Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane of endothelial cells leads to activation of Rap1. (A) Schematic representation of the optogenetic constructs CIBN–GFP–CAAX and CRY2–mCherry–talin expressed in immortalized mouse lung endothelial cells. The CIBN moiety is anchored to the plasma membrane and recruits CRY2–mCherry–talin upon exposure of the cells to 450 nm (blue) light. Previous work has shown that such recruitment leads to activation of integrin αVβ3 . Rap1 activation, the transition from Rap1–GDP to Rap1–GTP, can also be monitored during this process. (B) Time course of Rap1 activation in endothelial cells in response to blue light illumination. Rap1–GTP was selectively pulled down using agarose beads loaded with the Rap-binding domain of RalGDS and detected using an anti-Rap1 antibody. Upper panel: representative western blots of the Rap1–GTP pull-down assay and total Rap1 in whole-cell lysates (input: 5%). Lower panel: quantitative analysis of Rap1–GTP. The ratio of Rap1–GTP to total Rap1 was calculated and normalized to that observed at time zero when the cells were maintained in the dark. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test). (C) Time course of Rap1 activation in response to blue light in A5 CHO cells stably expressing integrin αIIbβ3, CIBN–GFP–CAAX and CRY2–mCherry–talin. Data represent means±s.e.m. of four experiments (* P <0.05; ** P <0.01; paired two-tailed Student's t -test). (D) Recruitment to the plasma membrane of a CRY2–mCherry–talin mutant (R118E) that cannot interact with Rap1 fails to activate Rap1 in A5 CHO cells. Data represent means±s.e.m. of four experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (E) Expression of a CRY2–mCherry–talin mutant (L325R) defective in activating integrins still enables Rap1 activation in these cells upon recruitment of CRY2–mCherry–talin to the plasma membrane in A5 CHO cells. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: Rap1 protein was then detected by immunoblotting with anti-Rap1 antibody (Proteintech, 67174; 1:500 dilution).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Construct, Binding Assay, Western Blot, Pull Down Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to cell edges. (A) Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to the cell periphery in suspended cells. Immortalized murine endothelial cells in suspension expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were illuminated using blue light for 30 min before Rap1–GTP was detected in situ as described in the Materials and Methods. Samples without GST–RalGDS were used as a control (column 1). White arrows indicate enrichment of active Rap1 and CRY2–mCherry–talin at the cell periphery, the former only in response to blue light. Scale bar: 10 µm. (B) Optogenetic recruitment of talin to the plasma membrane enriches active Rap1 localization at cell protrusions in adherent cells. Immortalized murine endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were plated on fibronectin-coated coverslips for 30 min before being illuminated with blue light or maintained in the dark for 30 min. Samples were fixed and in situ Rap1–GTP assay was performed as described in the Materials and Methods. Samples without GST–RalGDS incubation were used as a negative control and are shown in column 1. In these representative images, cell protrusions are highlighted by the small box in the main panel and presented as magnified insets on the bottom right of each image. Blue light illumination induces active Rap1 localization on cell lamellipodium-like protrusions (column 4) and pseudopodium-like protrusions (column 5). Note that such signal enrichment was not seen in cells maintained in the dark (columns 2 and 3) despite the formation of cell protrusions. Scale bars: 20 µm (main panel); 1 µm (inset). Images in A,B are representative of three independent experiments.

Article Snippet: Rap1 protein was then detected by immunoblotting with anti-Rap1 antibody (Proteintech, 67174; 1:500 dilution).

Techniques: Clinical Proteomics, Membrane, Suspension, Expressing, In Situ, Control, Incubation, Negative Control

