Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 4. Mutations that disrupt the AIR/Cdc25HD interaction activate C3G constitutively. (A) Conservation scores of C3G per residue (colored bars) and average values per domain (boxes). Alignment of the sequences of the P3 motif and part of the AIR from representative species; positions are colored according to their conservation. Secondary structure predictions by three methods are shown under the sequences. Secondary structure prediction for the com- plete SH3b domain is shown in fig. S2. (B and C) Binding of the Cdc25HD to GST-AIR, WT, and mutants, analyzed by PD assays. Cdc25HD in the PD was detected by Western blot. Two types of substitutions were assayed: reverse-charge replacements (B) and changes to alanine and mutations described in lymphomas (C). PDs are representative of two independent experiments. (D) Helical wheel representation of the predicted helix in the AIR-CBR. Met551, Tyr554, and Met555 define the putative binding site for the Cdc25HD. (E and F) Representative nucleotide exchange reactions of Rap1:mant-dGDP catalyzed by full-length C3G WT and the indicated point mutants (1 M) (E) and the exchange rates (kobs) (F). n = 3 to 5 independent experiments, as indicated, one shown in (E). (G) Schematic representation of auto inhibited C3G and the uninhibited conformation induced by mutations that destabilize the AIR/Cdc25HD interaction.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Residue, Binding Assay, Western Blot

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 5. Lymphoma-related mutations in C3G activate Rap1 and LFA-1 in cells. (A) Analysis of Rap1-GTP in HEK293T cells expressing mEGFP; C3G-mEGFP WT; the mutants M551R, Y554H, or M555K; or the membrane-targeted WT with a CAAX sequence. Expression of mEGFP and endogenous Rap1 and -actin in cell lysates were analyzed by Western blot. In the PD, Rap1-GTP was detected by Western blot, and GST-RalGDS-RBD was detected by Ponceau S staining. PD is representative of three independent experiments. (B) Scatterplot and bar chart of three independent measurements of Rap1 activation in HEK293T cells, as described and represented in (A). Rap1-GTP levels in cells expressing C3G-mEGFP-CAAX were used to normalize the data from different experiments. (C) Correlation between the exchange activity in vitro (kobs) of C3G WT and mutants (shown in Fig. 4F) and the Rap1-GTP levels that they induced in HEK293T cells [shown in (B)]. (D) Rap1-GTP levels in Ba/F3 cells expressing C3G-mEGFP, WT or the indicated mutants, or mEGFP alone and in uninfected cells. Proteins were detected in the cell lysates and in the GST-RalGDS- RBD PDs as described in (A). In addition, C3G (endogenous and mEGFP-tagged) was also detected using an antibody against C3G. PD is representative of two independent experiments. (E) Time course activation of Rap1 after stimulation with IL-3 of Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or Y554H. Rap1-GTP was detected as described in (A) and (D). PD is representative of two independent experiments. (F) Activation of the integrin LFA-1 in Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or mutants, determined by flow cytometry (n = 3, biological replicates, means ± SD). Statistical comparison to cells expressing mEGFP was analyzed using ANOVA followed by Dunnett’s test. ***P < 0.001; ****P < 0.0001.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Expressing, Membrane, Sequencing, Western Blot, Staining, Activation Assay, Activity Assay, In Vitro, Flow Cytometry, Comparison

