Figure 6.
Figure 6. KIF1C recruits Hook3 to the cell periphery. (A) 293T KIF1CKO cells (KO) were infected with viral particles encoding MSCV-driven KIF1C-tagRFP-3xFLAG or KIF1CΔ794-807-tagRFP-3xFLAG plasmids. Immunoblots were performed with the indicated antibodies. Low and high exposures with the KIF1C antibody are shown. β-Actin provided a loading control. 293T cells transfected with CRISPR-Cas9 (CTRL) were used as control cells. (B) Confocal microscopy of KIF1C and Hook3 localization in stable 293T cell lines expressing KIF1C-tagRFP-3xFLAG or KIF1CΔ794-807-tagRFP-3xFLAG. Cells were grown on glass coverslips, fixed, and stained for endogenous Hook3 (Endo-Hook3). The tagRFP and Hook3 signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across individual z-sections is shown to the right of the images. (C) The mean normalized Hook3 intensity within KIF1C foci for KIF1C-tagRFP-3xFLAG (n = 25) or KIF1CΔ794-807-tagRFP-3xFLAG (n = 24). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in the corresponding regions in maximum projection images. Box plots represent the maximum and minimum values. Statistical significance was calculated with an unpaired t test. ****, P < 0.0001. Representative data from three independent experiments is shown. (D) Confocal microscopy of KIF1C and Hook3 in U2OS cells. Cells were grown on glass coverslips and transiently cotransfected with the indicated sfGFP-tagged Hook3 (full-length Hook3, Hook3NT [aa 1–552], and Hook3CT [aa 553–718]) or control sfGFP constructs, and KIF1C-V5. 24 h after transfections, cells were fixed and stained with V5-specific antibody. The V5 and sfGFP signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across an individual z-section is shown to the right of the images. (E) The mean normalized Hook3 intensity within KIF1C foci for cells transfected with different Hook3 constructs (CTRL, n = 27; Hook3, n = 28; Hook3NT, n = 33; Hook3CT, n = 29). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in these corresponding regions in maximum projection images. Box plots represent maximum and minimum values. Statistical significance was calculated with one-way ANOVA with Tukey post-test, ****, P < 0.0001. Representative data from three independent experiments is shown. ns, not significant.

KIF1C recruits Hook3 to the cell periphery. (A) 293T KIF1CKO cells (KO) were infected with viral particles encoding MSCV-driven KIF1C-tagRFP-3xFLAG or KIF1CΔ794-807-tagRFP-3xFLAG plasmids. Immunoblots were performed with the indicated antibodies. Low and high exposures with the KIF1C antibody are shown. β-Actin provided a loading control. 293T cells transfected with CRISPR-Cas9 (CTRL) were used as control cells. (B) Confocal microscopy of KIF1C and Hook3 localization in stable 293T cell lines expressing KIF1C-tagRFP-3xFLAG or KIF1CΔ794-807-tagRFP-3xFLAG. Cells were grown on glass coverslips, fixed, and stained for endogenous Hook3 (Endo-Hook3). The tagRFP and Hook3 signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across individual z-sections is shown to the right of the images. (C) The mean normalized Hook3 intensity within KIF1C foci for KIF1C-tagRFP-3xFLAG (n = 25) or KIF1CΔ794-807-tagRFP-3xFLAG (n = 24). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in the corresponding regions in maximum projection images. Box plots represent the maximum and minimum values. Statistical significance was calculated with an unpaired t test. ****, P < 0.0001. Representative data from three independent experiments is shown. (D) Confocal microscopy of KIF1C and Hook3 in U2OS cells. Cells were grown on glass coverslips and transiently cotransfected with the indicated sfGFP-tagged Hook3 (full-length Hook3, Hook3NT [aa 1–552], and Hook3CT [aa 553–718]) or control sfGFP constructs, and KIF1C-V5. 24 h after transfections, cells were fixed and stained with V5-specific antibody. The V5 and sfGFP signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across an individual z-section is shown to the right of the images. (E) The mean normalized Hook3 intensity within KIF1C foci for cells transfected with different Hook3 constructs (CTRL, n = 27; Hook3, n = 28; Hook3NT, n = 33; Hook3CT, n = 29). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in these corresponding regions in maximum projection images. Box plots represent maximum and minimum values. Statistical significance was calculated with one-way ANOVA with Tukey post-test, ****, P < 0.0001. Representative data from three independent experiments is shown. ns, not significant.

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