Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Biophysical characterization of TACC3 629–838. (A–C) Sedimentation velocity traces and fits of TACC3 629–838 (2 mg/ml). (A) Fitting model: c(s)+1 discrete component, ME regularization at a confidence level of 0.68. (B) Residuals of fit shown in A. (C) Resulting c(s) distribution. (D–F) CW EPR and DEER of TACC3 TACC domain. (D) CW EPR spectra of TACC3 MTSL-C828 (black) and TACC3 MTSL-C662 (red) at 120 K. (E and F) Normalized four-pulse DEER trace at 50 K for (E) TACC3 MTSL-C828 and (F) TACC3 MTSL-C662. Inset, traces after subtraction of a mono-exponential decay. The weak oscillation is most likely from residual proton modulation.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Sedimentation

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Characterization of ch-TOG 1817–1957. (A) In vitro coprecipitation assays between immobilized NusA-TACC3 TACC domain and ch-TOG truncates. (B) Superposition of the top 20 ch-TOG 1817–1957 NMR structures using the structured core of helices, H1-H4 (residues 1826–1894) for alignment. Inset, structures aligned on ch-TOG residues 1905–1915. (C) Cartoon representation of the best NMR structure for ch-TOG 1817–1957 is shown in the same orientation as in B. Inset, stick representation of ch-TOG residues 1905–1915. Structures in B and C are colored by spectrum mode where the N-terminus is blue and the C-terminus is red. Source data are available for this figure: .

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: In Vitro

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Interactions of ch-TOG with TACC3. (A) Chemical shift perturbations observed on the interaction of 2 H/ 15 N labeled ch-TOG 1817–1957 with a threefold excess of TACC3 629–838 ∆699–765. Red lines mark different multiples in standard deviation (as stated) in chemical shift perturbation. Data extracted from the spectra shown in . (B) MST binding experiment of a synthetic ch-TOG H5 peptide with TACC3 629–838. (C) Cartoon representation of an AlphaFold2 Multimer model of the complex between the TACC3 629–838 ∆699–765 dimer (wheat) and ch-TOG 1817–1957 (green). Arrows indicate the position of the deletion in TACC3. The model is shown below colored according to per residue confidence score (pLDDT) in rainbow colors from high (blue) to low (red) confidence. (D) Cartoon representations of the AlphaFold2 model showing the interface between the H5 region of ch-TOG (green) and TACC3 (wheat). Sidechains contributing to the interface from TACC3 protomer A (orange), TACC3 protomer B (yellow), and ch-TOG H5 (green) are shown in stick representation.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Labeling, Standard Deviation, Binding Assay, Residue

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: NMR interaction and AlphaFold2 modeling of TACC3 and ch-TOG, the effect of ch-TOG H5 mutations on TACC3 binding and HDX-MS of TACC3 in the absence/presence of Affimers. (A) Portions of a TROSY experiment of 2 H/ 15 N labeled ch-TOG 1817–1957. Plots show the protein alone (blue) and in the presence of a threefold excess of TACC3 629–838 ∆699–765 (red). (B) Predicted aligned error (PAE) plot for a model of the complex between TACC3 629–838 ∆699–765 and ch-TOG 1817–1957 generated by AlphaFold2 Multimer. (C) In vitro co-precipitation assays between immobilized His-NusA-ch-TOG 1817–1957 constructs and TACC3 629–838 (top). Binding of TACC3 was resolved by western blot (bottom). (D) Sequence coverage map of TACC3 629–838 ∆699–765 in HDX-MS experiments. The yellow shaded regions in the thick bar at the top of the panel represent regions with sequence coverage while gray indicates regions that were not covered by detected peptides. Narrow yellow bars represent the individual peptides detected. (E–G) Woods plots showing the differences in deuterium uptake in TACC3 at three HDX timepoints (0.5, 5, 30 min of HDX), comparing TACC3 629–838 ∆699–765 alone with TACC3 629–838 ∆699–765 in the presence of Affimers E4 (E), E7 (F) and E8 (G). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides with no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. A summary of key details of the HDX-MS experiment is shown in . Source data are available for this figure: .

