To simultaneously observe MFs and the cell plate, tobacco BY-GF11 cells, in which MFs were visualized by expression of a GFP-ABD2 fusion protein 37, were stained with the styryl dye, FM4-64. After pulse-labeling with this endocytic tracer, the fluorescence moves from the PM to endosomes, and finally reaches the vacuolar membranes 394041. In addition, FM4-64 localizes to the cell plate membranes of Arabidopsis root cells 1042 and tobacco BY-2 cells 4043, in agreement with the previously suggested involvement of the endocytic pathway in membrane trafficking to the cell plate 10.

From 5 min to 3 h after pulse-labeling with 16 μM FM4-64, we observed that the fluorescence became localized mainly in the cell plate membranes and endosomes of the cytokinetic BY-GF11 cells (Fig. 1A), similar to that of wild type tobacco BY-2 cells (data not shown). At this time, there was still a slight remnant of fluorescence in the PM but not in the vacuolar membrane. For cell plate observations, all results presented in this report were, unless otherwise indicated, from those conducted 2 h after pulse-labeling.

At metaphase, no prominent MFs or FM4-64 fluorescence was observed in the mitotic apparatus (Fig. 1A, 0 min), whereas cortical MF twin peaks 37 were evident (Fig. 1A, 0 min, asterisks). From anaphase to telophase, the FM4-64 fluorescence gradually accumulated at the equatorial region, and the cell plate subsequently emerged (Fig. 1A, 4–6 min) as previously reported 10. Concomitantly, there was a gradual accumulation of MFs from the periphery of the daughter nuclei towards the emerging cell plate (Fig. 1A, 4–6 min). At telophase, the MFs became localized near the expanding cell plate (Fig. 1A, 8–13 min). Measurements of fluorescent intensity around the division plane illustrate the accumulation of MFs in parallel with development of the FM4-64-labelled cell plate (Fig. 1B). To evaluate the spatial changes in MFs, a kymograph was constructed by tracking the fluorescence across the developing cell plate (Fig. 1C). The kymograph suggests that the MFs gradually approached the cell plate (Fig. 1C, yellow broken lines).

To examine the structural relationship between MFs and cell plate during cytokinesis, the GFP-ABD2 and FM4-64 fluorescent intensities were measured along the cell plate at early (Fig. 2A, line segment PQ) and late (Fig. 2F, line segment RS) telophase. GFP-ABD2 was detected all along the equatorial plane, but its intensity was lower than that of FM4-64 in the cell plate at early telophase (Fig. 2B). In contrast, the intensity of GFP-ABD2 was high, especially at the edges of the cell plate, at late telophase (Fig. 2G). These results indicate that only a few MFs were localized in the mitotic apparatus at early telophase, but that they accumulated around the expanding cell plate at late telophase. A three-dimensional cross-sectional image, reconstructed from serial optical sections, confirmed these localization patterns at early (Fig. 2C, D and 2E) and late (Fig. 2H, I and 2J) telophase. These results suggest that MFs undergo dynamic changes in their arrangement during cell plate development.

As mentioned above, vital staining with FM4-64 allowed us to follow chromosomal separation (Fig. 3A, -1-0 min), the gradual accumulation of cell plate vesicles (Fig. 3A, 1–2 min), cell plate expansion (Fig. 3A, 3–9 min), and final division of the parental cell (Fig. 3A, 12 min). To study the role of MFs in cell plate development, these sequential events were monitored under MF-disrupted conditions induced by pre-treatment with the dimeric macrolide, bistheonellide A (BA), an inhibitor of actin polymerization 44. Treatment with 1 μM BA for 1 h almost completely destroyed the MF networks and resulted in the dispersion of free GFP-ABD2 fluorescence in the cytoplasm (Fig. 3B). A 1-h pre-treatment with 1 μM BA caused a temporary malformation of the emerging cell plate, which thus appeared to be discontinuous (as described further below; Fig. 3C, 2 min), and also increased the time needed for the expanding cell plate to complete its fusion with the parental cell wall independently of cell size (Fig. 3D). These results suggest that MFs partially contribute to cell plate formation and expansion.

