(A and B) Live-cell microtubule binding assay. COS cells expressing mCit-KIF1A were imaged, permeabilized with SLO, and treated with AMPPNP. (A) Still images. (B) Quantification of the percentage of KIF1A-mCit that changed from cytoplasmic to microtubule (MT)-associated localization (Relocation Index) over time. n = 16 cells each.
(C) Coimmunoprecipitation. Lysates of COS cells coexpressing mCit-KIF1A + Myc-KIF1A were immunoprecipitated (IP) with anti-Myc or control IgG antibodies, separated by SDS-PAGE, and immunoblotted.
(D) Crosslinking. Lysates of COS cells expressing Myc-KIF1A, mCit-KIF1A, mCit-KHC, or mCit were untreated or treated with DMP, separated by SDS-PAGE, and immunoblotted. Left, MW standards in kilodaltons.
(E and F) Motor-to-motor FRET in live COS cells.
(E) Still images of cells coexpressing mCit-KIF1A + mCFP-KIF1A (top panels) or Kinesin-1 (mCit-KHC + mCFP-KHC + HA-KLC; bottom panels).
(F) Quantification of E_AVE (30–40 cells, two to three experiments).
(G) Crosslinking. Extracts were treated with or without DMP, separated by SDS-PAGE, and immunoblotted with anti-KIF1A antibodies. Left, MW standards in kilodaltons.
(H) Sucrose gradient centrifugation. Fractions were removed from the top (lane 1), run on SDS-PAGE, and immunoblotted. Size standards: catalase (11.3 S) and bovine serum albumin (4.3 S). All data are mean ± SE.

(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).

(A and B) Live-cell microtubule binding assay. COS cells expressing mCit-tagged NC constructs were imaged during SLO permeabilization and AMPPNP treatment. (A) Representative images before and after AMPPNP treatment. Scale bar indicates 20 μm. (B) Percentage of cells whose NC constructs localize to the indicated cellular locations before and after AMPPNP treatment (averages of two to three experiments, ∼50 cells each). MT, microtubule.
(C) In vivo processive motility assays of mCit-tagged NC constructs in differentiated CAD cells. Scale bar indicates 20 μm.
(D and E) In vitro single-molecule motility assays of COS cell lysates expressing 3xmCit-tagged NC constructs. (D) Distribution of velocities. (E) Distribution of run lengths.

(A) CC prediction for MmKIF1A using windows of 14 (gray line) or 21 (blue line) amino acids (COILS, Lupas Method).
(B) Schematic of mCit-tagged FL and truncated KIF1A constructs.
(C–E) Live-cell microtubule binding assay. COS cells expressing mCit-tagged KIF1A truncations were imaged during SLO permeabilization and AMPPNP treatment. (C) Still images. Scale bar indicates 20 μm. (D) Quantification of the percentage of KIF1A-mCit that changed from cytoplasmic to microtubule (MT)-associated localization (Relocation Index) over time. (1–726), n = 17 cells; (1–491), n = 11 cells; (1–393), n = 12 cells. (E) Percentage of cells whose KIF1A truncations localize to the indicated cellular locations before and after AMPPNP treatment. (Average = three to five experiments, 50–100 cells each).
(F) Processive motility in vivo. Representative images of differentiated CAD cells expressing mCit-tagged constructs. Scale bar indicates 20 μm.
(G and H) Processive motility in vitro. Single-molecule motility assays were carried out using COS cell lysates expressing 3xmCit-tagged constructs. Individual spots were tracked, and population velocities (G) and run lengths (H) were plotted as histograms.

(A) Sequence comparison (top, ClustalW) of the NC region across species. Red, identical residues; orange, similar residues. Schematic (bottom) of NC constructs used in this study and their relation to KIF1A/Unc104 constructs used in previous studies.
(B) Crosslinking. Lysates from COS cells expressing Myc-tagged NC truncations were treated with or without DMP, separated by SDS-PAGE, and immunoblotted with anti-Myc antibodies. Arrow, reduced mobility of KIF1A(1–393) upon crosslinking.
(C and D) Photobleaching analysis. Lysates of COS cells expressing 3xmCit-tagged NC constructs were imaged by TIRF microscopy. (C) Stepwise photobleaching of representative spots. (D) Distribution of the number of photobleaching steps. (1–393), n = 171 spots, average = 3.91 ± 0.10 steps; (1–381), n = 181 spots, average = 2.40 ± 0.07 steps; (1–369), n = 171 spots, average = 2.49 ± 0.07 steps.

In vitro single-molecule motility assays of dimeric (1–393)-3xmCit or monomeric (1–369)-3xmCit KIF1A were carried out. Analysis includes motile events lasting at least 0.3 s. (A) Representative kymographs.
(B) Distribution of velocities.
(C) Distribution of run lengths.
(D and E) Comparison of motile properties of monomeric and dimeric motors in the presence of ATP or ADP. Distribution of velocities (D) or run lengths (E) for KIF1A(1–393), (1–369), or KHC(1–891) in the presence of ATP or ADP as a function of time spent in one-directional motility. Red ovals, 1D diffusion. Blue ovals, processive motility.