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Binding Assay, Labeling, Generated, In Vitro, Construct, Western Blot, Sequencing

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Isolation of Affimers that bind TACC3 and inhibit TACC3-ch-TOG interaction. (A) In vitro co-precipitation assay between Affimers, TACC3, and ch-TOG. C-terminal His-tagged Affimers were immobilized on Nickel Sepharose resin and incubated with TACC3 629–838 (TACC3 TD) or TACC3 629–838 ∆699–765 (TACC3 TD∆). Binding of ch-TOG 1517–1957 in the presence of Affimer was assessed by the addition of ch-TOG to TACC3 TD∆ reactions. (B) ELISAs to assess binding between Affimers and TACC3 629–838 ∆699–765. Biotinylated TACC3 629–838 ∆699–765 was immobilized on Streptavidin-coated plates and incubated with an Affimer dilution series (orange circles). Background binding of Affimers to the plate was measured by incubating the proteins in wells coated with PBS (gray squares). Data points are the mean ± standard error of the mean from two experiments. (C) Woods plots describing differences in deuterium uptake by residue, after 30 min of exchange, between TACC3 629–838 ∆699–765 in the absence of a binding partner and in the presence of Affimers (as indicated). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides exhibiting no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. (D) Cartoon representation the TACC3 629–838 ∆699–765–ch-TOG complex model with TACC3 colored according to HDX behavior as in C. The region of TACC3 protected from hydrogen/deuterium exchange in the presence of Affimers E4, E7, and E8 is colored blue, and ch-TOG H5 is colored pink. (E) Representative confocal micrographs of HeLa cells transfected with mCherry or mCherry-Affimers, as labelled; stained with ch-TOG and TACC3 antibodies for proximity ligation assay. Nuclei are indicated by DAPI staining (blue). Green foci indicate TACC3-ch-TOG protein complexes. Single ch-TOG antibody staining was used as a control for PLA interactions. Scale bars, 10 µm. (F) Dot plot graph displaying the number of TACC3-ch-TOG PLA signals per cell from E. Data represent counts from at least 20–30 cells, n = 4 . Error bars represent standard deviation of four biological replicates. ****P < 0.0001 in comparison with no transfected sample (−) by one-way ANOVA. (G) Western blot to show that ch-TOG and TACC3 levels are not reduced by Affimer expression. Source data are available for this figure: .

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Isolation, In Vitro, Incubation, Binding Assay, Residue, Generated, Transfection, Staining, Proximity Ligation Assay, Control, Standard Deviation, Comparison, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: TACC3 Affimers cannot be used to localize or inducibly relocalize endogenous TACC3 but do affect the mitotic spindle localization of clathrin or TACC3. (A) Representative confocal micrographs of metaphase HeLa cells expressing the indicated mCherry-Affimers (red). Cells were fixed and stained with anti-TACC3 (green). (B) Widefield micrographs of live HeLa cells in metaphase expressing GFP-TACC3 (green) and mCherry-Affimers (red). (C) Induced relocalization of TACC3 Affimers to mitochondria. Representative confocal micrographs of HeLa cells at metaphase expressing the indicated FKBP-GFP-Affimers (green) with dark-MitoTrap, that were either treated or not with rapamycin (200 nM, 30 min) prior to fixation. Cells were stained for tubulin (not shown in merge) and TACC3 (red). DNA (blue) is shown in the merge. Relocalization of the Affimer to mitochondria can be seen in the rapamycin-treated cells compared to control, but no relocation of TACC3 is observed, therefore no inactivation of TACC3 activity at the mitotic spindle. (D) Quantification of Affimer (x-axis) and TACC3 (y-axis) spindle localization in untreated cells (salmon) and rapamycin treated cells (turquoise). Spindle localization was calculated as the ratio of spindle to cytoplasmic fluorescence shown on a log 2 scale, n = 11–22 cells per condition. (E) Representative confocal micrographs of untreated or MLN8237-treated (0.3 μM, 40 min) knock-in CLTA-FKBP-GFP HeLa cells at metaphase. Cells were fixed in PTEMF and stained for TACC3 (red), DNA (blue), and a GFP-boost antibody was used to enhance the signal of CLTA-FKBP-GFP (green). Cells expressing the indicated mCherry-Affimers (gray, not shown in merge), and quantification of spindle recruitment of clathrin (CLTA) and TACC3. Scale bars, 10 μm.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Expressing, Staining, Control, Activity Assay, Fluorescence, Knock-In

