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Molecular encounters at microtubule ends in the plant cell cortex
Martine Pastuglia and David Bouchez
The cortical arrays that accompany plant cell division and
elongation are organized by a subtle interplay between intrinsic
properties of microtubules, their self-organization capacity and
a variety of cellular proteins that interact with them, modify their
behaviour and drive organization of diverse, higher order arrays
during the cell cycle, cell growth and differentiation. As a polar
polymer, the microtubule has a minus and a plus end, which
differ in structure and dynamic characteristics, and to which
different sets of partners and activities associate. Recent
advances in characterization of minus and plus end directed
proteins provide insights into both plant microtubule
properties and the way highly organized cortical arrays emerge
from the orchestrated activity of individual microtubules.
Addresses
Institut Jean-Pierre Bourgin, Station de Génétique et d’Amélioration
des Plantes UR254, INRA, Centre de Versailles, F-78000 Versailles,
France
Corresponding author: Pastuglia, Martine ([email protected])
Current Opinion in Plant Biology 2007, 10:1–7
This review comes from a themed issue on
Cell Biology
Edited by Ben Scheres and Volker Lipka
1369-5266/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2007.08.001
Introduction
Microtubules (MTs) are distinctive components of eukaryotic cells, where they play essential roles in spatially
controlled processes like organization of the cytoplasm,
cell shape and polarity, flagella/cilia and cell motility,
intracellular transport of vesicles and proteins, mitotic
and meiotic cell division and cell wall deposition (in
plants), to name a few. MTs are non-covalent, polar
polymers of a/b tubulin heterodimers assembled headto-tail into linear protofilaments. Side association of
(usually) 13 protofilaments forms a hollow cylinder of
25 nm in diameter, a rigid filamentous structure that is
intrinsically polar, with a rapidly growing plus end and a
slow growing minus end defined as the a-tubulin end
(Figure 1a). Properties of tubulin make MTs highly
dynamic, with stochastic transitions between growing
and shrinking phases, a phenomenon called ‘dynamic
instability’. When MT minus ends are anchored or capped,
only plus ends undergo dynamic instability, whereas unanchored MTs can display directional repositioning through
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‘treadmilling’ (net polymerization at the plus end, net
depolymerization at the minus end); for a recent review
on MT assembly, see reference [1].
Thanks to intrinsic properties of MTs and to activities of
many cellular proteins that can modify, (de)stabilize,
move, cut or bridge them, MTs generate higher order
networks that organize the cellular space and adopt
different forms during the cell cycle. In expanding plant
cells, MTs are cortical, tightly associated with the inner
face of the plasma membrane and arranged into parallel
arrays transverse to the elongation axis, guiding cellulose
deposition on the outside of the cell [2]. At G2/M
transition, cortical MTs arrange into a highly condensed
ring encircling the nucleus, with radial MTs connecting
nucleus and cortex. This transient structure, the preprophase band of MTs (PPB), is specific to land plants and
precisely predicts the site of division before disassembling at prophase.
To control the distribution and organization of MTs,
many eukaryotic cells rely on specific organelles called
MT-organizing centres (MTOCs), which are sites for
nucleation of new MTs [3]. MTs can spontaneously
assemble in vitro under conditions of high concentrations
of tubulin, but they form at a much lower tubulin concentration in living cells. MTOCs help overcome the
kinetic barrier of limiting tubulin concentration but are
also a means for spatial and temporal regulation of MT
initiation. In animals, the centrosome consists of a pair of
centrioles linked by a matrix and surrounded by a pericentriolar matrix, which promotes de novo nucleation of
MTs for the setup of interphase cytoplasmic MT arrays
and mitotic spindles. Its capacity to organize MT arrays
also depends on tight control of anchoring, capping and
release of nucleated MTs.
Unlike most other eukaryotes, cells of land plants lack a
conspicuous MTOC like a centrosome, except for basal
bodies formed in flagellate sperm cells of lower land
plants. The absence of centrosomes, centrioles and the
mechanisms of MT nucleation in higher plants have
prompted many questions as to whether plant cells have
conserved, revamped or reinvented functions associated
with MTOCs in other eukaryotes. The theory that centrosomes are flexible bodies that adopt a variety of forms
was formulated more than 20 years ago [4]. The idea of a
distributed centrosome at the membrane’s periphery in
plant cells, involved in nucleation, release and attachment of MTs at the cortex, has received wide support in
the field, but until recently this had limited direct experimental support.