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 6. The AIR/Cdc25HD interaction is the main autoinhibitory mechanism and is disrupted by CrkL for activation. (A) Phosphorylation of purified C3G-WT with SrcKD, analyzed by Western blot. Similar analysis of Cdc25HD and the C3G mutants M551R, Y554R, and Y554H are shown in fig. S3 (A to D). (B) Representative dissociation reactions of Rap1:mant-dGDP catalyzed by C3G (1 M), unmodified or phosphorylated with SrcKD (p-C3G), alone and in the presence of CrkL (10 M). (C) Nucleotide ex- change rates (kobs) catalyzed by full-length C3G WT and point mutants and the isolated Cdc25HD. Unphosphorylated and SrcKD-phosphorylated samples were analyzed alone and in the presence of CrkL, n = 3 to 10 independent experiments as indicated. Representative dissociation reactions are shown in (B) (WT) and in fig. S3 (A to D) (Cdc25HD and mutants). (D) Analysis by PD of the competition of CrkL with the Cdc25HD for binding to four constructs of the AIR that contain the P3 and P4 (GST-537- 646-WT), only the P3 (GST-537-646-P4A), only the P4 (GST-545-646-WT), or none of these proline-rich motifs (GST-545-646-P4A). PDs are representative of two independent experiments. (E) Time course of the in vitro phosphorylation of the AIR (537-646) by the SrcKD, analyzed by Western blot; representative of two independent experiments. (F) Binding of Cdc25HD to GST-AIR (537-646) phosphorylated with SrcKD in PD assays. PD is representative of two independent experiments. (G) Exchange activity (kobs) of the Cdc25HD alone and in the presence of AIR (untagged or GST-fusion) or the AIR mutants in (D). n = 3 to 4 independent experiments as indicated. Representative dissociation experiments are shown in fig. S3E. (H) Schematic representation of the effect of phosphorylation and CrkL binding to the AIR on the release of the inhibitory interaction when the AIR and the Cdc25HD are assayed as individual proteins.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activation Assay, Phospho-proteomics, Purification, Western Blot, Isolation, Binding Assay, Construct, In Vitro, Activity Assay

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 7. The NTD/REM interaction stimulates the GEF activity of the Cdc25HD. (A) Schematic representation of full-length C3G-WT, truncation fragments, and the mutant E731R/E784R (red crosses) in which the NTD/REM interaction is destabilized. (B to E) Nucleotide exchange activity (kobs) of the proteins depicted in (A), in the absence of stimuli (B), in the presence of CrkL (C), phosphorylated with SrcKD (D), and when CrkL and phosphorylation were combined (E). The number of independent experiments (n) is indicated. Lines are means ± SD. Representative nucleotide dissociation reactions are shown in fig. S4 (A, B, D, and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA followed by Dunnett’s test (B and C) or unequal variance Brown-Forsythe ANOVA followed by Dunnett’s test (D and E). (F) Analysis of Rap1 activation in HEK293T cells expressing C3G-mEGFP WT, the mutant E731R/E784R, or C3G with the membrane targeting CAAX-tag. Results of two independent experiments are shown. (G) Schematic illustration of the contribution of the NTD/REM interaction to the activation by CrkL and phosphorylation of C3G WT and mutants.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, Activation Assay, Expressing, Membrane

Journal: Journal of Cell Science

Article Title: Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

doi: 10.1242/jcs.177170

Figure Lengend Snippet: Spatiotemporal dynamics of RasG and Rap1 activities in pkaC null cells. (A) cAMP-induced RasG and Rap1 activation. Active RasG (Ras–GTP) and Rap1 (Rap1–GTP) were pulled down with GST–Raf1(RBD) and GST–RalGDS(RBD), respectively, and revealed by immunoblotting for pan-Ras and Rap1. (B,E) Localization of Raf1(RBD)–GFP (B) and RalGDS(RBD)–GFP (E) in resting cells. (C,F) Live imaging of Raf1(RBD)–GFP (C) and RalGDS(RBD)–GFP (F) in cells that had been exposed to an exponential gradient of cAMP. (D,G) Live imaging of Raf1(RBD)–GFP (D) and RalGDS(RBD)–GFP (G) in cells that had been uniformly stimulated with 5 μM cAMP for the indicated times. Relative reporter cytosolic fluorescence intensity is shown on the right, expressed as a fold over the basal level. Scale bars: 10 μm. Quantified data represent the mean fluorescence intensity±s.e.m. of 20 Raf1(RBD)–GFP-expressing WT cells, nine Raf1(RBD)–GFP-expressing pkaC null cells, 109 RalGDS(RBD)–GFP-expressing WT cells and 135 RalGDS(RBD)–GFP-expressing pkaC null cells. Data represent or are representative of at least three independent experiments.