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Affimer-mediated disruption of TACC3–ch-TOG interaction results in lower spindle localization of ch-TOG. (A) Representative confocal micrographs of untreated or MLN8237-treated (0.3 μM, 40 min) knock-in GFP-FKBP-TACC3 HeLa cells in metaphase. Cells were stained for ch-TOG (red), DNA (blue), and GFP-boost antibody was used to enhance the signal of GFP-FKBP-TACC3 (green). (B) Cells expressing mCherry or mCherry-Affimers, labeled as in A. Note, no specific enrichment of Affimers at the spindle (see ). Scale bars, 10 μm. (C) Quantification of spindle recruitment of TACC3 and ch-TOG. SuperPlots show single cell measurements as dots, experiment means as outlined markers, colors indicate experiments. Dashed line, no spindle enrichment. Analysis of variance (ANOVA) followed by Tukey’s post hoc test is shown above each group, using the untransfected and untreated cells (black) and untransfected MLN8237-treated cells (purple) for comparison. ***, P < 0.001; NS, >0.05. (D) Spindle microtubule intensity was similar in cells expressing mCherry or mCherry-Affimers. SuperPlot of three experiments with mean ± SD represented by the bar and error. (E) Averaging images from C shows the relative centrosomal localization of ch-TOG versus TACC3 (i) and the displacement of ch-TOG from the spindle to the cytoplasm in cells expressing Affimers E7 and E8 (ii).

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Disruption, Knock-In, Staining, Expressing, Labeling, Comparison

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: TACC3 Affimers do not affect mitotic spindle morphology or positioning in HeLa cells, and PCM fragmentation is not rescued by microtubule depolymerization. (A) Quantification of mitotic spindle parameters in HeLa cells expressing mCherry (mCh) or mCherry-Affimers (E4, E7, E8). Cells were fixed in PTEMF and stained for α-tubulin, pericentrin and DNA. Superplots show the spindle parameters that were measured using a semi-automated workflow. Spindle offset is the euclidean distance between the cell center and the spindle center. The distances d2 and d1 refer to the distance from each centrosome to the cell boundary (taken as a sphere that best fit the 3D perimeter of the cell). Spindle tilt and angle are the angle between the spindle axis and the imaging plane or the metaphase plate, respectively. Dots, single cell; outlined markers, mean independent experiments (indicated by color). Bars show overall mean ± SD; P values from Tukey’s HSD post hoc test. (B) Counts of PCNT foci in cells expressing mCherry (mCh), or mCherry-Affimers (E7 and E8) for 24 h. Nocodazole treatment (5 µM, 10 min) did not reduce the number of excess PCNT foci. (C) Nocodazole was active in these experiments. Example micrographs of PCNT foci show that in two PCNT cells was normal or expanded; while fragmented are cells with >2 PCNT foci. Scale bar, 10 µm. Pie charts of the fraction of 2 PCNT cells that had normal or expanded PCNT staining.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Expressing, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Expression of a ch-TOG mutant deficient in binding TACC3 results in fragmentation of pericentrin in mitotic HeLa cells. (A) Representative max intensity z projection images of HeLa cells co-expressing shRNA against ch-TOG, and either GFP, RNAi-resistant ch-TOG-GFP (WT) or ch-TOG(L1939,1942A)-GFP (LLAA). Cells were stained for pericentrin (green), α-tubulin (red), and DNA (blue). Scale bar, 10 μm. (B) Histograms to show how many cells in each condition had two or more PCNT foci. The percentage of cells with >2 foci is indicated. Fisher’s exact test was used to test for association between the protein expressed and the PCM foci category. Bonferroni adjustment was used to calculate P values. (C) Superplots show the total volume of pericentrin foci. Dots, single cells; markers, mean of each experiment, colors indicate experiments. In B and C, cells with exactly two pericentrin foc are shown in salmon/filled dots and those with >2, turquoise/empty dots; P value from two-way ANOVA, between expression conditions.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Expressing, Mutagenesis, Binding Assay, shRNA, Staining

