507

Expansion of the phragmoplast during plant cytokinesis:
a MAPK pathway may MAP it out
Ryuichi Nishihama* and Yasunori Machida†
Plant cytokinesis involves the formation of a cell plate. This isaccomplished with the help of the phragmoplast, a plantspecific cytokinetic apparatus that consists of microtubules
and microfilaments. During centrifugal growth of the cell plate,the phragmoplast expands to keep its microtubules at theleading edge of the cell plate. Recent studies have revealed
potential regulators of phragmoplast microtubule dynamics
and the involvement of a mitogen-activated protein kinasecascade in the control of phragmoplast expansion. Thesestudies provide new insights into the molecular mechanismsof plant cytokinesis.
Addresses*Department of Biology, University of North Carolina, Chapel Hill,
North Carolina 27599-3280, USA† Developmental Biology Group, Division of Biological Science,
Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan; e-mail: yas@biol1.bio.nagoya-u.ac.jp
Correspondence: Yasunori Machida
Current Opinion in Plant Biology 2001, 4:507–512
1369-5266/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
Ase1
Anaphase spindle elongation1
AtPAKRP1
Arabidopsis thaliana phragmoplast-associatedkinesin-related protein 1
BFA
brefeldin A
BY-2
bright yellow-2
CDK
cyclin-dependent kinase
DcKRP120-2 Daucus carota kinesin-related protein of 120 kDa-2
FTN
fusion-tube-generated membrane network
KLP
kinesin-like MT-based motor protein
MAP
MT-associated protein
MAPK
mitogen-activated protein kinase
MAPKK
MAPK kinase
MAPKKK
MAPKK kinaseMTmicrotubule
NPK1
nucleus- and phragmoplast-localized protein kinase1
Ntf6
Nicotiana tabacum MAPK 6
NtMAP65-1
Nicotiana tabacum MAP65-1
PRC1
Protein Regulator of Cytokinesis1
TKRP125
tobacco kinesin-related polypeptide of 125 kDa
TNtubular network
TVN
tubulo-vesicular network

Introduction
Cytokinesis is a physical process that distributes genetic
information and cytoplasm from a parent cell into two
daughter cells. In higher plants, cytokinesis is achieved
through the formation of a new cross-wall, called the cell
plate, which stretches from the interior to the periphery of
the cell [1]. Materials for the construction of the cell plate
are supplied from the Golgi complex through the vesicle
trafficking system [2,3•,4•]. Membrane vesicles that havebudded from the Golgi fuse with one another to form an
immature cell plate between the two daughter nuclei. The

details of cell-plate maturation have been revealed throughthe observation of cryofixed tobacco cells by Samuels et al.
[5] (Figure 1a). Vesicles fuse via long thin (i.e. 20 nm indiameter) curving fusion tubes to produce a fusion-tubegenerated membrane network (FTN). This network
undergoes a series of morphological and biochemicalchanges, including the formation of a tubulo-vesicular
network (TVN), a tubular network (TN), and a fenestrated
membrane sheet. The cell plate grows centrifugally,
through the continuous addition of vesicles to its edge, until
it reaches the parental cell walls. Therefore, the center ofthe growing cell plate is the most mature part.
The apparatus responsible for localization and fusion ofvesicles is the phragmoplast, whose formation is initiatedduring late anaphase [1]. A phragmoplast complex is composed of two bundles of anti-parallel microtubules (MTs)and actin filaments. MTs are, in general, orientated andconsist of a plus end and a minus end. The MTs overlap attheir plus ends in the center of the phragmoplast. The
initial shape of the phragmoplast is a cylinder- or barrellike structure. Golgi-derived vesicles are transported to theequator of the phragmoplast by the MTs, whose action isprobably assisted by MT motor proteins that have yet tobe identified [6]. Once cell-plate formation begins in its
equatorial zone, the phragmoplast changes into a ring-likestructure and centrifugally expands, maintaining localization of the MTs at the leading edge of the cell plate. Thischange in phragmoplast shape is crucial for the lateral
growth of the cell plate. This review focuses on the mechanism of lateral expansion of the phragmoplast andencompasses recent findings that shed light on the regulation of this process. For aspects of cytokinesis not coveredin this review, such as mechanisms of vesicle fusion, rolesof actin filaments, and control of the division plane, thereader is referred to other recent review articles [7–10].

