Computer Simulations and Image Processing Reveal Length-Dependent Pulling Force as the Primary Mechanism for C. elegans Male Pronuclear Migration  Akatsuki.

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Computer Simulations and Image Processing Reveal Length-Dependent Pulling Force as the Primary Mechanism for C. elegans Male Pronuclear Migration  Akatsuki Kimura, Shuichi Onami  Developmental Cell  Volume 8, Issue 5, Pages 765-775 (May 2005) DOI: 10.1016/j.devcel.2005.03.007 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Distribution of Microtubules during Pronuclear Migration in Caenorhabditis elegans Embryos Time-lapse series of images of a GFP::tubulin-expressing embryo recorded by spinning-disk confocal microscopy. “M” and “F” (in A) indicate positions of the male and female pronuclei, respectively. Elapsed time is shown in minutes and seconds. Scale bar equals 10 μm. Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Computer Modeling of Male Pronuclear Migration (A and B) Schematic of force generation in the pushing mechanism (A) and in the pulling mechanism (B). The large oval represents the fertilized egg, small light-blue circle the male pronucleus, and blue lines the MTs. (A) Forces are generated when growing MTs push the cortex. Reaction forces are shown as red arrows. As a result, the pronucleus moves to the cell center (light-blue arrow). (B) Pulling forces (red arrows) of MTs are proportional to the length of the MTs. (C) Example of the computer simulation of pronucleus migration. Snapshots of a simulated egg during the simulation are shown. The blue oval represents the fertilized egg, red sphere the pronucleus, and yellow lines the MTs. (D and E) Path of simulated pronuclear migration in the pushing model (D) and in the pulling model (E). Different colors of lines correspond to different parameter values used, as described in (F) and (G). (F and G) Distance-time graphs for simulated migration of the male pronucleus based on the pushing model (F) and pulling model (G). Distances of the center of the male pronucleus from the posterior end of the egg are plotted as a function of time. Different colors of lines correspond to different parameter values for the viscosity of cytosol, η (Ns/m2): 0.25 (orange), 0.5 (pink), 1.0 (black), 2.0 (blue), and 4.0 (green). For the other parameters, the “standard condition” shown in Table 1 was used. Inset shows the magnification (0.5–3.3 min for time and 10%–35% for distance) of the early stage of migration where the overall shape of the graph is convex upward in the pushing model and concave upward in the pulling model. The shape of each graph is convex in (F) but sigmoidal in (G). Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Parameter Value on Shape of the Distance-Time Graph for Simulated Migration Distance-time graph for simulations using various parameters for the strength of pushing/pulling forces, dynamic instability, and the numbers of MTs. Graphs of the pushing model simulation are shown at the left panel of each figure, and those of the pulling model simulation are shown at the right, as in Figures 2F and 2G. The parameters shown as “standard condition” in Table 1 were used, unless otherwise indicated. (A and B) Effect of strength parameters of the pushing/pulling forces. In the pushing model, MT rigidity, κ, was set to 50 (A left) or 2 (B left) × 10−24 Nm2. In the pulling model, the number of motors per unit length of MT, D, was set to 500 (A right) or 20 (B right) × 103/m. (C and D) Effect of dynamic instability parameters. (C) Set of maximum parameter values reported by Dhamodharan and Wadsworth (1995) was applied (Vg = 0.328 μm/s, Vs = 0.537 μm/s, fcat = 0.046/s, fres = 0.133/s) to represent the fastest dynamic instability. (D) Set of parameter values reported by Verde et al. (1992) was applied (Vg = 0.118 μm/s, Vs = 0.157 μm/s, fcat = 0.0115/s, fres = 0.018/s) to represent the slowest dynamic instability. (E and F) Effect of number of MTs. Number of MTs was set at 550 (E) and 30 (F). For better comparison of the graph shape between the models, the range of η was adjusted to 1.25 (orange), 2.5 (pink), 5.0 (black), 10.0 (blue), and 20.0 Ns/m2 (green) in (A) and (E); to 0.05 (orange), 0.1 (pink), 0.2 (black), 0.4 (blue), and 0.8 Ns/m2 (green) in (B); to 0.25 (orange), 0.5 (pink), 1.0 (black), 2.0 (blue), and 4.0 Ns/m2 (green) in (C) and (D); and to 0.125 (orange), 0.25 (pink), 0.5 (black), 1.0 (blue), and 2.0 Ns/m2 (green) in (F). Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Distance-Time Graph of Male Pronuclear Migration In Vivo (A) Example of objective pronuclear detection system. The original Nomarski DIC images are shown in the upper panels (darker shaded). The lower panels (lighter shaded) show the binary images processed using the algorithm (described in the Supplemental Data); the low-entropy regions (corresponding to “smooth” regions in the original images) are shown in gray. “M” and “F” indicate positions of the male and female pronuclei, respectively. Elapsed time is shown in minutes and seconds and corresponds to the time in (B). Scale bar equals 10 μm. (B) Distance-time graph of male pronuclear migration in wild-type eggs. Position of the pronucleus was detected objectively, and its resultant distance from a posterior end was plotted against time. Three independent measurements are represented as different colors. The time for the male pronucleus to reach the female pronucleus was normalized in the figure to occur at 7.5 min. The shape of the distance-time graph was sigmoidal for all embryos examined (n = 19). (C and D) Distance-time graph of male pronuclear migration in zyg-9 mutant embryos (C) and in embryos treated with nocodazole (2 μg/ml) (D). The original Nomarski DIC images are shown in Supplemental Figure S7. Because the distance of migration upon treatment with nocodazole varied from egg to egg, two representative results are shown in (D). The male pronucleus reached a point about 40% of the long axis of the egg and encountered the female pronucleus (blue curve in D). In another sample, the male pronucleus reached a point about 25% of the long axis and did not encounter the female pronucleus (black curve in D). Only short MTs were detected in the nocodazole-treated embryos under the experimental conditions used here (data not shown). Perturbations in the dynamics of MTs did not affect the shape of the distance-time graph of the in vivo migration (n = 8 for zyg-9 mutants and n = 6 for nocodazole-treated embryos). Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Relationship between the Direction of Male Pronuclear Migration and the Position of the MT Aster Time-lapse series of images of a GFP::tubulin-expressing embryo in zyg-1 (b1) mutant recorded by spinning-disk confocal microscopy. Longest axis is shown as dotted line, and positions of male and female pronuclei as “M” and “F” (in I), respectively. Red dots indicate the centers and yellow arrows indicate the velocity vectors of the male pronucleus in (A)–(I). In (J) the pronuclear envelope is breaking down. Asymmetry in the direction of the MTs was confirmed by immunostaining using anti-tubulin antibodies (data not shown). Initially, the center of the pronucleus was on the axis (A and B). When the centrosome and the associated MT aster moved to the lateral side of the pronucleus, the pronucleus migrated toward the MT-aster side of the pronucleus (i.e., left side of the longest axis in the figures) (C–H). When the centrosome returned to the long axis of the egg, the male pronucleus also approached this axis (I and J). This behavior of zyg-1 embryos was observed for all embryos whose centrosome could be tracked on a single focal plane (n = 10). A pulling model simulation with a single aster predicts this in vivo behavior (Supplemental Figure S8). Elapsed time is shown in minutes and seconds. Scale bar equals 10 μm. Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Absence of Pronuclear Migration in dhc-1 (RNAi) Embryos Independent of Centrosome Separation Time-lapse series of images of a GFP::tubulin-expressing embryo of zyg-1(b1); dhc-1(RNAi) (A–D) and zyg-1(b1) (E–H) recorded by spinning-disk confocal microscopy. Male pronuclear migration occurred in zyg-1(b1) embryos with a single aster (n = 12), whereas the migration was blocked in zyg-1(b1); dhc-1(RNAi) embryos with a single aster (n = 13). No apparent fluorescent signals of DHC-1 proteins were detected in dhc-1 (RNAi) embryos stained with anti-DHC-1 antibodies (Supplemental Figure S9). Times shown in minutes and seconds were normalized by the time of nuclear envelope breakdown (D and H). Positions of the male and female pronuclei are indicated by “M” and “F,” respectively. Scale bar equals 10 μm. Developmental Cell 2005 8, 765-775DOI: (10.1016/j.devcel.2005.03.007) Copyright © 2005 Elsevier Inc. Terms and Conditions