Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis
© Higaki et al; licensee BioMed Central Ltd. 2008
Received: 19 April 2008
Accepted: 17 July 2008
Published: 17 July 2008
Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown.
Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2) fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER) revealed that MFs were involved in ER accumulation in the phragmoplast at the late phase.
By expression of GFP-ABD2 and vital staining with FM4-64, the dynamics of MFs and the cell plate could be followed throughout plant cytokinesis in living cells. Pharmacological treatment and live imaging analysis also allowed us to quantify MF contribution to cell plate expansion during cytokinesis. Our results suggest that MFs play significant roles in cell plate formation and expansion via regulation of endomembrane dynamics.
Eukaryotic cell division is required for the separation of duplicated chromosomes and the subsequent step of physical division of the mother cells into two daughter cells. In plants, the process of chromosomal separation is generally similar to that of animal cells [1, 2], whereas the mechanism of physical cell division, called cytokinesis, differs strikingly between plants and animals. In plants, the cell membrane-enveloped juvenile cell wall, referred to as the cell plate, accumulates between the daughter nuclei in late anaphase, and the cell plate subsequently expands centrifugally so as to divide the parental cell into two [3, 4]. Cell plate formation and expansion are performed by structures that sandwich the developing cell plate, known as the phragmoplast, which is composed of membranes and cytoskeletons.
The cytokinetic membranes are generally thought to originate from Golgi-derived vesicles [3, 5–7]. During cytokinesis, Golgi stacks were seen to accumulate and to become associated with the cell plate in living tobacco BY-2 cells  and in cryopreserved shoot apical meristematic cells of Arabidopsis thaliana . It was recently proposed that endocytic vesicles also participate in cell plate formation , and this was based on the localization of several cell wall and/or plasma membrane (PM) materials in the cell plate; including xyloglucans [11, 12], pectin [10, 12], PM proteins, such as the auxin transport facilitators, PIN1  and PIN2, the water channel, PIP2, the integral PM protein, LTI6b, and the brassinolide receptor, BRI1 , dynamin , clathrin , and the endosomal marker proteins, GNOM-Myc, Ara6 and Ara7 . However, a more recent study demonstrated that endocytosis is not essential for cell plate formation . The role of endocytosis in cell plate formation thus remains controversial.
Microtubules (MTs) are a major component of the phragmoplast, and the MTs and predicted kinesin motor proteins play a crucial role in the transport of cell plate materials [15, 16]. The plus-ends of MTs face the cell plate  and terminate within the cell plate assembly matrix . Recent electron tomographic analysis has revealed, with nano-scale resolution, that the three-dimensional geometry of phragmoplast MTs undergoes dynamic changes .
Actin microfilaments (MFs) are also distributed throughout the phragmoplast and are aligned parallel to the MTs, as reported in endosperm cells of the blood lily, Haemanthus katherinae [19–22], and the kaffir lily, Clivia nobilis , in root cells of onion, Allium cepa [24, 25], in stamen hair cells of spiderwort plants, Tradescantia virginia [26–28], in cultured cells of carrot, Daucus carota , and alfalfa, Medicago sativa , as well as in tobacco BY-2 cells [31, 32]. Electron microscopy-based, nano-scale structures of phragmoplast-related MFs have not yet been reported, mainly because of technical limitations . Numerous studies have shown that, unlike MT inhibitors, actin inhibitors could not inhibit the initiation of the cell plate [27, 33–37]. However, an actin polymerization inhibitor was found to delay cell plate expansion in Haemanthus katherinae endosperm cells . Microinjection of profilin, a monomeric actin binding protein, into dividing Tradescantia stamen hair cells was found to disrupt phragmoplast-associated MFs and to inhibit cell plate expansion . Furthermore, treatment with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase, or with ML-7, a specific inhibitor of myosin light-chain kinase, could delay the complete fusion of the cell plate with the parental cell wall . Based on these results, MFs have been postulated to contribute to cell plate expansion at the late phase of cytokinesis. However, the mode of MF contribution to plant cytokinesis still remains unclear.
