Figure 6. Binding of the Kinesin Motor Domain to Microtubules is Inhibited by Ca⁺⁺-dependent Interactions with Miro.

(A) Immunoprecipitations in the presence or absence of Ca⁺⁺ of KHC-ECFP (with anti-GFP) from lysates of HEK cells, transfected as indicated. In the presence of milton, neither Ca⁺⁺ nor the EF-hands are required for Miro to precipitate with KHC, but in the absence of milton, both are needed (for Ca⁺⁺, compare third and fourth lanes; for the EF-hands compare fourth and eighth lanes). Low levels of coprecipitation in their absence may be mediated by endogenous milton. (B, C) Illustration of the two modes of association of Miro and KHC, a Ca⁺⁺-independent association via milton (B) and a Ca⁺⁺-dependent, milton-independent association (C). Blue boxes represent EF-hands; the black region represents the milton binding site in KHC. (D) Immunoprecipitations with anti-Myc from cells expressing Myc-tagged full length (FL, left two panels) or truncated KHC 1-682 (682, right two panels), and T7-tagged DMiroΔTM, but not milton. (E, F) Illustration of the persistence of the Ca⁺⁺-induced association of Miro and KHC, despite deletions of the KHC tail and TM domain of Miro. (G) Immunoprecipitations with anti-Myc from cells transfected with Miro-Myc and the kinesin motor domain (YFP-tagged KIF5C1-335). (H) Illustration of the dependence of motor domain/Miro interactions on Ca⁺⁺ and the EF hands of Miro. (I) Microtubule cosedimentations were performed in either 0 or 2 mM Ca⁺⁺ buffer and with Taxol-stabilized microtubules and lysates of the indicated transfected HEK cells. Equal volumes of supernatants (S) and microtubule pellets (P) were loaded and probed separately for KIF5C(1-335)-YFP, Miro-Myc or tubulin. (J) Illustration of the Ca⁺⁺-dependent, EF-hand-dependent shift of the motor domain from microtubules to Miro, as in (G-I). (K) Ca⁺⁺ dependence of the cosedimentation of the motor domain KIF5C(1-335)-YFP with microtubules, as in (I), in the presence of Miro-Myc, and the indicated free Ca⁺⁺ concentrations. The level of KIF5C(1-335)-YFP in each fraction was determined with anti-GFP. (L) Ca⁺⁺ dependence of the binding of Miro-Myc to KIF5C(1-335)-YFP, by precipitation from lysates of transfected HEK cells with anti-Myc and probing with anti-GFP (KIF5C(1-335)-YFP). (M) Quantification of Ca⁺⁺ dependence of motor-domain cosedimentation with microtubules (as in K) and coprecipitation with Miro (as in L). The percent bound to microtubules was calculated as the intensity of the band in the pellet divided by the summed intensity of bands in both pellet and supernatant, and was defined as 100% in 0 Ca⁺⁺. n=3 transfections. The percent of maximal binding to Miro was defined as the intensity of the precipitated band normalized to its input and set at 100% in 2 mM Ca⁺⁺. n=3 transfections. For each section, input lanes contained one fifth of the amounts used for immunoprecipitation, and anti-tubulin was used as a loading control.