Phragmoplast expansion driven by
MT turnovers
How do phragmoplast MTs move centrifugally toward thecortex as the cell plate grows? A clue came from an experiment performed by Yasuhara et al. [6] who used taxol, achemical that blocks depolymerization of MTs, to examine
the mechanism of cell-plate expansion. Taxol treatment of
tobacco bright yellow-2 (BY-2) cultured cells during
telophase inhibits the centrifugal expansion of theirphragmoplasts, producing cells with abnormally thick cell
plates that apparently result from the increased accumulation of vesicles (Figure 1d). The requirement for MT
depolymerization during phragmoplast expansion indicatesthat neither the pushing force of the cell plate nor thepulling force of the parental cell walls is responsible for the508

Cell biology

Figure 1(a)(b) Brefeldin A treatment−
Cell wall+
Fusiontube−(c) Caffeine treatment

Golgi-derived
vesicleCollapse of FTN∗(d) Taxol treatment or
kinase-negative NPK1
overexpression

Diagram of phragmoplast expansion andcell-plate formation in normal, drug-treated, or
genetically modified plant cells. (a) In a normal
plant cell, a phragmoplast is assembled with
anti-parallel MTs overlapping in the center attheir plus ends (+). Fusion of Golgi-derived
vesicles (open circles), through fusion tubes
in the equatorial zone of the phragmoplast,
gives rise to a FTN (orange), which
successively maturates into a TVN (red), a TN
(pink), and a fenestrated sheet/cell plate(blue). In (a), green bars represent MTs thathave recently been polymerized, or in (b,c),that are not yet destined for depolymerization,
whereas gray bars represent MTs that aredestined for depolymerization during the nextstage. Note that the shape of thephragmoplast changes during the transition
from a cylinder to a ring, which is marked byan asterisk. (b) Brefeldin A blocks the supplyof vesicles by disrupting the Golgi, resulting ina lack of cell-plate materials in thephragmoplast equator. (c) Caffeine blocks thematuration of the FTN into the TVN, which
eventually results in the collapse of the FTN.
(d) Taxol blocks the depolymerization of MTs,
which inhibits phragmoplast expansion but
allows the formation of an incomplete cell
plate. Overexpression of a kinase-negative
mutant of the NPK1 MAPKKK results in asimilar phenotype. Note that, in (b−d),phragmoplasts are never transformed intoring shapes.

Formation ofincomplete cell plate

Current Opinion in Plant Biology

mechanical enlargement of the phragmoplast. Rather, it
seems more likely that the supply of free tubulins, whichhave been depolymerized from preexisting MTs on the
inner side of the phragmoplast, forces the lateral expansionof the phragmoplast by constructing new MT arrays at its
outer edge. Thus, the phragmoplast appears to possess anactivity that can initiate MT polymerization at its outer edge.
The incorporation of free tubulins into the phragmoplastis known to occur at the plus ends of MTs located in theequatorial zone [11,12]. On the basis of the fluorescence
redistribution after photobleaching (FRAP) analysis performed by Hush et al. [13], phragmoplast MTs seem toexhibit dynamic instability [14]. Following the photobleaching of phragmoplast MTs that had incorporated

fluorescein-labeled tubulins, Hush et al. [13] observed a
rapid and uniform recovery of fluorescence. This observation is more consistent with a mechanism of dynamic
instability and exchange of tubulin dimers than withtreadmilling. Therefore, depolymerization of MTs mightalso occur at the plus end of MTs in the phragmoplast. If
this were the case, then MTs on the inner side of thephragmoplast would be more unstable than those on theouter side..

