EMBO/SourceData huggingface dataset:
Figure 2B Streptavidin pull-down assay with extracts of HEK293T cells expressing Bio-GFP-tagged liprin-α1 full length (FL) or its indicated fragments, analyzed by Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin.D Streptavidin pull down assay with extracts of HEK293T cells expressing Bio-GFP-tagged wild-type (WT) liprin-α1 fragment N1 or its NR/AA mutant, analyzed by Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. E Streptavidin pull down assay with extracts of HEK293T cells expressing Bio-GFP-tagged WT liprin-α1 N1 fragment or its NR/AA mutant (bait) together with GFP-CEP170B (prey), analyzed by Western blotting with GFP antibody.F Immunofluorescence staining of liprin-β1 in liprin-α1 KO HeLa cells transiently transfected with mCherry-tagged liprin-α1 full length or its NR/AA mutant. G, H Averaged intensity distributions for WT or NR/AA versions of mCherry-liprin-α1 (G) and liprin-β1 (H) for the experiments shown in (F). Distance from the cell edge is shown on the horizontal axis. n = 25-29 cells from three technical replicates. Data represent mean ± SEM. Data information: Scale bars, 5 μm.J, K Immunofluorescence staining of α-tubulin (blue) in liprin-α1 KO/Strep-GFP-CEP170B knock-in double-engineered HeLa cell line transiently transfected with mCherry-tagged liprin-α1 FL (J), fragment N1 (K), or their NR/AA mutant counterparts (J, K). Data information: Scale bars, 5 μm.
Figure 3A TIRF microscopy time-lapse images on the left show the accumulation of CEP170B along the distal segments of MT lattices in the cortical region of Strep-GFP-CEP170B knock-in HeLa cell line transiently transfected with EB3-TagRFP. Representative kymographs on the right show an MT plus end undergoing very short alternating growth and shrinking episodes (corresponding to the one marked as "1" in the images; upper right panel) and an MT plus end undergoing longer depolymerization followed by rescues (corresponding to the one marked as "2" in the images; bottom right panel ). Scale bars: horizontal, 2 μm; vertical, 30 s. Data information: Scale bars, 5 μm.D Immunofluorescence staining of CAMSAP2 (red) and α-tubulin (blue) in HeLa cells transiently transfected with GFP control or GFP-CEP170B. Lower panels show enlargements of boxed areas. E Quantification of the length of CAMSAP2 stretches for the experiments shown in (D). n = 12 cells from three technical replicates. Data information: Unless otherwise stated, Data represent mean ± SD. *, P<0.05; **, P < 0.01; ***, P < 0.001; two-tailed t test. Scale bars, 5 μm.I TIRF images showing distribution of TagRFP-Rab6 vesicles in indicated HeLa cell lines. Seven consecutive frames (0.3s / frame) were merged into a single image. J Representative images of TagRFP-Rab6 vesicle tracks in indicated HeLa cell lines. Data information: Scale bars, 5 μm.K-M Cartoon representation and quantification of the radiality of TagRFP-Rab6 vesicle tracks in indicated HeLa cell lines (K, L). Center: Golgi center; Start: starting position of the vesicle; End: ending position of the vesicle. A 0° or 180° angle means that the vesicle moves radially toward the cell periphery or center of the Golgi, respectively, with the Golgi center, starting position and ending position of the vesicle in a straight line. Percentage of tracks with the angle ranging from 0° to 45° is shown in (M). n = 10,000 vesicle tracks in 8-9 cells from two technical replicates in (L); n = 8-9 cells from two technical replicates in (M). Data information: Unless otherwise stated, Data represent mean ± SD. *, P<0.05; **, P < 0.01; ***, P < 0.001; two-tailed t test.