Article Snippet: The Rap1 (directed against amino acids 169-182 of Dictyostelium Rap1) and Pia antibodies (directed against amino acids 138-159 of Dictyostelium Pianissimo) were custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Activation Assay, Western Blot, Imaging, Fluorescence, Expressing

Journal: Journal of Cell Science

Article Title: Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

doi: 10.1242/jcs.177170

Figure Lengend Snippet: The expression of cAR1 and its role in RasG, Rap1 and TORC2 activation in pkaC null cells. (A) Expression of cAR1, Ras, Rap1, Pia, PKB and PKBR1 in wild-type (WT) and pkaC null (pkaC—) vegetative cells (0 h) and in cells pulsed with cAMP for 5.5 h, detected by immunoblotting using protein-specific antibodies. (B) cAR1–GFP expression in carA and pkaC null cells, revealed by GFP immunoblotting. (C) Chemotaxis phenotype of cAR1–GFP-expressing carA and pkaC null cells migrating in an exponential cAMP gradient created by a point source. Differential interference contrast (DIC) and fluorescence (GFP) images showing expressed cAR1–GFP are shown. (Right) Traces of the migration path for a subset of the migrating cells. *, position of the cAMP-filled micropipette. Scale bars: 50 μm. (D) cAMP-induced RasG and Rap1 activation. Active RasG (Ras–GTP) and Rap1 (Rap1–GTP) were pulled down with GST–Raf1(RBD) and GST–RalGDS(RBD), respectively, and revealed by immunoblotting for pan-Ras and Rap1. (E) cAMP-induced phosphorylation of the hydrophobic motif (HMP; TORC2 site) of PKB and PKBR1. CB, Coomassie Blue staining. Data are representative of at least three independent experiments.

Article Snippet: The Rap1 (directed against amino acids 169-182 of Dictyostelium Rap1) and Pia antibodies (directed against amino acids 138-159 of Dictyostelium Pianissimo) were custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Chemotaxis Assay, Fluorescence, Migration, Phospho-proteomics, Staining

Journal: Journal of Cell Science

Article Title: Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium

doi: 10.1242/jcs.177170

Figure Lengend Snippet: Proposed model for the role of PKA in regulating the chemoattractant signal transduction pathways in Dictyostelium. Although PKA controls gene expression during development, including that of cAR1, and this probably explains part of the observed severe chemotaxis phenotypes of pkaC null cells, our study suggests that PKA is also likely to play a direct role in controlling the directional migration of cells. In light of our findings, we propose that PKA controls chemotaxis, in part, by spatially and temporally regulating the activation of RasG, Rap1 and TORC2.

Article Snippet: The Rap1 (directed against amino acids 169-182 of Dictyostelium Rap1) and Pia antibodies (directed against amino acids 138-159 of Dictyostelium Pianissimo) were custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Transduction, Gene Expression, Chemotaxis Assay, Migration, Activation Assay

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and hTR by smiFISH in HeLa cells. The percentage of cells, in which at least 1 TERRA-hTR colocalization event is detected, is indicated (mean ± SD; n = 5; 298 cells analyzed). Scale bar, 5 μm. ( B ) Quantification of the number of TERRA-hTR colocalizations per nucleus (mean ± SD; n = 5; 298 cells analyzed). ( C ) Detection of TERRA, hTR, and telomeres by smiFISH/RAP1 IF in HeLa cells (mean ± SD; n = 2; 102 cells analyzed). Scale bar, 5 μm. ( D ) Quantification of the number of TERRA-hTR colocalizations per cell detected at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) (mean ± SD; n = 2; 102 cells analyzed). ( E ) Number of hTR-RAP1 colocalizations per cell with and w/o TERRA (mean ± SD; n = 2; 102 cells analyzed). ( F ) Quantification of the number of TERRA-hTR foci at telomeres (TERRA-hTR-RAP1) and outside telomeres (TERRA-hTR w/o RAP1) during G 1 , S, and G 2 phase in HeLa cells upon cell synchronization (mean ± SD; n = 2; total number of cells analyzed: 54 (G 1 phase), 46 (S phase), and 45 (G 2 phase). Fraction of hTR-TERRA foci at telomeres: 20.9 ± 3.3% in G 1 -phase cells, 40.2 ± 11% in S-phase cells, and 41.1 ± 5.5% in G 2 -phase cells.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques:

