Figure 5.
DCX-EMAP stabilizes microtubule by reducing koffof tubulin dimers from both growing and shrinking microtubule ends. (A) Schematic diagrams of various DCX-EMAP mutants. (B) Representative confocal images showing the microtubule-binding ability of full-length DCX-EMAP, ΔDCX1/2, and DCX1/2 in S2 cells. Scale bar, 10 µm. (C) Representative kymographs of microtubule dynamics (tubulin: 2 μM) in the presence 0 or 0.5 μM DCX1/2. Vertical bar, 1 min. Horizontal bar, 2 μm. (D) Plots of the microtubule plus end growth rate as a function of tubulin concentration in the presence of 0 (gray; n = 182, 212, and 202 events from three assays) or 0.5 μM (red; n = 138, 109, 135 events from three assays) DCX1/2. (E) Statistical quantification of microtubule catastrophe frequency (tubulin: 2 μM) in the presence 0 (n = 97 microtubules from three assays) or 0.5 μM (n = 87 microtubules from three assays) DCX1/2. (F) Statistical quantification of microtubule shrinking rate (tubulin: 2 μM) in the presence of 0 (n = 94 events from three assays) or 0.5 μM (n = 46 events from three assays) DCX1/2. (G) Representative TIRF images showing the binding of DCX1/2, DCX1, or DCX2 (0.5 μM) to the GMPCPP-stabilized (blue) and dynamic (red) microtubules. Scale bar, 2 μm. (H) Statistical quantification of the binding of DCX1/2, DCX1, or DCX2 (0.5 μM) on GMPCPP-stabilized and dynamic microtubules (n = 90 microtubules from three assays). (I) Representative kymographs of microtubule dynamics (tubulin: 2 μM) in the presence of DCX1 (0.5 μM), DCX2 (0.5 μM), or DCX1+DCX2 mixture (0.5 μM each). Vertical bar, 1 min; horizontal bar, 2 μm. (J) Statistical quantification of microtubule catastrophe frequency in the presence of DCX1 (0.5 μM, n = 95 microtubules from three assays), DCX2 (0.5 μM, n = 94 microtubules from three assays), or DCX1+DCX2 mixture (0.5 μM each, n = 67 microtubules from three assays). (K) Statistical quantification of microtubule shrinking rate in the presence of DCX1 (0.5 μM, n = 83 events from three assays), DCX2 (0.5 μM, n = 89 events from three assays), or DCX1+DCX2 mixture (0.5 μM each, n = 89 events from three assays). Data are presented as mean ± SD with scattered data points. Two-sided unpaired Student’s t test. *, P < 0.05. **, P < 0.01. ***, P < 0.001. n.s., no significance.

DCX-EMAP stabilizes microtubule by reducing k off of tubulin dimers from both growing and shrinking microtubule ends. (A) Schematic diagrams of various DCX-EMAP mutants. (B) Representative confocal images showing the microtubule-binding ability of full-length DCX-EMAP, ΔDCX1/2, and DCX1/2 in S2 cells. Scale bar, 10 µm. (C) Representative kymographs of microtubule dynamics (tubulin: 2 μM) in the presence 0 or 0.5 μM DCX1/2. Vertical bar, 1 min. Horizontal bar, 2 μm. (D) Plots of the microtubule plus end growth rate as a function of tubulin concentration in the presence of 0 (gray; n = 182, 212, and 202 events from three assays) or 0.5 μM (red; n = 138, 109, 135 events from three assays) DCX1/2. (E) Statistical quantification of microtubule catastrophe frequency (tubulin: 2 μM) in the presence 0 (n = 97 microtubules from three assays) or 0.5 μM (n = 87 microtubules from three assays) DCX1/2. (F) Statistical quantification of microtubule shrinking rate (tubulin: 2 μM) in the presence of 0 (n = 94 events from three assays) or 0.5 μM (n = 46 events from three assays) DCX1/2. (G) Representative TIRF images showing the binding of DCX1/2, DCX1, or DCX2 (0.5 μM) to the GMPCPP-stabilized (blue) and dynamic (red) microtubules. Scale bar, 2 μm. (H) Statistical quantification of the binding of DCX1/2, DCX1, or DCX2 (0.5 μM) on GMPCPP-stabilized and dynamic microtubules (n = 90 microtubules from three assays). (I) Representative kymographs of microtubule dynamics (tubulin: 2 μM) in the presence of DCX1 (0.5 μM), DCX2 (0.5 μM), or DCX1+DCX2 mixture (0.5 μM each). Vertical bar, 1 min; horizontal bar, 2 μm. (J) Statistical quantification of microtubule catastrophe frequency in the presence of DCX1 (0.5 μM, n = 95 microtubules from three assays), DCX2 (0.5 μM, n = 94 microtubules from three assays), or DCX1+DCX2 mixture (0.5 μM each, n = 67 microtubules from three assays). (K) Statistical quantification of microtubule shrinking rate in the presence of DCX1 (0.5 μM, n = 83 events from three assays), DCX2 (0.5 μM, n = 89 events from three assays), or DCX1+DCX2 mixture (0.5 μM each, n = 89 events from three assays). Data are presented as mean ± SD with scattered data points. Two-sided unpaired Student’s t test. *, P < 0.05. **, P < 0.01. ***, P < 0.001. n.s., no significance.

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