Fast microtubule dynamics in meiotic spindles measured by single molecule imaging: evidence that the spindle environment does not stabilize microtubules.
Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have proposed that microtubules are stabilized by the spindle environment. We used single molecule imaging to measure tubulin turnover in spindles, and nonspindle assemblies, in Xenopus laevis egg extracts. We observed many events where tubulin molecules spend only a few seconds in polymer and thus are difficult to reconcile with standard models of polymerization dynamics. Our data can be quantitatively explained by a simple, phenomenological model-with only one adjustable parameter-in which the growing and shrinking of microtubule ends is approximated as a biased random walk. Microtubule turnover kinetics did not vary with position in the spindle and were the same in spindles and nonspindle ensembles nucleated by Tetrahymena pellicles. These results argue that the high density of microtubules in spindles compared with bulk cytoplasm is caused by local enhancement of nucleation and not by local stabilization. It follows that the key to understanding spindle morphogenesis will be to elucidate how nucleation is spatially controlled.
PubMed ID: 19940016
PMC ID: PMC2808228
Article link: Mol Biol Cell.
Grant support: GM-39565 NIGMS NIH HHS , R01 GM039565-22 NIGMS NIH HHS , R01 GM039565 NIGMS NIH HHS
Genes referenced: kif2c
Article Images: [+] show captions
|Figure 1. (A) Raw images were filtered and smoothed, and local maxima above a threshold brightness were selected as candidate particles (Crocker and Grier, 1996). For tracking displacements, the subpixel location of molecules were determined by fitting the intensity around each particle to a two-dimensional Gaussian, but the pixel location of particles was sufficient for the measurement of tubulin lifetimes and subpixel refinement was not used in that analysis. (B, left) Particle locations were linked to form trajectories by a global optimization procedure (Crocker and Grier, 1996). (B, right) Individual particles appeared and disappeared in a step-like manner. A particle's lifetime is defined as the difference between the times of disappearance and appearance. (C) Raw image of a spindle with single molecule level labeling.|
|Figure 2. Trajectories of particles in spindles are illustrated by displaying the dynamics of particles located in a particular frame. A particle's future trajectory is colored red, its past trajectory is blue, and particles that were born in the selected frame are indicated by a green square. The shorter trajectories in the FCPT-treated spindle illustrate the reduced tubulin motion in this structure. Bar, 10 μm.|
|Figure 3. Distribution of tubulin lifetimes in spindles with FCPT (blue circles). A best fit to Equation 1 is included (red line, τ = 71 ± 3 s). The data and best fit were normalized to have an area of 1.|
|Figure 4. (A)Tubulin lifetimes are the same in unperturbed spindles (control, green symbols) and when microtubule translocation is blocked with FCPT (+FCPT, blue symbols). Best fits to Equation 1 are displayed without FCPT (black line, τ = 64 ± 6 s) and with FCPT (red line, τ = 71 ± 3 s). (B) Increasing concentration of MCAK, a microtubule depolymerizer, results in decreasing tubulin lifetimes. Spindles were assembled in extract with MCAK reduced to 50% native levels (green symbols and black line best fit to Equation 1, τ = 97 ± 11 s) or 200% native levels (blue symbols and red best fit, τ = 47 ± 8 s).|
|Figure 5. (A) Spindles were divided into fifths; two pole regions (green), two midregions (blue), and one central region (red). Bar, 10 μm. (B) Ratio of particle deaths to births is nearly equal to one in all regions. (C) Distributions of tubulin lifetimes in each region are the same.|
|Figure 6. (A) Microtubules growing off of pellicles in X. laevis egg extracts. (A, top) Heavy labeling with Alexa-488 tubulin allows all microtubules to be visualized, but individual filaments cannot be resolved. (A, bottom) The same region as in A with single molecule level Alexa-647 tubulin. Bar, 10 μm. (B) Distributions of tubulin lifetimes from pellicles (pellicles, blue) and unperturbed spindles (control spindles, green) are very similar. Best fits to Equation 1 are displayed for pellicles (red, τ = 71 ± 9 s) and spindles (black, τ = 64 ± 6 s).|