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. 2016 Dec 6;113(49):14157-14162.
doi: 10.1073/pnas.1613979113. Epub 2016 Nov 22.

Cytoskeleton Dynamics Control the First Asymmetric Cell Division in Arabidopsis Zygote

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Free PMC article

Cytoskeleton Dynamics Control the First Asymmetric Cell Division in Arabidopsis Zygote

Yusuke Kimata et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The asymmetric cell division of the zygote is the initial and crucial developmental step in most multicellular organisms. In flowering plants, whether zygote polarity is inherited from the preexisting organization in the egg cell or reestablished after fertilization has remained elusive. How dynamically the intracellular organization is generated during zygote polarization is also unknown. Here, we used a live-cell imaging system with Arabidopsis zygotes to visualize the dynamics of the major elements of the cytoskeleton, microtubules (MTs), and actin filaments (F-actins), during the entire process of zygote polarization. By combining image analysis and pharmacological experiments using specific inhibitors of the cytoskeleton, we found features related to zygote polarization. The preexisting alignment of MTs and F-actin in the egg cell is lost on fertilization. Then, MTs organize into a transverse ring defining the zygote subapical region and driving cell outgrowth in the apical direction. F-actin forms an apical cap and longitudinal arrays and is required to position the nucleus to the apical region of the zygote, setting the plane of the first asymmetrical division. Our findings show that, in flowering plants, the preexisting cytoskeletal patterns in the egg cell are lost on fertilization and that the zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration.