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2 Cell Biology
Figure 1
Microtubules from nucleation to establishment of cortical arrays. (a) Microtubule assembly and disassembly. MTs are polymers of a/b tubulin
heterodimers. A typical MT is composed of 13 linear protofilaments resulting from head-to-tail arrangement of heterodimers. Lateral association
of protofilaments forms a hollow cylinder of 25 nm in diameter. The orientation of a/b tubulin dimers in the MT lattice confers a polarity that is
crucial for MT organization, with b-tubulin pointing towards the fast growing plus end, and a-tubulin pointing towards the slow growing minus
end. a and b-tubulins have N-terminal GTP-binding pockets, but GTP is exchangeable only in b-tubulin in un-incorporated heterodimers. The GTP
of the b subunit is buried upon MT polymerization and is rapidly hydrolyzed by a-tubulin catalytic residues into non-exchangeable GDP. The
hydrolysis of GTP in b-tubulin is coupled to incorporation in the MT lattice and affects the structure of the a/b heterodimer, which in turn
introduces tensions in the protofilament that provide energy for rapid disassembly of the MT. The MT structure is stabilized by a cap of a few
layers of GTP-tubulin subunits at the plus end; when the cap is lost, the MT undergoes a rapid depolymerization (termed a catastrophe). Recent
models propose that the plus end of a growing MT is an open sheet of protofilaments with lateral contacts, gradually closing into a hollow cylinder [1].
Such a sheet conformation of growing plus ends is expected to provide an open surface for specific interactions at the plus end. Typical curled
protofilaments (’protofilament peeling’) are produced during MT shortening. MTs undergo dynamic instability, showing stochastic transitions
between growing, pause and shrinking phases. When the minus end is anchored at its nucleation site, only the free plus end exhibits dynamic
instability, but free minus ends are also dynamic in non-anchored MTs. This seems to be the rule in the plant cell cortex, where MTs are detached
from their nucleation sites and translocated by a ‘hybrid treadmilling’ mechanism [9]. (b) Microtubule nucleation and the g-tubulin complex. The
basic process of MT nucleation is probably highly conserved in eukaryotes including plants, though this has to be further confirmed experimentally.
In animal cells, g-tubulin is present in a large ring-shaped 2 MDa multi-protein complex, approximately the diameter of a MT (25 nm), called
a g-TuRC. The g-TuRC accelerates the rate of MT initiation, and could also have a capping function, inhibiting minus end dynamics. A widely
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Molecular encounters at microtubule ends in the plant cell cortex Pastuglia and Bouchez 3
This review is an overview of some progress on the
characterization of protein networks at the minus and
plus ends of MTs in plant cells. Recent results show that
such interactions at MT ends are instrumental in producing organized cortical arrays from concerted activity of
individual MTs.
g-Tubulin complexes and MT nucleation at the
cortex
The absence of a well-defined, central MT organizer has
long delayed characterization of the molecular basis of
MT nucleation in higher plant cells. The third member of
the tubulin superfamily, g-tubulin, is a key component of
eukaryotic MTOCs where it is found associated to minus
ends of MTs in large multi-protein complexes that serve
as templates for MT initiation (Figure 1b). Although gtubulin is conserved in plant genomes, its involvement in
MT nucleation in acentrosomal plant cells has been
controversial until recently. The first evidence of MT
nucleation by plant g-tubulin came from heterologous
expression of Arabidopsis g-tubulin in fission yeast (Schizosaccharomyces pombe) lacking endogenous g-tubulin.
Arabidopsis g-tubulin was properly targeted to MTOCs
and able to nucleate MTs in fission yeast [5]. In plant
cells, g-tubulin was not restricted to MT ends as expected
but seen as a punctate staining along MT walls [6,7].