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Telomere length measurement by TRF through Southern blot in HeLa cells in the indicated conditions. NT, untreated; CTR, DMSO-treated. Estimated average telomere length and elongation rate for each sample are indicated in the table below. Average ± SD from two technical replicates. ( B ) Detection of TERRA and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( C ) Quantification of the number of telomeric TERRA foci detected per nucleus by smiFISH/IF (each dot represents a nucleus) (mean ± SD; n = 2; 124 CTR cells, 111 7PD rescue cells, and 112 24PD rescue cells analyzed). Unpaired nonparametric Kruskal-Wallis test coupled with post hoc Dunn’s multiple-comparison test: ** P < 0.01, *** P < 0.001. ( D and E ) Distribution analyses of the number of telomeric TERRA foci per cell. Two-way analysis of variance (ANOVA) test: ** P < 0.01. Post hoc Tukey’s multiple-comparison test: group 1 to 4 P values = 0.07 (CTR versus 7PD rescue), 0.04 (7PD rescue versus 24PD rescue), not significant (ns) (CTR versus 24PD rescue). ( F ) Detection of TERRA, hTR, and telomeres by smiFISH/IF in CTR, 7PD rescue, and 24PD rescue cells. Scale bar, 5 μm. ( G ) Quantification of the percentage of TERRA-hTR foci colocalizing at telomeres and extratelomeric (mean ± SD; n = 2; 124 CTR cells, 111 7PDs rescue cells, and 112 24PDs rescue cells analyzed). Two-way ANOVA test: **** P < 0.0001. ( H ) Distribution analyses of the number of TERRA-hTR-RAP1 colocalizing foci per nucleus. Two-way ANOVA test: **** P < 0.0001; multiple-comparison test: P = 0.0003 for 7PD versus 24PD rescue, P < 0.0001 for CTR versus 7PD rescue, P = 0.001 CTR versus 24PD rescue. ( I ) Number of telomeric hTR foci not colocalizing with TERRA per cell detected by smiFISH/IF. Kruskal-Wallis test: P = 0.0025.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Southern Blot, Comparison

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and RAP1 by smiFISH/IF in HeLa cells. An example of a colocalization event between TERRA and RAP1 is shown in the image magnifications. DAPI is used to stain nuclei. Scale bar, 5 μm. ( B ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in HeLa cells as detected by smiFISH/IF. Each dot represents a single RAP1 focus. Mean ± SD is shown. A total of 110 cells were analyzed in three independent biological replicates. Statistical significance was assessed by Mann-Whitney test. **** P < 0.0001. ( C ) Integrated density quantification of RAP1 foci colocalizing and not colocalizing with TERRA foci in the indicated samples. Each dot represents a single RAP1 focus. Mean ± SD is shown from the following number of samples and biological replicates: 64 CTR cells ( n = 2), 67 BIBR1532 cells (123PDs of treatment with BIBR 1532) ( n = 2), 111 7PD rescue cells ( n = 2), 151 POT1 WT cells ( n = 3), and 148 POT1-ΔOB cells ( n = 3). The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Staining, MANN-WHITNEY

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Detection of TERRA and telomeres in HeLa cells by smiFISH/IF. Image acquisitions were performed using three-dimensional structured illumination microscopy (3D-SIM). Examples of TERRA foci colocalizing with a single telomere (top images) or telomere doublet (bottom images) are displayed. TERRA is shown in red; telomeres are in green. Scale bar is indicated in the rotated view images. ( B ) Quantification of the fraction of single telomeres versus telomere doublets colocalizing with TERRA. Data are shown as percentage of TERRA-colocalizing telomeres and represent mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. ( C and D ) Quantification of the integrated density (C) and volume (D) of RAP1 foci colocalizing (with TERRA) and not colocalizing (without TERRA) with TERRA. Both TERRA-single telomere and TERRA-telomere doublet colocalizations were considered. Data are shown as arbitrary units (a.u.), in (C), and μm 3 , in (D), and represents mean ± SD from three independent biological replicates for a total of 30 cells and 663 TERRA-colocalizing RAP1 foci analyzed. The Mann-Whitney test was used to assess statistical significance. **** P < 0.0001. ( E ) Quantification of the integrated density of all TRF1-mCherry foci and TRF1-mCherry foci colocalizing with MS2-tagged telomere 15q TERRA transcripts per nucleus. Forty nuclei corresponding to 3690 telomeres and 73 telomeres colocalizing with MS2-TERRA transcripts were analyzed from imaging datasets obtained in . Statistical analysis was performed with a Kolmogorov-Smirnov test: P ≤ 0.0001.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Microscopy, MANN-WHITNEY, Imaging