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Blocking TACC3–ch-TOG interaction with Affimers results in fragmentation of PCM and mitotic delay. (A) Stills from live cell imaging experiments to track the number of γ-tubulin foci in cells expressing mEmerald-γ-tubulin (green) and the indicated mCherry or mCherry-Affimers constructs (not shown), SiR-DNA staining is shown (red). Scale bar, 10 μm. (B) Sankey diagrams to show the number cells containing supernumerary γ-tubulin foci at the indicated stages of cell division. The number of γ-tubulin foci was tracked from G2-prometaphase, prometaphase-metaphase and metaphase-anaphase. Numbers in each node represent the number of cells observed at each stage, as labeled. Node color represents the number of γ-tubulin foci in the cell, those with two are shown in salmon and those >2, turquoise. Data is pooled from four independent overnight experiments. (C) Mitotic progression of HeLa cells expressing mCherry or mCherry-Affimers. Cumulative histograms of prometaphase to metaphase (i) and prometaphase to anaphase (ii) timings. Number of cells analyzed: mCherry, 75; Affimer E4, 81; Affimer E7, 82; Affimer E8, 72. (D) Frequencies of Affimer E7- or Affimer E8-expressing cells shown in A, comparing timings of cells with two γ-tubulin foci (2; salmon) during metaphase with cells that undergo PCM fragmentation during metaphase (>2; turquoise). Number of cells: (2 and >2 foci, respectively): Affimer E7, 65 and 11; Affimer E8, 58 and 11.

Article Snippet: In brief, 1 μg of biotinylated TACC3 629–838 ∆699–765-Avi was bound to streptavidin-coated wells (Pierce), washed, and then 1 × 10 12 cfu phage was added for 2 h with shaking.

Techniques: Blocking Assay, Live Cell Imaging, Expressing, Construct, Staining, Labeling

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Biophysical characterization of TACC3 629–838. (A–C) Sedimentation velocity traces and fits of TACC3 629–838 (2 mg/ml). (A) Fitting model: c(s)+1 discrete component, ME regularization at a confidence level of 0.68. (B) Residuals of fit shown in A. (C) Resulting c(s) distribution. (D–F) CW EPR and DEER of TACC3 TACC domain. (D) CW EPR spectra of TACC3 MTSL-C828 (black) and TACC3 MTSL-C662 (red) at 120 K. (E and F) Normalized four-pulse DEER trace at 50 K for (E) TACC3 MTSL-C828 and (F) TACC3 MTSL-C662. Inset, traces after subtraction of a mono-exponential decay. The weak oscillation is most likely from residual proton modulation.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Sedimentation

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Characterization of ch-TOG 1817–1957. (A) In vitro coprecipitation assays between immobilized NusA-TACC3 TACC domain and ch-TOG truncates. (B) Superposition of the top 20 ch-TOG 1817–1957 NMR structures using the structured core of helices, H1-H4 (residues 1826–1894) for alignment. Inset, structures aligned on ch-TOG residues 1905–1915. (C) Cartoon representation of the best NMR structure for ch-TOG 1817–1957 is shown in the same orientation as in B. Inset, stick representation of ch-TOG residues 1905–1915. Structures in B and C are colored by spectrum mode where the N-terminus is blue and the C-terminus is red. Source data are available for this figure: .