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Overexpression of SHANK3 blocks Rap1 activation induced by talin recruitment to the plasma membrane. (A–C) SHANK3 tagged with Myc–mAzurite was transfected into A5 CHO cells stably expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells triple positive for mAzurite, GFP and mCherry were sorted by flow cytometry. Cells expressing Myc–mAzurite without SHANK3 served as a control. (A) Western blot analysis of SHANK3–Myc–mAzurite expression in these cells. β-actin served as a loading control. SHANK3 overexpression did not affect the levels of CRY2–mCherry–talin. The images represent two independent experiments. (B) SHANK3 overexpression inhibits Rap1 activation in response to the optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of five experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (C) SHANK3 blunts activation of integrin αIIbβ3 in response to the optogenetic recruitment of talin to the plasma membrane. Activation of integrin αIIbβ3 was monitored by flow cytometry using the PAC-1 antibody and expressed as the fold increase relative to that observed when cells were maintained in the dark. Data represent means±s.e.m. of five experiments (* P <0.05; paired two-tailed Student's t -test). (D–G) Lentiviruses encoding the WT SPN domain of SHANK3 [mAzurite–FLAG–SPN (WT)], the R12C SPN mutant [mAzurite–FLAG–SPN (R12C)] or the L68P SPN mutant [Myc–mAzurite–FLAG–SPN (L68P)] were transduced into immortalized murine lung endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells infected with empty lentiviral vector served as controls. (D) WT SPN, but not R12C or L68P SPN, inhibits Rap1 activation following optogenetic recruitment of talin to the plasma membrane. Data represent mean±s.e.m. of four experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). (E) WT SPN, but not the R12C or L68P SPN mutants, inhibits specific fibrinogen binding to integrin αVβ3 upon optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of eight experiments (N.S., not significant; * P <0.05; ** P <0.01; paired two-tailed Student's t -test). (F,G) Duolink proximity ligation assay (PLA) was performed to examine the effects of SHANK3 SPN on the association of Rap1 with CRY2–mCherry–talin in endothelial cells. (F) Schematic representation of the Duolink PLA. Created in BioRender by Liao, Z., 2025. https://BioRender.com/m47c469 . This figure was sublicensed under CC-BY 4.0 terms. (G) After 30 min of incubation at room temperature in the absence or presence of blue light illumination, cells were fixed, permeabilized and stained with rabbit anti-mCherry and mouse anti-Rap1 antibodies. Then, Duolink PLA flow cytometry was performed to assess the interaction between CRY2–mCherry–talin and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: Rap1 protein was then detected by immunoblotting with anti-Rap1 antibody (Proteintech, 67174; 1:500 dilution).

Techniques: Over Expression, Activation Assay, Clinical Proteomics, Membrane, Transfection, Stable Transfection, Expressing, Flow Cytometry, Control, Western Blot, Two Tailed Test, Mutagenesis, Infection, Plasmid Preparation, Binding Assay, Proximity Ligation Assay, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane impairs Rap1 interaction with SHANK3. (A) Schematic representation of the Duolink proximity ligation assay (PLA). Cells were fixed, permeabilized and stained with rabbit anti-SHANK3 and mouse anti-Rap1 antibodies before Duolink PLA was performed to assess the proximity of SHANK3 to Rap1. Created in BioRender by Liao, Z., 2025. https://BioRender.com/b40n281 . This figure was sublicensed under CC-BY 4.0 terms. (B) Immortalized murine lung endothelial cells expressing CRY2–mCherry–talin and CIBN–GFP–CAAX were plated on fibrinogen overnight, fixed, permeabilized and stained with anti-SHANK3 and anti-Rap1 antibodies. PLA was performed to evaluate colocalization of endogenous SHANK3 and Rap1. Cell nuclei were counterstained with DAPI and cells were imaged by confocal microscopy. Cells kept in the dark and untreated with primary antibodies served as controls. Two areas within the images with merged signals for PLA and CIBN–GFP–CAAX are highlighted with boxes and presented as magnified insets on the bottom. PLA signals were observed within the cytoplasm (inset on the left) and on the plasma membrane (inset on the right). Scale bars: 35 µm (main panel); 10 µm (inset). Images represent two independent experiments. (C) Immortalized murine lung endothelial cells in suspension were either kept in the dark or exposed to blue light illumination for the indicated times, before being subjected to Duolink PLA assay and analyzed by flow cytometry to quantitatively assess the interaction of endogenous SHANK3 and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of five experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). Cells transduced with lentivirus encoding shRNA to knock down SHANK3 were also used as a further control to demonstrate the specificity of the PLA signal in four out of the five experiments (* P <0.05; unpaired two-tailed Student's t -test).

Article Snippet: Rap1 protein was then detected by immunoblotting with anti-Rap1 antibody (Proteintech, 67174; 1:500 dilution).

Techniques: Clinical Proteomics, Membrane, Proximity Ligation Assay, Staining, Expressing, Confocal Microscopy, Suspension, Flow Cytometry, Two Tailed Test, Transduction, shRNA, Knockdown, Control

Journal: The Journal of Biological Chemistry

Article Title: GoLoco/GPR motif-dependent regulation of Rap1GAP1 by Gα o is disrupted by Gα o encephalopathy variants