Figure 4C Immunofluorescence staining of CAMSAP2 (green) and α-tubulin (red) in control or CEP170B KO MCF7 monolayer. Left panels: basal plane; middle panels: apical plane; right panels: side view. D Immunofluorescence staining of α-tubulin (red) in control CAMSAP3-GFP knock-in Caco-2 monolayer or CEP170B KO/CAMSAP3-GFP knock-in double engineered Caco-2 monolayer. Left panels: basal plane; middle panels: apical plane; right panels: side view. Data information: Scale bars: 5 μmG Caco-2 cells were cultured in Matrigel for 5 days, and subsequently treated with Cholera toxin for 12 hours. Phase-contrast images of thus formed control or CEP170B KO Caco-2 cysts were shown. H, I Representative images and quantification of four categories of lumen formation for experiments shown in (G). n = 100 cysts from three technical replicates. J Quantification of the diameter of control and CEP170B KO Caco-2 cysts. n = 30 cysts from three technical replicates. Data information: Data represents the mean ± SD. ***, P < 0.001; two-tailed t test. Scale bars: 25 μm
Figure 5A, TIRF images of live KIF2A-GFP or KIF2C-GFP knock-in HeLa cells transiently transfected with mCherry-KANK1 (A) Insets show enlargements of boxed areas. Data information: Scale bars: 5 μm.F Immunofluorescence staining of CAMSAP2 (green) and α-tubulin (red) in control or indicated KO HeLa cell lines. Insets show enlargements of boxed areas. G Averaged intensity distributions for CAMSAP2 in the indicated cell lines for the experiments shown in (F). Distance from the cell edge is shown on the horizontal axis. n = 38-40 cells from three technical replicates. H Quantification of the length of CAMSAP2 stretches for the experiments shown in (F). n = 11-14 cells from three technical replicates. Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test. Scale bars: 5 μm.K, L Averaged intensity distributions for KIF2A (K) or CEP170B (L) in the indicated cell lines for the experiments shown in (I, J). Distance from the cell edge is shown on the horizontal axis. n = 20-38 cells from three technical replicates. Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test.M, N Quantification of intensities of KIF2A at the cell edge (M) or CEP170B along cortical MT(N) in the indicated cell lines for the experiments shown in (I, J). n = 22-26 cells from three technical replicates in (M); n = 16 cells from three technical replicates in (N). Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test.
Figure 6A Images and corresponding kymographs of GFP-tagged full length CEP170B tracking or blocking dynamic MT minus ends at different concentrations. B, C Quantification of MT minus-end growth rate (B) or plus-end depolymerization rate (C) in the presence of GFP-CEP170B at indicated concentrations. n = 20-30 MTs from 3 independent experiments. N.D., not detected. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.J Quantification of MT minus-end growth rate in the presence of indicated CEP170B fragments at indicated concentrations. n = 20-43 MTs from 3 independent experiments. N.D., not detected. Data information: Data represent mean ± SD.L Images and corresponding kymographs showing the behavior of indicated GFP-tagged CEP170B fragments on dynamic MTs at 10 nM. M Quantification of intensity of indicated CEP170B fragments along MT lattice at 10 nM. n = 16 MTs from 2 independent experiments. N.D., not detected. N, O Quantification of MT plus-end depolymerization rate (N) or minus-end growth rate (O) in the presence of indicated CEP170B fragments at 10 nM. n = 20-30 MTs from 2 independent experiments. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.
Figure 7A Images and corresponding kymographs showing that KIF2A-SNAP labelled with Alexa Fluor 647 (shown here as green) tracks growing MT minus ends at 0.5-2.5 nM but induces depolymerization from minus-end at 5 nM. Quantification of MT minus-end (B) or plus-end (C) catastrophe frequency in the presence of KIF2A-SNAP at indicated concentrations. N.A., not available. n = 20-39 MTs from 2-3 independent experiments. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.J-L Images and corresponding kymographs showing the behaviors of 30 nM TagBFP-CAMSAP2 alone, 3 nM GFP-CEP170B alone, 5 nM KIF2A-SNAP alone, 30 nM TagBFP-CAMSAP3 alone (J), 30 nM TagBFP-CAMSAP2 in combination with 3 nM GFP-CEP170B (K), or 30 nM TagBFP-CAMSAP2 in combination with 5 nM KIF2A-SNAP (L) on dynamic MTs. Data information: Data represent mean ± SD.M Quantification of MT minus-end depolymerization rate in the presence of GFP- CEP170B and KIF2A-SNAP at indicated concentrations. n = 22-35 MTs from 2-3 independent experiments. Data information: Data represent mean ± SD.P, Q Kymographs showing that 5 nM KIF2A-SNAP in complex with 3 nM but not 1 nM GFP-CEP170B could depolymerize preassembled MT stretches decorated by 30 nM TagBFP-CAMSAP2 (P) or TagBFP-CAMSAP3 (Q) in the flow-in experiments. Red arrows indicate time points of flow-in. Data information: Scale bars: horizontal, 2 μm; vertical, 1 min.