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: In Vitro

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Interactions of ch-TOG with TACC3. (A) Chemical shift perturbations observed on the interaction of 2 H/ 15 N labeled ch-TOG 1817–1957 with a threefold excess of TACC3 629–838 ∆699–765. Red lines mark different multiples in standard deviation (as stated) in chemical shift perturbation. Data extracted from the spectra shown in . (B) MST binding experiment of a synthetic ch-TOG H5 peptide with TACC3 629–838. (C) Cartoon representation of an AlphaFold2 Multimer model of the complex between the TACC3 629–838 ∆699–765 dimer (wheat) and ch-TOG 1817–1957 (green). Arrows indicate the position of the deletion in TACC3. The model is shown below colored according to per residue confidence score (pLDDT) in rainbow colors from high (blue) to low (red) confidence. (D) Cartoon representations of the AlphaFold2 model showing the interface between the H5 region of ch-TOG (green) and TACC3 (wheat). Sidechains contributing to the interface from TACC3 protomer A (orange), TACC3 protomer B (yellow), and ch-TOG H5 (green) are shown in stick representation.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Labeling, Standard Deviation, Binding Assay, Residue

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: NMR interaction and AlphaFold2 modeling of TACC3 and ch-TOG, the effect of ch-TOG H5 mutations on TACC3 binding and HDX-MS of TACC3 in the absence/presence of Affimers. (A) Portions of a TROSY experiment of 2 H/ 15 N labeled ch-TOG 1817–1957. Plots show the protein alone (blue) and in the presence of a threefold excess of TACC3 629–838 ∆699–765 (red). (B) Predicted aligned error (PAE) plot for a model of the complex between TACC3 629–838 ∆699–765 and ch-TOG 1817–1957 generated by AlphaFold2 Multimer. (C) In vitro co-precipitation assays between immobilized His-NusA-ch-TOG 1817–1957 constructs and TACC3 629–838 (top). Binding of TACC3 was resolved by western blot (bottom). (D) Sequence coverage map of TACC3 629–838 ∆699–765 in HDX-MS experiments. The yellow shaded regions in the thick bar at the top of the panel represent regions with sequence coverage while gray indicates regions that were not covered by detected peptides. Narrow yellow bars represent the individual peptides detected. (E–G) Woods plots showing the differences in deuterium uptake in TACC3 at three HDX timepoints (0.5, 5, 30 min of HDX), comparing TACC3 629–838 ∆699–765 alone with TACC3 629–838 ∆699–765 in the presence of Affimers E4 (E), E7 (F) and E8 (G). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides with no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. A summary of key details of the HDX-MS experiment is shown in . Source data are available for this figure: .

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Binding Assay, Labeling, Generated, In Vitro, Construct, Western Blot, Sequencing

