Nesprin-2 needs to bind to both dynein and kinesin-1 to drive nuclear migration in CGCs. (A) Nissl staining of the cerebellum lobe IV from WT and mutant mice at P9 and P12. Post-mitotic CGCs migrate from the external granule layer (EGL), through the molecular layer (ML), eventually reaching the inner granule layer (IGL). (B) Immunofluorescence of Ki-67 (green) and p27-Kip1 (magenta) in the EGL from WT and Nesprin-2 mutant mice. (C) Quantitative analysis of the thickness of the outer and inner EGL of the cerebellar lobe IV (left) and lobe V (right). n = 5 (WT) and 7 (mutant) littermate mice from three independent experiments. Unpaired t test was used to compare WT and mutant. (D) Representative time-lapse sequences of CGCs in reaggregate culture from WT and mutant mice. Cells were transfected with EGFP and DsRed-NLS. (E) Quantitative analysis of nuclear net displacement. n = 36 (WT), 38 (Nesp2 mutant), 45 (Mutant + SR48), 36 (Mutant + SR48-5A-AAVV), and 41 (Mutant + SR48-LEAA) cells from four to five independent experiments per group. Unpaired t test. (F) Representative time-lapse sequences of CGCs from Nesprin-2 mutant mice, which were transfected with DsRed-NLS together with mNG-SR48-KASH, mNG-SR48-5A-AAVV-KASH, or SR48-LEAA-KASH. (G) Trajectories of nuclear centroid in cells that are shown in D and F. Bars show mean ± SEM. Scale bars, 50 μm in A and B; 5 μm in D and F. Related to Video 3.