doi: 10.1016/j.jbc.2025.110446

Figure Lengend Snippet: RaP1GAP1a preferentially interacts with active Gα o . A , normalized abundance (shown as mean ± SEM) of endogenous Rap1GAP1 detected by mass spectrometry in PC12 cells transduced with TurboID-Gα o (WT) , TurboID-Gα o (Q205L) , and TurboID-CaaX (N = 3) (∗ p < 0.05, calculated using one-way ANOVA with Tukey's multiple comparisons test). B , cell lysates from HEK cells transfected with indicated constructs were subjected to immunoprecipitation with Flag antibody followed by gel electrophoresis and immunoblotting. Representative Western blot from 3 independent experiments shows Gα o co-immunoprecipitated with Rap1GAP1a. C , quantification from three independent experiments (shown as mean ± SEM) of Gα o pulled down with Rap1GAP1a (N = 3) (∗∗ p < 0.01, calculated using an unpaired two-tailed t test). D , schematic of nanoluciferase-based complementation assay with C-terminally tagged Rap1GAP1a with SmBiT (Rap1GAP1a SmBiT) with Gα o fused to LgBiT. E , HEK cells transfected with cDNAs encoding Rap1GAP1a SmBiT in conjunction with Gα o (WT) LgBiT, Gα o (Q205L) LgBiT, or Lyn LgBiT. 24 h after transfection, cells were transferred to a 96-well plate. 10 min after incubation with furimazine, luminescence generated from the complementation of the split-nanoluciferase pairs was measured. Data from eight independent experiments with a minimum of four technical replicates were plotted as mean ± SEM and analysed using one-way ANOVA with Tukey's multiple comparisons test (∗ p < 0.05, ∗∗∗∗ p < 0.0001). After the assay, cells were lysed and western blotted. A representative Western blot highlights the expression of all LgBiT-fused constructs.

Article Snippet: The following primary antibodies were utilized: V5 (Cell Signaling Technologies, D3H8Q), rabbit anti-HA (Cell Signaling Technologies, C29F4), Flag (Invitrogen, PA1-984B and Cell Signaling, 14793S), mouse anti-flag (Sigma-Aldrich, F1804), Rap1GAP1 (Proteintech, 19174-I-AP), GAPDH (Invitrogen, MA5-15738), and mouse anti-GM-130 (BD Transduction Laboratories, 610,822).

Techniques: Mass Spectrometry, Transduction, Transfection, Construct, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Two Tailed Test, Incubation, Generated, Expressing

Journal: The Journal of Biological Chemistry

Article Title: GoLoco/GPR motif-dependent regulation of Rap1GAP1 by Gα o is disrupted by Gα o encephalopathy variants

doi: 10.1016/j.jbc.2025.110446

Figure Lengend Snippet: The N terminus of Rap1GAP1 drives differential interaction with different nucleotide-bound states of Gα o . A , schematic representation of Rap1GAP1a-RlucII constructs used in BRET assays, including N-terminal truncations. The GAP domain is highlighted in dark blue and the GoLoco motif in orange . B , A293 cells were co-transfected with Rap1GAP1-RlucII and N-terminal truncated constructs along with rGFP-CaaX and Gα o . BRET signal was measured 24 h after transfection following the addition of coelenterazine 400a. Data are shown as means ± SEM of three independent experiments (∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, calculated using one-way ANOVA with Tukey's multiple comparisons test). C , sequence alignment of human Rap1GAP1a, Rap1GAP1b N-termini and the RGS14 GoLoco/GPR motif. Stars represent amino acids either identical or conserved relative to the RGS14 GoLoco/GPR motif. D , A293 cells were transfected with cDNAs encoding Rap1GAP1b-RlucII along with Gα o and acceptors, rGFP-CaaX ( left panel ), rGFP-Giantin ( middle panel ), and rGFP-FYVE ( right panel ). ebBRET experiments were performed in three biological replicates, with each condition in four technical replicates. Data represent means ± SEM (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, calculated using one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: The following primary antibodies were utilized: V5 (Cell Signaling Technologies, D3H8Q), rabbit anti-HA (Cell Signaling Technologies, C29F4), Flag (Invitrogen, PA1-984B and Cell Signaling, 14793S), mouse anti-flag (Sigma-Aldrich, F1804), Rap1GAP1 (Proteintech, 19174-I-AP), GAPDH (Invitrogen, MA5-15738), and mouse anti-GM-130 (BD Transduction Laboratories, 610,822).

Techniques: Construct, Transfection, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: GoLoco/GPR motif-dependent regulation of Rap1GAP1 by Gα o is disrupted by Gα o encephalopathy variants

doi: 10.1016/j.jbc.2025.110446

Figure Lengend Snippet: Model of Rap1GAP1 interactions with, and regulation by Gα o and Gα o -linked pathologic variants . A , structural model of Gα i1 bound to the GoLoco/GPR motif from RGS14 (PDB: 3ONW ). The RGS14 GoLoco motif is shown in blue and light green . The light green region represents amino acids analogous to the amino acids that are missing in the truncated Rap1GAP1a GoLoco motif. The α-helical domain from Gα i1 is in gray and the Ras-like domain is in orange with Switch II in dark green and the α-3 helix in magenta . B , Rap1GAP1 isoforms engage with Gα o differentially. ( Left ) Rap1GAP1b preferentially engages with Gα o -GDP via a full GoLoco/GPR motif. ( Right ) Rap1GAP1a selectively engages with active Gα o through its partial GoLoco/GPR motif. This results in reduced Rap1 activation at the plasma membrane. This functional interaction is impaired by pathogenic Gα o variants.