To study the time-dependency of MF contribution to cell plate expansion, we sequentially measured the cell plate diameters from time-lapse images. Measurements of cell plate diameter and estimations of cell plate area showed that the expansion rate decreased with time in BA-treated cells (Table 1, BA treatment), whereas the rate was roughly constant in control cells (Table 1, control). These results suggest that the contribution of MFs to cell plate expansion increases with the progress of cytokinesis.

To quantitatively estimate this contribution of MFs, we used a simple mathematical model for cell plate expansion, based on the well-known concept of a constant increase in cell plate area 45. In turn, the diameter increased with the square root of time, as follows:

D = a t MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGaemiraqKaeyypa0Jaemyyae2aaOaaaeaacqWG0baDaSqabaaaaa@3111@

where D is the cell plate diameter, a is the expansion rate, and t is time. The time-lapse data of the diameter fit equation 1 well for the control cells (Fig. 4A, control) but not for the BA-treated cells (Fig. 4A, BA treatment), as demonstrated by the chi-square goodness of fit test (Fig. 4C, model 1). This result suggests that disruption of MFs affected the constancy of cell plate expansion.

Consequently, we developed a new model equation as given by:

D = ( a − b t n ) t MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGaemiraqKaeyypa0JaeiikaGIaemyyaeMaeyOeI0IaemOyaiMaemiDaq3aaWbaaSqabeaacqWGUbGBaaGccqGGPaqkdaGcaaqaaiabdsha0bWcbeaaaaa@380A@

where a, b and n are constants. In this model, the expansion rate, a-btⁿ, gradually slows if both b and n are positive. In BA-treated cells, the time-lapse data precisely fit this model 2 (Fig. 4B, BA treatment, Fig. 4C). The mean values of a, b and n were 6.77, 0.01 and 1.81, respectively (Fig. 4B, BA treatment). These results indicate that the expansion rate was reduced in BA-treated cells and suggest that MFs make a gradual contribution to cell plate expansion.

However, to compare the contributions of MFs to cell plate expansion in control and BA-treated cells, both data sets need to be applied to the same model equation. When we applied time-lapse data from control cells to model equation 2, n became underspecified because b came close to 0 (data not shown). We, therefore, assumed that n is invariant at 1.81, even in the control cells, and applied the control data to model equation 2 (Fig. 4B, control). Consequently, the control data precisely fit model 2 as well as model 1 (Fig. 4C). The mean values of a and b were 7.61 and 0.006, respectively (Fig. 4B, control). Simulating the expansion rate of the cell plate with model equation 2 quantitatively estimated the changes in the expansion rate (i.e. a-btⁿ in model equation 2) during cytokinesis in control and BA-treated cells (Fig. 4D). Finally, the contribution of MFs to cell plate expansion could be time-sequentially estimated by the differences between expansion rates in control and BA-treated cells (Fig. 4E). The contribution of MFs to cell plate expansion was found to be about 10% just after cell plate formation and gradually increased up to about 25% at the end of cytokinesis (Fig. 4E).

Interestingly, a 1 h pre-treatment with 1 μM BA caused malformation of the emerging cell plate, which thus appeared discontinuous (Fig. 3C, 2 min). This aberration was only temporary, in that it could be observed for a maximum of only 1–2 min (Fig. 3C, 2 min), after which the blank areas of the cell plate were filled in so that it attained a normal appearance (Fig. 3C, 3–5 min). Cross-sectional images revealed abnormal structures with several holes (Fig. 5A, upper panels). To assess their morphology, we extracted the cell plate area from the cross-sections (Fig. 5A, lower panels), and measured their area, perimeter and complexity using ImageJ software 46. Complexity was defined as a morphological parameter and calculable from the area and perimeter (see Methods) 47. A circle has the lowest value, 1.0, of complexity and, as the structure becomes more complicated, the complexity reaches appropriately higher values 47. The mean value of complexity of cell plates in BA-treated cells was higher than that of control cells (Fig. 5B), whereas their mean areas did not differ significantly (Fig. 5C). In addition, aniline blue staining revealed that the deformed cell plate contained callose (Fig. 5D) as in the control cells (data not shown). These results suggest that MFs modulated cell plate formation/expansion at the early phase of cytokinesis, but did not affect callose synthesis.

