Figure 2.
Nek7 Knockdown Accelerates Microtubule Growth
(A) Diagram of the Cre-dependent constructs expressing mCherry and shRNA. The plasmids were co-transfected with EB3-YFP, and mNek7 for the rescue, in primary cortical cultures from Nkx2-1Cre mice at 4 DIV, and axons were recorded at 7 DIV.
(B–D) Confocal Z projection frames from control shRNA (B), Nek7 shRNA (C), and rescue with mNek7 (D) Nkx2-1Cre growth cones expressing mCherry (red, before the time-lapse) and EB3-YFP (gray, time-lapse). The path of EB3-YFP comets is tracked with a red line. Scale bar represents 2 μm.
(E–G) Kymographs of EB3 comets in control (E), Nek7 shRNA (F), and rescue (G) growth cones showing their existence time as a function of distance.
(H and I) Average speed (H) and speed distribution of EB3 comets (I) comparing control (n = 31 growth cones), Nek7-depleted cells (n = 35 growth cones), and mNek7 rescue cells (n = 35 growth cones) from three independent cultures. All growth cones with EB3 comets were quantified. One-way ANOVA (H) and χ2 test (I). ∗p < 0.05 and ∗∗p < 0.01. Data are represented as mean ± SEM (H) or total cell percentage (I).
See also Figures S5–S7 and Videos S1, S2, and S3.