Phragmoplast MT-associated proteins (MAPs)
Recently, two classes of MT-associated proteins (MAPs),
which are possible regulators of MT dynamics, have beenshown to localize to the phragmoplast. One of these classes
is comprised of the kinesin-like MT-based motor proteinsExpansion of the phragmoplast during plant cytokinesis Nishihama and Machida(KLPs). Plus-end-directed KLPs, including Xenopus kinesin
central motor1 (XKCM1) [15] and Xenopus kinesin
superfamily2 (XKIF2) [16], and the minus end-directed
KLP KAR3 [17] are known to depolymerize MTs at theplus and minus end, respectively (reviewed in [18]).
Arabidopsis thaliana phragmoplast-associated kinesinrelated protein1 (AtPAKRP1) is localized to the center ofthe phragmoplast, and microinjection of anti-AtPAKRP1
antibodies or truncated AtPAKRP1 proteins into tobacco
BY-2 cells leads to the disorganization of phragmoplastMTs [19••]. Carrot DcKRP120-2 (Daucus carota kinesinrelated protein of 120 kDa-2) is a KLP homologue of
tobacco TKRP125 (tobacco kinesin-related polypeptide of
125 kDa) [20•], which possesses an activity that translocates phragmoplast MTs toward their minus ends.
TKRP125 is localized to whole arrays of phragmoplast
MTs except for their plus ends [21]. Unlike TKRP125,
DcKRP120-2 is also localized to the MT-interdigitatingzone of the phragmoplast [20•]. The minus end-directed
KLPs, KCBP (kinesin-like calmodulin-binding protein) [22]and KatA (kinesin-like protein in Arabidopsis thaliana A)[23], localize along the length of phragmoplast MTs. It will
be intriguing to test the effects of these KLPs on MT disassembly. The roles of these KLPs in the progression of M
phase or the expansion of phragmoplasts remain unknown.
Another MAP family is comprised of the structural MAPs
recently identified by Smertenko et al. [24••]. They identified a member of the tobacco NtMAP65-1 (Nicotiana
tabacum MAP65-1) family that is able to bind to taxolstabilized MTs. This MAP promotes polymerization of
MTs in vitro but does not promote the bundling of MTs[24••]. This polymerizing activity is sufficient to classify
the protein as a MT-dynamics regulator. Antiserum thatrecognizes multiple members of the NtMAP65-1 family
does not stain all MT arrays. At metaphase, the antiserum
stains an area of spindle MTs proximal to the metaphase
plate but not those at the pole [24••]. The antibody also
stains the spindle midzone and the phragmoplast equatorial zone [24••]. These patterns of localization suggest a rolefor NtMAP65-1 proteins in the regulation of MT dynamics
in specific areas of the spindle and the phragmoplast where
MTs overlap at their plus ends.
It is intriguing to note that NtMAP65-1 proteins havesignificant amino-acid-sequence homology to two other
MAPs: human PRC1 (Protein Regulator of Cytokinesis1),
which is required for cytokinesis [25], and yeast Ase1
(Anaphase spindle elongation1), which is required forspindle assembly, elongation and disassembly [26]. PRC1
and Ase1 share two notable sequence features: a consensus
cyclin-dependent kinase (CDK) phosphorylation site anda sequence that is similar to a mitotic cyclin destruction
box. It has been proven both in vivo and in vitro that PRC
is phosphorylated at the consensus CDK phosphorylationsites by CDK2 [25]. It has also been proven that Ase1 is atarget of proteolysis mediated by the anaphase-promoting
complex [27]. NtMAP65-1 proteins also exhibit these509

characteristic sequences. Our computer-based analysis also
predicted that NtMAP65-1 proteins may form a highly
α-helical structure similar to that of PRC1 (R Nishihama,
Y Machida, unpublished data).
After anaphase, PRC1 is restricted to plus-end MT-overlapping regions [25], a pattern that is reminiscent of
NtMAP65-1 localization in plant cells. Remarkably, themicroinjection of anti-PRC1 antibodies into HeLa cells
blocks the completion of cytokinesis [25]. Although it isstill unclear what PRC1 actually does to MTs, this study
on PRC1 raises the possibility that NtMAP65-1 proteins
also control the progression of cytokinesis in plants byregulating phragmoplast-MT dynamics. Whether PRC1
and Ase1 actively promote MT polymerization is now an
open question.
Recent studies have revealed the existence of plant
proteins in addition to the NtMAP65-1 family that havehomologies to animal MAPs [28•,29•]. Furthermore, ourbrief search of the complete Arabidopsis genome sequence
revealed two putative MAPs with similarity to humanEB1 protein and MAP1A/B light chain 3 (R Nishihama,
Y Machida, unpublished data). Interestingly, EB1 isknown to localize to MT plus ends and is involved in theregional control of MT dynamics and capture (see [30,31]
for reviews). Further research should be performed to test
the involvement of various MAPs in the regulation ofphragmoplast MT dynamics.