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Isolation of Affimers that bind TACC3 and inhibit TACC3-ch-TOG interaction. (A) In vitro co-precipitation assay between Affimers, TACC3, and ch-TOG. C-terminal His-tagged Affimers were immobilized on Nickel Sepharose resin and incubated with TACC3 629–838 (TACC3 TD) or TACC3 629–838 ∆699–765 (TACC3 TD∆). Binding of ch-TOG 1517–1957 in the presence of Affimer was assessed by the addition of ch-TOG to TACC3 TD∆ reactions. (B) ELISAs to assess binding between Affimers and TACC3 629–838 ∆699–765. Biotinylated TACC3 629–838 ∆699–765 was immobilized on Streptavidin-coated plates and incubated with an Affimer dilution series (orange circles). Background binding of Affimers to the plate was measured by incubating the proteins in wells coated with PBS (gray squares). Data points are the mean ± standard error of the mean from two experiments. (C) Woods plots describing differences in deuterium uptake by residue, after 30 min of exchange, between TACC3 629–838 ∆699–765 in the absence of a binding partner and in the presence of Affimers (as indicated). Woods plots were generated using Deuteros 2.0. Peptides colored in blue are protected from hydrogen/deuterium exchange in the presence of Affimers. Peptides exhibiting no significant difference in exchange between conditions, determined using a 99% confidence interval and a hybrid statistical test (dotted line), are shown in gray. (D) Cartoon representation the TACC3 629–838 ∆699–765–ch-TOG complex model with TACC3 colored according to HDX behavior as in C. The region of TACC3 protected from hydrogen/deuterium exchange in the presence of Affimers E4, E7, and E8 is colored blue, and ch-TOG H5 is colored pink. (E) Representative confocal micrographs of HeLa cells transfected with mCherry or mCherry-Affimers, as labelled; stained with ch-TOG and TACC3 antibodies for proximity ligation assay. Nuclei are indicated by DAPI staining (blue). Green foci indicate TACC3-ch-TOG protein complexes. Single ch-TOG antibody staining was used as a control for PLA interactions. Scale bars, 10 µm. (F) Dot plot graph displaying the number of TACC3-ch-TOG PLA signals per cell from E. Data represent counts from at least 20–30 cells, n = 4 . Error bars represent standard deviation of four biological replicates. ****P < 0.0001 in comparison with no transfected sample (−) by one-way ANOVA. (G) Western blot to show that ch-TOG and TACC3 levels are not reduced by Affimer expression. Source data are available for this figure: .

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Isolation, In Vitro, Incubation, Binding Assay, Residue, Generated, Transfection, Staining, Proximity Ligation Assay, Control, Standard Deviation, Comparison, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: TACC3 Affimers cannot be used to localize or inducibly relocalize endogenous TACC3 but do affect the mitotic spindle localization of clathrin or TACC3. (A) Representative confocal micrographs of metaphase HeLa cells expressing the indicated mCherry-Affimers (red). Cells were fixed and stained with anti-TACC3 (green). (B) Widefield micrographs of live HeLa cells in metaphase expressing GFP-TACC3 (green) and mCherry-Affimers (red). (C) Induced relocalization of TACC3 Affimers to mitochondria. Representative confocal micrographs of HeLa cells at metaphase expressing the indicated FKBP-GFP-Affimers (green) with dark-MitoTrap, that were either treated or not with rapamycin (200 nM, 30 min) prior to fixation. Cells were stained for tubulin (not shown in merge) and TACC3 (red). DNA (blue) is shown in the merge. Relocalization of the Affimer to mitochondria can be seen in the rapamycin-treated cells compared to control, but no relocation of TACC3 is observed, therefore no inactivation of TACC3 activity at the mitotic spindle. (D) Quantification of Affimer (x-axis) and TACC3 (y-axis) spindle localization in untreated cells (salmon) and rapamycin treated cells (turquoise). Spindle localization was calculated as the ratio of spindle to cytoplasmic fluorescence shown on a log 2 scale, n = 11–22 cells per condition. (E) Representative confocal micrographs of untreated or MLN8237-treated (0.3 μM, 40 min) knock-in CLTA-FKBP-GFP HeLa cells at metaphase. Cells were fixed in PTEMF and stained for TACC3 (red), DNA (blue), and a GFP-boost antibody was used to enhance the signal of CLTA-FKBP-GFP (green). Cells expressing the indicated mCherry-Affimers (gray, not shown in merge), and quantification of spindle recruitment of clathrin (CLTA) and TACC3. Scale bars, 10 μm.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Expressing, Staining, Control, Activity Assay, Fluorescence, Knock-In

