Commentary
2805
Microtubule assembly during mitosis – from distinct
origins to distinct functions?
Sylvain Meunier1 and Isabelle Vernos1,2,*
1
Microtubule Function and Cell Division group, Cell and Developmental Biology Program, Centre for Genomic Regulation (CRG) and UPF,
Dr. Aiguader 88, 08003 Barcelona, Spain
2
Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluis Companys 23, 08010 Barcelona, Spain
*Author for correspondence (isabelle.vernos@crg.eu)
Journal of Cell Science
Journal of Cell Science 125, 2805–2814
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.092429
Summary
The mitotic spindle is structurally and functionally defined by its main component, the microtubules (MTs). The MTs making up the
spindle have various functions, organization and dynamics: astral MTs emanate from the centrosome and reach the cell cortex, and thus
have a major role in spindle positioning; interpolar MTs are the main constituent of the spindle and are key for the establishment of
spindle bipolarity, chromosome congression and central spindle assembly; and kinetochore-fibers are MT bundles that connect the
kinetochores with the spindle poles and segregate the sister chromatids during anaphase. The duplicated centrosomes were long thought
to be the origin of all of these MTs. However, in the last decade, a number of studies have contributed to the identification of noncentrosomal pathways that drive MT assembly in dividing cells. These pathways are now known to be essential for successful spindle
assembly and to participate in various processes such as K-fiber formation and central spindle assembly. In this Commentary, we review
the recent advances in the field and discuss how different MT assembly pathways might cooperate to successfully form the mitotic
spindle.
Key words: Microtubule, Nucleation, Spindle assembly
Introduction
Microtubules (MTs) are involved in a large number of processes,
such as protein and organelle transport, cell polarity, cell shape,
cell motility and cell division. They are assemblies of a- and btubulin heterodimers that are arranged in a head-to-tail fashion
into protofilaments. In mammalian cells, thirteen protofilaments
then interact laterally to form a tubular structure of ,25 nm in
diameter. MT nucleation is facilitated by c-tubulin, which,
together with gamma tubulin complex component (GCP; or
TUBGPC) 2 and 3, forms the so-called c-tubulin small complex
(c-TuSC). Several c-TuSCs form the large c-tubulin ring
complex (c-TuRC), which includes additional GCPs (namely
GCP4, GCP5 and GCP6). The c-TuRC provides a template for
the addition of tubulin heterodimers and thereby promotes MT
polymerization (reviewed by Kollman et al., 2011).
The orientation of the a- and b-tubulin heterodimers within the
MT confers an intrinsic polarity that is defined by a minus-end
(where a-tubulin is exposed) and a plus-end (where b-tubulin is
exposed). Interestingly, each end has distinct dynamic properties
and the plus-end is the dominant site for the addition of tubulin
subunits and MT elongation. MTs are dynamic filaments that
undergo successive cycles of growth and shrinkage, a property
called dynamic instability (Mitchison and Kirschner, 1984). A
large number of factors are involved in mechanisms that favor
MT stabilization or destabilization. This allows the cell to alter
the parameters of MT dynamics and to reorganize its MT
network. This occurs, for example, during cell division, when
MTs form the mitotic spindle. As the cell commits to divide, MTs
nucleated by the centrosomes become shorter and more dynamic,
which leads to the disassembly of the interphase MT network
(Belmont et al., 1990). In addition, after nuclear envelope
breakdown, the chromosomes direct de novo MT assembly and
a separate mechanism drives MT amplification. The local
stabilization and organization of these centrosomal and noncentrosomal MTs lead to the assembly of a bipolar spindle that
aligns the chromosomes on the metaphase plate and segregates
them into the two daughter cells (Walczak and Heald, 2008).
Here, we will first review the recent findings on the specific
characteristics (organization, dynamic properties and function) of
the different MT subclasses in the spindle. We will then describe
our current knowledge of the mechanisms underlying the noncentrosomal MT assembly pathways and their contribution to
spindle assembly. Finally, we will discuss whether the specific
MT origins might confer specific properties to the spindle MTs
that contribute to spindle functionality and, therefore, successful
cell division.
