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Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

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Abstract

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.

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Acknowledgements

We thank Prof. S. Hasezawa (The University of Tokyo) for providing BY-GF11 cells. We also thank Dr. E. Yokota (University of Hyogo) and Dr. S. Nonaka (National Institute for Basic Biology) for fruitful discussions. We also thank Mr. Katsumoto Umano (Mitani Corporation) and Mr. Kazuyuki Ishiwata (Nikon Instech Co. Ltd.) for their collaboration for the development of the GLIM system. This work was supported by JST SENTAN.

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Author notes

  1. Kengo Arima and Daisuke Tamaoki contributed equally.

Authors and Affiliations

  1. Graduate School of Life Science, University of Hyogo, Kamigori-cho, Ako-gun, Hyogo, 678-1297, Japan

    Kengo Arima, Teruo Shimmen, Tohru Yoshihisa & Seiji Sonobe

  2. Graduate School of Science and Engineering, University of Toyama, Gofuku 3190, Toyama, Toyama, 930-8555, Japan

    Daisuke Tamaoki

  3. Graduate School of Life Science, University of Hyogo, 267 Shosha, Himeji, Hyogo, 671-2280, Japan

    Daisuke Tamaoki, Yoshinobu Mineyuki & Tomonori Nakai

  4. JST SENTAN, 267 Shosha, Himeji, Hyogo, 671-2280, Japan

    Daisuke Tamaoki & Yoshinobu Mineyuki

  5. Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka, 564-8680, Japan

    Hiroki Yasuhara

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  1. Kengo Arima
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Correspondence to Seiji Sonobe.

Electronic supplementary material

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Supplementary material 1 (PDF 812 KB)

10265_2018_1047_MOESM2_ESM.avi

Supplementary Movie S1 Returning movement of a mitotic/cytokinetic apparatus in a centrifuged BY-GF11 cell. Time-lapse images of a centrifuged BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. Green and magenta indicate GFP-fimbrin and FM 4-64, respectively. See Fig. 2 in detail (AVI 910 KB)

10265_2018_1047_MOESM3_ESM.avi

Supplementary Movie S2 A BY-GF11 cell with an incomplete returning cytokinetic apparatus. Time-lapse images of a BY-GF11 cell were taken at a 1-min interval, and the movie plays at 30 frames per second. Green and magenta indicate GFP-fimbrin and FM 4-64, respectively. See Fig. S4a in detail (AVI 569 KB)

10265_2018_1047_MOESM4_ESM.avi

Supplementary Movie S3 A BY-GF11 cell forming a tilted phragmoplast near the cell center. Time-lapse images of a centrifuged BY-GF11 cell were taken at a 2-min interval, and the movie plays at 5 frames per second. Green and magenta indicate GFP-fimbrin and FM 4-64, respectively. See Fig. S4b in detail (AVI 931 KB)

10265_2018_1047_MOESM5_ESM.avi

Supplementary Movie S4 A BY-GF11 cell forming an actin cable between the CDZ and the phragmoplast. Time-lapse images of GFP-fimbrin in a centrifuged BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. See Fig. 3 in detail (AVI 1352 KB)

10265_2018_1047_MOESM6_ESM.avi

Supplementary Movie S5 An actin band appeared at the CDZ in BY-GF11 cells with non-returning cytokinetic apparatus. Time-lapse images of a centrifuged BY-GF11 cell with non-returning cytokinetic apparatus were taken at a 2-min interval, and the movie plays at 5 frames per second. Green and magenta indicate GFP-fimbrin and FM 4-64, respectively. See Fig. 4a in detail (AVI 290 KB)

10265_2018_1047_MOESM7_ESM.avi

Supplementary Movie S6 A 3-D movie of the actin band in the BY-GF11 cell in Fig. 4a. In the first part, 3-D images of a region of the cell bounded by two yellow dotted lines in Fig. 4a are reconstituted from z-stacks of images, and viewed as a movie from the yellow dotted arrow in Fig. 4a according to progression of observing time points. White numbers in the top left indicate the time after starting observation (min). The movie plays at 5 frames per second. In the second part, the 3-D structure of the actin band in the last frame of Fig. 4a is viewed from various angles Yellow numbers in the bottom left indicate rotation angles (°). Scale bar 10 µm (AVI 526 KB)

10265_2018_1047_MOESM8_ESM.avi

Supplementary Movie S7 Transient appearance of an actin band at the CDZ in a BY-GF11 cell. Time-lapse images of GFP-fimbrin in a centrifuged BY-GF11 cell were taken at a 1-min interval, and the movie plays at 15 frames per second. See Fig. 4b in detail (AVI 375 KB)

Supplementary Movie S8 Global and local images of a BY-GF11 cell obtained with the GLIM. Time-lapse DIC images (left) and the fluorescence images of cell cortex (right) of a BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. See Fig. S6 in detail (AVI 2703 KB)

10265_2018_1047_MOESM10_ESM.avi

Supplementary Movie S9 Global and local images of a centrifuged BY-GF11 cell with an un-returned cytokinetic apparatus. Time-lapse DIC images (left) and the fluorescent images of cell cortex (right) of a centrifuged BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. See Fig. 5 in detail (AVI 1349 KB)

10265_2018_1047_MOESM11_ESM.avi

Supplementary Movie S10 Global and local images of a centrifuged BY-GF11 cell with a returned cytokinetic apparatus. Time-lapse DIC images (left) and the fluorescent images of cell cortex (right) of a centrifuged BY-GF11 cell were taken at a 30-sec interval, and the movie plays at 10 frames per second. See Fig. S7 in detail (AVI 2042 KB)

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Arima, K., Tamaoki, D., Mineyuki, Y. et al. Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells. J Plant Res 131, 803–815 (2018). https://doi.org/10.1007/s10265-018-1047-4

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