Journal: Science Advances

Article Title: TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends

doi: 10.1126/sciadv.adk4387

Figure Lengend Snippet: ( A ) Northern blot analysis of TERRA in HeLa cells upon TERRA-ASO or control ASO (ASO SCR) transfection. Bottom image shows 18 S rRNA band upon gel run. ( B ) Quantification of TERRA signal from Northern blot analyses of TERRA-ASO–transfected cells shown as fold over ASO SCR (dashed line). * P < 0.05; mean ± SD, n = 2. ( C ) RT-qPCR analyses of TERRA expression from the indicated telomeres in TERRA-ASO cells shown as fold over ASO SCR. Mean ± SD from four independent biological replicates. Unpaired t test: ** P < 0.01, *** P < 0.001. ( D ) Integrated density quantification of TERRA foci colocalizing with RAP1 foci. Each dot represents a single TERRA signal (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: ** P < 0.01. ( E ) Quantification of the number of hTR foci detected per nucleus in TERRA-ASO and ASO SCR cells (mean ± SD; n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( F ) RT-qPCR quantification of hTR levels using primer pairs detecting the precursor or mature RNA ( , ). Results are shown as fold change over ASO SCR (dashed line) (mean ± SD, n = 2). U6 gene was used for normalization . ( G ) Detection of hTR and telomeres by smiFISH/IF. Scale bar, 5 μm. ( H ) Quantification of the number of telomeric hTR foci detected per nucleus. Data are shown as number of RAP1-hTR colocalizations per cell (each dot represents a cell) (mean ± SD, n = 2; 131 ASO SCR cells and 122 TERRA-ASO cells analyzed). Mann-Whitney test: **** P < 0.0001. ( I and J ) Distribution analysis of the number of RAP1-hTR colocalizations detected per cell. Two-way ANOVA test: ** P < 0.01.

Article Snippet: Cells were blocked by incubation in 1× PBG buffer (0.2% fish gelatin, 0.5% BSA, 1× PBS) for 1 to 6 hours and incubated for 1 hour with anti-RAP1 primary antibody (rabbit anti–TERF2-IP antibody, from Novus Biological, catalog no. NB100-292; RRID: AB_10000825) diluted 1:500 in 1× PBG.

Techniques: Northern Blot, Control, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

doi: 10.1101/2022.11.24.515150

Figure Lengend Snippet: ( A ) Schematic representation of the PRM mutants used to study the activation of C3G by CrkL. ( B ) Representative exchange reactions of Rap1:mant-dGDP (200 nM) catalyzed by C3G wild type and mutants (1 µM) in the presence of CrkL (40 µM). Lines are the single exponential decay models fitted to obtain the k obs . ( C ) Nucleotide exchange rates of C3G wild type and mutants (1 µM) alone and in the presence of 40 µM CrkL. Data are shown as scatter plots with bars, means ± standard deviation. The number of independent measurements is indicated in parentheses. Statistical comparison was analyzed using ANOVA followed by Tukey’s multiple comparisons test; *** P < 0.001, **** P < 0.0001, ns P > 0.05. ( D ) Dose-dependent effect of CrkL on the GEF activity of C3G (1 µM) wild type and two mutants. Lines are the fitted sigmoidal models. ( E ) Nucleotide exchange rates of Src-phosphorylated C3G (pC3G, 0.2 µM) wild type and mutants, alone and in the presence of 5 µM CrkL. Statistical comparisons were done as in C. ( F ) Dose-dependent effect of CrkL on the GEF activity of pC3G (0.2 µM) wild type and mutants. Dissociation rate constants in C-F are referred to 1 µM C3G for comparison.

Article Snippet: The following primary antibodies were used for western blot, at 1/1000 dilutions unless otherwise specified: mouse monoclonal antibody (mAb) G9 against C3G (sc-393836), rabbit polyclonal antibody (pAb) C-20 against CrkL (sc-319), mouse mAb B-2 against GFP (sc-9996), rabbit pAb 121 against Rap1 (sc-65) were from Santa Cruz Biotechnology, mouse mAb 4G10 against phospho-Tyr (05-321, Millipore), and mouse mAb against β-actin (AC-15, Sigma-Aldrich, used at 1/2000).