In this study, we have quantitatively estimated the time-sequential changes in MF contribution to cell plate development using live cell image analysis of tobacco BY-2 cell lines. In addition, we examined the relationship between MFs and endomembrane dynamics, including the endosomes and endoplasmic reticulum (ER). Our results detail new features of MFs in plant cytokinesis via regulation of the endomembrane system.
Dynamic changes in MF localization during plant cytokinesis
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 , 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 [39–41]. In addition, FM4-64 localizes to the cell plate membranes of Arabidopsis root cells [10, 42] and tobacco BY-2 cells [40, 43], in agreement with the previously suggested involvement of the endocytic pathway in membrane trafficking to the cell plate .
At metaphase, no prominent MFs or FM4-64 fluorescence was observed in the mitotic apparatus (Fig. 1A, 0 min), whereas cortical MF twin peaks  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 . 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).
Quantitative analysis of the effects of an actin polymerization inhibitor on cell plate expansion
Cell plate diameter and estimated cell plate area in tobacco BY-2 cells during cytokinesis.
Time after initiation of cell plate formation (min)
ΔArea (μm2 min-1)
ΔArea (μm2 min-1)
6.9 ± 0.3
37.9 ± 3.4
37.9 ± 3.6
9.5 ± 0.5
72.2 ± 7.2
34.3 ± 6.2
12.2 ± 0.5
119.2 ± 10.0
47.0 ± 6.6
11.2 ± 0.4
99.9 ± 8.2
14.7 ± 0.6
172.1 ± 12.7
52.9 ± 5.2
13.7 ± 0.5
149.4 ± 11.9
49.5 ± 8.1
17.0 ± 0.6
229.3 ± 16.9
57.2 ± 5.6
15.3 ± 0.6
187.5 ± 16.2
38.2 ± 9.4
18.6 ± 0.6
274.0 ± 18.0
44.8 ± 6.1
16.5 ± 0.8
219.0 ± 20.0
31.5 ± 8.1
19.9 ± 0.6
313.5 ± 18.6
39.5 ± 4.6
17.7 ± 0.8
251.0 ± 23.0
31.9 ± 4.6
21.1 ± 0.7
352.5 ± 22.9
39.0 ± 7.8
18.4 ± 0.8
270.3 ± 23.4
19.3 ± 3.9
22.1 ± 0.9
389.2 ± 29.3
36.7 ± 7.4
18.9 ± 0.8
287.2 ± 24.7
16.9 ± 4.7
22.8 ± 1.0
427.3 ± 24.6
38.1 ± 7.7
19.6 ± 0.8
306.5 ± 27.1
19.3 ± 5.0
20.0 ± 0.9
321.0 ± 28.6
14.5 ± 3.0
20.3 ± 0.9
330.1 ± 29.0
9.1 ± 1.9
20.7 ± 0.9
342.0 ± 33.6
11.9 ± 3.0
21.1 ± 1.0
359.1 ± 32.1
17.1 ± 6.0
21.7 ± 0.9
376.4 ± 32.5
17.3 ± 7.5
21.9 ± 0.9
383.4 ± 34.3
7.0 ± 3.4
where a, b and n are constants. In this model, the expansion rate, a-bt n , 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-bt n 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).
