TY - JOUR
AU - Higaki,, Takumi
AB - Abstract Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis. Introduction Plant cells are separated by new cross walls known as cell plates that centrifugally expand from the cell center to the parental cell wall during cytokinesis. The cell plate is formed and expanded by the phragmoplast, a complex structure composed of microtubules, actin filaments and membrane vesicles (Müller and Jürgens 2016, Smertenko et al. 2018). Microtubules have crucial roles in cell plate formation and expansion. In the phragmoplasts, microtubules are oriented with their plus ends toward the cell plates (Asada et al. 1991, Austin et al. 2005) and Golgi-derived membrane vesicles are transported to the equatorial planes to form and expand the cell plates (Samuels et al. 1995, Seguí-Simarro et al. 2004, Reichardt et al. 2007, van Oostende-Triplet et al. 2017). The mitogen-activated protein kinase (MAPK) kinase kinase NPK1 and its activators NACK1 and NACK2 kinesins were identified as key regulators of cell plate expansion in tobacco (Nishihama et al. 2001, Nishihama et al. 2002). NACK1 colocalizes with NPK1 on the cell plates and triggers the MAPK cascade that finally phosphorylates the microtubule-associated protein MAP65-1 (Nishihama et al. 2001, Nishihama et al. 2002, Sasabe et al. 2006, Sasabe et al. 2011). The MAPK cascade-induced phosphorylation of MAP65-1 decreases its microtubule bundling activity, resulting in the stimulation of phragmoplast microtubule turnover (Sasabe et al. 2006). Phragmoplast microtubule turnover and phragmoplast microtubule branching and flow (Murata et al. 2013) have been suggested to be driving forces for cell plate expansion (Sasabe et al. 2006). The contribution of microtubule turnover to cell plate expansion was supported by the effect of a microtubule stabilizer taxol, which delays cell plate expansion (Yasuhara et al. 1993). In Arabidopsis thaliana, a similar MAPK cascade pathway, named the NACK–PQR pathway (Soyano et al. 2003), for cell plate expansion has been identified (Kosetsu et al. 2010). The homolog of AtNACK1 (also known as HINKEL), AtNACK2 (also known as STUD or TETRASPORE), has functional redundancy in cytokinesis because the atnack1/atnack2 double knockout mutant showed severe phenotype compared with each single mutant (Tanaka et al. 2004). It has been shown that ANP MAPK kinase kinase, which is the homolog of NPK1 in A. thaliana, directly binds with both AtNACK1 and AtNACK2 (Takahashi et al. 2010), similar to the case of tobacco (Nishihama et al. 2002). The distribution of actin filaments in phragmoplast has been examined by various techniques, including phalloidin staining and immunostaining (Clayton and Lloyd 1985, Seagull et al. 1987, Traas et al. 1987, Kakimoto and Shibaoka 1987, Palevitz 1987, Schmit and Lambert 1987, Schmit and Lambert 1988, Molè‐Bajer et al. 1988, Hasezawa et al. 1991), microinjection of fluorescent probes (Schmit and Lambert 1990, Cleary et al. 1992, Valster et al. 1997) and expression of fluorescent protein-tagged probes (Sano et al. 2005, Yu et al. 2006, Higaki et al. 2008). Using live cell imaging and quantitative image analysis, we previously showed that actin filaments appeared from the periphery of the daughter nuclei toward the emerging cell pate at the early phase of cytokinesis and gradually moved closer to the expanding cell plate at the late phase of cytokinesis (Higaki et al. 2008). Perturbation of actin filaments delayed cell plate expansion (Schmit and Lambert 1988, Valster et al. 1997, Higaki et al. 2008, van Oostende-Triplet et al. 2017), showing the contribution of actin filaments to cell plate expansion. Especially in the late phase of plant cytokinesis, actin microfilaments play supporting roles in cell plate expansion by regulating the dynamics of endoplasmic reticulum and endocytic vesicles around the expanding cell plate (Higaki et al. 2008). In addition, actin filaments in transvacuolar strands connecting the cortical division zone and the edge of the phragmoplast were frequently observed in vacuolated cells, suggesting that they guide cell plate edges to the future cell division sites (Lloyd and Traas 1988, Sano et al. 2012, Arima et al. 2018). Despite the accumulated knowledge about actin filaments at the late phase of the cytokinesis, their roles in cell plate formation at the early phase are still enigmatic. We previously found that pharmacological disruption of actin filaments caused temporary malformation of the emerging cell plate, which appeared discontinuous, at the early phase of cytokinesis (Higaki et al. 2008). The temporary malformation of the cell plate suggested that actin filaments are involved in the organization of the initial phragmoplast microtubules. However, the relationship between actin filaments and microtubules at the initial phase of plant cytokinesis is still unclear. In this study, we focused on the effects of pharmacological disruption of actin filaments on the initial organization of phragmoplast microtubules and examined the dynamics of NACK1, NACK2 and NPK1 as functional markers for phragmoplast microtubules. Our findings reveal possible roles of actin filaments in regulating initial phragmoplast microtubules. Results Disruption of actin filaments caused dispersion of cell plate vesicles To examine the impacts of pharmacological disruption of actin filaments on the ultrastructure of plant cytokinetic apparatus, cell cycle-synchronized tobacco BY-2 cells treated with or without latrunculin B (LatB), which is an actin polymerization inhibitor (Kojo et al. 2013), were observed under a transmission electron microscope (TEM). Cells at the early telophase were identified by the morphology of the daughter nuclei. In the control, cell plate membrane vesicles were clustered and aligned along the equatorial plane (Fig. 1A), whereas in the LatB-treated cells, the clusters of cell plate vesicles were not fully connected and gaps between sub-clusters of cell plate membrane vesicles were observed (Fig. 1B). Although it had been suggested that chemical fixation can affect the contiguous nature of the cell plate by disrupting the membrane structures (Samuels et al. 1995), these TEM images are fully consistent with the results of our previous live cell imaging of cells stained with the fluorescent dye FM4-64, which labels cell plate membranes (Higaki et al. 2008). The disruption of actin filaments by LatB treatment was confirmed by the simplified vacuolar structures and