Fig. 4: hKIF21B variants induce abnormal migration through enhanced KIF21B motor activity. | Nature Communications

Fig. 4: hKIF21B variants induce abnormal migration through enhanced KIF21B motor activity.

From: Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity

Fig. 4

a, c, i Coronal sections of E18.5 cortices, 4 days after IUE with the indicated NeuroD-IRES-GFP constructs. GFP-positive electroporated cells are depicted in green. Nuclei are stained with DAPI. b, d, j Percentage (means ± S.E.M.) of electroporated cells in upper (Up CP) and lower (Lo CP) cortical plate, intermediate (IZ) and subventricular zone (SVZ), showing the effect of increasing (b) amount or (d) activity of hKIF21B and (j) contribution of the processive activity to the phenotype. Data were analyzed by two-way ANOVA (Bonferroni’s multiple comparisons test). Number of embryos analyzed: b Empty vector and WT 2 µg/µL, n = 3; WT 1 and 1.5 µg/µL, n = 4; d n = 3 for each condition; j Empty vector, n = 9; WT, n = 6; p.Gln313Lys, n = 8; p.Ile678Leu, n = 5; p.Ala1001Thr, n = 8; p.Gln313LysΔATP, n = 5; p.Ile678LeuΔATP, n = 8; p.Ala1001ThrΔATP, n = 6. e Immunolabeling of ST cells transfected with indicated HA-tagged hKIF21B constructs showing impaired localization of p.Ile678Leu variant. Histogram (means ± S.E.M.) represents the percentage of cells with a diffuse versus an impaired localization of the HA-tagged proteins. Data were analyzed by two-way ANOVA (Bonferroni’s multiple comparisons test). Number of cells analyzed: WT, n = 467; p.Gln313Lys, n = 429; p.Ile678Leu, n = 540; p.Ala1001Thr, n = 387; from three independent experiments. f Immunolabeling of ST cells transfected with the indicated Myc- and HA-tagged hKIF21B constructs showing that the p.Ile678Leu variant alters the localization of the WT protein. g, h Live imaging of Cos7 cells transfected with the indicated GFP-tagged hKIF21B constructs. Histograms (means ± S.E.M.) represent (g) velocities distribution and (h) mean velocities. 20–32 cells from 3–5 independent experiments were analyzed by (g) two-way ANOVA or h one-way ANOVA (Bonferroni’s multiple comparisons test). Total number of particles analyzed: WT, n = 203; p.Gln313Lys, n = 182; p.Ile678Leu, n = 195; p.Ala1001Thr, n = 237. ns non-significant; *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Scale bars: (a, c, i) 50 μm; (e, f) 20 µm. k Model: WT KIF21B switches between an autoinhibition and an active (purple arrows) state. hKIF21B variants (marked by a star) conformation favors the active state. KIF21B hyper-motility (pink arrow) leads to migration defects. Source data are provided in the Source Data file.

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