Keywords: Arabidopsis thaliana; actin filament; apical–basal axis; microtubule; zygote polarity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Live-cell imaging and quantification of MT dynamics during zygote polarization. (A) Schematic diagram of the Arabidopsis zygote that develops deep in the flower. (B–J) 2PEM images of the (B) egg cell and (C–J) time-lapse observations of the zygote in in vitro-cultivated ovules expressing the MT/nucleus marker. The images are representative of three time-lapse images. Numbers indicate the time (hours:minutes) from the first frame. The dotted yellow lines show the site where the egg cell and the zygote attach to the maternal tissue. Arrowheads and brackets show the nucleus and the subapical transverse MT ring, respectively. The lengths from the center of the nucleus to the apical edge and the basal end of the cell are shown as A and B, respectively, in C. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 µm.) (K) Illustrations showing a summary of the respective stages in B–J. (L) Graph of ∆θ (the average angle of the fibers against the longitudinal axis) in the indicated cells. The illustrations show the correlation between the values and the cytoskeleton patterns. *P < 0.05. (M) Graph of cell area in the indicated cells. (N) Time course of A and B shown in C until H. (O) Graph of ∆θ of MTs in the apical and basal compartments. Error bars represent the SD of 8–10 samples. Significant differences from the values of young zygotes were determined by Dunnett’s test in L, and the letters in M and O indicate significant differences among stages (P < 0.01 by the Tukey–Kramer test). Elong, elongating zygote; ns, not significant; PPB, preprophase band.
Fig. 2.
Fig. 2.
Live-cell imaging and quantification of F-actin dynamics during zygote polarization. (A–G) 2PEM images of the (A) egg cell and (B–G) time-lapse observation of the zygote in in vitro-cultivated ovules expressing the F-actin/nucleus marker. The images are representative of five time-lapse images. Numbers indicate the time (hours:minutes) from the first frame. The dotted yellow lines show the site where the egg cell and zygote attach to the maternal tissue. Arrowheads and brackets show the nucleus and apical cap, respectively. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 µm.) (H) Illustrations showing a summary of the respective stages in A–G. (I and J) Graphs of (I) parallelness and (J) ∆θ of each fiber in the indicated cells. Error bars represent the SD of 8–15 samples. Significant differences from the values of young zygotes were determined by Dunnett’s test. ns, Not significant. *P < 0.05; **P < 0.01.
Fig. S1.
Fig. S1.
Facile discrimination of unfertilized and fertilized ovules using the MT/nucleus marker. (A and B) Wide-field epifluorescence images of ovules expressing the MT/nucleus marker. (A) In the unfertilized ovule, the egg cell nucleus (arrowhead) was positioned at the apex, and the central cell contained one nucleus (arrow). (B) However, in the fertilized ovule, the young zygote nucleus was detached from the apex, and the endosperm harbored multiple nuclei. The egg cell and zygote are outlined, and A, Right and B, Right show the differential interference contrast (DIC) images without fluorescence. (Scale bars: 30 µm.)
Fig. S2.
Fig. S2.
2PEM is more suitable than confocal microscopy for zygote imaging. Images of the same zygote expressing the MT/nucleus marker, which were taken using (A) confocal microscopy and (B) 2PEM. Arrowheads indicate the zygote nucleus. Maximum intensity projection (MIP) images generated by serial optical sections are shown. (Scale bars: 10 µm.)
Fig. S3.
Fig. S3.
Skeletonized images and quantification of MTs. (A–D) Image processing of 2PEM images of an egg cell expressing the MT/nucleus marker. (A) MIP image generated by serial optical sections. (B) Mask image of the cell area of A. (C) Image generated by skeletonization of A and masking with B. (D) MIP image generated by skeletonized serial optical sections and masking with B. (E–G) MIP images generated by skeletonized serial optical sections and masking of indicated cells. (H–J) Graphs of the (H) parallelness of each fiber, (I) density (occupancy of the cytoskeleton in the whole-cell area), and (J) skewness of the intensity distribution (a metric for the appearance of bundled cables) of the MTs in the indicated cells. H, Right, I, Right, and J, Right show the correlation between the cytoskeleton features and the respective values. Error bars represent the SD of 8–10 samples, and significant differences from the values of young zygotes were determined by Dunnett’s test. Parallelness and density were measured using the images as shown in C. Skewness was calculated based on the images as shown in D. Elong, elongating zygote; ns, not significant. *P < 0.05; **P < 0.01. (K–N) Image processing for the elongating zygote expressing the MT/nucleus marker. (K) MIP image. (L) Image generated by skeletonization of K and masking. (M) Cropped image of the circled region of L as the apical area. (N) Cropped image excluding the circled region of L as the basal area. (Scale bars: 10 µm.)
Fig. S4.
Fig. S4.
Skeletonized images and quantification of F-actin. (A–C) MIP images generated by skeletonized serial optical sections and masking of the indicated cells expressing the F-actin/nucleus marker. (Scale bar: 10 µm.) (D and E) Graphs of the (D) skewness and (E) density of F-actin in the indicated cells. Error bars represent the SD of 8–15 samples. Significant differences from the values of young zygotes were determined by Dunnett’s test. ns, Not significant. *P < 0.05.
Fig. S5.
Fig. S5.
The specific effects of oryzalin and LatB in the zygote. (A–F) 2PEM images of mature zygotes expressing the (A–C) MT/nucleus and (D–F) F-actin/nucleus markers after incubation for 1 h with (A and D) control DMSO and (B and E) 1 μM oryzalin or (C and F) 1 μM LatB. MIP images generated by serial optical sections are shown. (Scale bars: 10 µm.) (G) Graph of the cell width in Fig. 3A. Error bars represent the SD of 13–14 samples, and significant differences from the values of DMSO-treated zygotes were determined by Dunnett’s test. ns, Not significant. *P < 0.05.
Fig. 3.
Fig. 3.
Roles of MT and F-actin in zygote polarization. (A–C) Confocal time-lapse observations of the MT/nucleus marker in the presence of (A) control DMSO and polymerization inhibitors for (B) MTs (1 μM oryzalin) and (C) actin (1 μM LatB). Numbers indicate the time (hours:minutes) from the first frame. The mature zygote was set as one frame before the nuclear division, and the cell shape and nuclear position in the mature zygotes are summarized in column 4. The lengths from the center of the nucleus to the apical edge and the basal end of the cell are shown as A and B, respectively. The width of the zygote is shown as W. Brackets on the images show the lengths of the apical and basal cells after the zygotic division. Note that the oryzalin-treated zygote failed to complete cell division. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 µm.) (D and E) Graphs of (D) zygote cell length (A + B) and (E) asymmetry (A/B). Error bars represent the SD of 13–14 samples, and significant differences from the values of DMSO-treated zygotes were determined by Dunnett’s test. ns, Not significant. *P < 0.05; **P < 0.01. (F) Schematic representation of the patterns and roles of MTs and F-actin in zygote polarization.

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