Article Figures & Data
Figures
- Figure 1.
The effect of microtubule inhibitors on cell proliferation and cell migration. Paclitaxel (A), vinblastine (B), and colchicine (C) were tested for their ability to inhibit cell migration using a wound-healing scratch assay (open squares, solid lines), and for their ability to block cell division measured as an increase in the mitotic index (solid circles, dotted lines).
- Figure 2.
Changes in select parameters of dynamic instability in cells treated with microtubule inhibitors. HUVEC were transfected with EGFP-MAP4 and microtubule behavior was measured in varying concentrations of paclitaxel (A), vinblastine (B), and colchicine (C). The plots show the effects of drugs on various parameters of dynamic instability (colored lines) superimposed on the drug effects on cell motility determined from a wound-healing scratch assay (solid circles, black line).
- Figure 3.
Asymmetric distribution of dynamic microtubules. A, HUVEC cell migrating into a scratch wound. The cell was transfected with EGFP-MAP4 to visualize the microtubules. Left, the leading edge; and right, the trailing edge. Areas that were monitored by time lapse are outlined in red. Scale bar, 7 μm. B, time-lapse sequences of regions a and b from the leading edge. Photographs shown have been taken at intervals of 10 seconds apart. A few microtubules are traced in red to aid the viewer. C, time-lapse sequences of regions c and d from the trailing edge. Photographs shown have been taken at intervals of 10 seconds. In contrast to the static microtubules at the leading edge, many of the microtubules in the trailing edge were seen to grow (arrows) or shorten (arrowheads). D, quantification of the percentage of the time microtubules were growing, shortening, or pausing at the leading or trailing edges of the cell. The calculated dynamicity of the microtubules is also plotted on a separate scale. All the differences shown between the leading and trailing edges were found to be significant. *, P < 0.05; **, P < 0.01.
- Figure 4.
Membrane activity in HUVEC. A time-lapse series of images taken at intervals of 1 minute were analyzed by measuring the distance of the leading (solid line) and trailing (dotted line) edges from the nucleus. Untreated cells (A), cells treated with 1 nmol/L paclitaxel to suppress microtubule dynamics (B), cells treated with 2 nmol/L colchicine to suppress microtubule dynamics (C), and cells treated with 100 nmol/L vinblastine to eliminate the microtubules (D) are shown. Both edges exhibited relatively rapid fluctuations in the distance of the membrane from the nucleus. However, the trailing edge of control cells [dotted line in (A)] also exhibited a periodic rise and fall that we interpret as stretching and release of the trailing edge during cell migration. We define the time between successive peaks as the periodicity. Membrane activity, defined as the average rate of membrane displacement from the nucleus is plotted in E. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 5.
Migration paths of individual cells. Time-lapse movies of HUVEC moving into a scratch wound were analyzed by plotting the nuclear positions of individual cells at 15-minute intervals. The direction of the wound is toward the right. A, untreated cells. Circles represent the position of the nucleus at time zero and every 15 minutes thereafter. B, cells treated with 1 nmol/L paclitaxel to suppress microtubule dynamics. Shaded circles indicate the lack of any movement for one or more 15-minute intervals. Gray, no change in position for two to three 15-minute intervals; black, no change in position for four or more 15-minute intervals. C, cells treated with 2 nmol/L colchicine to suppress microtubule dynamics. Shaded circles have the same meaning as in the previous panel. D, cells treated with 100 nmol/L vinblastine to eliminate the microtubules. Circles were omitted because of extensive overlap.
- Figure 6.
In vitro angiogenesis assay showing capillary-like tube formation on Matrigel. HUVECs were plated at a subconfluent density on Matrigel and their ability to form tubes was measured after 5 hours without (A) and with (B) 1 nmol/L paclitaxel. The cells were stained with calcein for 30 minutes and observed with the aid of a ×4 fluorescence objective. Quantification of tube formation by measuring tube length or area gave similar results that are plotted in C as a function of paclitaxel concentration. Closed circles and solid line, drug effects on cell migration using a wound-healing assay; open circles and dotted line, drug effects on tube formation.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file - 56K
- Supplementary Figure 1 - PDF file - 94K, Supplementary figure 1. Structures of drugs used in these studies.
- Supplementary Figure 2 - PDF file - 187K, Supplementary figure 2. Immunofluorescence microscopy of drug treated HUVEC.
- Supplementary Tables 1 - 4 - PDF file - 78K, Table S1. Microtubule dynamics in HUVEC in the presence and absence of paclitaxel. Table S2. Microtubule dynamics in HUVEC in the presence and absence of vinblastine. Table S3. Microtubule dynamics in HUVEC in the presence and absence of colchicine. Table S4. Microtubule dynamics in the leading and trailing edges of HUVEC.
- Supplementary Video 1 - MOV file - 3617K, Migration of HUVEC.
- Supplementary Video 2 - MOV file - 3602K, Migration of HUVEC in the presence of 1 nM paclitaxel.
- Supplementary Video 3 - MOV file - 3630K, Migration of HUVEC in the presence of 100 nM vinblastine.
Volume 12, Issue 12