However, a recent study established that this pattern
reflects bona fide MT-dependent nucleation sites [8]
and that in the plant cell cortex, MTs are mostly
nucleated from the surface of extant MTs, resulting in
a branched MT network (Figure 1c). Furthermore,
recruitment of cytoplasmic g-tubulin onto existing
MTs was necessary to initiate new MTs. Several studies
already reported MT nucleation at cortical sites linked to
pre-existing MTs in plant cells [9,10], and such branched
organization occurs in animal cells [11]. Knocking down
both g-tubulin genes in Arabidopsis further supported the
involvement of g-tubulin in MT nucleation [12,13].
Regrowth of cortical MTs was particularly impaired in
cells with depleted RNAi g-tubulin [12].
In animal cells, g-tubulin is part of large ring-shaped
protein complexes called g-tubulin ring complexes
(g-TuRCs) (Figure 1b). The g-TuRC contains 10–13 gtubulin molecules per complex and additional subunits
named gamma complex proteins (GCPs 2–6) in humans
and Dgrip in Drosophila [3] (Table 1). The Arabidopsis
genome encodes putative orthologues for all GCPs, including the recently discovered GCP-WD/Nedd1 (Dgp71WD). An antibody directed towards plant Spc98 (GCP3)
inhibits MT assembly from isolated tobacco (Nicotiana
tabacum) nuclei [14]. However, the absence of co-localization of AtSpc98 and g-tubulin in the cortex raises questions on its role in MT assembly of cortical arrays. Further
characterization of the putative plant g-TuRC and functional analysis of these proteins are needed to clarify the
processes of MT nucleation in plant cells.
The organization of highly structured arrays strongly
depends on the spatial and temporal distribution of
MT nucleation sites in a cell. Recruitment of nucleation
complexes at the surface of extant MTs is not restricted to
plant cells. For example, in Drosophila, g-tubulin transiently associates to the spindle and midbody MTs during
mitosis, and this interaction requires cap components
Dgrip 75, 128, 163 and Dgp71WD and a fully assembled
g-TuRC [15]. Whether this recruitment requires specific
docking proteins on the MT wall is not known. Such
proteins recruiting g-tubulin complexes to cytoplasmic
MTs were recently identified in fission yeast [16], but no
obvious homologues have been identified in any other
eukaryotes.
A distributed centrosome at the plant cell
cortex?
Vertebrate centrosomes are involved in a range of cellular
events in addition to MTOCs. They are notably involved
in cell cycle transitions, cellular responses to stress and
organization of signal transduction pathways. Organization of the plant cortical cytoskeleton is undoubtedly
tightly coordinated with the cell cycle, though most
players involved in molecular crosstalk between the cell
cycle machinery and the cytoskeleton are not identified.
Nevertheless, in addition to g-TuRC components, there
is converging evidence that a number of functions and
proteins are conserved between the animal centrosome
(Figure 1 Legend Continued )accepted view of the molecular arrangement of the g-TuRC (the so-called ‘template’ model) is presented here.
In this model, 6–7 g-TuSC subcomplexes (each composed of 2 g-tubulins, one GCP2 and one GCP3) are held together in a helicoidal conformation
by other GCP subunits that form the cap structure of the g-TuRC. Arrangement of g-tubulin molecules in the complex provides a template for
initiating a/b-tubulin heterodimer polymerization and MT assembly. The genome of Arabidopsis (and other plants) possesses clear orthologues
of g-TuSC components, as well as equivalents of most cap components of the g-TuRC (Table 1). (c) Emergence of parallel MT arrays at the cortex.
The cortex is shown as a two-dimension plane.
Stochastic MT dynamic instability. MT Plus ends alternate between phases of polymerization
and depolymerization [9]. Shown is a MT undergoing catastrophe, rescued (middle panel), then switching to a polymerization phase (right panel). MT
nucleation from pre-existing MT producing branched structures [8].
Encounter between two MTs growing in opposite directions [37]. The MT
growing in the minority direction undergoes catastrophe (middle panel) and complete depolymerization (right panel).
Release of MT from
nucleation site by a putative katanin severing activity.
Shallow angle MT encounters usually result in MT co-alignment and bundling [45]
(right panel).