elimination of tubular vacuoles that was observed around the expanding cell plate (Fig. 1B), similar to what was found previously by confocal imaging (Supplementary Fig. S1) (Kutsuna et al. 2003, Higaki et al. 2006). Fig. 1 Open in new tabDownload slide Effects of LatB treatment on the ultrastructure of the cytokinetic apparatus in wild-type tobacco BY-2 cells at the early telophase. Cell cycle-synchronized BY-2 cells treated with dimethyl sulfoxide (DMSO) (A) or 2.5 μM LatB (B) for 60–90 min were observed under a TEM. Middle and right panels show the enlarged image of the insets on the left and middle panels, respectively. Representative images from three independent observations are shown. Note that the clusters of cell plate vesicles were not fully connected in the cells treated with LatB. CP, cell plate membrane; MTs, microtubules; V, vacuole. Scale bars indicate 1 μm. Fig. 1 Open in new tabDownload slide Effects of LatB treatment on the ultrastructure of the cytokinetic apparatus in wild-type tobacco BY-2 cells at the early telophase. Cell cycle-synchronized BY-2 cells treated with dimethyl sulfoxide (DMSO) (A) or 2.5 μM LatB (B) for 60–90 min were observed under a TEM. Middle and right panels show the enlarged image of the insets on the left and middle panels, respectively. Representative images from three independent observations are shown. Note that the clusters of cell plate vesicles were not fully connected in the cells treated with LatB. CP, cell plate membrane; MTs, microtubules; V, vacuole. Scale bars indicate 1 μm. In addition, the LatB-induced dispersed accumulation of cell plate membranes was visualized by green fluorescent protein (GFP)-tagged PATROL1, which is a membrane trafficking factor involving H+-ATPase delivery to plasma membranes (Hashimoto-Sugimoto et al. 2013, Higaki et al. 2014) (Supplementary Fig. S2). PATROL1 has an MUN domain that is structurally similar to vesicle tethering factors including EXOCYST (Hashimoto-Sugimoto et al. 2013), and the GFP fusions localized on the early cell plate membranes (Supplementary Fig. S2). Disruption of actin filaments perturbed midzone constriction of phragmoplast microtubules The TEM observations revealed that the distribution of phragmoplast microtubules also seemed to be affected by LatB treatment because they connected with the sub-clusters of cell plate membrane vesicles (Fig. 1), so next we monitored the formation and expansion of phragmoplast microtubules by live cell imaging. To simultaneously visualize the microtubules and actin filaments, we used transgenic BY-2 cells in which the microtubules and actin filaments were labeled with yellow fluorescent protein (YFP)-TUB and red fluorescent protein (tdTomato)-actin-binding domain of the AtFIM1 fusion protein (tdTomato-ABD2) (BY-YTRF1 cells; Kojo et al. 2013). The transgenic BY-2 cells were stained with FM4-64 and then time-sequentially observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the tdTomato channel in our optical setting, so the tdTomato-ABD2-specific signals were identified by comparison of both channel images. In the control cells, when the FM4-64-labeled cell plate membranes accumulated at the equatorial plane (Fig. 2A, 0 min, arrowhead), phragmoplast microtubules appeared inside the anaphase spindle with midzone constriction (Fig. 2A, 0 min, arrows), as previously reported (Granger and Cyr 2000). At the same time, actin filaments appeared from the periphery of the daughter chromosomes toward the emerging cell plate (Fig. 2A, 0 min, asterisks), as previously reported (Higaki et al. 2008). We confirmed that no major localization of actin filaments was present at the phragmoplast midline by examining cytokinetic BY-2 cells without FM4-64 staining (Supplementary Fig. S3). The midzone constriction of phragmoplast microtubules was eased within 1 min, and then, the microtubules centrifugally expanded to separate the cells (Fig. 2A, 1–10 min). Fig. 2 Open in new tabDownload slide Time-sequential images of YFP-TUB-labeled microtubules, FM4-64-labeled cell plate membranes and tdTomato-ABD2-labeled actin filaments during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the tdTomato channel in our optical setting. Representative images from 10 independent observations are shown. Arrows, arrowheads and asterisks indicate midzones, onsets of accumulation of cell plate membranes and appearance of actin filaments, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Note that the midzone constriction of phragmoplast microtubules did not occur in the cells treated with LatB. Scale bars indicate 10 μm. Fig. 2 Open in new tabDownload slide Time-sequential images of YFP-TUB-labeled microtubules, FM4-64-labeled cell plate membranes and tdTomato-ABD2-labeled actin filaments during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the tdTomato channel in our optical setting. Representative images from 10 independent observations are shown. Arrows, arrowheads and asterisks indicate midzones, onsets of accumulation of cell plate membranes and appearance of actin filaments, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Note that the midzone constriction of phragmoplast microtubules did not occur in the cells treated with LatB. Scale bars indicate 10 μm. In the cells treated with LatB, the cell plate membranes were dispersed and unconnected compared with the control (Fig. 2B, 0 min, arrowhead), which is consistent with our TEM observations (Fig. 1B). The midzone constriction of phragmoplast microtubules did not occur at the early phase of cytokinesis, resulting in splayed phragmoplast microtubules (Fig. 2B, 0 min, arrows). In accordance with expanding phragmoplast microtubules, the cell plate membranes gradually connected and expanded to separate the cells (Fig. 2B, 1–15 min), although cytokinesis was slightly delayed, as previously reported (Higaki et al. 2008). Kymograph analysis along the equatorial planes showed that microtubule midzone constriction and cell plate membrane accumulation occurred simultaneously in the control cells, whereas cell plate membranes dispersedly accumulated without microtubule midzone constriction in the cells treated with LatB (Fig. 3). In addition, our image analysis quantitatively and statistically confirmed that LatB treatment inhibited midzone constriction of phragmoplast microtubules at the onset of cell plate membrane accumulation (Fig. 4 and Supplementary Fig. S4). These results indicate that the initial phragmoplast microtubules with