Article Snippet: The following primary antibodies were utilized: V5 (Cell Signaling Technologies, D3H8Q), rabbit anti-HA (Cell Signaling Technologies, C29F4), Flag (Invitrogen, PA1-984B and Cell Signaling, 14793S), mouse anti-flag (Sigma-Aldrich, F1804), Rap1GAP1 (Proteintech, 19174-I-AP), GAPDH (Invitrogen, MA5-15738), and mouse anti-GM-130 (BD Transduction Laboratories, 610,822).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Functional Assay

Journal: Journal of ethnopharmacology

Article Title: Anemarrhena asphodeloides Bunge and Phellodendri Chinensis Cortex inhibits the PTGS2/EP2/cAMP/Epac1 signaling pathway to reduce microglial M1 polarization, thereby blocking chronic stress-induced depression-like behavior.

doi: 10.1016/j.jep.2025.119792

Figure Lengend Snippet: Fig. 8. Neomangiferin alleviates the release of proinflammatory factors in BV2 cells by inhibiting PTGS2/EP2/cAMP/Epac1 signals. (A) representative immunofluorescence staining of EP2 and Epac1, and (B) fluorescence area (n = 3). (C–D) Protein expression changes of EP2 and Epac1 (n = 3). The contents of PGE2 and cAMP in BV2 cells were determined by E-F Elisa kit (n = 3). (G) Representative images of zebrafish embryos in Neomangiferin experiment (n = 10). Data are expressed as mean±SD. *** means P < 0.001,** means P < 0.01,* means P < 0.05, ns means P > 0.05.

Article Snippet: Seal with 5 % skim milk powder at room temperature for 2 h. Primary antibody β-actin (rabbit, 1:5000; bs-00R,Bioss, China), GAPDH(rabbit, 1:10,000; 10494- 1-AP,Proteintech, China), HSP90(rabbit, 1:5000; 13171-1-AP,Proteintech, China), Arg-1 (rabbit, 1:1000; 16001-AP, Proteintech, China), iNOS (rabbit, 1:1000; ab283655, Abcam, UK), PTGS2 (mouse, 1:400; 66351-1-Ig, Proteintech, China), EP2 (rabbit, 1:1000; HA721380, HUABIO, China), EPAC1 (rabbit, 1:1000; A02483-3, Boster, China), incubated overnight at 4 ◦C.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1

doi: 10.3390/vaccines10020203

Figure Lengend Snippet: VLPs characterization. MSP-8 ( A ), MSP-9 ( B ) and RAP1 ( C ) VLP morphologies were visualized under TEM. MSP-8 ( D ), MSP-9 ( E ) and RAP1 ( F ) VLPs were loaded (8, 40 µg) for SDS-PAGE, and bands corresponding to each antigen were observed using either the polyclonal P. berghei antibody or the monoclonal M1 antibody.

Article Snippet: Nucleotide sequences of codon-optimized (RAP1) were obtained from GenScript, and compared with the original non-codon optimized RAP1 (XM_034565422.1) sequence acquired from NCBI database.

Techniques: SDS Page

Journal: Vaccines

Article Title: Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1

doi: 10.3390/vaccines10020203

Figure Lengend Snippet: Experimental schedule. Mice were immunized twice with the combined VLP vaccines expressing MSP-8, MSP-9, and RAP1 of P. berghei at 4 weeks intervals, and blood samples were drawn 3 weeks after each immunization. Challenge infection was performed 4 weeks after boost immunization and half of the mice in each group were sacrificed at 1 week post-challenge.

Article Snippet: Nucleotide sequences of codon-optimized (RAP1) were obtained from GenScript, and compared with the original non-codon optimized RAP1 (XM_034565422.1) sequence acquired from NCBI database.

Techniques: Vaccines, Expressing, Infection

Journal: Vaccines

Article Title: Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1

doi: 10.3390/vaccines10020203

Figure Lengend Snippet: IgG antibody response. Mice were intramuscularly immunized twice with the mixture of MSP-8, MSP-9 and RAP1 VLPs. Sera ( n = 6) were collected at regular intervals after each immunization with the VLPs. VLPs immunization induced IgG antibodies reacted with P. berghei Ag ( A ) and VLPs ( B ) upon prime and boost compared to naïve sera (** P < 0.01, *** P < 0.001).

Article Snippet: Nucleotide sequences of codon-optimized (RAP1) were obtained from GenScript, and compared with the original non-codon optimized RAP1 (XM_034565422.1) sequence acquired from NCBI database.

Techniques: