(A) Two-color TIRF imaging of a COS cell coexpressing KHC(1-560)-3xmCit and EB3-mCherry. After imaging, a SD Map of KHC(1-560)-3xmCit motility events (KHC SD Map) and an average map of the EB3-mCherry fluorescence (EB3 AVG Map) were created and merged (Merged). Yellow line, edge of cell. Scale bar, 4 µm. In the boxed region, multiple KHC(1-560)-3xmCit motility events can be observed to occur adjacent to an EB3-mCherry-marked microtubule. (B) The KHC(1-560)-3xmCit motility events in the boxed region of (A) are depicted in the kymograph (left) and schematic representation (right). (C) Movement of EB3-mCherry in the boxed region of (A) is depicted in the kymograph (left) and schematic representation (right). Vertical scale bar, 0.5 s. Horizontal scale bar, 2 µm. (D) Schematic summary of the displacement over time of multiple KHC(1-560)-3xmCit motors and EB3-mCherry plus-ends in the cell in (A).

(A) Schematic illustration of the domain structure of full length KIF17 (top) and the truncated, constitutively active version KIF17(1-490)-3xmCit (bottom). (B) Two-color TIRF imaging of KIF17(1-490)-3xmCit motors and mCherry-tubulin in live COS cells. An SD Map of the KIF17(1-490)-3xmCit motility events was created from the time series and compared to the average map of mCherry-tubulin fluorescence. Scale bar, 4 µm. (C) Two-color TIRF imaging of KIF17(1-490)-3xmCit motors and EB3-mCherry in live COS cells. An SD Map of the KHC(1-560)-3xmCit motility events was created from the time series and compared to the average map of EB3-mCherry fluorescence. Arrow, microtubule utilized by KIF17(1-490)-3xmCit but not EB3-mCherry. Yellow line, edge of cell. Scale bar, 4 µm. (D) Kymograph of the boxed region in (C) showing multiple KIF17(1-490)-3xmCit motility events along a single EB3-marked microtubule. Horizontal scale bar, 2 µm. Vertical scale bar, 0.5 s. (E) Schematic diagram of the KIF17 (green) and EB3 (red) tracks in (D).

(A) After live cell imaging of Golgi-to-plasma membrane transport of VSVG-GFP, the cells were fixed and stained for retrospective immunofluorescence. An SD Map of the VSVG-GFP vesicle tracks was compared to the image of acetylated microtubules in the same cell. Scale bar, 10 µm. (B) Kymograph of the yellow boxed region in (A). Arrowheads show four VSVG-GFP-containing vesicles being transported along an acetylated microtubule. Horizontal scale bar, 4 µm. Vertical scale bar, 2 s. (C) Schematic diagram summarizing VSVG-GFP-containing vesicles (yellow, white, and cyan traces) undergoing transport on acetylated microtubule (red trace) in (B).

(A) Schematic illustration of the domain structure of the KHC subunit of Kinesin-1 (left) and the constitutively active, truncated version KHC(1-560)-3xmCit (right). (B) Phase contrast image of a COS cell expressing KHC(1-560)-3xmCit. Scale bar, 10 µm. TIRF microscopy (C) was performed on a small region in the periphery of the cell (e.g., boxed area in B). From the image series, a SD Map (D) was created that provides a visual readout of all of the Kinesin-1 motility events. Scale bar, 3 µm. (E) TIRF image of a COS cell expressing mCherry-tubulin. Scale bar, 3 µm. (F) Two-color TIRF imaging. (1) COS cells coexpressing (2) KHC(1-560)-3xmCit and mCherry-tubulin were (3) imaged in the TIRF microscope. A SD Map of KHC(1-560)-3xmCit motility (KHC SD Map) and an average map of mCherry-tubulin (mChe-tub AVG Map) were created. The two images were (4) aligned and merged (Merged). Scale bar, 4 µm. (G) Retrospective immunofluorescence. (1) COS cells expressing (2) KHC(1-560)-3xmCit were (3) imaged by TIRF microscopy. The cells were (4) fixed immediately, stained with antibodies to total tubulin, and then (5) the same cell was imaged again in the TIRF microscope. A SD Map of the KHC(1-560)-3xmCit motility events was (6) aligned with the total tubulin image to create a merged image (Merged). Scale bar, 4 µm. Yellow lines in (D–G) indicate the edge of the cell.

Single KHC(1-560)-3xmCit motors in live COS cells were imaged by TIRF microscopy. The cells were fixed and stained for retrospective immunofluorescence. SD Maps of the KHC(1-560)-3xmCit motility events were created from the time series (far left images) and compared to the fixed images of (A) total tubulin (middle panel) and acetylated tubulin (right panel), (B) total tubulin (middle panel) and detyrosinated tubulin (right panel), or (C) total tubulin (middle panel) and polyglutamylated tubulins (right panel). Scale bar, 4 µm. The far right panels indicate schematic representations of the overlap between the Kinesin-1 motility events in the SD Map (green lines) and the modified microtubules (red lines).

(A) Schematic illustration of the domain structure of full length KIF1A (top) and the truncated, constitutively active version KIF1A(1-393)-3xmCit (bottom). (B) Two-color TIRF imaging of KIF1A(1-393)-3xmCit motors and mCherry-tubulin in live COS cells. An SD Map of the KIF1A(1-393)-3xmCit motility events was created from the time series and compared to the average map of mCherry-tubulin fluorescence. Scale bar, 4 µm. (C) Two-color TIRF imaging of KIF1A(1-393)-3xmCit motors and EB3-mCherry in live COS cells. An SD Map of the KHC(1-560)-3xmCIt motility events was created from the time series and compared to the average map of EB3-mCherry fluorescence. Yellow line, edge of cell. Scale bar, 4 µm. (D) Kymograph of the boxed region in (C) showing multiple KIF1A(1-393)-3xmCit motility events along a single EB3-marked microtubule. Horizontal scale bar, 2 µm. Vertical scale bar, 0.3 s. (E) Schematic diagram of the KIF1A (green) and EB3 (red) tracks in (D).

(A–E) Coexpression of a DN KIF17 construct inhibits Kv1.5-GFP channel movement in HL-1 cells. HL-1 cells expressing (A) Kv1.5-GFP, (B) Kv1.5-GFP+mCherry-KIF17-DN, or (C) Kv1.5-GFP+mCherry-KHC-DN were imaged by live cell microscopy. SD Maps (top panels) and schematic drawings (red lines in bottom panels) were generated to visualize Kv1.5-GFP vesicle tracks over time. Yellow line, edge of cell. Black line, nucleus. The average (D) distance traveled and (E) net velocity (includes pauses) of Kv1.5-GFP vesicles under each condition was calculated. Data are mean±SE. Kv1.5-GFP, n = 13 cells; Kv1.5-GFP+KIF17-DN, n = 17 cells; Kv1.5-GFP+KHC-DN, n = 13 cells. *p<0.05, **p<0.001 as compared to Kv1.5-GFP alone. (F,G) Retrospective immunofluorescence of HL-1 cells expressing Kv1.5-GFP. After live cell imaging, an SD Map of the Kv1.5 vesicle tracks was made and compared to the fixed cell images of (F) total microtubules or (G) acetylated microtubules in the same cells. Arrow in (F) indicates microtubule that Kv1.5 vesicle moved on. Yellow line in (G), edge of cell. White line in (G), nucleus. All scale bars, 10 µm.