Steep angle MT encounters usually result in depolymerization of the encountering MT [45] (right panel).
Depolymerization of the
original MTs after MT nucleation [8].
Recruitment of cytoplasmic g-tubulin on the side of MTs and nucleation of new MT [8].
Cortical
arrays contain around 80% of MTs growing in the same direction [37]. A MT growing in the opposite direction can still be incorporated into
bundles formed of MTs of opposite polarity.
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4 Cell Biology
Table 1
g-TuRC components in Arabidopsis.
AGI name
Name
At3g61650
At5g05620
At5g17410
At5g06680
At3g53760
At1g80260
At1g20570
At3g43610
At5g05970
AtTUBG1
AtTUBG2
AtSpc97
AtSpc98
AtGCP4
AtGCP5a
AtGCP5b
AtGCP6 ?
At-Nedd1
MW (kDa)
Drosophila
Xenopus
Human
53
53
77
95
86
106
109
127
85
g-tubulin
(2 genes)
Dgrip84
Dgrip91
Dgrip75
Dgrip128
g-tubulin
Xgrip110
Xgrip109
Xgrip76
Xgrip133
g-tubulin
(2 genes)
hGCP2
hGCP3
hGCP4
hGCP5
Dgrip163
Dgp71WD
Xgrip210
X-Nedd1
hGCP6
GCP-WD (NEDD1)
Saccharomyces
cerevisiae
Schizosaccharomyces
pombe
Tub4p
Tubg1
Spc97p
Spc98p
Alp4p
Alp6p
Gfh1p
Alp16
The AGI names refer to standard nomenclature for Arabidopsis genome annotation. g-TuRC proteins are conserved across animal species and
contain conserved g-ring protein (grip) motifs. AtSpc97 and AtSpc98, as well as AtGCP4, 5a and 5b are clear orthologues of their animal and yeast
counterparts on the basis of sequence similarity and phylogeny reconstruction. Although a bona fide member of the GCP family, the position of
At3g43610 is less clear with respect to human and Drosophila proteins. Arabidopsis is the only species to have two distinct GCP5 isoforms, sharing
80% similarity. Despite this duplication, it is interesting to note that mutations in AtGCP5a result in embryo lethality (http://www.seedgenes.org/;
emb1427-1 and emb1427-2). Depletion of g-tubulin in Arabidopsis also results in gametophytic lethality [12,13]. Other g-TuRC components have
not been functionally analyzed up to now in plants.
and the organization of the cortical cytoskeleton in plants.
For example, several proteins located on, or involved in
formation of the PPB have centrosomal counterparts; the
FASS protein is a protein phosphatase PP2A regulatory
subunit involved in the setup of the PPB [17]. Its homologue in Caenorhabditis elegans, RSA-1, was recently
shown to recruit a PP2A complex to the centrosome,
where it is involved in spindle assembly and MT outgrowth from the centrosome [18]. Similarly, the cyclindependent kinase CDKA is recruited to the late PPB in
plant cells entering mitosis [19]. In mammalian cells,
the initial activation of cyclin B1-Cdk1 in early prophase
takes place at the centrosome, before spreading to the rest
of the cell to induce nuclear and cytoplasmic mitotic
events [20]. Interestingly, TONNEAU has been recently
shown to co-purify with Arabidopsis CDKA [21]. The
TON protein is required for PPB formation and shares
a common domain with a human centrosomal protein,
FOP [22,23] (Azimzadeh et al., unpublished). Therefore,
the view that higher plant cells, being devoid of centrosomes, centrioles, cilia and flagella, have lost the corresponding proteomes (e.g. [24]) may deserve more
attention. In particular, it should be considered that
centrosomal proteins, especially pericentriolar matrix
ones, are notably difficult to analyze with standard bioinformatic methods because of strong compositional biases
and richness in coil-coiled regions that prevent efficient
similarity searches.
MT density by creating seeds for new MT growth [25].
Katanin is a heterodimeric enzyme containing a p60
catalytic subunit and a p80 regulatory subunit and uses
energy from ATP hydrolysis to sever MTs. The Arabidopsis p60 subunit is sufficient to sever MTs in vitro [26].
