Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition
Figure 3
Deletion of CC1 Restores the Dimer State and Processive Motility
(A) Crosslinking. Immunoblot of lysates from COS cells expressing the indicated Myc-tagged KIF1A truncations treated with or without DMP. The positions of the truncated proteins in the absence (arrowheads on left) and presence (arrows on right) of DMP are indicated.
(B) Coimmunoprecipitation. Lysates of COS cells coexpressing Myc- and mCit-tagged KIF1A constructs of the same length were analyzed directly (input lysate) or after immunoprecipitation (IP) with anti-Myc or control IgG antibodies. Shown are nonadjacent lanes from the same gel.
(C and D) Photobleaching analysis. Lysates of COS cells expressing 3xmCit-tagged KIF1A or Kinesin-1 constructs were imaged by TIRF microscopy. (C) Stepwise photobleaching of representative fluorescent spots. AU, arbitrary units. (D) Distribution of the number of photobleaching steps. FL: n = 204 traces, average = 3.77 ± 0.10 steps; (1–726): n = 173 traces, average = 3.72 ± 0.10 steps; (1–491): n = 171 traces, average = 2.83 ± 0.07 steps; (1–393): n = 164 traces, average = 3.91 ± 0.10 steps; KHC(1–891): n = 159 traces, average = 4.03 ± 0.11 steps.
(E) Two-color single-molecule (SM) motility assay. Two-color TIRF imaging of COS cell lysates coexpressing KIF1A(1–393)-3xmCit + KIF1A(1–393)-3xmCherry. Representative time series showing motility of a dual-labeled/dimerized KIF1A(1–393) motor. y-axis, distance (yellow bar = 2 μm); x-axis, time (white bar = 0.3 s).