Co-ordination of phragmoplast expansion withcell-plate formation
As described above, expansion of the phragmoplast is
essential for the lateral growth of the cell plate. Cell-plate
formation and phragmoplast expansion are co-dependent:
without formation of the cell plate the phragmoplast never
expands. For example, the use of brefeldin A (BFA), a
chemical that disrupts the Golgi apparatus, to block the
supply of vesicles carrying cell-plate materials prevents theformation of the cell plate and results in the generation of
binucleate cells [2], but does not disturb phragmoplast
formation [32]. Interestingly, under appropriate conditions,
BFA treatment completely arrests the lateral expansion ofphragmoplasts at the cylindrical stage after only an initialslight enlargement [32] (Figure 1b). These phragmoplasts
no longer become ring-shaped and no MT disassemblytakes place in their central region. Thus, the process ofphragmoplast expansion is tightly linked to cell-plate
formation. This finding suggests that there may be amechanism that induces MT depolymerization in
response to cell-plate formation.
What is the cue for MT depolymerization? Caffeine is
known to disrupt cell-plate formation in plants ([33] andreferences therein). In Tradescantia stamen-hair cells treatedwith caffeine, cell-plate formation is initiated but theimmature cell plate ceases to grow and is ultimately
degraded after it has expanded to about three-quarters

510

Cell biologyof the cell’s width [34]. Although the initiation of thephragmoplast is not inhibited in this system, the phragmoplast fails to adopt a ring structure and remains fixed
as a cylindrical structure (Figure 1c). Electron microscopyof cryofixed BY-2 cells shows that caffeine disrupts theconversion of the FTN into the TVN during the development of the cell plate [33,35]. This implies that a minimallevel of TVN formation is required for the induction of
MT disassembly. Consistent with this finding, it has been
observed that phragmoplast MTs are associated with the
TVN but not with the TN [5] (Figure 1a). Taken together,
these results suggest that MT depolymerization is induced
during the TVN→TN transition. An ultrastructural study
has also revealed several features of the TVN, such asassembly of the fuzzy membrane coat, widening of theconnecting tubules, formation of clathrin-coated buds, andinitiation of callose synthesis [5]. These features are goodcandidates for events that constitute ‘MT-disassembly
signals’. In conclusion, cell-plate expansion is driven by
successive rounds of the following events: vesicle transport
to the phragmoplast equator, formation of the FTN byvesicle fusion, maturation of the FTN into the TVN, andMT disassembly on the inside and its reassembly on theoutside of phragmoplast.

A mitogen-activated protein kinase(MAPK) cascade as a regulator ofphragmoplast expansion
A coupling mechanism should exist between the events
involved in cell-plate formation and the disassembly ofphragmoplast MTs. Recent studies have suggested the
involvement of a mitogen-activated protein kinase(MAPK) cascade in the regulation of such a couplingmechanism [36–38,39••]. The MAPK cascade is a signaling
pathway that is conserved in all eukaryotes and thatconsists of members of three protein kinase families. In
their respective order in the signaling cascade, they are the
MAPK kinase kinase (MAPKKK) family, the MAPK
kinase (MAPKK) family and the MAPK family [40].
On the basis of the sites of its transcript accumulation, atobacco MAPKKK, Nucleus- and Phragmoplast-localized
Protein kinase1 (NPK1) [41], has been suggested to play arole in cell division [42]. The accumulation of NPK1 protein
is cell-cycle dependent; proteins are present in the cellfrom S to M phase, with a peak in accumulation during
M phase, after which NPK1 is degraded [39••]. NPK1 is
confined to the nucleus from S phase to prophase [39••].
NPK1 kinase activity is transiently increased late in M
phase [39••]. Upon nuclear envelope breakdown, NPK1
proteins are dispersed into the cytoplasm and localize inpatches until early anaphase. By late anaphase, NPK1
begins to concentrate at the spindle midzone before
becoming localized to the equatorial zone of the cylindricalphragmoplast. During cytokinesis, NPK1 is consistently
found at the equator of the ring-shaped phragmoplast, but
not at the cell plate. Some NPK1 proteins relocate back to
the reforming daughter nuclei.