Its overexpression in vivo leads to numerous short
bundled MTs, probably resulting from increased severing
activity of p60 subunits [27]. Genetic analysis indicated a
role of p60 katanin in organizing cortical MTs; in absence
of katanin, MT organization at the cortex is impaired,
both in rice [28] and Arabidopsis [29,30,31], and MTs are
longer and sparser [31].
Proteins at MT plus ends
MT assembly/disassembly does not occur by simple
helical addition/subtraction of individual a/b subunits,
and growing/shrinking MT ends adopt particular conformations, providing structural cues for specific recognition
by cellular proteins [1] (Figure 1a). Since their first
discovery in 1999 because of improvements in imaging
technologies, a growing number of proteins that track MT
plus ends (+TIPs) have been identified in animal and
yeast cells [32]. Some bind directly to MTs, others locate
at MT plus ends via interaction with the first ones. Owing
to their specific localization, +TIPs influence MT
dynamics, and also enable the growing plus end to search
for specific cellular targets like organelles, membranes,
microfilaments or other MTs. In animals, these proteins
have been involved in MT attachment to the cortex [32].
Severing releases new MT ends
Time-lapse observations of individual MTs revealed that
most newly nucleated MTs do not remain anchored at
their nucleation sites but are released from initiation sites
[9] (Figure 1c). The MT-severing katanin is likely to
perform such activity. Severing of newly formed MTs
then recycles free g-tubulin complexes able to nucleate
new MTs. Katanin severing-activity could also increase
Current Opinion in Plant Biology 2007, 10:1–7
The study of +TIPs is just beginning in plants, where
homology searches indicate that some animal +TIPs are
conserved [33]. The most studied +TIP in plants is EB1, a
protein now considered a key element among plus end
players in animals [32]. In addition to its localization at
MT plus end, EB1 is also present at the centrosome of
animal cells, independently of MTs [32]. The localization
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Molecular encounters at microtubule ends in the plant cell cortex Pastuglia and Bouchez 5
pattern in plants of the EB1 protein family is still debated.
Overexpression of AtEB1a-GFP fusions labels not only
MT plus ends [34] but also MT minus ends [35] as well as
endosomes [36]. Using an inducible promoter to tightly
control EB1-GFP expression, Dixit et al. showed that two
out of three Arabidopsis EB1 proteins (AtEB1a and
AtEB1b) display comet-like structures, whereas AtEB1c,
the most distant from human EB1, localized to the
nucleus [37]. Beyond their use as markers for time-lapse
imaging of MTs, the functional characterization of EB1
proteins in plants still awaits. In this respect, recent
isolation of an Arabidopsis mutant in the a-tubulin 5 gene
clarifies the interaction of plant EB1 with MTs [38].
This mutation affects a critical residue at the a/b interface and stabilizes mutant MTs by preventing GTP
hydrolysis in b-tubulin. Interestingly, in mutant cells,
EB1b associates to lateral walls of MTs, indicating that
EB1b recognizes the presence of GTP-b-tubulins in the
MT lattice, normally restricted to GTP-capped plus ends
[38].
Apart from EB1, very few proteins localized at MT plus
ends have been characterized in plants. One is SPR1, a
member of a plant-specific protein family involved in
cortical MT organization and anisotropic cell growth [39].
SPR1 associates to MTs [40] with a preferred accumulation at MT growing ends in interphase cortical arrays
[41]. Whether SPR1 binds directly to MT plus ends or
rather via interaction with another +TIP is not known.
Another plant +TIP is ATK5, a kinesin primarily important for spindle morphogenesis. In addition to spindle
assembly, ATK5 may be involved in cortical array organization during PPB formation [42]. Indeed, ATK5 localizes to MTs of the PPB and mutation in atk5 gene affects
PPB narrowing [42,43]. In vitro, ATK5 can co-align and
bundle MTs after their encounter, suggesting a role in
MT capture and co-alignment [42].