midzone constriction were sensitive to LatB treatment. Fig. 3 Open in new tabDownload slide Time relations of phragmoplast microtubule midzone constriction and cell plate membrane accumulation. (A) A single snapshot from time-sequential images of YFP-TUB-labeled microtubules (yellow) and FM4-64-labeled cell plate membranes (magenta) in BY-2 cells. (B) Kymographs along the a–b axis ([he 2.5-μm wide white line in (A)]. Solid lines indicate the onset time of accumulation of cell plate membranes, which was defined as 0 min. Scale bars indicate 10 μm. Fig. 3 Open in new tabDownload slide Time relations of phragmoplast microtubule midzone constriction and cell plate membrane accumulation. (A) A single snapshot from time-sequential images of YFP-TUB-labeled microtubules (yellow) and FM4-64-labeled cell plate membranes (magenta) in BY-2 cells. (B) Kymographs along the a–b axis ([he 2.5-μm wide white line in (A)]. Solid lines indicate the onset time of accumulation of cell plate membranes, which was defined as 0 min. Scale bars indicate 10 μm. Disruption of actin filaments shortened lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-tagged NACK1 kinesin in equatorial planes To determine the importance of LatB-sensitive microtubule organization in phragmoplast function, we examined the impact of LatB treatment on functional markers for phragmoplast microtubules. We chose GFP-tagged NACK1 and NACK2 kinesins and NPK1 MAPK kinase kinase as functional markers for phragmoplast microtubules because of their cell plate localization and crucial roles in cell plate expansion. We used a microtubule marker TagRFP-TUA6 and the cell plate membrane marker dye FM4-64 to identify localization dynamics of the three markers during cytokinesis under the control condition. Although some of the FM4-64 fluorescence leaked into the TagRFP channel, as was the case for the tdTomato channel (Fig. 2), we were able to confirm the LatB-induced cell plate vesicle dispersion and splayed phragmoplast microtubules (Figs. 5–7; TagRFP-TUA6/FM4-64 and FM4-64). Fig. 4 Open in new tabDownload slide Morphometry of cytokinetic apparatus. (A) Representative images of YFP-TUB-labeled microtubules in cytokinetic apparatus at the onset of cell plate membrane accumulation (corresponds to 0 min in Fig. 2). The control cell (left) was treated with DMSO, and the LatB cell (right) was treated with 2.5 μM LatB for 60–90 min. Width and length are defined as the range occupied by microtubules along the equatorial plane and pole-to-pole distance of the cytokinetic apparatus, respectively. Scale bars indicate 10 μm. The width (B) and length (C) of the cytokinetic apparatus in the cells treated with DMSO or LatB. Data are presented as the mean ± SD (n = 9–10). Significance was determined using the Mann–Whitney’s U-test (*P < 0.01). Fig. 4 Open in new tabDownload slide Morphometry of cytokinetic apparatus. (A) Representative images of YFP-TUB-labeled microtubules in cytokinetic apparatus at the onset of cell plate membrane accumulation (corresponds to 0 min in Fig. 2). The control cell (left) was treated with DMSO, and the LatB cell (right) was treated with 2.5 μM LatB for 60–90 min. Width and length are defined as the range occupied by microtubules along the equatorial plane and pole-to-pole distance of the cytokinetic apparatus, respectively. Scale bars indicate 10 μm. The width (B) and length (C) of the cytokinetic apparatus in the cells treated with DMSO or LatB. Data are presented as the mean ± SD (n = 9–10). Significance was determined using the Mann–Whitney’s U-test (*P < 0.01). Fig. 5 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NACK1 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 10 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NACK1, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. Fig. 5 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NACK1 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 10 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NACK1, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. Fig. 6 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NACK2 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 10 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NACK2, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. Fig. 6 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NACK2 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 10 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NACK2, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. Fig. 7 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NPK1 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 7 to 8 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NPK1, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. Fig. 7 Open in new tabDownload slide Time-sequential images of TagRFP-TUA6-labeled microtubules, FM4-64-labeled cell plate membranes and GFP-NPK1 during cytokinesis. Cell cycle-synchronized cells treated with DMSO (A) or 2.5 μM LatB (B) for 60–90 min were observed under a spinning-disk confocal laser scanning microscope. Some of the FM4-64 fluorescence leaked into the TagRFP channel in our optical setting. Representative images from 7 to 8 independent observations are shown. Arrowheads and arrows indicate the onset of accumulation of cell plate membranes and GFP-NPK1, respectively. The time when the cell plate membranes began to accumulate was defined as 0 min. Scale bars indicate 10 μm. GFP-NACK1 was not detected at the equatorial plane when FM4-64-labeled cell plate membranes appeared (Fig. 5A, 0 min, arrowhead) with midzone constriction of phragmoplast microtubules (Fig. 5A, 0 min). Fluorescence of GFP-NACK1 began to be detected at the cell plate about 3 min after the onset of the accumulation of FM4-64-labeled cell plate membranes (Fig. 5A, 3 min, arrow). For GFP-tagged NACK2, which is a NACK1 homolog, the GFP signal began to be observed at the cell plate about 2 min after the onset of FM4-64 accumulation (Fig. 6A). We also examined NPK1 MAPK kinase kinase, which is activated by direct binding with NACK1 or NACK2 and triggers the MAPK cascade to expand the cell plate (Nishihama et al. 2001, Nishihama et al. 2002). GFP-NPK1 appeared at the cell plate about 1 min after the onset of the FM4-64 accumulation (Fig. 7A). These results suggest that the functional markers sequentially accumulated at the cell plate during the early phase of cytokinesis, in the order GFP-tagged NPK1, NACK2 and NACK1. We also examined the