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. 2018 Jul 17;24(3):685–700. doi: 10.1016/j.celrep.2018.06.071

Figure 4.

Figure 4

KIF1A-Driven DCVs Transport Is Regulated by Ca2+/CaM

(A) Representative dendrites of rat hippocampal neurons (11–14 DIV) co-transfected with GFP-KIF1A_WT or GFP-KIF1A_5∗Ala (green) with NPY-RFP (red). Arrows point to co-localizing puncta. Scale bar, 5 μm.

(B) Line scans of fluorescence intensity of GFP-KIF1A and NPY-RFP channels shown in (A).

(C) Dendrites of neurons co-transfected with GFP-KIF1A_WT or GFP-KIF1A_5∗Ala (green) with mCherry-Syt4 (red). Arrows point to co-localizing puncta. Scale bar, 5 μm.

(D) Line scans of fluorescence intensity of GFP-KIF1A and mCherry-Syt4 channels shown in (C).

(E) Quantifications of GFP-KIF1A_WT and GFP-KIF1A_5∗Ala puncta in dendrites. The bars show mean ± SEM (n = 20–28 dendrites; ∗∗∗p < 0.001, t test).

(F) Ca2+ dependence of the in vitro binding of CaM to KIF1A. AP of CaM (anti-CaM beads) from lysates of cells transfected with HA-KIF1A(657-1105_WT) or HA-KIF1A(657-1105_5∗Ala). WB detection was performed using anti-HA antibody.

(G) Quantification of KIF1A binding to CaM shown in (F). The percentage of maximal binding to CaM was defined as the intensity of the co-precipitated band of KIF1A and set at 100% in 2 mM Ca2+. n = 3 experiments per condition. The bars show mean ± SEM.

(H and I) Representative kymographs showing trajectories of GFP-KIF1A and NPY-RFP vesicles in selected neurites of co-transfected neurons pre- and posttreatment with DMSO (H) or with 40 μm of bicuculline (I).

(J and K) Quantifications of trajectories of GFP-KIF1A co-localizing with NPY-RFP (see H and I) pre- and posttreatment with DMSO (J) or with bicuculline (K). The bars show mean ± SEM (n = 12 dendrites in J and n = 34 dendrites in K; ∗∗∗p < 0.001, paired t test).

(L) Intracellular calcium levels in a representative dendrite treated with bicuculline and visualized with the calcium indicator GCaMP6. Scale bar, 5 μm.

(M) GFP-KIF1A clustering in a representative dendrite treated with bicuculline. Scale bar, 5 μm.

(N–P) Quantifications of the percentage of mobile GFP-KIF1A (N), NPY-GFP vesicles (O), or mCherry-Syt4 vesicles (P) in neurons (11–14 DIV) treated with DMSO (CTR) or 10 μm of BAPTA-AM. Bars show the mean (n = 21–29 dendrites in N, n = 20–28 dendrites in O, and n = 41–50 dendrites in P; ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001, t test).