Assembly and positioning of microtubule asters in microfabricated chambers

Abstract

Intracellular organization depends on a variety of molecular assembly processes; while some of these have been studied in simplified cell-free systems, others depend on the confined geometry of cells and cannot be reconstructed using bulk techniques. To study the latter processes in vitro, we fabricated microscopic chambers that simulate the closed environment of cells. We used these chambers to study the positioning of microtubule asters. Microtubule assembly alone, without the action of molecular motors, is sufficient to position asters. Asters with short microtubules move toward the position expected from symmetry; however, once the microtubules become long enough to buckle, symmetry is broken. Calculations and experiments show that the bending-energy landscape has multiple minima. Microtubule dynamic instability modifies the landscape over time and allows asters to explore otherwise inaccessible configurations.

Figure 1

Positioning of MTOCs in microfabricated chambers. (a) Three differential interference contrast images of a centrosome in a square chamber. The chamber is 4 μm deep and the tubulin concentration is 3.2 mg/ml. Images are 3 min apart. The slope of the well dominates the signal close to the edges of the chamber. (b) Three fluorescence images of an AMTOC in a square chamber. The chamber is 6 μm deep and the tubulin concentration is 1.4 mg/ml. Time is indicated in each frame. (c) An aster regrown from an AMTOC in 2.3 mg/ml tubulin, stabilized by diluting with 30% (vol/vol) glycerol/BRB80, spun down through a cushion of 40% glycerol/BRB80 (17) onto a coverslip coated with 3-aminopropyltriethoxysilane, and fixed with glutaraldehyde. Image taken by confocal fluorescence microscopy. (d) Two centrosomes in a square chamber. (All bars are 10 μm.)

Figure 2

(a) Centrosome moving in a dumbbell-shaped chamber. (Bar is 10 μm.) (b) Schematic view of the chamber. Only the region indicated by the broken lines is shown in a. (c) Centrosome position vs. time. The zero-line corresponds to the geometric center of the square section of the chamber. Arrows indicate the times where the images were taken.

Figure 3

Symmetry breaking in aster positioning and evolution of aster configurations. (a) Track of AMTOC position over a 63-min period in a 6-μm-deep circular chamber of radius 18 μm (10). The chamber edge is indicated by the circle. The tubulin concentration is 1.6 mg/ml. Each dot represents a single measurement, taken at 1-s intervals. During the interval shown here microtubules were polymerized. The longest arc was followed from the center toward the edge, increasing the displacement of the AMTOC from the center. (b) Track of aster position in a numerical simulation of bending and dynamic instability (24). Note the long periods of directed motion, similar to the experimental findings. (c) Contour plot of the total bending energy of the 20 microtubules in an aster as the position of the aster is varied (microtubule lengths and angles held constant). The aster is shown at the position of lowest energy.