effect of LatB treatment on the localization dynamics of GFP-NACK1, GFP-NACK2 and GFP-NPK1. The effects of LatB were confirmed by splayed phragmoplast microtubules labeled with TagRFP-TUA6 and disconnected cell plate membranes at 0 min (Figs. 5B, 6B, 7B). GFP-NACK1 quickly appeared at the malformed cell plate in the cells treated with LatB (Fig. 5B). Shortening of the lag time between accumulations of FM4-64-labeled cell plate membranes and GFP-NACK1 was quantitatively and statistically confirmed by kymograph analysis (Fig. 8). However, the lag-time shortening was not observed for GFP-NACK2 or GFP-NPK1 (Figs. 6B, 7B, 8C). These results suggest that sensitivity to LatB treatment distinguishes phragmoplast microtubules involved in the cell plate localization of GFP-NACK1 and of GFP-NACK2 and GFP-NPK1. Fig. 8 Open in new tabDownload slide Lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1, GFP-NACK2 and GFP-NPK1. (A) A single snapshot from time-sequential images of FM4-64-labeled cell plate membranes and GFP-NACK1 in BY-2 cells. (B) Kymographs along the a–b axis shown in (A). Solid and dashed lines indicate the onset time of accumulation of cell plate membranes and GFP-NACK1, respectively. Lag time was defined as the time between both onset times. (C) Lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1, GFP-NACK2 and GFP-NPK1. Shortening of the lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1 was observed. Data are presented as the mean ± SD (n = 7–10). Significance was determined using the Mann–Whitney’s U-test (*P < 0.015). Fig. 8 Open in new tabDownload slide Lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1, GFP-NACK2 and GFP-NPK1. (A) A single snapshot from time-sequential images of FM4-64-labeled cell plate membranes and GFP-NACK1 in BY-2 cells. (B) Kymographs along the a–b axis shown in (A). Solid and dashed lines indicate the onset time of accumulation of cell plate membranes and GFP-NACK1, respectively. Lag time was defined as the time between both onset times. (C) Lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1, GFP-NACK2 and GFP-NPK1. Shortening of the lag time between accumulation of FM4-64-labeled cell plate membranes and GFP-NACK1 was observed. Data are presented as the mean ± SD (n = 7–10). Significance was determined using the Mann–Whitney’s U-test (*P < 0.015). Discussion Actin filaments regulated the organization of initial phragmoplast microtubules In this study, we examined the effects of pharmacological disruption of actin filaments on phragmoplast microtubules at the early phase of cytokinesis. We found that LatB caused splayed phragmoplast microtubules at the onset of cell plate membrane accumulation [Figs. 2 (0 min), 3, 4]. Although the possibility that LatB had off-target effects could not be eliminated, this result suggests that the midzone constriction of phragmoplast microtubules depends on actin filaments. The LatB-induced splayed organization of phragmoplast microtubules may be responsible for the unconnected clusters of cell plate membrane vesicles (Figs. 1B, 2B, 0 min, arrowhead). The midzone constriction of initial phragmoplast microtubules seems to be rational for cell plate formation because membrane vesicles can be concentratedly transported to the center of the equatorial plane and effectively fused together (Seguí-Simarro et al. 2004). These results suggest that actin microfilaments contribute to efficient cell plate formation via regulation of phragmoplast microtubule orientation. However, it is still unclear how actin filaments regulate the orientation of initial phragmoplast microtubules. When the midzone constriction of phragmoplast microtubules occurred, actin filaments appeared from the periphery of daughter nuclei toward the emerging cell plate (Higaki et al. 2008) (Fig. 2A, 0 min, asterisks). These actin filaments near the daughter chromosomes/nuclei seem to colocalize with the minus-end regions of the initial phragmoplast microtubules (Fig. 2A, 0 min). This partial colocalization suggests the involvement of interacting proteins between phragmoplast microtubule and actin filaments in the midzone constriction process. A plant-specific minus-end-directed kinesin KinG, which localizes both microtubules and actin filaments, was observed in phragmoplasts in tobacco BY-2 cells (Buschmann et al. 2011, Spiegelman et al. 2018). Such a kinesin linker between actin filaments and microtubules may be a promising candidate key regulator of actin filament-dependent regulation of phragmoplast microtubule orientation. Interactions between actin filaments and microtubules have been reported in cells other than cytokinetic cells. The preprophase band of microtubules, which is believed to determine the cell division site, appears at the G2 phase and narrows during prophase (Mineyuki 1999). The narrowing can be inhibited by the pharmacological disruption of actin filaments, suggesting that actin microfilaments contribute to narrowing of cortical microtubules during preprophase band formation (Mineyuki and Palevitz 1990, Eleftheriou and Palevitz 1992). In addition, the reorganization of cortical microtubules at the M/G1 interphases involves emerging cortical microtubules that elongate from around daughter nuclei toward the end distal to the division plane along with the actin filaments (Hasezawa et al. 1998). Treatment with actin polymerization inhibitors suspended the appearance of the longitudinal cortical microtubules at the early G1 phases (Hasezawa et al. 1998). In cortical microtubule reorientation in response to developmental and environmental cues, pharmacological perturbation of actin filaments inhibited cortical microtubule reorientation in the epidermal cells of azuki (Vigna angularis Ohwi et Ohashi) bean epicotyl segments (Takesue and Shibaoka 1998), root cells in Zea mays L. (Blancaflor 2000), leaf epidermal cells in Allium porrum L. (Sainsbury et al. 2008) and hypocotyl cells in A. thaliana (Sampathkumar et al. 2011). These reports suggested that actin filaments are used for rapid and dynamic changes in microtubule organization at any cell cycle phase, though the key molecules have not been identified. However, the possibility that actin filaments are involved in the dynamics of the cytokinetic vesicles and the appropriate vesicle organization is needed to establish the midzone constriction of phragmoplast microtubules cannot be overlooked. Despite this, it is still important to clarify the molecular basis of the interactions among actin