Techniques: Activation Assay, Standard Deviation, Comparison, Activity Assay

Journal: bioRxiv

Article Title: Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms

doi: 10.1101/2022.11.24.515150

Figure Lengend Snippet: ( A ) Analysis by co-immunoprecipitation (coIP) in Jurkat cells of the interaction between stably expressed exogenous C3G-mEGFP variants and endogenous CrkL. Proteins were immunoprecipitated with affinity resin against GFP. CrkL and mEGFP-tagged proteins were detected in cell lysates and in the IP by western blot (WB). ( B ) Analysis of the interaction between C3G-mEGFP and CrkL in HEK293T cells. mEGFP-tagged C3G WT and mutants were transiently expressed and the interaction with endogenous CrkL was analyzed by coIP as in A. ( C ) Imaging of C3G-mEGFP (upper panels), and cortical actin (stained with phalloidin-iFluor 647, middle panels) in Jurkat cells expressing C3G wild type or the indicated mutants. Nuclei were stained with DAPI. Cells were plated on coverslips treated with poly-L-lysine and were unstimulated (-anti-CD3) or stimulated with OKT-3 antibody against CD3. Representative fields are shown, in which the median of the Manders’ overlap coefficient (MOC) for the cells in each field is similar to the value observed for the total of cells analyzed. Scale bars, 20 μm. ( D ) Quantification of the co-localization of C3G-mEGFP with phalloidin-stained actin in confocal microscopy images of Jurkat cells as shown in C. MOC values are shown as scatter plots (from left to right, n = 637, 466, 346, 565, 423, and 459 cells). Middle bars mark the median and whiskers are the 25th and 75th percentiles. Statistical analysis were done with non-parametric Kruskal-Wallis and Dunn’s multiple comparisons tests (**** P < 0.0001, n.s. P > 0.05). ( E ) Fluorescence intensity of C3G-mEGFP and phalloidin staining along the yellow dashed lines in panel C, which run across representative cells. ( F ) Analysis of Rap1 activation in Jurkat cells stably expressing C3G-mEGFP WT and mutants, or isolated mEGFP as control, before and after stimulation with antibody against CD3.

Article Snippet: The following primary antibodies were used for western blot, at 1/1000 dilutions unless otherwise specified: mouse monoclonal antibody (mAb) G9 against C3G (sc-393836), rabbit polyclonal antibody (pAb) C-20 against CrkL (sc-319), mouse mAb B-2 against GFP (sc-9996), rabbit pAb 121 against Rap1 (sc-65) were from Santa Cruz Biotechnology, mouse mAb 4G10 against phospho-Tyr (05-321, Millipore), and mouse mAb against β-actin (AC-15, Sigma-Aldrich, used at 1/2000).

Techniques: Immunoprecipitation, Stable Transfection, Western Blot, Imaging, Staining, Expressing, Confocal Microscopy, Fluorescence, Activation Assay, Isolation, Control

Journal: Genes & development

Article Title: Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

doi: 10.1101/gad.349039.121

Figure Lengend Snippet: Figure 1. Rap1 maintains gene transcription independent of telomeres. (A) Schematic representation of CRISPR/Cas9 gene-editing strat- egy to substitute Rap1 isoleucine 312 with arginine. Successful targeting creates an AciI restriction site to be used during genotyping. Sin- gle-stranded oligo donor (ssODN, black line) and cut site (red scissors) are indicated. (B) PCR genotyping of tail tip DNA from two Rap1I312R/+ heterozygous mice following gene targeting. (C) Example Sanger sequencing of a Rap1I312R/+ heterozygous mouse. (D) Rep- resentative image of IF-FISH in Rap1+/+, Rap1−/−, and Rap1I312R/I312R MEFs for Rap1 (green) and telomeres (red) using Rap1 antibody and a TTAGGG PNA probe, respectively. DAPI (blue) was used as a counterstain. (E) Rap1 Western blot from Rap1−/−, Rap1+/+, and Rap1I312R/I312R MEFs following subcellular fractionation into cytoplasmic (Cyt), nucleoplasmic (Nuc), and chromatin-bound (CBFs) frac- tions. α-Tubulin and histone H3 were loading controls for Cyt and CBFs, respectively. The blot is representative of n = 2 biological repli- cates. (F) Immunoblot for Rap1 from whole-cell lysates obtained from Rap1−/−(n = 2), Rap1+/+ (n = 3), and Rap1I312R/I312R (n = 3) MEFs. Rap1 relative abundance was determined by normalizing to γ-tubulin. (G) Hierarchically clustered heat map representing RNA-seq data for differentially expressed genes (DEGs) between Rap1+/+ and Rap1−/−MEFs (FC > 1.5; FDR < 0.1; n = 3 biological replicates per ge- notype). (Rap1IR/IR) Rap1I312R/I312R.