Cortical array organization: action at MT
ends
During interphasic cell growth, MT arrays in the plant
cell cortex are typically unfocused, associated to the
plasma membrane and organized in parallel arrays transverse to the elongation axis. Surprisingly, this organization can be switched to a radial MT array emanating
from a single particle located in the vicinity of the
nucleus, simply by overexpression of a particular kinesin
fused to GFP [44]. This observation indicates that acting
on a limited number of key activities may form profoundly different MT arrays.
Studying dynamics of individual MTs in the plant cell
cortex has shed light on the way cortical arrays are set up
[37,45,46] (Figure 1c). Interactions at MT ends appear
to play an important role in addition to the influence of a
variety of MT-associated proteins that act on specific
parameters of MT dynamic instability and/or favour
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bundling [47,48]. MTs at the cell cortex are tightly
connected to the plasma membrane, and hence grow in
a two-dimensional cortical space; in such a molecularly
crowded space, encounters between growing MT plus
ends and pre-existing MTs are frequent. A steep angle of
collision usually induces disassembly of the encountering
MT; when the angle is shallow, the growth trajectory of
the encountering MT is adjusted to align to the existing
MT [45]. This property is sufficient to explain selforganization of MTs into parallel arrays [45]. Within
cortical arrays, MTs tend to acquire a common polarity
by selective stabilization of concordant MTs [37]. Discordant MTs branching from pre-existing MTs [8] at a
408 angle may counterbalance this tendency to homogeneity, allowing exploration of the cortical space and
adaptation of MT arrays to cellular or environmental cues.
Group behaviour of MTs within a cell has recently
revealed another level of complexity to cortical MT array
organization [46]. According to this study, the plant cell
cortex is a mosaic of polarized MTs domains, rotating and
reorienting during elongation.
Plus end dynamics appear all the more important during
PPB formation. At the onset of division, MTs are progressively condensed and bundled into a narrow band,
concomitant with gradual disappearance of other cortical
MTs. During this stage, MTs outside the PPB become
more dynamic and longer, which has been proposed to
contribute to PPB formation via a ‘search and capture’
mechanism [49]. This change in dynamics could allow
MT plus ends to probe the cortex more efficiently, and
the MTs to be selectively stabilized and bundled as they
reach the division site [49]. In addition, MT nucleating
sites as revealed by g-tubulin localization in PPB microtubules [7] probably insert new MTs into the developing
PPB. A search and capture mechanism of MT plus ends
may also exist at this stage, for endoplasmic MTs establishing connections between the nucleus and the PPB
[50].
Conclusions
Despite significant advances concerning proteins that
interact with MTs, information is still scattered and
disconnected, and there is much to learn about key factors
that organize the plant cell cortical cytoskeleton. Many
mechanistic, molecular and cellular aspects remain poorly
understood, such as connections and crosstalk of the MT
cytoskeleton with the cell cycle, the plasma membrane
and the cell wall. There are undoubtedly many more
players at MT ends than the few identified. Searching for
interacting partners of +TIPs could help identify new
proteins acting on MT dynamics and connecting MT
ends to other cellular structures such as actin, organelles
and membranes. At the minus end, beyond g-tubulin
complexes, the regulation of the distribution and activity
of MT nucleation sites in the cortical space is crucial.
Future progress relies on combining approaches such as
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6 Cell Biology
genetic and in vivo cytologic analyses, and the use of cellfree systems. Comparative genomics will help identify
shared components of the distributed MT organizer of
plant cells and eukaryotic MTOCs but will not replace
designing methods to identify plant-specific cellular
proteins that interact with the MT wall and the minus
and plus ends. In this respect, developing high-throughput methods to characterize subcellular proteomes such
as what was achieved for the human centrosome or
mitotic spindle proteomes would be of tremendous significance for plant cell biology.
Acknowledgements
Work in our laboratory is supported by grants from the French Ministry for
Research and by the Région Île-de-France for imaging facilities.
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www.sciencedirect.com
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Current Opinion in Plant Biology 2007, 10:1–7
Please cite this article in press as: Pastuglia M, Bouchez D, Molecular encounters at microtubule ends in the plant cell cortex, Curr Opin Plant Biol (2007), doi:10.1016/j.pbi.2007.08.001