filaments, microtubules and cytokinetic vesicles at the initial phase of plant cytokinesis. LatB-induced acceleration of NACK1 kinesin accumulation suggested two distinct phragmoplast microtubules Although caution is required when comparisons are made among different transgenic cell lines expressing GFP-fusion molecules, our live cell imaging and kymograph-based image analysis suggested that the GFP-tagged functional markers for phragmoplast microtubules sequentially accumulated at the cell plate after the accumulation of FM4-64-labeled cell plate membranes (Figs. 5A, 6A, 7A, 8C), in the order GFP-NPK1, GFP-NACK2 and GFP-NACK1. Interestingly, the lag time between FM4-64 and GFP-NACK1 accumulation was shortened by LatB treatment (Figs. 5B, 8C), whereas the accumulation times of GFP-NACK2 and GFP-NPK1 were insensitive to LatB treatment (Figs. 6B, 7B, 8C). The shortened lag time between FM4-64 and GFP-NACK1 accumulation cannot be explained only by splaying of phragmoplast microtubules. By assuming there are two distinct phragmoplast microtubules, LatB-sensitive initial phragmoplast microtubules that do not associate with NACK1 and later LatB-insensitive phragmoplast microtubules that associate with NACK1, the lag-time shortening can be explained. That is, LatB treatment might skip initial phragmoplast microtubules and accelerate the appearance of later phragmoplast microtubules. Recently, measurements of the diameter of expanding cell plates labeled with GFP-SYP111 (also known as KNOLLE) in tobacco BY-2 cells indicated that two distinct phases, initial plate assembly (IPA) and primary centrifugal growth (PCG), sequentially occurred at early telophase (van Oostende-Triplet et al. 2017). IPA and PCG are regarded as subphases of cell plate biogenesis (Rybak et al. 2014, van Oostende-Triplet et al. 2017, Smertenko et al. 2017). In IPA, membrane vesicles accumulated at equatorial planes and formed the initial cell plate (van Oostende-Triplet et al. 2017) with a diameter of about 5.5-μm diameter that is sustained for about 1 min. In PCG, the cell plate diameter rapidly expanded to about 15.5 μm (van Oostende-Triplet et al. 2017). This model combined with our observations indicates that the actin filament-dependent and NACK1-negative phragmoplast microtubules and actin filament-independent and NACK1-positive phragmoplast microtubules may be responsible for IPA and PCG, respectively. Overexpression of a dominant-negative mutant of NACK1 showed defective cell plate expansion and caused incomplete cell plates but did not result in the absence of cell plates (Nishihama et al. 2002). The incomplete cell plates may have been produced by the initial phragmoplast microtubules in IPA, independently of NACK1. Unlike GFP-NACK1, GFP-NPK1 and GFP-NACK2 were insensitive to the LatB treatment (Figs. 6B, 7B, 8C). Therefore, GFP-NPK1 and GFP-NACK2 might be associated with phragmoplast microtubules in both IPA and PCG. It has been reported that both NACK1 and NACK2 transcripts were specifically accumulated during M phase in synchronized BY-2 cells and both proteins interact with NPK1 in yeast cells (Nishihama et al. 2002). The binding site of NACK1 to NPK1 is highly conserved in NACK2 (Nishihama et al. 2002), suggesting that both NACK1 and NACK2 are responsible for the localization of NPK1 in tobacco cells. Thus, NPK1/NACK2 and NPK1/NACK1 complexes could possibly regulate IPA + PCG and PCG, respectively. Assuming that NPK1 and NACK2 accumulated to the cell plate almost simultaneously with about 1 min lag time and were followed later by NACK1 (Fig. 8C), NACK2 could be considered to be a transporter of NPK1 to cell plate in IPA. In A. thaliana, the cytokinetic defects in atnack1 (hinkel)/atnack2 (tetraspore) double knockout mutants were more prominent than they were in the single mutants, suggesting that the functions of NACK1 and NACK2 are redundant (Tanaka et al. 2004). Both AtNACK1/HINKEL and TETRASPORE/AtNACK2 also interact with ANP1 and ANP3 MAPKKK in yeast cells (Takahashi et al. 2010). It is also possible that they are subfunctionalized for sequential procedures of cell plate formation and expansion. However, if functionally distinct phragmoplast microtubules are spatiotemporally coordinated for cell plate formation and expansion, it is not clear how microtubule-associated proteins, including NACK1 kinesin, recognize them. This could be an important topic for future research to help understand the molecular mechanisms of plant cytokinesis. Materials and Methods Plant materials and cell synchronization Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells were diluted 95-fold with modified Linsmaier and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid at weekly intervals (Kumagai-Sano et al. 2006). The cell suspensions were agitated on a rotary shaker at 130 rpm at 27°C in the dark. For cell synchronization, 3 ml of 7-day-old cells were transferred to 30 m of fresh medium and cultured for 24 h with 5 mg l−1 aphidicolin (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan) (Kumagai-Sano et al. 2006). The cells were washed with 10 volumes of fresh medium and resuspended in the same medium. A transgenic BY-2 cell line stably expressing YFP-TUB and tdTomato-ABD2 under the cauliflower mosaic virus (CaMV) 35S promoter was established as previously described (BY-YTRF1 cells; Kojo et al. 2013). We also established a transgenic BY-2 cell line stably expressing N-terminal GFP fusions with PATROL1 (Hashimoto-Sugimoto et al. 2013, Higaki et al. 2014) under the CaMV 35S promoter using the Agrobacterium tumefaciens-mediated method (An 1985). Full-length cDNA fragments of A. thaliana PATROL1 were subcloned into the binary vector pGWB552 (Nakagawa et al. 2007). PATROL1 cDNA was amplified by PCR with specific primers and cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA) before subcloning into the pGWB552 vector. To monitor the dynamics of microtubules and NACK1, NACK2 and NPK1, we also established transgenic BY-2 cell lines expressing both tagRFP-TUB6 and GFP-NACK1, GFP-NACK2 or GFP-NPK1 by the A. tumefaciens-mediated method (An 1985). To express the GFP constructs in cells, the full-length cDNA fragments of tobacco NACK1, NACK2 and NPK1 were subcloned into the binary vector pER8-GFP:GW (Sasabe et al. 2011, Sasabe et al. 2015). NACK2 cDNA was amplified by PCR with specific primers from the tobacco cDNA library and cloned into the pENTR vector before subcloning into the pER8-GFP:GW vector. All of these constructs were