the experimental findings on NR selectivity: the differential binding profile between the targeted cell line (MDA MD 468) and the non-targeted cell line (Hek 293T) with the targeted NRs (NRs_18Ab and NRs_18Apt). For the MDA MD 468 cells, binding events with the targeted NRs are characterized by very long trajectories, indicating specific binding, while the binding trajectories with the Hek 293T are typically much shorter, indicating non-specific binding. b) Representative image of NRs_18Ab trajectories at 60 minutes after their incubation with MDA MD 468 and Hek 293T cells. Cell contours are indicated by the dotted line. Color bar indicates the diffusion coefficients (ranging from 0 to 4 µm 2 /s). c) Scatter plot of all the binding events for non-functionalized NRs, NR_18Ab and NR_18Apt (i.e. all the trajectories with D ≤ 1), plotted against their respective trajectory length (y-axis). d) Bar plot displaying the differential specific binding percentage between MDA MD 468 and Hek 293T for the different NR designs (NR_18Ab and NR_18Apt) at 3 distinct time points (10 min, 30 min and 60 min). Results are shown as the mean +-standard error of the mean. n = 3 biological replicates (per biological replicate, the trajectories of 5 different movies were combined, i.e. 5 technical replicates) *: significant difference between groups with p ≤ 0.05, ** : significant difference between groups with p ≤ 0.01, *** : significant difference between groups with p ≤ 0.001.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Incubation, Diffusion-based Assay

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Scheme of the binding kinetics between targeted NRs (NR_18Ab and NR_18Apt) and cells (left panel). The corresponding formula of the binding kinetics is displayed in the right panel. b) Comparison of the total number of specific binding events between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 and Hek 293T cells. The amount of binding events can be directly related to k on . c) Example of the exponential decay fitting for the binding time of NR functionalized with 18 antibodies in MDA MD 468 cells, where dotted line represents the fitted courve and the corresponding equation is displayed. d) τ B values obtained via an exponential decay fitting of all the binding events for each NR design and cell type. This value is inversely proportional to k off . A comparison was made between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 or Hek 293T. Results are shown as mean fitted value, where error bars represent the standard error of the fitting for each NR design in the different cell lines.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Comparison