The bipolar spindle consists of different MT
subclasses
In animal cells, a number of criteria (position, organization and
functionality) define three distinct subclasses of MTs in the
mature mitotic spindle: the astral MTs, the interpolar MTs and
the kinetochore-fibers (K-fibers) (Fig. 1). Astral MTs emanate
from the two centrosomes and extend all the way to the cell
cortex. They have a major role in centrosome separation during
prophase and in spindle positioning (Rosenblatt, 2005) and,
therefore, largely mediate the important role of the centrosomes
in polarized cells and during asymmetric cell division (Knoblich,
2010). Mitosis can, however, occur without astral MTs
2806
Journal of Cell Science 125 (12)
B
+
+
+
+
A
–
+
Interpolar MTs
+
t1/2= 30 seconds–1 minute
C
+
+
+
+
+
+
+
+
+
+
+
–
+
K-fibers
+
+
t1/2= 4–8 minutes
D
Journal of Cell Science
+
+
+
+
–
+
Astral MTs
t1/2= 30 seconds–1 minute
(Khodjakov et al., 2000; Mahoney et al., 2006), and we will
therefore focus on interpolar MTs and K-fibers.
Interpolar MTs
The interpolar MTs are the most dynamic class of spindle MTs
and have an average half-life (t1/2) of less than 1 minute (Zhai
et al., 1995). They are the most abundant class of MTs and
comprise the main body of the mature spindle in many systems.
Interpolar MTs emanate from the centrosome and extend towards
the center of the spindle, where they interact in an antiparallel
fashion with interpolar MTs emanating from the opposite spindle
pole (Fig. 1). However, some interpolar MTs are shorter and do
not extend half-way through the spindle (Mastronarde et al.,
1993). In spindles assembled in Xenopus egg extracts, interpolar
MTs are particularly abundant and comprise 95% of the spindle
MTs (Ohi et al., 2007). They form a ‘polar’ array of parallel MTs
near the spindle poles and a ‘barrel’ array of antiparallel,
overlapping MTs close to the chromosomes (Yang et al., 2007).
During anaphase, interpolar MTs are rearranged and become the
major component of the central spindle, where they form an array
of antiparallel MTs with bundled overlapping plus-ends (Glotzer,
2009).
Interpolar MTs have multiple functions, including the
establishment and maintenance of spindle bipolarity. In addition,
they have a role in chromosome congression because they interact
dynamically with the chromosomes through the chromokinesins
[KID (also known as KIF22), kinesin family member (KIF) 4 and
KLP2 (also known as KIF15) (Vanneste et al., 2011)] or through
lateral interactions with the kinetochores (Cai et al., 2009;
Magidson et al., 2011; Wignall and Villeneuve, 2009). However,
the interpolar MTs are not directly responsible for chromosome
segregation. This function is provided by the K-fibers.
K-fibers
K-fibers are bundles of 20–40 parallel MTs (McEwen et al.,
1997; Rieder, 1981) whose main function is to attach the
chromosomes to the two spindle poles and to segregate the sister
Fig. 1. The three subclasses of spindle MTs. (A) Schematic
metaphase spindle, showing its three different MT subclasses:
interpolar MTs (orange), K-fibers (red) and astral MTs (green). The
‘+’ indicates the plus-ends of the MTs. (B–D) Representation of the
three kinds of spindle MTs and their dynamic properties (see the text
for more details). Astral MTs have dynamic properties that are
similar to those of the interpolar MTs (Mitchison et al., 1986). Kfibers are less dynamic and undergo a constant polewards tubulin
flux (arrows).
chromatids into the daughter cells. K-fibers are much less
dynamic than the interpolar MTs. Depending on the experimental
system, their half-life is 4–8 minutes, which is closer to that of
interphase MTs (t1/259–10 minutes) (Bakhoum et al., 2009; Zhai
et al., 1995). Consistent with this, K-fibers are the last MT
subclass to depolymerize following the exposure of cells to cold
or nocodazole.
The mechanism driving the assembly of the K-fibers is still not
fully understood. The attachment of MTs to the kinetochore occurs
progressively as the cell moves from prometaphase to anaphase, in
a process called K-fiber maturation (McEwen et al., 1997). The
characteristic bundling of the K-fiber MTs seems to be, in part,
achieved by a protein complex that includes clathrin, TACC3 (for
transforming, acidic coiled-coil containing protein 3, also known as
maskin) and chTOG (also known as CKAP5 and, in Xenopus,
XMAP215) (Booth et al., 2011). It is possible that some K-fiberassociated proteins that show MT-bundling activities in vitro might
also be important players in K-fiber formation (Ribbeck et al.,
2006; Silljé et al., 2006; Yokoyama et al., 2009). Further work is
required to understand how MTs organize into the K-fiber bundles.
