Figure 2. Simultaneous depletion of EB1... | Rockefeller University Press

$Figure 2. Simultaneous depletion of EB1 and EB3 disrupts persistent MT growth. (A and B) Time-lapse sequence of YFP-CLIP-170 in cells expressing shRNAs either to luciferase (control) (A) or to EB1 and EB3 (B). Images were acquired every 2 s. Three tips of CLIP-170 “comets” are indicated by an arrow, an arrowhead, and a black-and-white arrowhead. Projection analysis (20 successive frames) and diagrams of trajectories of individual CLIP-170 comets illustrate episodes of uninterrupted MT growth. The centrosome region is indicated by a dashed circle; the cell border is shown by a dotted line. Time in seconds is in the top right corner. Bar, 10 µm. (C and D) Histograms of MT growth rates and the lengths of YFP-CLIP-170 tracks in control (luciferase) and EB1/EB3 shRNA-expressing cells. (E) Distribution of the growing MT plus-ends along the cell radius in control and EB1/EB3-depleted cells; error bars indicate SD. The cell radius was divided into five zones (each zone was a 0.2 fraction of the cell radius) and the number of YFP-CLIP-170–positive tips were scored for each zone (∼600 MT tips in 8–12 cells for each condition). The results are represented as percentage of the MTs within each zone where 100% is a total number of the scored growing plus-ends in the cell. (F and G) Rescue of EB1/EB3 depletion by EB1ΔAc. (F) EB1/EB3-depleted cells expressing EB1ΔAc are indicated by dotted lines. EB3 was detected with rabbit antibodies (green in overlay); EB1 was stained with the mouse antibody from BD Biosciences, which detects both the endogenous EB1 and EB1ΔAc (left panel; red in overlay); and the rat antibody KT51 (Absea), specific for the acidic tail of EB1, was used to stain the endogenous EB1 but not EB1ΔAc (right panel; blue in overlay). (G) Intensity distribution shows that the levels of endogenous EB1 and EB3 in EB1ΔAc-rescued cells were negligible (green and blue lines in panel g′′); level of EB1ΔAc (red line in panel g′′) was similar to the level of endogenous EB1 (red line in panel g′). (H) Time-lapse sequence of YFP-CLIP-170 in the cells coexpressing shRNAs to EB1/EB3 and EB1ΔAc rescue construct; images were acquired every 2.5 s. Tips of CLIP-170 comets are indicated by white and black-and-white arrowheads. Projection analysis and trajectories of individual YFP-CLIP-170 comets are generated in the same way as in panel A. Time in seconds is in the top right corner. Bar, 10 µm.$

Figure 2.

Simultaneous depletion of EB1 and EB3 disrupts persistent MT growth. (A and B) Time-lapse sequence of YFP-CLIP-170 in cells expressing shRNAs either to luciferase (control) (A) or to EB1 and EB3 (B). Images were acquired every 2 s. Three tips of CLIP-170 “comets” are indicated by an arrow, an arrowhead, and a black-and-white arrowhead. Projection analysis (20 successive frames) and diagrams of trajectories of individual CLIP-170 comets illustrate episodes of uninterrupted MT growth. The centrosome region is indicated by a dashed circle; the cell border is shown by a dotted line. Time in seconds is in the top right corner. Bar, 10 µm. (C and D) Histograms of MT growth rates and the lengths of YFP-CLIP-170 tracks in control (luciferase) and EB1/EB3 shRNA-expressing cells. (E) Distribution of the growing MT plus-ends along the cell radius in control and EB1/EB3-depleted cells; error bars indicate SD. The cell radius was divided into five zones (each zone was a 0.2 fraction of the cell radius) and the number of YFP-CLIP-170–positive tips were scored for each zone (∼600 MT tips in 8–12 cells for each condition). The results are represented as percentage of the MTs within each zone where 100% is a total number of the scored growing plus-ends in the cell. (F and G) Rescue of EB1/EB3 depletion by EB1ΔAc. (F) EB1/EB3-depleted cells expressing EB1ΔAc are indicated by dotted lines. EB3 was detected with rabbit antibodies (green in overlay); EB1 was stained with the mouse antibody from BD Biosciences, which detects both the endogenous EB1 and EB1ΔAc (left panel; red in overlay); and the rat antibody KT51 (Absea), specific for the acidic tail of EB1, was used to stain the endogenous EB1 but not EB1ΔAc (right panel; blue in overlay). (G) Intensity distribution shows that the levels of endogenous EB1 and EB3 in EB1ΔAc-rescued cells were negligible (green and blue lines in panel g′′); level of EB1ΔAc (red line in panel g′′) was similar to the level of endogenous EB1 (red line in panel g′). (H) Time-lapse sequence of YFP-CLIP-170 in the cells coexpressing shRNAs to EB1/EB3 and EB1ΔAc rescue construct; images were acquired every 2.5 s. Tips of CLIP-170 comets are indicated by white and black-and-white arrowheads. Projection analysis and trajectories of individual YFP-CLIP-170 comets are generated in the same way as in panel A. Time in seconds is in the top right corner. Bar, 10 µm.

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