Inhibition of cell migration and cell division correlates with distinct effects of microtubule inhibiting drugs

Abstract

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a "search and capture" behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.

FIGURE 1.

Tubulin immunofluorescence. CHO cells were treated for 2 days with the indicated concentrations of colcemid or vinblastine and stained with DM1A for tubulin (green) and DAPI for DNA (red). Bar = 10 μm. Upper insets show representative mitotic cells at each of the indicated drug concentrations. Lower insets show 1.5-fold enlargements of a part of the cell to more clearly indicate the presence of microtubule fragments (arrowheads); bar = 5 μm.

FIGURE 2.

Microtubule life history plots. CHO cells were transfected with EGFP-MAP4, grown overnight, and examined by time-lapse fluorescence microscopy at 37 °C 30 min after the addition of colcemid or vinblastine. Images were taken every 5 s, and the microtubule length, measured from an arbitrary fixed point, was plotted against time. Each line represents a different microtubule. The drug concentrations were chosen to maximally suppress microtubule dynamics without affecting cell division.

FIGURE 3.

Effect of drugs on cell migration. A, CHO cells were treated with colcemid or vinblastine, and the distance they moved was measured using a wound healing assay. Each line is labeled with the nm drug concentration used in the experiment. Each data point and error bar represents the mean ± S.D. of at least four independent experiments. B, control CHO cells or cells treated with 4 nm vinblastine were tracked while moving into a wound (situated toward the right). Each line represents a single cell. The x and y coordinates of the cell nucleus were measured every 15 min relative to a fixed position on the dish and plotted onto the graphs. Each small open circle represents a single 15-min time point. When movement did not occur after 15 min, the time points were assigned symbols according to the legend to indicate the number of 15-min intervals that passed before cell migration resumed. Thus, filled circles represent 2 intervals or 30 min, open triangles represent 3 intervals or 45 min, etc. Note that untreated cells exhibited few instances where they did not move in a 15-min time span, but vinblastine-treated cells had many such instances.

FIGURE 4.

The appearance of microtubule fragments correlates with inhibition of cell division in wild-type and mutant CHO cells. A, wild-type cells, CV 2-8, a colcemid-resistant mutant that is also 2-fold resistant to vinblastine, and Tax 5-6, a paclitaxel-resistant mutant that is 2-fold more sensitive to vinblastine, were incubated in varying concentrations of vinblastine for 2 days and stained for tubulin immunofluorescence. The percentage of cells with >10 microtubule fragments was plotted against drug concentration for each cell line. B, cell proliferation (●) was measured using a clonogenic assay and expressed relative to untreated cells set at 100%. The percent of cells with >10 microtubule fragments (○) was counted after treating with colcemid or vinblastine at the indicated concentrations for 2 days. The rate of cell migration relative to untreated control cells (♦) was measured using a wound healing assay that began 30 min after adding the drug. Microtubule dynamicity (□) was calculated relative to untreated cells using the values found in supplemental Tables S1 and S2. The percent of total cellular tubulin found in microtubules (▴) was measured by lysing cells in a microtubule stabilizing buffer, centrifuging to separate polymerized from non-polymerized tubulin, and quantifying the tubulin in each fraction.

FIGURE 5.

Microtubule detachment during interphase. CHO cells transfected with EGFP-MAP4 were left untreated (upper panels) or were treated 30 min with 15 nm vinblastine, the IC₅₀ concentration for inhibition of cell division (lower panels). Fluorescence microscopic images, taken 5 s apart at 37 °C, were deconvolved to improve contrast. Arrows point to microtubule minus-ends before and after detachment. The numbers represent the time (in seconds) the images were taken relative to an arbitrary zero time point. Bar = 5 μm.

FIGURE 6.

Microtubule detachment during mitosis. A, EGFP-MAP4-transfected CHO cells were left untreated (upper panels) or were treated 30 min with 15 nm vinblastine (lower panels) and were then viewed by live cell fluorescence microscopy. Prophase cells were located and photographed every 5 s. The images were deconvolved to improve contrast. Arrows point to the minus-ends of microtubules that detached from the spindle poles. Bar = 5 μm. B, the number of detachments seen in control- and drug-treated prophase and interphase cells were counted from captured images and divided by the total time of observation. The mean ± S.D. was calculated from 11–20 cells during a total observation time of 50–80 min.