N-terminally GFP-fused. The CaMV 35S promoter was used for tagRFP-TUB6, and an estradiol-inducible promoter was used to express GFP-NACK1, GFP-NACK2 and GFP-NPK1. To express the GFP-tagged cell plate-localized proteins, 0.1 μM 17-β-estradiol (Sigma-Aldrich, St Louis, MO, USA) was added to the cell cultures just after the aphidicolin release (>8 h before observation). All the transgenic cell lines were maintained and synchronized by procedures similar to those used for the wild-type BY-2 cell line. TEM observations To investigate the ultrastructure of the cytokinetic apparatus, we observed cell cycle-synchronized wild-type BY-2 cell under a TEM. The samples were fixed with equal amounts of 4% paraformaldehyde and 4% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) at room temperature and then refrigerated at 4°C to lower the temperature. They were fixed with 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) at 4°C overnight. After fixation, the samples were washed three times with 0.05 M cacodylate buffer for 10 min each and postfixed with 2% osmium tetroxide in 0.05 M cacodylate buffer at 4°C for 2 h. The samples were dehydrated in graded ethanol solutions (50% ethanol for 30 min at 4°C, 70% ethanol for 30 min at 4°C, 90% ethanol for 30 min at room temperature and four changes of 100% ethanol for 30 min each at room temperature). After these dehydration processes, the samples were dehydrated continuously with 100% ethanol at room temperature overnight. Then, the samples were infiltrated with propylene oxide twice for 30 min each, put into a 70:30 mixture of propylene oxide and resin (Quetol-651; Nisshin EM Co., Tokyo, Japan) for 1 h and kept in tubes with the cap open so that the propylene oxide was volatilized overnight. The samples were transferred to fresh 100% resin and polymerized at 60°C for 48 h. The polymerized resins were ultra-thin sectioned at 80 nm with a diamond knife using an ultramicrotome (Ultracut UCT; Leica, Vienna, Austria), and the sections were mounted on copper grids. The sections were stained with 2% uranyl acetate at room temperature for 15 min, washed with distilled water and secondary-stained with lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) at room temperature for 3 min. The grids were observed under a TEM (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Images were taken with a charge-coupled device camera (EM-14830RUBY2; JEOL Ltd.). Confocal microscopy The transgenic BY-2 cells were transferred into φ35-mm Petri dishes with φ14-mm coverslip windows at the bottom (Matsunami, Osaka, Japan). The dishes were placed onto the inverted platform of a fluorescence microscope (IX-70; Olympus, Tokyo, Japan) equipped with a CSU-X1 scanning head (Yokogawa, Tokyo, Japan) and a complementary metal oxide semiconductor camera (Zyla; Andor, Belfast, UK). YFP/GFP/FM4-64 and tdTomato/tagRFP were excited with a 488-nm laser and a 561-nm laser, respectively. Fluorescence was detected through a 510–550-nm band-pass filter for YFP/GFP or a 624–640-nm band-pass filter for FM4-64/tdTomato/tagRFP. Cell plate staining To observe the cell plate, cells were treated with 16.5 μM N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64; Thermo Fisher Scientific, Waltham, MA, USA) for >5 min. Inhibitor To disrupt the actin filaments, cells were treated with 2.5 μM LatB (Fujifilm Wako Pure Chemical Corporation) for 60–90 min before observations. Image analysis To measure the width of the cytokinetic apparatus of the cells in the early phase of cytokinesis, the equatorial plane was defined as the long axis of the fitted ellipse of the binarized FM4-64-labeled cell plate (Supplementary Fig. S4). YFP-TUB images were cropped with regions of the equatorial planes with 1.9-μm width, and then the intensities were averaged in the direction perpendicular to the equatorial plane. The high-intensity regions detected by Otsu’s thresholding were defined, and the width was measured as the width of the cytokinetic apparatus. To measure the lag time between accumulation of FM4-64 and GFP-NACK1, GFP-NACK2 or GFP-NPK1, a kymograph was obtained using a straight line with 3.9-μm width, which was set manually at the center of the cell in the direction perpendicular to the equatorial plane. Starting time of the fluorescence accumulation was detected by the appearance of an intensity peak of intensity profile along the straight line, and the lag time was calculated. All image processing and analyses were performed using ImageJ software (Schneider et al. 2012). Funding Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science [Grant numbers 15H01223 and 17K07432 to M.S., 16H04802 and 24114007 to S.H. and 16H06280, 17K19380 and 18H05492 to T.H.]. Acknowledgments We thank Mr. Keisuke Okubo (The University of Tokyo) and Mr. Ken Sakuma (The University of Tokyo) for technical assistance with the microscopic observations and Ms. Kanako Fujita (The University of Tokyo), Ms. Megumi Takahashi (The University of Tokyo), Ms. Hiroko Shibata (Kumamoto University) and Ms. Hitomi Okada (Kumamoto University) for their support in cell culture maintenance. We thank Margaret Biswas, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript and helping to draft the abstract. Disclosures The authors have no conflicts of interest to declare. References An G. ( 1985 ) High efficiency transformation of cultured tobacco cells . Plant Physiol . 79 : 568 – 570 . Google Scholar Crossref Search ADS PubMed WorldCat Arima K. , Tamaoki D. , Mineyuki Y. , Yasuhara H. , Nakai T. , Shimmen T. , et al. ( 2018 ) 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 . Google Scholar Crossref Search ADS PubMed WorldCat Asada T. , Sonobe S. , Shibaoka H. ( 1991 ) Microtubule translocation in the cytokinetic apparatus of cultured tobacco cells . Nature 350 : 238 – 241 . Google Scholar Crossref Search ADS WorldCat Austin J.R. , Seguí-Simarro J.M. , Staehelin L.A. ( 2005 ) Quantitative analysis of changes in spatial distribution and plus-end geometry of microtubules involved in plant-cell cytokinesis . J. Cell Sci . 118 : 3895 – 3903 . Google Scholar Crossref Search ADS PubMed WorldCat Blancaflor E.B. ( 2000 ) Cortical actin filaments potentially interact with cortical microtubules in regulating polarity of cell expansion in primary roots of maize (Zea mays L.) . J. Plant Growth Regul. 19 : 406 – 414 . Google Scholar Crossref Search ADS PubMed WorldCat Buschmann H. , Green P. , Sambade A. , Doonan J.H. , Lloyd C.W. ( 2011 ) Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium‐mediated transient transformation of tobacco BY‐2 cells . New Phytol . 