Figure 1C, D) Low-angle Pt/C shadowing of Mis12:Ndc80 (panel C) and Mis12:Ndc80Δloop (Δ431-463) (panel D) complexes. The Mis12 complex appears as a 20 nm rod-like extension and marks the SPC24:SPC25 side of the Ndc80 complex.E) Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) profiles of fluorescently labelled. Ndc80 and Ndc80Δloop. Calculated and (theoretical) masses are indicated.F) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes on taxol-stabilized microtubules that were attached to a passivated glass surface. Kymographs show Ndc80 complexes at a concentration of 0.2 nM with (FL, blue) or without (ΔL, orange) the loop. Scale bars: vertical (5 µm), horizontal (5 s). G) Quantification of Ndc80 residence times for data as in panel F. Solid line represents a single exponential fit.J) Typical fields of view showing decoration of taxol-stabilized microtubules (cyan) incubated with full-length or loopless Ndc80 (yellow) at the indicated concentration. Images show an average projection of 200 frames. The contrast between individual fluorescent channels (inverted grayscale) was fixed. Auto-contrast was used for the composite images to highlight the differences in the uniformity of Ndc80 decoration.
Figure 2TMR-labelled Ndc80 trimers with (FL, blue) or without (ΔL, orange) the loop were added to taxol-stabilized microtubules to measure their residence time (panel C) and one-dimensional diffusion (panel D). Ndc80 trimers with and without the loop are shown. Scale bars: vertical (1000 s), horizontal (5 µm).F) Kymographs of full-length and loopless Ndc80 trimers (10 pM) that reside on dynamically growing and shortening microtubules. Trimers remain bound to the ends of shortening microtubules. Scale bars: vertical (100 s), horizontal (5 µm). G) Distribution of initial brightnessH) Fraction of Ndc80 trimers that switches from lateral microtubule binding to tracking shortening ends. Data from four repeats (total 140 events) for full-length Ndc80 trimers (blue), and two repeats (total 158 events) for loopless Ndc80 trimers (orange). Horizontal lines indicate average values.I) End-tracking speed of full-length (blue) and loopless (orange) Ndc80 trimers that follow shortening microtubule ends. Compared to Ndc80-free shortening ends in the same field of views (gray). Horizontal lines indicate median values.
Figure 3B) Correlations of stall duration and stall force. Each datapoint represents a single stall event. Filled squares: stalls resulting in microtubule rescue. Open squares: stalls resulting in bead detachment from the microtubule.
Figure 4B) Immunoblot of NDC80 levels following depletion of the Ndc80 complex by RNAi.C) Quantification of the time that cells spent in mitosis following various treatments. Each dot represents a cell and the red lines indicate median values. Nocodazole was added 17 hours after electroporation and 1 hour before microscopy. A minimum of 30 cells were analysed for each condition.
Figure 5A) Schematic of a cold-shock assay following an electroporation experiment. Immunofluorescence images showing the attachment status of kinetochores to microtubules in cells electroporated with recombinant Ndc80-wt, Ndc80-∆L, or Ndc80-M5 complexes. The number of cells with multipolar spindles and the total number of analysed cells are shown. Some signal from the tubulin channel is visible in the CENP-C channel. Scale bar: 5 µm.B) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes (0.6 nM) added to trimeric Ndc80TMR (10 pM) on fluorescent taxol-stabilized microtubules that were attached to a passivated glass surface. Typical kymographs showing virtually motionless Ndc80 trimers (magenta) and transiently binding Ndc80 monomers (yellow). Wild-type (wt) monomers associate with wt trimers (left), but not with M5 trimers (right). Scale bars: vertical (100 s), horizontal (5 µm).C) Quantification of the intensity of the monomeric Ndc80 associating with microtubule-bound trimeric Ndc80. A threshold for binding was set at an intensity equivalent to one Alexa488 copy. Intensities well above 1 (yellow) could thus reflects multiple monomers binding simultaneously.