To investigate the interactive mechanisms of MFs and cell plate formation/expansion, the relationship between MFs and endosomes, which have been implicated in cell plate development 10, was examined. In a control cell at the late phase, there was active movement of numerous endosomes (Fig. 6C and 6F) around the edge of the expanding cell plate (Fig. 6A). Some endosomes were found to interact with the edge of the expanding cell plate (Fig. 6B). To examine the role of actin-myosin systems on endosomal movement around the cell plate, we treated the cells with BA or 2,3-butanedion monoxime (BDM), a general myosin ATPase inhibitor 4849. The movements were discernibly inhibited by BA (Fig. 6D and 6G) and BDM pre-treatments (Fig. 6E and 6H). These inhibitory effects were confirmed statistically by measuring the velocity of the endosomal movements (Fig. 6I, J and 6K). Dual observations of MFs and endosomes showed that the endosomes moved towards the cell plate along the MFs (Fig. 6L, M and 6N). These results suggest that endosomal movement around the edge of the expanding cell plate depends on the actin-myosin systems.

To further test the involvement of MFs in endomembrane dynamics during cytokinesis, we analyzed the effects of BA treatment on ER-organization during cytokinesis in tobacco BY-2 cells expressing SP-GFP-HDEL, which is retained within the ER 50. ER accumulated in the spindle poles in meta-/anaphase (Fig. 7A, 0–3 min), sandwiched the cell plate in telophase (Fig. 7A, 6–21 min), and accumulated in the phragmoplast in late telophase (Fig. 7A, 18–21 min, yellow arrowheads), as previously reported 6851. A 1 h BA pre-treatment did not induce marked changes in ER-organization at metaphase (Fig. 7C, 0 min), but did inhibit ER-accumulation in the phragmoplast at late telophase (Fig. 7C, 48–63 min, yellow arrows). Three-dimensional images also showed a significant decrease in the amount of ER in the phragmoplast (Fig. 7B and 7D). In addition, time-sequential measurements of fluorescent intensity around the division plane illustrated the inhibition of ER-accumulation in the phragmoplast (Fig. 7E).

It is widely accepted that there are two stages in plant cytokinesis that can be distinguished by cell morphological features and sensitivity to caffeine 28. The first stage is the early initiation phase, which starts immediately after chromosomal separation and involves the deposition and early expansion of the cell plate until it reaches the width of the daughter nuclei. The second stage is the late expansion phase that involves growth of the cell plate beyond the width of the nuclei until it reaches and fuses with the parental cell wall. The late expansion phase is completely inhibited by caffeine 28, which also inhibits callose deposition and the organization of phragmoplast MTs 58.

MFs are thought to be also involved in the late expansion phase of the cell plate. In cytochachalasin-treated Haemanthus katherinae endosperm cells, the cell plate often remains incomplete 20. Similar cell plate inhibition has been reported in profilin-microinjected Tradescantia virginiana stamen hair cells 28, and in Tradescantia stamen hair cells treated with myosin inhibitors 38. In this study, the increase in MF contribution to the late expansion phase of the cell plate, which was confirmed by time-sequential measurements and data fitting analysis (Fig. 4), concurs with previous reports and also with the gradual accumulation of MFs near the cell plate found in this study (Fig. 1C). On the other hand, our results revealed that MF-disruption decreased the expansion rate by about 10%, even just after cell plate formation, but by about 25% at the end of cytokinesis (Fig. 4E) and, in addition, caused malformation of the emerging cell plate (Fig. 5). These results suggest that MFs not only facilitate cell plate expansion in the late expansion phase but also in the early initiation phase.