190 : 258 – 267 . Google Scholar Crossref Search ADS PubMed WorldCat Clayton L. , Lloyd C.W. ( 1985 ) Actin organization during the cell cycle in meristematic plant cells: actin is present in the cytokinetic phragmoplast . Exp. Cell Res . 156 : 231 – 238 . Google Scholar Crossref Search ADS PubMed WorldCat Cleary A.L. , Gunning B.E. , Wasteneys G.O. , Hepler P.K. ( 1992 ) Microtubule and F-actin dynamics at the division site in living Tradescantia stamen hair cells . J. Cell Sci . 103 : 977 – 988 . OpenURL Placeholder Text WorldCat Eleftheriou E.P. , Palevitz B.A. ( 1992 ) The effect of cytochalasin D on preprophase band organization in root tip cells of Allium . J. Cell Sci . 103 : 989 – 998 . OpenURL Placeholder Text WorldCat Granger C.L. , Cyr R.J. ( 2000 ) Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD . Planta 210 : 502 – 509 . Google Scholar Crossref Search ADS PubMed WorldCat Hasezawa S. , Marc J. , Palevitz B.A. ( 1991 ) Microtubule reorganization during the cell cycle in synchronized BY‐2 tobacco suspensions . Cell Motil. Cytoskeleton 18 : 94 – 106 . Google Scholar Crossref Search ADS WorldCat Hasezawa S. , Sano T. , Nagata T. ( 1998 ) The role of microfilaments in the organization and orientation of microtubules during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells . Protoplasma 202 : 105 – 114 . Google Scholar Crossref Search ADS WorldCat Hashimoto-Sugimoto M. , Higaki T. , Yaeno T. , Nagami A. , Irie M. , Fujimi M. , et al. ( 2013 ) A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses . Nat. Commun. 4 : 2215 . Google Scholar Crossref Search ADS PubMed WorldCat Higaki T. , Hashimoto-Sugimoto M. , Akita K. , Iba K. , Hasezawa S. ( 2014 ) Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells . Plant Cell Physiol . 55 : 773 – 780 . Google Scholar Crossref Search ADS PubMed WorldCat Higaki T. , Kutsuna N. , Okubo E. , Sano T. , Hasezawa S. ( 2006 ) Actin microfilaments regulate vacuolar structures and dynamics: dual observation of actin microfilaments and vacuolar membrane in living tobacco BY-2 cells . Plant Cell Physiol . 47 : 839 – 852 . Google Scholar Crossref Search ADS PubMed WorldCat Higaki T. , Kutsuna N. , Sano T. , Hasezawa S. ( 2008 ) Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis . BMC Plant Biol. 8 : 80 . Google Scholar Crossref Search ADS PubMed WorldCat Kakimoto T. , Shibaoka H. ( 1987 ) Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells . Protoplasma 140 : 151 – 156 . Google Scholar Crossref Search ADS WorldCat Kojo K.H. , Higaki T. , Kutsuna N. , Yoshida Y. , Yasuhara H. , Hasezawa S. ( 2013 ) Roles of cortical actin microfilament patterning in division plane orientation in plants . Plant Cell Physiol . 54 : 1491 – 1503 . Google Scholar Crossref Search ADS PubMed WorldCat Kosetsu K. , Matsunaga S. , Nakagami H. , Colcombet J. , Sasabe M. , Soyano T. , et al. ( 2010 ) The MAP kinase MPK4 is required for cytokinesis in Arabidopsis thaliana . Plant Cell 22 : 3778 – 3790 . Google Scholar Crossref Search ADS PubMed WorldCat Kumagai-Sano F. , Hayashi T. , Sano T. , Hasezawa S. ( 2006 ) Cell cycle synchronization of tobacco BY-2 cells . Nat. Protoc. 1 : 2621 – 2627 . Google Scholar Crossref Search ADS PubMed WorldCat Kutsuna N. , Kumagai F. , Sato M.H. , Hasezawa S. ( 2003 ) Three-dimensional reconstruction of tubular structure of vacuolar membrane throughout mitosis in living tobacco cells . Plant Cell Physiol . 44 : 1045 – 1054 . Google Scholar Crossref Search ADS PubMed WorldCat Lloyd C.W. , Traas J.A. ( 1988 ) The role of F-actin in determining the division plane of carrot suspension cells. Drug studies . Development 102 : 211 – 221 . OpenURL Placeholder Text WorldCat Mineyuki Y. ( 1999 ) The preprophase band of microtubules: its function as a cytokinetic apparatus in higher plants . Int. Rev. Cytol . 187 : 1 – 49 . Google Scholar Crossref Search ADS WorldCat Mineyuki Y. , Palevitz B.A. ( 1990 ) Relationship between preprophase band organization, F-actin and the division site in Allium: fluorescence and morphometric studies on cytochalasin-treated cells . J. Cell Sci . 97 : 283 – 295 . OpenURL Placeholder Text WorldCat Molè‐Bajer J. , Bajer A.S. , Inoué S. ( 1988 ) Three‐dimensional localization and redistribution of F‐actin in higher plant mitosis and cell plate formation . Cell Motil. Cytoskeleton 10 : 217 – 228 . Google Scholar Crossref Search ADS PubMed WorldCat Müller S. , Jürgens G. ( 2016 ) Plant cytokinesis—no ring, no constriction but centrifugal construction of the partitioning membrane . Semin. Cell Dev. Biol . 53 : 10 – 18 . Google Scholar Crossref Search ADS PubMed WorldCat Murata T. , Sano T. , Sasabe M. , Nonaka S. , Higashiyama T. , Hasezawa S. , et al. ( 2013 ) Mechanism of microtubule array expansion in the cytokinetic phragmoplast . Nat. Commun. 4 : 1967 . Google Scholar Crossref Search ADS PubMed WorldCat Nakagawa T. , Suzuki T. , Murata S. , Nakamura S. , Hino T. , Maeo K. , et al. ( 2007 ) Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants . Biosci. Biotechnol. Biochem . 71 : 2095 – 2100 . Google Scholar Crossref Search ADS PubMed WorldCat Nishihama R. , Ishikawa M. , Araki S. , Soyano T. , Asada T. , Machida Y. ( 2001 ) The NPK1 mitogen-activated protein kinase kinase kinase is a regulator of cell-plate formation in plant cytokinesis . Genes Dev . 15 : 352 – 363 . Google Scholar Crossref Search ADS PubMed WorldCat Nishihama R. , Soyano T. , Ishikawa M. , Araki S. , Tanaka H. , Asada T. , et al. ( 2002 ) Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex . Cell 109 : 87 – 99 . Google Scholar Crossref Search ADS PubMed WorldCat Palevitz B.A. ( 1987 ) Accumulation of F-actin during cytokinesis in Allium. Correlation with microtubule distribution and the effects of drugs . Protoplasma 141 : 24 – 32 . Google Scholar Crossref Search ADS WorldCat Reichardt I. , Stierhof Y.D. , Mayer U. , Richter S. , Schwarz H. , Schumacher K. , et al. ( 2007 ) Plant cytokinesis requires de novo secretory trafficking but not endocytosis . Curr. Biol . 17 : 2047 – 2053 . Google Scholar Crossref Search ADS PubMed WorldCat Rybak K. , Steiner A. , Synek L. , Klaeger S. , Kulich I. , Facher E. , et al. ( 2014 ) Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes . Dev. Cell 29 : 607 – 620 . Google Scholar Crossref Search ADS PubMed WorldCat Sainsbury F. , Collings D.A. , Mackun K. , Gardiner J. , Harper J.D. , Marc J. ( 2008 ) Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin‐based streaming and partial microtubule‐membrane detachment . Plant J . 