Tanimoto H et al. (2016), Shape-motion relationships of centering microtu...

ECB-ART-44599

J Cell Biol 2016 Mar 28;2127:777-87. doi: 10.1083/jcb.201510064.

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Shape-motion relationships of centering microtubule asters.

Tanimoto H , Kimura A , Minc N .

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Although mechanisms that contribute to microtubule (MT) aster positioning have been extensively studied, still little is known on how asters move inside cells to faithfully target a cellular location. Here, we study sperm aster centration in sea urchin eggs, as a stereotypical large-scale aster movement with extreme constraints on centering speed and precision. By tracking three-dimensional aster centration dynamics in eggs with manipulated shapes, we show that aster geometry resulting from MT growth and interaction with cell boundaries dictates aster instantaneous directionality, yielding cell shape-dependent centering trajectories. Aster laser surgery and modeling suggest that dynein-dependent MT cytoplasmic pulling forces that scale to MT length function to convert aster geometry into directionality. In contrast, aster speed remains largely independent of aster size, shape, or absolute dynein activity, which suggests it may be predominantly determined by aster growth rate rather than MT force amplitude. These studies begin to define the geometrical principles that control aster movements.

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Genes referenced: dnah3 impact LOC100887844 LOC115919910 LOC115925415

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	Figure 1. Sperm asters move to the egg center with persistent directionality and constant speed, in a MT- and dynein-dependent manner. (A) Time-lapse confocal images superimposed with differential interference contrast (DIC) of a male pronucleus (white arrowhead) at the center of a sperm MT aster. (B) 3D trajectories of 10 individual asters and enlarged trajectory of an aster that migrates mostly in-plane. Time is color-coded. The centration is subdivided into three phases: an initial penetration phase (P), a rapid centration phase with straight path and constant speed (C), and a final slowing-down phase (S). (C) Aster traveling distance and orientation toward the cell center as a function of time, for the sample enlarged in B. T1 and T2 denote the beginning and end of the rapid centration phase. (D and E) Distance and orientation time plots for 10 individual asters. Red line, mean; gray section, SD. The broken line is a guide for the eyes. (F) Time-lapse images of eggs treated with different inhibitors 3 min after sperm entry. (G) 3D trajectories corresponding to the eggs and conditions from F. Time is color-coded as in B. (H) Mean aster traveling distance as a function of time in the presence of inhibitors. The gray region indicates the period during which inhibitors are present. Red, DMSO (n = 5); blue, 20 µM latrunculin B (n = 5); green, 20 µM nocodazole (n = 7); purple, 50 µM ciliobrevin D nonactive analog (n = 6); and orange, 50 µM ciliobrevin D (n = 6). (I) Aster speed computed using the distance–time curve between 5 and 7 min. Error bars represent SD. Bars, 50 µm.
	Figure 2. Aster centration is driven by MT-pulling forces in the cytoplasm. (A) Time-dependent immunostaining of centering MT asters (MT, green; DNA, red). The indicated time is taken in reference with sperm entry by accounting for a mean 3-min delay between sperm addition and entry. (B) Centering MT asters are ablated parallel to the centration trajectory. A drift in the trajectory toward (away from) the ablation line suggests that MTs are pushing (pulling). (C) In situ MT aster immunostaining performed immediately after laser ablation along the red broken line. (D and E) Time-lapse (D) and time projection (E) of a centering aster ablated as indicated. Time 0 is the time of ablation. White arrowhead, male pronucleus. (F) Definition of aster velocity and drift after ablation. (G) Aster drift in the indicated conditions (n = 10 for control; n = 28 for side ablation; n = 7 for nocodazole; n = 10 for nocodazole + side ablation). Bar, 10 μm. (H) Aster velocity vectors after ablation at the indicated locations. (I) Aster speed along the centration path after ablation in the indicated conditions. (J) Distance–time plot of a front-ablated aster. (K) Aster velocity along the centration path after ablation (V2) as a function of the velocity before (V1). n.s., nonsignificant; **, P < 10−4 (Student’s t test). Error bars represent SD. Bars, 50 µm.
	Figure 3. Aster geometry determines aster directionality. (A) Time lapses of aster centration in shape-manipulated eggs. (B) Centering trajectories for the time lapses presented in A. (C) Corresponding numerical simulations. (D) 3D centering trajectory of a sperm aster exhibiting two subsequent turning points (black arrowheads). The plot volume corresponds to a cell quarter with X = Y = Z = 0 marking the cell center. (E) Numerical simulation corresponding to D. (F) Distance–time plot for the centration trajectory presented in D, with black arrowheads marking the turning points. (G) Distance–time curves for 35 centering asters in various cell geometries. Shape aspect ratio: red, ≥1; blue, <1. The black line is an averaged distance–time curve for normal spherical cells. (H) Aster speed in different cell geometries. Error bars represent ±SD. Bars, 100 µm.
	Figure 4. Speed determination in growing asters. (A) 1D model of a centering aster. Each MT exerts a pulling force F that scales to its length L. The aster moves with a speed V. (B) Time evolution of MT lengths in the model. Note that Lrear is equal to aster position in the model. τ and T2 correspond to the time needed to reach constant speed and to the time at which the front MT contacts the opposite cortex. (C and D) 3D simulations for various force parameter values and for the two scaling conditions linking α and β. (E and F) Simulations assessing the impact of abruptly reducing the force amplitude in the two different scaling conditions. The force parameter was decreased by a factor 20 in the simulation at 5 min (G) Confocal time lapse of a centering aster treated with a low dose of 10 µM ciliobrevin D at 5 min after sperm entry. (H and I) Aster speed before (V1) and after (V2) ciliobrevin D treatment. (I) V2 plotted as a function of V1 for seven individual eggs. Broken line marks V1 = V2. Bars, 50 µm.
	Figure 5. Aster shape–motion relationships. (A) Proposed model for how centering MT asters may determine their speed and directionality. Each MT exerts a pulling force on the centrosome that scales to MT length. Aster shape asymmetry, which corresponds to the difference between centrosome position and aster geometrical center, is characterized by a unit vector e→ corresponding to aster directionality. Asters migrate with a constant speed determined by the growth rate Vp. Therefore, the aster velocity vector can be simply represented as Vp⋅e→. (B) These shape–motion relationships enable asters to probe local cell geometry to faithfully find the center in any cell shape.

References [+] :

Brito, Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium. 2005, Pubmed