Centrosome reorientation in wound-edge cells is cell type specific

Abstract

The reorientation of the microtubule organizing center during cell migration into a wound in the monolayer was directly observed in living wound-edge cells expressing gamma-tubulin tagged with green fluorescent protein. Our results demonstrate that in CHO cells, the centrosome reorients to a position in front of the nucleus, toward the wound edge, whereas in PtK cells, the centrosome lags behind the nucleus during migration into the wound. In CHO cells, the average rate of centrosome motion was faster than that of the nucleus; the converse was true in PtK cells. In both cell lines, centrosome motion was stochastic, with periods of rapid motion interspersed with periods of slower motion. Centrosome reorientation in CHO cells required dynamic microtubules and cytoplasmic dynein/dynactin activity and could be prevented by altering cell-to-cell or cell-to-substrate adhesion. Microtubule marking experiments using photoactivation of caged tubulin demonstrate that microtubules are transported in the direction of cell motility in both cell lines but that in PtK cells, microtubules move individually, whereas their movement is more coherent in CHO cells. Our data demonstrate that centrosome reorientation is not required for directed migration and that diverse cells use distinct mechanisms for remodeling the microtubule array during directed migration.

Figure 1

(a) Time-lapse sequence of a living wound-edge CHO cell expressing GFP-γ–tubulin; time, in minutes, is given in the lower corner of each panel. Bar, 5 μm. The direction of cell motility is toward the top of the page. Note the movement of the centrosome to the front of the nucleus between panels 0 and 54 and its subsequent persistence in that position. Cells behind the wound-edge cell were not expressing GFP–γ-tubulin and thus are not visible in the fluorescence image. (b) Histogram of rates of movement of the centrosome and nucleus for the cell shown in a; the end of the time interval used to measure the rate is shown on the x axis. (c–e) Plots of the distance the centrosome and nucleus moved from their initial positions over time. (b) Graph corresponds to the cell shown in a. In each example, the centrosome moves forward a greater distance than the nucleus such that it reorients and remains in front of the nucleus.

Figure 2

(a) Time-lapse sequence of a living wound-edge PtK cell expressing GFP–γ-tubulin; time, in minutes, is given in the lower corner of each panel. Bar, 5 μm. The direction of cell motility is toward the top of the page. Note the lagging of the centrosome behind the nucleus over time (small white dot). Other cells in this field of view were not expressing GFP–γ-tubulin and thus are not visible in the fluorescence image. (b) Histogram of rates of movement of the centrosome and nucleus for the cell shown in a; the end of the time interval used to determine the rates is shown on the x axis. (c–e) Plots of the distance the centrosome and nucleus moved from their initial positions over time. (b) Graph corresponds to the cell shown in a. In a and b, the nucleus moves a greater distance than the centrosome, leaving it behind. In c, the initial position of the centrosome is in front of the nucleus, and it remains there throughout the course of observation.

Figure 3

Centrosome behavior in wound-edge CHO and PtK cells. Phase images (a and d) are shown, along with corresponding immunolocalization of γ-tubulin (b and e) and microtubules (c and f). Bar, 5 mm. Note the greater proportion of centrosomes oriented toward the wound edge in CHO cells than in PtK cells. (g) Diagram of cellular regions used to score centrosome position. Front, sides, rear and center regions are labeled F, S, R, and C, respectively. The wounds are toward the top of the figure; images were acquired ∼ 2.5 h after wounding.

Figure 4

Microtubule transport in live wound-edge CHO (a) and PtK (b) cells. Cells were comicroinjected with rhodamine-labeled tubulin to visualize all the microtubules and with caged fluorescein tubulin for local photoactivation. The pairs of larger panels show all microtubules on the right (rhodamine fluorescence) and photoactivated microtubules on the left (fluorescein fluorescence) immediately and 15 (a) or 35 (b) minutes after photoactivation. Bars, 10 μm. The smaller panels are magnified views of photoactivated microtubules; time after photoactivation is given, in minutes, in the upper corner of each panel. The lines are provided as a reference; arrowheads in b mark individual microtubules. Bars, 5 μm. The direction of movement is upward for both cells. Note the extensive movement of individual microtubules in PtK cells and the coherent motion of microtubules in CHO cells.

Figure 5

Cadherin expression in CHO cells alters centrosome behavior. Immunolocalization of cadherin (a and b) and γ-tubulin (c). (a) Control CHO cells do not express detectable levels of cadherin. CHO cells transiently transfected with cadherin and expressing cadherin at sites of cell-to-cell contact (b); the position of the centrosome is detected after staining for γ-tubulin. Compare the position of the centrosome in these cells with those shown in Figure 3. The wound is at the top of the figure. Bar, 5 mm.

Figure 6

Model for centrosome reorientation in wound edge cells. (a) In CHO cells, most microtubules (blue lines) are focused at the centrosome (pink circle) and extend to the cell periphery. Activated motors in the cortex (red squares) contribute to centrosome repositioning; the microtubules and centrosome reorient as a unit. Photoactivated marks on the microtubules are shown in green. Blue arrow indicates location of wound. (b) In PtK cells, noncentrosomal microtubules persist due to the presence of stabilizing minus-end caps (not illustrated). Slower turnover results in fewer interactions between centrosomal microtubules and cortical motors. Microtubule are transported individually to the front of the cell.