Figure 1.
Figure 1. Impaired MT dynamics in TTL KO MEFs. (A) Analysis of nocodazole effects on WT or TTL KO MEFs. Data are expressed as the ratio of MT signals measured after nocodazole treatment versus control conditions (mean ± SEM). MT signals were estimated for a minimum of 39 MEFs from three independent experiments for each genotype and treatment condition. ***, P < 0.001 with a t test. (B) Video microscopy examination of MTs in WT or TTL KO MEFs expressing m-cherry α-tubulin and GFP-EB3 close to the leading edge of lamellipodial extensions. Colored, closed arrowheads indicate localization of the MT tip at different time points. Open arrowheads of the corresponding color indicate the initial MT tip localization. Most WT MTs underwent extensive depolymerization after membrane contact (white and yellow arrowheads). Some MTs followed the leading edge for a short period of time before depolymerization (pink arrowheads). Most TTL KO MTs continued to grow after membrane touch, running along the plasma membrane or even growing inward (white arrowheads). Occasionally, MTs seemed to push the membrane forward before disassembly, rescue, and regrowth (yellow arrowheads). Similar phenotypes were observed when MT ends were labeled with other TIPs or in experiments run with fluorescent tubulin alone (not depicted). (C) Measurement of the time spent by MTs exploring the area near the leading edge (persistence time). Experiments were run under control conditions or after cell transfection with either MCAK cDNA or MCAK siRNAs. At least 42 MTs were examined in each condition. With MCAK siRNAs, persistence times generally exceeded 100 s and overran the observation time (Videos 5 and 6). Results are shown in other conditions as mean values ± SEM. ***, P < 0.001 with a t test. (D) Analysis of MT dynamic instability. MTs whose growing plus tip was located within 10 µm of the leading edge at time 0 were followed over time after various cell treatments, as indicated. Mean values ± SEM are shown. Statistically significant differences between WT and KO cells are described in Results and discussion; in all cases, p-values were <0.01 with a Mann and Whitney U test. Bars, 10 µm.

Impaired MT dynamics in TTL KO MEFs. (A) Analysis of nocodazole effects on WT or TTL KO MEFs. Data are expressed as the ratio of MT signals measured after nocodazole treatment versus control conditions (mean ± SEM). MT signals were estimated for a minimum of 39 MEFs from three independent experiments for each genotype and treatment condition. ***, P < 0.001 with a t test. (B) Video microscopy examination of MTs in WT or TTL KO MEFs expressing m-cherry α-tubulin and GFP-EB3 close to the leading edge of lamellipodial extensions. Colored, closed arrowheads indicate localization of the MT tip at different time points. Open arrowheads of the corresponding color indicate the initial MT tip localization. Most WT MTs underwent extensive depolymerization after membrane contact (white and yellow arrowheads). Some MTs followed the leading edge for a short period of time before depolymerization (pink arrowheads). Most TTL KO MTs continued to grow after membrane touch, running along the plasma membrane or even growing inward (white arrowheads). Occasionally, MTs seemed to push the membrane forward before disassembly, rescue, and regrowth (yellow arrowheads). Similar phenotypes were observed when MT ends were labeled with other TIPs or in experiments run with fluorescent tubulin alone (not depicted). (C) Measurement of the time spent by MTs exploring the area near the leading edge (persistence time). Experiments were run under control conditions or after cell transfection with either MCAK cDNA or MCAK siRNAs. At least 42 MTs were examined in each condition. With MCAK siRNAs, persistence times generally exceeded 100 s and overran the observation time (Videos 5 and 6). Results are shown in other conditions as mean values ± SEM. ***, P < 0.001 with a t test. (D) Analysis of MT dynamic instability. MTs whose growing plus tip was located within 10 µm of the leading edge at time 0 were followed over time after various cell treatments, as indicated. Mean values ± SEM are shown. Statistically significant differences between WT and KO cells are described in Results and discussion; in all cases, p-values were <0.01 with a Mann and Whitney U test. Bars, 10 µm.

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