Furthermore, MF-disruption-induced malformation of the cell plate suggests that MFs regulate the early disc-like morphology of the cell plate. In the early phase, the MFs had not yet accumulated near the cell plate, but a thin, widely-spread MF-meshwork could be observed in the mitotic apparatus and near the cell plate (Fig. 1A, Fig. 2A–E). The existence of MFs near the cell plate in the early phase was also observed by rhodamine-phalloidin staining of fixed cells of onion Allium cepa 24. BA-induced cell plate malformation is reminiscent of the mini-phragmoplasts, which appear during Arabidopsis endosperm cellularization 5960 and in post-meiotic cytokinesis during pollen development 61. Mini-phragmoplasts consist of 2 to12 overlapping MT clusters, assembled between duplicated nuclei, and spaced on average 0.6 μm apart from each other across a given division plane 61. BA-induced cell plate blank regions were approximately of the same size (Fig. 5A, BA treatment). The fact that similar structures occur by MF-disruption suggests that MFs are involved in the spread and flattening of the cell plate at the early phase.

In this study, we found MF-disruption had parallel, aberrant effects on cell plate development and endosomal movement around the expanding cell plate (Fig. 6D, G and 6J). Treatment with the general myosin inhibitor, BDM, revealed that endosomal movements depended on the actin-myosin system (Fig. 6E, H and 6K). MF-dependent endosomal dynamics were also reported in root cells of Arabidopsis thaliana 13626364, Zea mays 6566, Medicago truncatula 67 and pollen tubes of Picea meyeri 68. Endosome association with MFs was observed in cytokinetic BY-2 cells (Fig. 6L, M and 6N) as well as in root hair cells 67. From these results, we hypothesize that the actin-myosin systems are partially involved in endocytic membrane traffic to the cell plate. This notion is supported by recent studies demonstrating that the plant myosin class VIII, ATM1, is localized in the expanding cell plate of tobacco BY-2 cells 69 and in FM4-64-labelled endosomes of Nicotiana benthamiana leaves 70. Although the endocytic pathway might participate in membrane trafficking to the cell plate 10, it is not essential for completion of cytokinesis 7. Indeed, as Golgi-derived vesicles transported by MTs and unidentified kinesins are necessary and sufficient for cell plate development 715, the contribution of MFs to cell plate formation and expansion may be rather limited. Unfortunately, we could not examine the effects of BDM on cell plate expansion, because BDM-pretreatment inhibited cell division in the BY-2 cells (data not shown), possibly through their side effects on MTs 48. Future studies, using combinations of genetic inhibition of myosin activity and time-sequential morphometry as shown in this study, will reveal the changes in contribution of myosins to cell plate expansion.

The ER is one of the first organelles to be visualized during plant cytokinesis. Numerous electron microscopic studies have shown that the tubular ER is first arranged parallel to the spindle MTs in metaphase, and then gradually accumulates near the cell plate and phragmoplast region 67172. Recent studies on the expression of ER-targeted fluorescence proteins have further described the dynamic behavior of the ER in living cells 851. Generally, the complex cortical ER-network structures are found to depend on MFs in plant cells 7374 but on MTs in animal cells 7576. On the other hand, ER-organization in mitotic spindles is associated mainly with MTs rather than MFs in mitotic cells of the gymnosperms, Pinus brutia and Pinus nigra 77, and in tobacco NT-1 cells 51. Our observation that the tubular ER aligned with mitotic spindles in BA-treated cells (Fig. 7C, 0 min) supports these earlier reports. In addition, we found that BA treatment inhibited ER-accumulation in the phragmoplast, especially in the late expansion phase (Fig. 7C, 48–63 min, D and E), suggesting that MFs are tightly associated with ER recruitment in the phragmoplast. In dividing tobacco mesophyll protoplasts, an unbiased ER redistribution was also found to be dependent on MFs 78.

