Figure 2.
Figure 2. Dimerized NOD485 exhibits ATP-dependent, plus end–directed motility in cells and in vitro. (A) TIRF microscopy of an interphase cell expressing NOD485CC. (B) Select frames from a TIRF time-lapse zoomed on the region in the black box in A showing a motile NOD485CC dimer (marked by arrow). (C) Kymograph of NOD485CC dimer from the black box in A walking on MTs toward the cell periphery (right side of kymograph). (D) Histogram of velocities of motile NOD485CC dimers in cells, with a mean velocity of 8.70 ± 3.61 µm/min; n = 55. (E) TIRF microscopy of NOD dimers (green) on taxol-stabilized MTs (red) using a cell lysate from NOD485CC-mCherry–expressing cells in the presence of ATP (top) or AMP-PNP (bottom). (F) Histogram of the velocities of motile NOD485CC dimers in cell lysates, with a mean velocity of 8.62 ± 2.32 µm/min; n = 47. (G) TIRF microscopy of taxol-stabilized MTs (red) incubated with Strep-tagged NOD485CC-mCherry (green) purified from S2 cells in the presence of ATP (top) or AMP-PNP (bottom). (H) Histogram of the velocities of purified Strep-tagged NOD485CC puncta, with a mean velocity of 5.79 ± 1.56 µm/min; n = 58. (I) Silver staining of SDS-PAGE gel (left) and Western blot (right) showing the purification of NOD485CC-mCherry from Drosophila S2 cells and mock purification from wild-type cell extracts. Arrows indicate the NOD bands. (J) Kymograph from a TIRF time-lapse of NOD485CC lysate (red) plus purified kinesin-1-EGFP (green) on taxol-stabilized MTs (blue). Horizontal bars: 10 µm (A); 1 µm (B, C, E, G, and J). Vertical bars: 10 s (A–C, E, and G); 20 s (J). Means ± SD.

Dimerized NOD485 exhibits ATP-dependent, plus end–directed motility in cells and in vitro. (A) TIRF microscopy of an interphase cell expressing NOD485CC. (B) Select frames from a TIRF time-lapse zoomed on the region in the black box in A showing a motile NOD485CC dimer (marked by arrow). (C) Kymograph of NOD485CC dimer from the black box in A walking on MTs toward the cell periphery (right side of kymograph). (D) Histogram of velocities of motile NOD485CC dimers in cells, with a mean velocity of 8.70 ± 3.61 µm/min; n = 55. (E) TIRF microscopy of NOD dimers (green) on taxol-stabilized MTs (red) using a cell lysate from NOD485CC-mCherry–expressing cells in the presence of ATP (top) or AMP-PNP (bottom). (F) Histogram of the velocities of motile NOD485CC dimers in cell lysates, with a mean velocity of 8.62 ± 2.32 µm/min; n = 47. (G) TIRF microscopy of taxol-stabilized MTs (red) incubated with Strep-tagged NOD485CC-mCherry (green) purified from S2 cells in the presence of ATP (top) or AMP-PNP (bottom). (H) Histogram of the velocities of purified Strep-tagged NOD485CC puncta, with a mean velocity of 5.79 ± 1.56 µm/min; n = 58. (I) Silver staining of SDS-PAGE gel (left) and Western blot (right) showing the purification of NOD485CC-mCherry from Drosophila S2 cells and mock purification from wild-type cell extracts. Arrows indicate the NOD bands. (J) Kymograph from a TIRF time-lapse of NOD485CC lysate (red) plus purified kinesin-1-EGFP (green) on taxol-stabilized MTs (blue). Horizontal bars: 10 µm (A); 1 µm (B, C, E, G, and J). Vertical bars: 10 s (A–C, E, and G); 20 s (J). Means ± SD.

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