Journal: Molecular and Cellular Biology

Article Title: TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

doi: 10.1128/mcb.00240-10

Figure Lengend Snippet: FIG. 6. The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1OB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

Article Snippet: Next, cells were incubated with one of several combinations of the following primary antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin (1:5,000, ) (1), mouse anti-TRF2 (1:1,000; Imgenex Corp., San Diego, CA), rabbit anti-hTERT (1:400; Rockland, Gilbertsville, PA), mouse anti-FLAG (1:500; Sigma-Aldrich, St. Louis, MO), rabbit anti-RAP1(1: 2,000; Novus Biologicals, Littleton, CO), and rabbit anti-53BP1(1:500; Bethyl, Montgomery, TX).

Techniques: shRNA, Marker

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: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Retrovirally-induced HRGβ2 regulates TRF2 and RAP1 expression and telomere length in MCF-7 breast cancer cells. Left. Representative immunoblot ( n = 3 ) showing upregulation of TRF2 and RAP1 in MCF-7 cells retrovirally engineered to overexpress HRGβ 2 . PD 0 represents the first sub-passage after selection for retroviral infection. At the indicated PDs, cells were washed with cold PBS and solubilized in lysis buffer containing phosphatase and protease inhibitors. Fifty μg of protein per sample was resolved by SDS-PAGE and subjected to Western blotting for TRF2 and hRap1 as described above (see “Materials and Methods” for details). Middle panel. MCF-7/HRGβ 2 cells do not exhibit significant changes in telomerase activity as assessed by the TRAP assay. Three thousand cell equivalents were used for each reaction and a representative experiment is shown ( n = 3 ). Right panel. Telomere length changes in MCF-7 cells upon retrovirally-induced HRGβ 2 overexpression. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from retrovirally-infected MCF-7 cells probed with a TTAGGG repeat fragment. B. HRG knockdown reduces the presence of TRF2 and RAP1 on telomeres and promotes telomere lengthening. Top left. Depletion of HRG with siRNA significantly affects TRF2 and RAP1 protein levels. A representative Western blot of MDA-MB-231 cell lysates 72 h after transfection of siRNA to HRG or siRNA control is shown ( n = 3 ). Top right. TRF length analysis in HRG knockdown MDA-MB-231 cells. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from MDA-MB-231 cells transiently transfected with control siRNA or graded concentrations of HRG siRNA (PD 4) probed with a TTAGGG repeat fragment ( n = 3 ). Bottom. Reduced TRF2 and RAP1 telomeric signals after HRG siRNA treatment. Western blot analysis showing the significant depletion of HRGβ 2 protein with a specific siRNA. The panel shows representative immunofluorescence images of MDA-MB-231 cells 48 h after introduction of HRG siRNA: staining with anti-TRF2 ( red ) and anti-RAP1 ( green ) antibodies is shown ( n = 3 ). DAPI was used to visualize nuclear DNA ( blue ).

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Expressing, Western Blot, Selection, Retroviral, Infection, Lysis, SDS Page, Activity Assay, TRAP Assay, Over Expression, Knockdown, Transfection, Control, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Subnuclear localization of HRGβ 2 and its relationship with telomeric loci. A431 cells naturally overexpressing HRGβ 2 were permeabilized twice and sequentially stained with an antibody against HRGβ 2 ( red ) and the fluorescein-conjugated telomere-specific peptide nucleic acid (PNA) probe ( green ). DAPI was used to visualize nuclear DNA ( blue ). Merged images reveal partial co-localization of endogenous HRGβ 2 and telomeres ( yellow ). B. Representative immunofluorescent images of A431 cells co-stained for endogenous HRGβ 2 ( green ) using anti-TRF2 or anti-RAP1 ( red ) antibodies is shown ( n = 3 ). Merged images, including DAPI staining of DNA, reveal a prominent co-localization ( yellow ) of endogenous HRGβ 2 with TRF2 and RAP1.

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Staining