The ER has been implicated in the control of the cytosolic calcium ion concentrations 79 and in facilitation of membrane exchange between the ER and cell plate during cell plate maturation 6. Therefore, it is likely that MFs contribute to cell plate expansion via recruitment of ER into the phragmoplast, and this is supported by the observed increase in MF contribution at the late phase. Taken together with the results from endosomal dynamics, these findings thus imply that MFs have multiple functions in the endomembrane systems during cytokinesis. Future studies will need to focus on molecular dissection of the complicated interactions between MFs and endomembranes to gain further insights into MF contribution to plant cytokinesis.

Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells and BY-GF11 cells expressing GFP-ABD2 37 were diluted 95-fold with a modified Linsmaier and Skoog medium at weekly intervals 80. The cell suspensions were agitated on a rotary shaker at 130 rpm at 27°C in the dark. For cell synchronization, 10 ml of 7-day-old cells were transferred to 95 ml fresh medium and cultured for 24 h with 5 mg liter^-1 aphidicolin (Sigma Chemical Co., St. Louis, MO, USA) 81. The cells were washed with 10 volumes of fresh medium and resuspended in the same medium.

The 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) equipped with a CSU10 scanning head (Yokogawa, Tokyo, Japan) and a cooled CCD camera head system (CoolSNAP HQ, PhotoMetrics). For multifocal observations, serial optical sections were taken at 0.5-μm intervals.

To observe the cell plate and endosomes, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64; Molecular Probes, Invitrogen) was added to the cell suspension at a final concentration of 16 μM. The cells were incubated for 2 min, washed with fresh culture medium, and then monitored. To simultaneously stain the cell plate and callose, the FM4-64-pre-stained cells were fixed with 3.7% (w/v) formaldehyde dissolved in PIPES buffer (pH 6.8) containing 1 mM MgSO₄, 5 mM EGTA and 1% glycerol (PMEG) for 1 h, and then stained with 0.05% aniline blue (Biosupplies Australia, Parkville, Victoria, Australia) in PMEG for 30 min.

For disruption of MFs, cells were treated with 1 μM bistheonellide A (BA, Wako Pure Chemical Ind., Osaka, Japan) for 60 min before observations. Myosin activity was inhibited by treatment with 20 mM BDM (Sigma Chemical Co., St. Louis, MO, USA).

Indirect quantification of fluorescence levels, maximum intensity projections and kymograph construction were performed using ImageJ software 46. Kymographs were constructed with our in-house developed plug-in package for ImageJ software, which is freely available from our website http://hasezawa.ib.k.u-tokyo.ac.jp/zp/Kbi/ImageJKbiPlugins.

To measure the diameter of an expanding cell plate, a binary image was obtained by intensity thresholding and used for measurements on ImageJ. A Gaussian filter was applied to the cross-sectional images before binarization. Cell plate diameters were calculated with ImageJ software. Regression analysis of the cell plate diameter was performed using Kaleida-graph software (Synergy Software, Reading, PA).

To assess the cell plate structures, we first obtained cross-sectional images from 70 serial optical sections taken at 0.5 μm intervals of each cell with view orthogonal planes function in MetaMorph image-analysis software (Universal Imaging Co., Downingtown, Panama). Subsequently, we obtained the binary images from the cross-sectional images by intensity thresholding using ImageJ software as shown in Figure 5A. To improve the signal to noise ratio, a Gaussian filter was applied to the cross-sectional images before binarization. Cell plate area and perimeter were calculated from the binary images with ImageJ software. Complexity, a secondary morphological parameter, was calculated by P²/4πA, where P is the perimeter and A is the area, according to Tanaka et al. (2007) 47.

To estimate the velocity of endosomal movement, we performed time-sequential observations at 1-sec intervals for 30 sec by spinning-disc confocal laser microscopy. From the time-sequential images, we measured the distance and time traveled by endosomes using the ImageJ plug-in MTrack2, which was developed for multiple particle detection and tracking, and which is freely available from the website http://valelab.ucsf.edu/~nico/IJplugins/MTrack2.html. Subsequently, endosomal velocity was calculated by dividing the distance traveled by the traveling time.