Figure 6A) Representation of the peptides used to raise AB-849 and AB-850 and immunoblots showing their recognition of wild-type, M5 and loopless Ndc80 complexes. 9G3 is a commercially available monoclonal antibody raised against NDC8056-642, later shown to recognize NDC80200-215. Asterisk shows the non-specific recognition of another protein, presumably NUF2.B) Diffusion of full-length and loopless Ndc80 trimers in absence and presence of AB-849. The primary rabbit polyclonal AB-849 was detected using a Alexa650-labelled anti-rabbit secondary IgG antibody. Scale bars: vertical (100 s), horizontal (5 µm).D) Summary showing diffusion coefficients (µm2/minute) that follow from the data shown in panel C. Mean values, SEM, and number of diffusion traces (n) are indicated. Statistically significant differences were determined using a two-tailed t-test. P-values: no AB, FL vs ΔL: 0.0180 (*); ΔL, no AB vs AB-849: 0.0108 (*); M5, no AB vs AB-849: 0.0458 (*); ΔL, AB849 vs AB-850: 0.0156 (*); AB-850, ΔL vs FL: 0.0461 (*); AB-850, ΔL vs M5: 0.0494 (*).
Figure 7Experimental workflow to investigate the time that cells, electroporated with recombinant Ndc80 complexes, spent in mitosis in the presence and absence of mitotic kinases inhibitors. Every dot represents a cell and red lines indicate median values. At least 30 cells were analysed for each condition.
Figure 8A) Effects of M13 and M14 on the time spent in mitosis following the experimental setup outlined in Figure 4. All other datapoints are also shown in Figure 4E. Colors indicate whether cells divided normally, sometimes with delayed chromosome congression (green), or showed long arrests followed by a catastrophic division (orange). Each dot represents a cell and the red lines indicate median values. A minimum of 30 cells were analysed for each condition.
Figure 1Proteoliposomes containing human ADP/ATP carrier were loaded with either 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) or 2.50 mM (dark blue traces) ATP (ATPint), and transport was initiated by the externally added radiolabeled [33P]-ATP (ATPext) at either 0.50 μM (filled circles), 0.67 μM (filled squares), 1.00 μM (filled upward triangles), 1.50 μM (filled downward triangles), 2.50 μM (open circles), 5.00 μM (open squares) or 15.0 μM (open upward triangles). A schematic representation of the transport assays is shown. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for the assays with 0.10, 0.25, 0.5 and 1.0 mM of internal ATP, and one biological repeat (based on the average of three technical repeats) for the 2.50 mM ATP internal assay.
Figure 2Initial rates were estimated by fitting the data with the model in Equation 1. A. Michaelis-Menten plots of 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) and 2.50 mM (dark blue traces) internally-loaded ATP. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.Initial rates were estimated by fitting the data with the model in Equation 1. B. Lineweaver-Burk analysis of [33P]-ATP/ATP homo-exchange (same color scheme as A). Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.Initial rates were estimated by fitting the data with the model in Equation 1. C. Km plotted against Vmax for the various substrate gradients. The kinetic parameters were determined by fitting of the Michaelis-Menten curves through iteration. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.