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Affimer-mediated disruption of TACC3–ch-TOG interaction results in lower spindle localization of ch-TOG. (A) Representative confocal micrographs of untreated or MLN8237-treated (0.3 μM, 40 min) knock-in GFP-FKBP-TACC3 HeLa cells in metaphase. Cells were stained for ch-TOG (red), DNA (blue), and GFP-boost antibody was used to enhance the signal of GFP-FKBP-TACC3 (green). (B) Cells expressing mCherry or mCherry-Affimers, labeled as in A. Note, no specific enrichment of Affimers at the spindle (see ). Scale bars, 10 μm. (C) Quantification of spindle recruitment of TACC3 and ch-TOG. SuperPlots show single cell measurements as dots, experiment means as outlined markers, colors indicate experiments. Dashed line, no spindle enrichment. Analysis of variance (ANOVA) followed by Tukey’s post hoc test is shown above each group, using the untransfected and untreated cells (black) and untransfected MLN8237-treated cells (purple) for comparison. ***, P < 0.001; NS, >0.05. (D) Spindle microtubule intensity was similar in cells expressing mCherry or mCherry-Affimers. SuperPlot of three experiments with mean ± SD represented by the bar and error. (E) Averaging images from C shows the relative centrosomal localization of ch-TOG versus TACC3 (i) and the displacement of ch-TOG from the spindle to the cytoplasm in cells expressing Affimers E7 and E8 (ii).

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Disruption, Knock-In, Staining, Expressing, Labeling, Comparison

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: TACC3 Affimers do not affect mitotic spindle morphology or positioning in HeLa cells, and PCM fragmentation is not rescued by microtubule depolymerization. (A) Quantification of mitotic spindle parameters in HeLa cells expressing mCherry (mCh) or mCherry-Affimers (E4, E7, E8). Cells were fixed in PTEMF and stained for α-tubulin, pericentrin and DNA. Superplots show the spindle parameters that were measured using a semi-automated workflow. Spindle offset is the euclidean distance between the cell center and the spindle center. The distances d2 and d1 refer to the distance from each centrosome to the cell boundary (taken as a sphere that best fit the 3D perimeter of the cell). Spindle tilt and angle are the angle between the spindle axis and the imaging plane or the metaphase plate, respectively. Dots, single cell; outlined markers, mean independent experiments (indicated by color). Bars show overall mean ± SD; P values from Tukey’s HSD post hoc test. (B) Counts of PCNT foci in cells expressing mCherry (mCh), or mCherry-Affimers (E7 and E8) for 24 h. Nocodazole treatment (5 µM, 10 min) did not reduce the number of excess PCNT foci. (C) Nocodazole was active in these experiments. Example micrographs of PCNT foci show that in two PCNT cells was normal or expanded; while fragmented are cells with >2 PCNT foci. Scale bar, 10 µm. Pie charts of the fraction of 2 PCNT cells that had normal or expanded PCNT staining.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Expressing, Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Expression of a ch-TOG mutant deficient in binding TACC3 results in fragmentation of pericentrin in mitotic HeLa cells. (A) Representative max intensity z projection images of HeLa cells co-expressing shRNA against ch-TOG, and either GFP, RNAi-resistant ch-TOG-GFP (WT) or ch-TOG(L1939,1942A)-GFP (LLAA). Cells were stained for pericentrin (green), α-tubulin (red), and DNA (blue). Scale bar, 10 μm. (B) Histograms to show how many cells in each condition had two or more PCNT foci. The percentage of cells with >2 foci is indicated. Fisher’s exact test was used to test for association between the protein expressed and the PCM foci category. Bonferroni adjustment was used to calculate P values. (C) Superplots show the total volume of pericentrin foci. Dots, single cells; markers, mean of each experiment, colors indicate experiments. In B and C, cells with exactly two pericentrin foc are shown in salmon/filled dots and those with >2, turquoise/empty dots; P value from two-way ANOVA, between expression conditions.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Expressing, Mutagenesis, Binding Assay, shRNA, Staining

