Figure 5.
Tks5 is associated with MAP1B and microtubules. (A) Coimmunoprecipitation of GFP-Tks5 with FLAG-MAP1B-LC1 in latrunculin A- and nocodazole-treated 293T cells. (B) PLA between MAP1B-LC1 and GFP-Tks5 in latrunculin A- and nocodazole-treated MDA-MB-231 cells. Left: Representative immunofluorescence images. Right: Quantification of the PLA dots. n = 15 cells from three independent experiments. (C) Immunofluorescence staining of cortactin and α-tubulin together with stably expressing GFP-Tks5 in PFA- and methanol-fixed MDA/GFP-Tks5 cells, respectively. (D) Left: Live-cell imaging of stably expressing GFP-Tks5 in MDA/GFP-Tks5 cells. The white arrowheads indicate microtubule-like structures of GFP-Tks5. Right: Sequential images of GFP-Tks5 that rides on a trafficking vesicle-like structure. The yellow arrowheads indicate a stable invadopodium-like structure. The white arrows indicate the movement of a GFP-Tks5 vesicle to the invadopodium-like structure. (E) Left: Proximity ligation assay of α-tubulin with stably expressing GFP-Tks5, cortactin-GFP, or Bet1-GFP. Right: Quantification of PLA dots. Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. Parental MDA-MB-231, n = 32 cells; MDA/GFP-Tks5, MDA/cortactin (CTTN)-GFP and MDA/Bet1-GFP, n = 34 cells from three independent experiments. (F) PLA between endogenous MAP1B-LC1 and stable GFP-Tks5 in MDA/GFP-Tks5 cells. (G) Top: The domain structure of Tks5 and its deletion and point mutants, and the summary of the mutants’ localization and microtubule bundling. Ser-rich and CC stand for serine-rich and coiled-coil domains, respectively. Bottom: Quantification of the cells with bundled microtubules and GFP-Tks5. The values indicate the mean ± SEM (n = 3–9 experiments). (H and I) Immunofluorescence staining of α-tubulin together with transiently expressed GFP-Tks5 mutants (H) and FLAG-MAP1B-LC1 (I). (B, E, and G) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (B–F and H) Scale bars, 10 μm. Source data are available for this figure: SourceData F5.

Tks5 is associated with MAP1B and microtubules. (A) Coimmunoprecipitation of GFP-Tks5 with FLAG-MAP1B-LC1 in latrunculin A- and nocodazole-treated 293T cells. (B) PLA between MAP1B-LC1 and GFP-Tks5 in latrunculin A- and nocodazole-treated MDA-MB-231 cells. Left: Representative immunofluorescence images. Right: Quantification of the PLA dots. n = 15 cells from three independent experiments. (C) Immunofluorescence staining of cortactin and α-tubulin together with stably expressing GFP-Tks5 in PFA- and methanol-fixed MDA/GFP-Tks5 cells, respectively. (D) Left: Live-cell imaging of stably expressing GFP-Tks5 in MDA/GFP-Tks5 cells. The white arrowheads indicate microtubule-like structures of GFP-Tks5. Right: Sequential images of GFP-Tks5 that rides on a trafficking vesicle-like structure. The yellow arrowheads indicate a stable invadopodium-like structure. The white arrows indicate the movement of a GFP-Tks5 vesicle to the invadopodium-like structure. (E) Left: Proximity ligation assay of α-tubulin with stably expressing GFP-Tks5, cortactin-GFP, or Bet1-GFP. Right: Quantification of PLA dots. Data are presented as boxes containing the first and third quartiles. The whiskers indicate the maxima and minima after outlier removal. Parental MDA-MB-231, n = 32 cells; MDA/GFP-Tks5, MDA/cortactin (CTTN)-GFP and MDA/Bet1-GFP, n = 34 cells from three independent experiments. (F) PLA between endogenous MAP1B-LC1 and stable GFP-Tks5 in MDA/GFP-Tks5 cells. (G) Top: The domain structure of Tks5 and its deletion and point mutants, and the summary of the mutants’ localization and microtubule bundling. Ser-rich and CC stand for serine-rich and coiled-coil domains, respectively. Bottom: Quantification of the cells with bundled microtubules and GFP-Tks5. The values indicate the mean ± SEM (n = 3–9 experiments). (H and I) Immunofluorescence staining of α-tubulin together with transiently expressed GFP-Tks5 mutants (H) and FLAG-MAP1B-LC1 (I). (B, E, and G) P values were determined using one-way ANOVA with Dunnett’s multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant. (B–F and H) Scale bars, 10 μm. Source data are available for this figure: SourceData F5.

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