These findings suggest a role for NPK1 in cytokinesis.
Indeed, inhibition of NPK1 signaling has been shown toresult in a defect in cytokinesis [39••]. Overexpression of akinase-negative mutant of NPK1 (NPK1KW) induces theformation of multinucleate cells that contain incomplete
disc-shaped cell plates. This mutant protein still localizes
to the equator of the phragmoplast. The phragmoplast
does not expand, however, in NPK1KW-overexpressing
plants (Figure 1d). Thus, NPK1 regulates the expansion ofboth the cell plate and the phragmoplast.
It should be noted that NPK1KW-induced incomplete cell
plates that are formed in NPK1KW-expressing cells contain callose [39••], a polysaccharide whose synthesis isinitiated at the TVN stage [5] (see above). Thus, it would
appear that NPK1 is primarily involved in a step that takes
place after the formation of the TVN, that is, in the outward redistribution of phragmoplast MTs. Consistent with
this, the effect provoked by blocking NPK1 signaling issimilar to that of taxol treatment (see above; Figure 1d).
NPK1 might regulate MT depolymerization at plus ends
in response to MT-disassembly signals generated in the
TVN/TN. There is, however, still a possibility that NPK1
might be involved in some of the earlier steps if inhibitionby the dominant-negative mutant were not complete.
The identification of downstream targets of NPK1 will bedecisive in completing our understanding of the molecularmechanism of phragmoplast expansion. It will be intriguing
to examine whether NPK1 regulates the MAPs that are
mentioned above and, if so, whether this regulation ismediated directly by NPK1 or indirectly through a putative
MAPK cascade initiated by NPK1. In light of this, it should
be noted that the p43Ntf6 MAPK from tobacco [36] andalfalfa MMK3 (Medicago MAPK3) [37] are activated during
cytokinesis. Both of these proteins are localized to theequatorial zone of the phragmoplast, and MMK3 is also
localized to the cell plate. Most recently, a tobacco
MAPKK, Nicotiana tabacum MAPK/extracellular signalregulated protein kinase kinase (NtMEK1), that activates
p43Ntf6 has been identified as a possible component of a
MAPK cascade involved in phragmoplast expansion [38],although its sub-cellular localization remains to be determined. Thus, phragmoplast expansion in tobacco may beregulated by a MAPK cascade, involving NPK1 MAPKKK,NtMEK1 MAPKK and p43Ntf6 MAPK, although evidence
for the involvement of NtMEK1 and p43Ntf6 remains to
be found.
In animal cells, active MAPKs have been shown to belocalized to the spindle midzone and midbody, a cytokinetic
bridge composed of MTs [43,44]. Although direct evidence
for the involvement of MAPKs in cytokinesis is still lacking, we speculate that animal MAPKs might also regulate
MT dynamics. In animal cells, cytokinesis proceeds in an
outside-in mode by the constriction of the cortex.
Therefore, spindle MTs must change their positions tomove inwardly during cortex constriction because the widthExpansion of the phragmoplast during plant cytokinesis Nishihama and Machida511

of the spindle is limited by the cortex. This redistribution
may involve MT turnovers. In addition, midbody MTs
should disassemble upon cell separation. Thus, the mechanism that regulates MT dynamics during cytokinesis may
be conserved in plants and animals.

7.

Heese M, Mayer U, Jürgens G: Cytokinesis in flowering plants:
cellular process and developmental integration. Curr Opin Plant
Biol 1998, 1:486-491.

8.

Smith LG: Divide and conquer: cytokinesis in plant cells. Curr Opin
Plant Biol 1999, 2:447-453.

9.

Nacry P, Mayer U, Jürgens G: Genetic dissection of cytokinesis.
Plant Mol Biol 2000, 43:719-733.

Conclusions

10. Verma DPS: Cytokinesis and building of the cell plate in plants.
Annu Rev Plant Physiol Plant Mol Biol 2001, 52:751-784.

In this review, we highlight the molecular mechanism forthe expansion of the phragmoplast during plant cytokinesis.
Phragmoplast expansion is a process of repeated depolymerization and repolymerization, during which MT
disassembly is linked to the progression of cell-plate
formation. Changes in MT stability may be mediated by
MAPs, such as various KLPs and NtMAP65-1 proteins that
localize to the equator of the phragmoplast. The NPK1MAPKKK, and probably a MAPK cascade initiated by
NPK1, is involved in phragmoplast expansion. One of thepossible functions of this pathway would be to regulate
MT depolymerization in response to signals generated by
the forming cell plate. Further research in this field will
aim to elucidate the MT-disassembly signals, identify thetarget proteins of the MAPK pathway, and examine
whether MAP activities are spatially regulated at the innerand outer edges of the phragmoplast.

Acknowledgements
We thank Hiroki Yasuhara, Tetsuhiro Asada, and members of the Machidalaboratory for helpful discussion. Work performed by the authors was
supported by Grants-in-Aid for Scientific Research on Priority Areas (Award
numbers 0678101-3 and 10182101-2) from the Japanese Ministry of
Education, Science, Culture and Sports; and by a grant from the Research
for the Future Program of the Japan Society for the Promotion of Science.
RN was supported by Research Fellowships from the Japan Society for the
Promotion of Science for Young Scientists.

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