MF-disruption-induced a temporary malformation of the emerging cell plate
Actin-myosin-dependent endosomal movement around the cell plate
MF-dependent accumulation of ER in the phragmoplast
In this study, we have quantitatively examined the temporary changes in MF contribution to cell plate expansion. To visualize the MFs and cell plate, we used GFP-ABD2 expression and FM4-64 vital staining, respectively. Although GFP-ABD2 is recognized as a more reliable MF-marker than GFP-mouse talin (GFP-mTn) [37, 52–54], recent studies have shown that overexpression of GFP-ABD2 can cause growth inhibition of pollen tubes  and organelle motility , suggesting the need for care and attention in the use of such GFP markers. Our BY-GF11 cell line shows a normal growth rate compared with wild-type BY-2 cells (data not shown) and appears to have minimal side effects. Furthermore, FM4-64, which has been widely used as an endocytosis marker, was found to induce abnormal membrane systems in fungal cells . However, FM4-64 cytotoxicity appears limited in plant cells  and, at least under our experimental conditions , abnormal membrane structures or an inhibition of cell division in the BY-2 cells were not observed. These labeling techniques, which cause minimal cell damage, have allowed us to study MF-dynamics and their roles in cell plate development.
MFs contribute to cell plate expansion in late expansion phase, but even in early initiation phase
It is widely accepted that there are two stages in plant cytokinesis that can be distinguished by cell morphological features and sensitivity to caffeine . 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 , which also inhibits callose deposition and the organization of phragmoplast MTs .
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 . Similar cell plate inhibition has been reported in profilin-microinjected Tradescantia virginiana stamen hair cells , and in Tradescantia stamen hair cells treated with myosin inhibitors . 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 . BA-induced cell plate malformation is reminiscent of the mini-phragmoplasts, which appear during Arabidopsis endosperm cellularization [59, 60] and in post-meiotic cytokinesis during pollen development . 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 . 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.
Actin-myosin system is possibly involved in plant cytokinesis via endosomal dynamics
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 [13, 62–64], Zea mays [65, 66], Medicago truncatula  and pollen tubes of Picea meyeri . Endosome association with MFs was observed in cytokinetic BY-2 cells (Fig. 6L, M and 6N) as well as in root hair cells . 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  and in FM4-64-labelled endosomes of Nicotiana benthamiana leaves . Although the endocytic pathway might participate in membrane trafficking to the cell plate , it is not essential for completion of cytokinesis . Indeed, as Golgi-derived vesicles transported by MTs and unidentified kinesins are necessary and sufficient for cell plate development [7, 15], 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 . 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.
MFs regulate ER-accumulation in the phragmoplast at late expansion phase
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 [6, 71, 72]. Recent studies on the expression of ER-targeted fluorescence proteins have further described the dynamic behavior of the ER in living cells [8, 51]. Generally, the complex cortical ER-network structures are found to depend on MFs in plant cells [73, 74] but on MTs in animal cells [75, 76]. 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 , and in tobacco NT-1 cells . 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 .
The ER has been implicated in the control of the cytosolic calcium ion concentrations  and in facilitation of membrane exchange between the ER and cell plate during cell plate maturation . 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.
Plant material and cell synchronization
Tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) cells and BY-GF11 cells expressing GFP-ABD2  were diluted 95-fold with a modified Linsmaier and Skoog medium at weekly intervals . 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) . 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).
General Image analysis
Indirect quantification of fluorescence levels, maximum intensity projections and kymograph construction were performed using ImageJ software . 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.
Time-sequential measurements of cell plate diameter
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).
Assessment of cell plate morphology
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) .
Analysis of endosomal movement
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.
actin binding domain 2
green fluorescent protein
The authors thank Prof. Ikuko Hara-Nishimura and Mr. Atsushi J Nagano of Kyoto University for transgenic BY-2 cells expressing SP-GFP-HDEL and advice on its use. We acknowledge Nico Stuurman of the University of California for developing Mtrack2, an ImageJ plug-in for particle tracking. The authors also thank Ms. Tomomi Hayashi and Ms. Atsuko Era of The University of Tokyo for preliminary observations of the cell plate and endoplasmic reticulum. This work was supported by a Scientific Research Grant from the Japan Society for the Promotion of Science to TH (No. 04392), a Grant-in-Aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, Culture, Sports and Technology to SH (Nos. 19039008 and 20061008) and Institute for Bioinformatics Research and Development (BIRD) from Japan Science and Technology Agency to SH.
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