FIGURE 5.

Microtubule polymerization in CDT- and MβCD-treated cells. Caco-2 cells transfected with EB3-GFP were monitored in fluorescence time-lapse microscopy. Cells were recorded 1.5 h after treatment with CDT (200 ng/ml CDTa and 400 ng/ml CDTb) or with an additional pretreatment with 5 mm MβCD. A, quantification of microtubule polymerization rate. Growing microtubules (EB3-GFP comets) were tracked over 10 s after CDT treatment. Data from ≥50 comets (five experiments) are given ±S.E. B, quantification of microtubule time in growth. The lifetime of ≥25 EB3-GFP comets from appearance to fading was measured (data are given ±S.E., ≥4 independent experiments). C, projections of EB3-GFP time-lapse microscopy. Images from 3 min movies (one picture every 3 s) were projected in one image to show tracks of polymerizing microtubules (treated as in A and B). In control cells, microtubules polymerize equally in the cell to form an ordered microtubule array. In CDT-treated cells, polymerization occurs mainly at the cell cortex and in formed protrusions. In MβCD-treated cells, microtubule polymerization takes place in the entire cell similar to control cells. In MβCD and CDT-treated cells, microtubules mainly polymerize at the cell cortex but without the formation of processes.