Article Snippet: The following primary antibodies were used: mouse Rap1 (1252, rabbit polyclonal), TRF2 (Novus Biologicals NB110-57130, rabbit polyclonal), Tip60 (Cell Signaling 12058, rabbit polyclonal), γ-tubulin (Millipore Sigma T6557, mouse monoclonal), α-tubulin (Abcam ab7291, mouse monoclonal), histone H3 (Abcam ab1791, rabbit polyclonal), Flag (Cell Signaling 14793, rabbit monoclonal), c-Myc (Santa Cruz Biotechnology sc789, rabbit polyclonal), HA (Abcam ab9110, rabbit polyclonal), and streptavidin-HRP (Invitrogen 1953050).

Techniques: CRISPR, Sequencing, Western Blot, Fractionation, RNA Sequencing

Journal: Genes & development

Article Title: Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

doi: 10.1101/gad.349039.121

Figure Lengend Snippet: Figure 2. Rap1-I312R does not bind to genomic loci or DNA. (A) Dot blot for telomere repeats following ChIP using anti-HA antibody in cells expressing HA-tagged Rap1-WT, Rap1-I312R, or empty vector (EV) control (n = 2 biological replicates). Signal intensity was deter- mined by normalizing to input. (B) High-throughput sequencing of ChIP samples as in A. Telomere binding was determined by calculating the proportion of reads with at least three telomere (TTAGGG)3/(CCCTAA)3 repeats. Data are the mean ± standard deviation of n = 4 bi- ological replicates. (C) Summary of ChIP-seq peak calling analysis that identified 109 peaks in HA-Rap1-WT samples (MACS2; q < 0.05). Peaks were classified as subtelomeric if localized within 500 kb from the telomere. (D) Heat map representing ChIP-seq profiles of HA- Rap1-WT peaks at chromosome ends. (E) Heat map representing ChIP-seq profiles of HA-Rap1-WT interstitial peaks. (F) Electrophoretic mobility shift assay (EMSA) using His-tagged RAP (0–15 µM) and a 74-bp TTAGGG/AATCCC double-stranded DNA (100 nM). (G) EMSA using non-His-tagged RAP (0–15 µM) and a 74-bp TTAGGG/AATCCC double-stranded DNA (100 nM).

Article Snippet: The following primary antibodies were used: mouse Rap1 (1252, rabbit polyclonal), TRF2 (Novus Biologicals NB110-57130, rabbit polyclonal), Tip60 (Cell Signaling 12058, rabbit polyclonal), γ-tubulin (Millipore Sigma T6557, mouse monoclonal), α-tubulin (Abcam ab7291, mouse monoclonal), histone H3 (Abcam ab1791, rabbit polyclonal), Flag (Cell Signaling 14793, rabbit monoclonal), c-Myc (Santa Cruz Biotechnology sc789, rabbit polyclonal), HA (Abcam ab9110, rabbit polyclonal), and streptavidin-HRP (Invitrogen 1953050).

Techniques: Dot Blot, Expressing, Plasmid Preparation, Control, Next-Generation Sequencing, Binding Assay, Standard Deviation, ChIP-sequencing, Electrophoretic Mobility Shift Assay