Journal: The Journal of Cell Biology

Article Title: Structural characterization and inhibition of the interaction between ch-TOG and TACC3

doi: 10.1083/jcb.202407002

Figure Lengend Snippet: Blocking TACC3–ch-TOG interaction with Affimers results in fragmentation of PCM and mitotic delay. (A) Stills from live cell imaging experiments to track the number of γ-tubulin foci in cells expressing mEmerald-γ-tubulin (green) and the indicated mCherry or mCherry-Affimers constructs (not shown), SiR-DNA staining is shown (red). Scale bar, 10 μm. (B) Sankey diagrams to show the number cells containing supernumerary γ-tubulin foci at the indicated stages of cell division. The number of γ-tubulin foci was tracked from G2-prometaphase, prometaphase-metaphase and metaphase-anaphase. Numbers in each node represent the number of cells observed at each stage, as labeled. Node color represents the number of γ-tubulin foci in the cell, those with two are shown in salmon and those >2, turquoise. Data is pooled from four independent overnight experiments. (C) Mitotic progression of HeLa cells expressing mCherry or mCherry-Affimers. Cumulative histograms of prometaphase to metaphase (i) and prometaphase to anaphase (ii) timings. Number of cells analyzed: mCherry, 75; Affimer E4, 81; Affimer E7, 82; Affimer E8, 72. (D) Frequencies of Affimer E7- or Affimer E8-expressing cells shown in A, comparing timings of cells with two γ-tubulin foci (2; salmon) during metaphase with cells that undergo PCM fragmentation during metaphase (>2; turquoise). Number of cells: (2 and >2 foci, respectively): Affimer E7, 65 and 11; Affimer E8, 58 and 11.

Article Snippet: The following plasmids were generated in the course of previous work: mNeonGreen-EB3, pMito-mCherry-FRBK70N, GFP-TACC3, pBrain-GFP-shch-TOG, pBrain-ch-TOGKDP-GFP-shch-TOG, and pBrain-ch-TOGDPGFP(LL1939,1942A)-shch-TOG ( ); pETM6T1 TACC3 629–838 and pETM6T1 ch-TOG 1517–1957 ( ); and mEmerald-γ-tubulin was from Addgene #54105. ch-TOG 1467–2032 was made by amplifying the corresponding cDNA region using the following primers, digestion of the TOPO-cloned PCR product using Nco I and Mfe I, and ligation into pETM6T1 cut with Nco I and Eco RI: chTOG_1467_NcoI_F (5′-CCA TGG CCC GAA GCA TGA GTG GGC ATC CTG AGG CAG CCC AGA TGG-3′), chTOG_2032_MfeI_R (5′-CAA TTG TCA TTT GCG ACT GCT CTT TAT TCT CTC CAG TCT TTT TTT CAA GTC GTC-3′). ch-TOG truncates were produced by amplifying the corresponding cDNA regions from ch-TOG 1467–2032 using the following primers, digestion of the PCR products with Nco I and Xho I, and ligation into pETM6T1 cut with the same restriction enzymes: chT1517start (5′-CGC GCC ATG GTC CTT ATT CCT GAA CCC AAG ATC-3′), chT1817startNco (5′-CGC GCC CAT GGC ATC TCG AAT AGA TGA AAA ATC ATC AAA GGC-3′), chT1827startNco (5′-CGC GCC ATG GCC AAA GTG AAT GAT TTC TTA GCT GAG-3′), chT1804Xhoend (5′-CGC GCT CGA GTC ACT GGT CCA TAC TGT GCT TC-3′), chT1815Xhoend (5′-CGC GCT CGA GTC ACT TTT CTG TTT CCT TAT CAG ACT TGC-3′), chT1957Xhoend (5′-CGC GCT CGA GTC AAG GTC GGT CAT CTT GC-3′). pETM6T1 TACC3 629–838 ∆699–765 was produced by deletion QuikChange site-directed mutagenesis (Agilent) of the TACC3 629–838 construct using primers: T3d699/765F (5′-GCA GAA GGA ACT TTC CAA AGC CCT GAA GGC CCA CGC G-3′), T3d699/765R (5′-CGC GTG GGC CTT CAG GGC TTT GGA AAG TTC CTT CTG C-3′).

Techniques: Blocking Assay, Live Cell Imaging, Expressing, Construct, Staining, Labeling