Figure 1A HERC3 increases the protein levels of active YAP, p-YAP (S381), YAP and TAZ. active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 cells stably expressing HERC3-WT or HERC3-CA. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.B HERC3 reverses the reduction in the active YAP and TAZ levels induced by serum starvation. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were cultured in medium with or without fetal bovine serum (FBS) for 1 h. Cells were harvested for Western blotting analysis with appropriate antibodies as indicated. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.C Depletion of HERC3 decreases the steady-state levels of YAP/TAZ. Levels of active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 and SUM159 cells stably expressing shCtrl (Control shRNA), sh-1 and sh-2 (two shRNAs against HERC3).E Depletion of HERC3 has no effects on mRNA levels of YAP/TAZ. Total mRNA levels of YAP/TAZ in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to YAP/TAZ. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. n.s., not significant.G Depletion of HERC3 attenuates YAP- or TAZ-induced 8xGTIIC-Luc reporter activity. HEK293T cells were transfected with expression plasmids for YAP or TAZ, shHERC3 or shCtrl, and reporter plasmids 8xGTIIC-Luc and Renilla-Luc as indicated. Relative luciferase activity was measured as described in the text. Western blots showing expression of indicated proteins were shown. H Depletion of HERC3 attenuates YAP- or TAZ-induced CTGF-Luc reporter activity. Cell transfection and luciferase assay were carried out as described in Panel G. Western blots showing expression of indicated proteins were shown. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01.I Depletion of HERC3 attenuates transcription of YAP/TAZ target genes. Total mRNA levels of CTGF and ANKRD1 in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to the indicated target gene. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.
Figure 2A HERC3 and HERC3-CA block β-TrCP-mediated degradation of TAZ. HEK293T cells were transfected with expression plasmids for HA-β-TrCP, Myc-HERC3 or Myc-HERC3-CA, FLAG-TAZ or FLAG-GFP as indicated. Cells were harvested 24 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.D HERC3 and HERC3-CA extend the half-life of TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 4, 8 or 12 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).F Depletion of HERC3 decreases the half-life of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 2, 4 or 6 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).G Depletion of β-TrCP reverses shHERC3-induced degradation of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 were transfected with siCtrl or siβ-TrCP as indicated. Cells were harvested 48 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.
Figure 3A HERC3 interacts with β-TrCP, but not YAP or TAZ. HEK293T cells were transfected with expression plasmids for FLAG-HERC3, HA-YAP or HA-TAZ or HA-β-TrCP. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA and FLAG antibodies. B HERC3 interacts with endogenous β-TrCP. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were harvested and subjected to IP with IgG or HA antibodies. Protein levels were detected by Western blotting with β-TRCP, HA and β-Actin antibodies. CH HERC3 blocks the interaction between β-TrCP and YAP. HEK293T cells were transfected with expression plasmids for FLAG-YAP, HA-β-TRCP, Myc-HERC3 and Myc-HERC3-CA as indicated. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA, FLAG and Myc antibodies.L HERC3 displaces TAZ on β-TRCP. Increasing concentrations of purified HERC3 or HERC3-CA proteins were added to the β-TrCP and TAZ complex, and followed by Western blotting analysis with indicated antibodies. The competitive binding assay in vitro was described in materials and methods.
Figure 4A Depletion of HERC3 reduces cell proliferation. Control or HERC3-deficient MDA-MB-231 were analyzed for indicated days by CCK8 assay as described in the materials and methods. Data are shown as mean ± SEM; n = 3 biological replicates.C Depletion of HERC3 inhibits colony formation in MDA-MB-231 cells. Control or HERC3-deficient MDA-MB-231 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J. D Depletion of HERC3 inhibits colony formation in SUM159 cells. Control or HERC3-deficient SUM159 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J.Depletion of HERC3 attenuates cell migration. Control or HERC3-deficient MDA-MB-231 cells (upper panel) and SUM159 cells (lower panel) were analyzed by transwell migration assay. E, migrated were fixed in 4% PFA and stained with 0.1% crystal violet and photographed. data quantification of migrated MDA-MB-231 cells (left panel) and SUM159 (right panel) cells counts by Image J.H RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on colony formation. Ectopic expression of RNAi-resistant HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to colony formation assay as described in Panel C.I, J RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on cell migration. I, ectopic expression of resistant variants of HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to transwell migration assay. Scale bar, 100 μm. J, graphic representation of quantitated cell migration in Panel I.