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. 2016 Oct 2;11(10):e1238547.
doi: 10.1080/15592324.2016.1238547.

Cytokinesis Defect in BY-2 Cells Caused by ATP-competitive Kinase Inhibitors

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Free PMC article

Cytokinesis Defect in BY-2 Cells Caused by ATP-competitive Kinase Inhibitors

Elena Kozgunova et al. Plant Signal Behav. .
Free PMC article

Abstract

Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

Keywords: 5-Iodotubercidin; actomyosin role in plant cytokinesis; Haspin kinase; ML-7; cytokinesis defect; inhibition of the cell plate expansion; kinesin NACK1.

Figures

Figure 1.
Figure 1.
Distribution of kinesin NACK1 throughout the cell cycle. Live-cell imaging was performed on BY-2 cells expressing GFP-NACK1 after a 1-h treatment with DMSO (control) or 1 µM 5-ITu. Images for GFP-NACK1 and brightfield are a single focal plane acquired every 10 min. Numbers indicate time (hh:mm); the starting point of cytokinesis, when GFP-NACK1 was first observed on the cell plate, is shown in the 00:00 column. Scale bars = 10 µm.
Figure 2.
Figure 2.
Cell plate expansion defect in BY-2 cells caused by ATP-competitive inhibitors 5-ITu and ML-7. (A) Live-cell imaging of the BY-2 cells expressing cell plate marker GFP-KNOLLE after a 1-h treatment with DMSO (control), 1 µM 5-ITu or 200 µM ML-7. Numbers indicate time (hh:mm). First column (time frame 00:00) is first appearance of GFP-KNOLLE on the cell plate, and is considered a starting point of the cell plate expansion. Second column (time frame 60:00) shows same cell plate after 1 hr. Images for GFP-KNOLLE are the maximum projection of Z-planes. Scale bar = 10 µm. (B) Quantitative data for cytokinesis defect phenotype. BY-2 cells expressing GFP-KNOLLE were treated with DMSO (control; n = 19), 1 µM 5-ITu (n = 14) or 200 µM ML-7 (n = 20). Cytokinesis defect was categorized into 2 types: cell plate orientation defect (tilted cell plates, gray bars) and cell plate expansion defect (cell plates that did not complete expansion within 2-h, black bars).
Figure 3.
Figure 3.
Schematic representation of cytokinesis process in plant cells. Cell plate expansion is guided by phragmoplast, which consists of MTs and AFs. Kinesin proteins are associated with MTs, facilitating vesicle transport (unidentified kinesin) or promoting MT turnover (kinesin NACK1). Myosin proteins are present on the cell plate and cell borders, presumably using AFs as a bridge to guide cell plate toward cell borders.

Erratum for

  • This manuscript is article addenda for the following article: Kozgunova E, , Suzuki T, , Ito M, , Higashiyama T, , Kurihara D. Haspin has Multiple Functions in the Plant Cell Division Regulatory Network. PCP. 2016; 57(4):848–861. http://dx.doi.org/10.1093/pcp/pcw030.

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