56 : 116 – 131 . Google Scholar Crossref Search ADS PubMed WorldCat Sampathkumar A. , Lindeboom J.J. , Debolt S. , Gutierrez R. , Ehrhardt D.W. , Ketelaar T. , et al. ( 2011 ) Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis . Plant Cell 23 : 2302 – 2313 . Google Scholar Crossref Search ADS PubMed WorldCat Samuels A.L. , Giddings T.H. , Staehelin L.A. ( 1995 ) Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants . J. Cell Biol . 130 : 1345 – 1357 . Google Scholar Crossref Search ADS PubMed WorldCat Sano T. , Hayashi T. , Kutsuna N. , Nagata T. , Hasezawa S. ( 2012 ) Role of actin microfilaments in phragmoplast guidance to the cortical division zone . Curr. Top. Plant Biol . 13 : 87 – 94 . OpenURL Placeholder Text WorldCat Sano T. , Higaki T. , Oda Y. , Hayashi T. , Hasezawa S. ( 2005 ) Appearance of actin microfilament ‘twin peaks’ in mitosis and their function in cell plate formation, as visualized in tobacco BY‐2 cells expressing GFP-fimbrin . Plant J . 44 : 595 – 605 . Google Scholar Crossref Search ADS PubMed WorldCat Sasabe M. , Boudolf V. , De Veylder L. , Inzé D. , Genschik P. , Machida Y. ( 2011 ) Phosphorylation of a mitotic kinesin-like protein and a MAPKKK by cyclin-dependent kinases (CDKs) is involved in the transition to cytokinesis in plants . Proc. Natl. Acad. Sci. USA 108 : 17844 – 17849 . Google Scholar Crossref Search ADS WorldCat Sasabe M. , Ishibashi N. , Haruta T. , Minami A. , Kurihara D. , Higashiyama T. , et al. ( 2015 ) The carboxyl-terminal tail of the stalk of Arabidopsis NACK1/HINKEL kinesin is required for its localization to the cell plate formation site . J. Plant Res. 128 : 327 – 336 . Google Scholar Crossref Search ADS PubMed WorldCat Sasabe M. , Soyano T. , Takahashi Y. , Sonobe S. , Igarashi H. , Itoh T.J. , et al. ( 2006 ) Phosphorylation of NtMAP65-1 by a MAP kinase down-regulates its activity of microtubule bundling and stimulates progression of cytokinesis of tobacco cells . Genes Dev . 20 : 1004 – 1014 . Google Scholar Crossref Search ADS PubMed WorldCat Schmit A.C. , Lambert A.M. ( 1987 ) Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis . J. Cell Biol . 105 : 2157 – 2166 . Google Scholar Crossref Search ADS PubMed WorldCat Schmit A.C. , Lambert A.M. ( 1988 ) Plant actin filament and microtubule interactions during anaphase-telophase transition: effects of antagonist drugs . Biol. Cell 64 : 309 – 319 . Google Scholar Crossref Search ADS PubMed WorldCat Schmit A.C. , Lambert A.M. ( 1990 ) Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells . Plant Cell 2 : 129 – 138 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Schneider C.A. , Rasband W.S. , Eliceiri K.W. ( 2012 ) NIH Image to ImageJ: 25 years of image analysis . Nat. Methods 9 : 671 – 675 . Google Scholar Crossref Search ADS PubMed WorldCat Seagull R.W. , Falconer M.M. , Weerdenburg C.A. ( 1987 ) Microfilaments: dynamic arrays in higher plant cells . J. Cell Biol . 104 : 995 – 1004 . Google Scholar Crossref Search ADS PubMed WorldCat Seguí-Simarro J.M. , Austin J.R. , White E.A. , Staehelin L.A. ( 2004 ) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing . Plant Cell 16 : 836 – 856 . Google Scholar Crossref Search ADS PubMed WorldCat Smertenko A. , Assaad F. , Baluška F. , Bezanilla M. , Buschmann H. , Drakakaki G. , et al. ( 2017 ) Plant cytokinesis: terminology for structures and processes . Trends Cell Biol . 27 : 885 – 894 . Google Scholar Crossref Search ADS PubMed WorldCat Smertenko A. , Hewitt S.L. , Jacques C.N. , Kacprzyk R. , Liu Y. , Marcec M.J. , et al. ( 2018 ) Phragmoplast microtubule dynamics—a game of zones . J. Cell Sci. 131 : jcs203331 . Google Scholar Crossref Search ADS PubMed WorldCat Soyano T. , Nishihama R. , Morikiyo K. , Ishikawa M. , Machida Y. ( 2003 ) NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is required for plant cytokinesis . Genes Dev . 17 : 1055 – 1067 . Google Scholar Crossref Search ADS PubMed WorldCat Spiegelman Z. , Lee C.M. , Gallagher K.L. ( 2018 ) KinG is a plant-specific kinesin that regulates both intra- and intercellular movement of SHORT-ROOT . Plant Physiol. 176 : 392 – 405 . Google Scholar Crossref Search ADS PubMed WorldCat Takahashi Y. , Soyano T. , Kosetsu K. , Sasabe M. , Machida Y. ( 2010 ) HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana . Plant Cell Physiol . 51 : 1766 – 1776 . Google Scholar Crossref Search ADS PubMed WorldCat Takesue K. , Shibaoka H. ( 1998 ) The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle . Planta 205 : 539 – 546 . Google Scholar Crossref Search ADS PubMed WorldCat Tanaka H. , Ishikawa M. , Kitamura S. , Takahashi Y. , Soyano T. , Machida C. , et al. ( 2004 ) The AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes, which encode functionally redundant kinesins, are essential for cytokinesis in Arabidopsis . Genes Cells 9 : 1199 – 1211 . Google Scholar Crossref Search ADS PubMed WorldCat Traas J.A. , Doonan J.H. , Rawlins D.J. , Shaw P.J. , Watts J. , Lloyd C.W. ( 1987 ) An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus . J. Cell Biol . 105 : 387 – 395 . Google Scholar Crossref Search ADS PubMed WorldCat Valster A.H. , Pierson E.S. , Valenta R. , Hepler P.K. , Emons A.M.C. ( 1997 ) Probing the plant actin cytoskeleton during cytokinesis and interphase by profilin microinjection . Plant Cell 9 : 1815 – 1824 . Google Scholar Crossref Search ADS PubMed WorldCat van Oostende-Triplet C. , Guillet D. , Triplet T. , Pandzic E. , Wiseman P.W. , Geitmann A. ( 2017 ) Vesicle dynamics during plant cell cytokinesis reveals distinct developmental phases . Plant Physiol. 174 : 1544 – 1558 . Google Scholar Crossref Search ADS PubMed WorldCat Yasuhara H. , Sonobe S. , Shibaoka H. ( 1993 ) Effects of taxol on the development of the cell plate and of the phragmoplast in tobacco BY-2 cells . Plant Cell Physiol . 34 : 21 – 29 . OpenURL Placeholder Text WorldCat Yu M. , Yuan M. , Ren H. ( 2006 ) Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells . Biol. Cell 98 : 295 – 306 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Actin Filament Disruption Alters Phragmoplast Microtubule Dynamics during the Initial Phase of Plant Cytokinesis
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcaa003
DA - 2020-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/actin-filament-disruption-alters-phragmoplast-microtubule-dynamics-cDyhwqjVPO
DP - DeepDyve
ER -