Microtubule release from the centrosome

Abstract

Although microtubules (MTs) are generally thought to originate at the centrosome, a number of cell types have significant populations of MTs with no apparent centrosomal connection. The origin of these noncentrosomal MTs has been unclear. We applied kinetic analysis of MT formation in vivo to establish their mode of origin. Time-lapse fluorescence microscopy demonstrated that noncentrosomal MTs in cultured epithelial cells arise primarily by constitutive nucleation at, and release from, the centrosome. After release, MTs moved away from the centrosome and tended to depolymerize. Laser-marking experiments demonstrated that released MTs moved individually with their plus ends leading, suggesting that they were transported by minus end-directed motors. Released MTs were dynamic. The laser marking experiments demonstrated that plus ends of released MTs grew, paused, or shortened while the minus ends were stable or shortened. Microtubule release may serve two kinds of cellular function. Release and transport could generate the noncentrosomal MT arrays observed in epithelial cells, neurons, and other asymmetric, differentiated cells. Release would also contribute to polymer turnover by exposing MT minus ends, thereby providing additional sites for loss of subunits. The noncentrosomal population of MTs may reflect a steady-state of centrosomal nucleation, release, and dynamics.

Figure 1

Fluorescently labeled MTs in PtK₁ cells. (A) Fluorescent MTs in a living PtK₁ cell. The nucleus excludes MTs and tubulin monomers, creating a region in the center of the cell with little or no background fluorescence. In the cell shown here, the centrosome is in the center of this darkened region, making it easier to see single MTs. (Bar = 5 μm.) (B) Diagram depicting the geometrical relationship among the ventral cortex, the centrosome, MTs, and the nucleus as seen in side view; the components are not necessarily drawn to scale. The dashed lines show the approximate region of focus.

Figure 2

Microtubule nucleation and release from the centrosome. In all three examples, the centrosome is in the upper left corner of the frame and the MT traverses the nuclear–ventral zone. Time (seconds) at which the image was collected is shown in the lower left corner of each frame. The MT of interest is highlighted by a semitransparent red overlay. Minus and plus ends of the MT are labeled − and +, respectively. Graphs below each sequence show the distance of the MT plus (+, circles) and minus (−, triangles) ends from the centrosome vs. time. Vertical dotted lines indicate the times at which the frames were acquired. The distance of the plus end from the centrosome was measured along the contour of the MT. (A) MT is shown shortly after its nucleation at the centrosome until after its release (between 85 and 89 s) and movement across the nuclear–ventral region (>89 s). The plus end of the MT bends (105 s). (B) MT is released, and the minus end moves away from centrosome while the plus end remains in the same position. (C) MT is released and moves away from the centrosome. The MT buckles (135 s) and begins to shorten, presumably from its minus end. (Bar = 5 μm.)

Figure 2

Microtubule nucleation and release from the centrosome. In all three examples, the centrosome is in the upper left corner of the frame and the MT traverses the nuclear–ventral zone. Time (seconds) at which the image was collected is shown in the lower left corner of each frame. The MT of interest is highlighted by a semitransparent red overlay. Minus and plus ends of the MT are labeled − and +, respectively. Graphs below each sequence show the distance of the MT plus (+, circles) and minus (−, triangles) ends from the centrosome vs. time. Vertical dotted lines indicate the times at which the frames were acquired. The distance of the plus end from the centrosome was measured along the contour of the MT. (A) MT is shown shortly after its nucleation at the centrosome until after its release (between 85 and 89 s) and movement across the nuclear–ventral region (>89 s). The plus end of the MT bends (105 s). (B) MT is released, and the minus end moves away from centrosome while the plus end remains in the same position. (C) MT is released and moves away from the centrosome. The MT buckles (135 s) and begins to shorten, presumably from its minus end. (Bar = 5 μm.)

Figure 3

Marking of MTs by laser bleaching. MTs were marked by a pulse of laser light (514 nm, 480 mW, and 200 ms) producing a 3-μm wide bleached zone. Numbers in the upper left corner of each image correspond to the time (seconds) after bleaching. The minus (−) and plus (+) ends of the MTs, as well as the proximal (p) and distal (d) ends of the bleach zones, are labeled in the first panel of each sequence. Graphs below each sequence show the distance from the centrosome of the MT minus (−, triangles) and plus (+, circles) ends, as well as the proximal (p, diamonds) and distal (d, inverted triangles) ends of the bleached zone vs. time after bleaching. Dotted lines correspond to the times at which the frames were acquired. (A) MT was released (155–160 s) and moves away from the centrosome (≥160 s), with the plus end stopping as it reaches the edge of the nuclear–ventral region (185 s). The minus end continues moving (≥185 s), with the MT buckling as a result. (B) The MT plus end grows while the minus end shortens (beginning at 215 s), producing treadmilling. This is superimposed on motor-based movement of the MT, as indicated by movement of the bleached bar. The MT was released before laser bleaching was performed. The centrosome is out of the field of view, above the upper left corner. (Bar = 5 μm.)

Figure 3

Marking of MTs by laser bleaching. MTs were marked by a pulse of laser light (514 nm, 480 mW, and 200 ms) producing a 3-μm wide bleached zone. Numbers in the upper left corner of each image correspond to the time (seconds) after bleaching. The minus (−) and plus (+) ends of the MTs, as well as the proximal (p) and distal (d) ends of the bleach zones, are labeled in the first panel of each sequence. Graphs below each sequence show the distance from the centrosome of the MT minus (−, triangles) and plus (+, circles) ends, as well as the proximal (p, diamonds) and distal (d, inverted triangles) ends of the bleached zone vs. time after bleaching. Dotted lines correspond to the times at which the frames were acquired. (A) MT was released (155–160 s) and moves away from the centrosome (≥160 s), with the plus end stopping as it reaches the edge of the nuclear–ventral region (185 s). The minus end continues moving (≥185 s), with the MT buckling as a result. (B) The MT plus end grows while the minus end shortens (beginning at 215 s), producing treadmilling. This is superimposed on motor-based movement of the MT, as indicated by movement of the bleached bar. The MT was released before laser bleaching was performed. The centrosome is out of the field of view, above the upper left corner. (Bar = 5 μm.)

Figure 4

Distribution of peak rates of translocation of released MTs. Although some released MTs moved at rates >35 μm/min, others reached peak rates of <10 μm/min. The average peak rate was 20.1 μm/min (n = 15).

Figure 5

(A) Three examples of the behavior of minus ends of released MTs. One MT (top, squares) was stable during much of the sequence but shortened toward the end. The second MT (middle, circles) was stable during the entire sequence. The third MT (bottom, triangles) was observed to shorten beginning at the time of release. (B) Distribution of shortening rates of released MTs. Rates of shortening between pairs of points were determined. The average rate of shortening was 5.3 μm/min (n = 20).

Figure 6

Minus end turnover pathway. (A) MT (open rectangle) attached to the centrosome (shaded circle) with a cap preventing depolymerization at the minus end (filled square). (B) MT is released from the centrosome, but the minus end remains capped. (C) Cap is stochastic, not permanent; MT has some probability of becoming uncapped. (D) MT minus end depolymerizes. k_R, k_U, and k_D are the rate constants for release, uncapping, and depolymerization, respectively.