Journal: Genes & development

Article Title: Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

doi: 10.1101/gad.349039.121

Figure Lengend Snippet: Figure 3. Proximity-dependent biotinylation reveals extratelomeric Rap1 binding partners. (A) Representative image of IF-FISH in Rap1−/−MEFs expressing BioID-Rap1-WT stained for BioID-Rap1 (magenta), biotin (green), and telomeres (red) using anti-Flag antibody, streptavidin, and TTAGGG PNA probe, respectively. DAPI (blue) was used as a counterstain. (B) Immunoblot for Rap1 and TRF2 follow- ing streptavidin pull-down in Rap1−/−MEFs expressing BioID-Rap1-WT (n = 3 biological replicates; clones C1, C2, and C3), BioID-Rap1- I312R (n = 4 biological replicates; clones C1, C2, C3, and C4), and BioID alone (EV). Where indicated, cells were treated with 50 µM biotin for 20 h prior to harvest. (C) Scatter plot representing log2 fold change in peptide spectrum match (PSM) of proteins identified by BioID- Rap1-WT versus BioID-Rap1-I312R. Fold change values were calculated relative to no biotin and BioID-alone controls. Each dot represents a unique protein. Known Tip60/p400 (green and purple) and shelterin (red) complex members are indicated. (D) Graphical representation of the top 10 biological processes overrepresented in BioID-Rap1-I312R streptavidin pull-down (FC > 2) using the PANTHER classification system statistical overrepresentation test. (E) Co-IP of Flag-tagged Tip60/p400 subunits (Tip60, Epc1, Epc2, Dmap1, Brd8, Ruvbl1, and Actl6a) and HA-Rap1 following cotransfection in HEK293T cells. (F) Reciprocal co-IP of HA-Rap1-I312R and Myc-tagged Epc1 or Epc2. Co-IP of HA-Rap1 or HA-Rap1-I312R with TRF2 was used as a control. (G) Reciprocal co-IP of HA-Rap1 and Myc-Tip60.

Article Snippet: The following primary antibodies were used: mouse Rap1 (1252, rabbit polyclonal), TRF2 (Novus Biologicals NB110-57130, rabbit polyclonal), Tip60 (Cell Signaling 12058, rabbit polyclonal), γ-tubulin (Millipore Sigma T6557, mouse monoclonal), α-tubulin (Abcam ab7291, mouse monoclonal), histone H3 (Abcam ab1791, rabbit polyclonal), Flag (Cell Signaling 14793, rabbit monoclonal), c-Myc (Santa Cruz Biotechnology sc789, rabbit polyclonal), HA (Abcam ab9110, rabbit polyclonal), and streptavidin-HRP (Invitrogen 1953050).

Techniques: Binding Assay, Expressing, Staining, Western Blot, Clone Assay, Co-Immunoprecipitation Assay, Cotransfection, Control

Journal: Genes & development

Article Title: Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

doi: 10.1101/gad.349039.121

Figure Lengend Snippet: Figure 5. Rap1 suppresses the 2C-like state by enhancing Tip60/p400 repression of 2C genes. (A) MA plot of log2 fold changes in gene expression in Rap−/−versus Rap1+/+ mESCs. Up-regulated (light red) and down-regulated (light blue) genes are indicated (FC > 1.5; FDR < 0.1; n = 5 biological replicates). 2C-stage genes are highlighted in dark red. (B) Hierarchically clustered heat map representing RNA- seq data of 2C genes up-regulated in Rap−/−versus Rap1+/+ mESCs (n = 5 biological replicates). (C) Volcano plot of differential repeat ex- pression analysis in Rap1−/−versus Rap1+/+ mESCs. Up-regulated (red) and down-regulated (blue) repeats are indicated (FC > 2; FDR < 0.1; n = 5 biological replicates). (D) Kmeans (k = 5) clustered heat map representing RNA-seq data of 2C genes in Rap1+/+ (gray bar) and Rap1−/−

Article Snippet: The following primary antibodies were used: mouse Rap1 (1252, rabbit polyclonal), TRF2 (Novus Biologicals NB110-57130, rabbit polyclonal), Tip60 (Cell Signaling 12058, rabbit polyclonal), γ-tubulin (Millipore Sigma T6557, mouse monoclonal), α-tubulin (Abcam ab7291, mouse monoclonal), histone H3 (Abcam ab1791, rabbit polyclonal), Flag (Cell Signaling 14793, rabbit monoclonal), c-Myc (Santa Cruz Biotechnology sc789, rabbit polyclonal), HA (Abcam ab9110, rabbit polyclonal), and streptavidin-HRP (Invitrogen 1953050).

Techniques: Gene Expression, RNA Sequencing