The K-fiber plus-ends become attached to the outer kinetochore
through the KMN complex (for KNL1–MIS12-complex–NDC80)
network (Joglekar et al., 2010), which couples force generation to
MT plus-end polymerization and depolymerization (Salmon et al.,
2005). Erroneous K-fiber–kinetochore attachments are released for
error correction through a mechanism involving the targeting and
regulation of various kinetochore factors by the kinase Aurora B
(Kelly and Funabiki, 2009). The mechanism controlling the
polymerization at the K-fiber plus-ends is largely dependent on
microtubule plus-end tracking proteins, including CLASP (for
cytoplasmic linker associated protein, also known as orbit) (Maiato
et al., 2005) and EB1 (also known as MAPRE1) (Tirnauer et al.,
2002). The activities of kinesin-8 and -13 negatively regulate Kfiber plus-end stability (Joglekar et al., 2010; Manning et al.,
2010).
It has still not been fully resolved how the individual MT
dynamics in the K-fibers result in directional kinetochore
Microtubule assembly in mitosis
(Khodjakov and Rieder, 1999). This process, called centrosome
maturation, results in a dramatic increase in MT nucleation at the
centrosomes during mitosis (Piehl et al., 2004).
The presence of two centrosomes actively nucleating MTs
from opposite sides of the nucleus in cells entering mitosis was
central to the ‘search-and-capture’ model proposed by Kirschner
and Mitchison in 1986 (Fig. 2). This model postulated that
dynamic centrosomal MTs emanating from the two centrosomes
grow and shrink, thereby exploring the cellular space until their
plus-ends become captured by the paired sister kinetochores. This
mechanism would naturally lead to the assembly of the bipolar
spindle (Kirschner and Mitchison, 1986). In the past decade, both
experimental and theoretical models have established that
additional mechanisms are at work to help the search-andcapture process to connect all 92 kinetochores to the spindle
poles in human cells (Magidson et al., 2011; Paul et al., 2009;
Wollman et al., 2005).
Early observations made in systems lacking centrosomes, such
as plant cells (De Mey et al., 1982) and female germ cells
undergoing meiosis in some animal species (Manandhar et al.,
2005), have shown that acentrosomal cell divisions can also take
place. More recently, different experimental approaches in cells
containing centrosomes (Khodjakov et al., 2000; Mahoney et al.,
2006), as well as in whole organisms (Basto et al., 2006), have
lent additional support to the idea that spindle assembly and
mitosis can occur in the absence of centrosomes (Fig. 2). This
implies that additional non-centrosomal source(s) of MTs that
drive spindle formation and chromosome segregation must exist
in dividing cells, even when they naturally contain centrosomes.
Karsenti and Kirschner observed in 1984 that the injection of
bacteriophage lambda or E. coli DNA into Xenopus eggs is
sufficient to trigger MT formation, thereby obtaining the first
experimental evidence that chromatin has a role in MT assembly
(Karsenti et al., 1984). The demonstration that DNA-coated
beads incubated in Xenopus egg extract induce the assembly of a
bipolar spindle provided further support to the idea that
chromatin itself is sufficient to induce MT assembly and
Journal of Cell Science
movements that can abruptly switch from a polewards to an antipolewards direction (Skibbens et al., 1993; VandenBeldt et al.,
2006). It has been shown, however, that the loss of tubulin
subunits at the K-fiber minus-end occurs without spindle pole
detachment and drives the polewards tubulin flux. This
mechanism is sufficient for generating pulling forces at the
kinetochores that stretch the centromeres in metaphase (Waters
et al., 1996). In anaphase, dramatic changes in K-fiber dynamics
at the plus-end, and also at the minus-end in some systems,
promote the shortening of the K-fibers and chromosome
segregation (Ganem and Compton, 2006; Kwok and Kapoor,
2007; Cheerambathur et al., 2007; Rogers et al., 2004).
Various observations in mammalian and Drosophila cells
suggest that the initial step of K-fiber formation occurs close to
the kinetochores (Maiato et al., 2004; Tulu et al., 2006). A
possible scenario is that the plus-ends of these MTs would be
efficiently captured and stabilized by the kinetochore, and their
minus-ends would connect with interpolar MTs before becoming
focused at the spindle poles in a dynein- and nuclear mitotic
apparatus protein 1 (NUMA1)-dependent manner (Khodjakov
et al., 2003). These observations suggest that some MTs, and
especially those forming the K-fibers, are not nucleated at the
centrosomes.
Moving towards non-centrosomal MT assembly
pathways
Since the initial description of the centrosome and its ‘radiating
aster’ in the late 19th century (Paweletz, 2001), detailed
structural studies have shown that centrosomes comprise two
centrioles surrounded by pericentriolar material (PCM) (Chrétien
et al., 1997; Ibrahim et al., 2009; Paintrand et al., 1992; Vorobjev
and Chentsov, 1982). Centrosome biogenesis follows a cycle that
is closely coordinated with the cell cycle, during which the single
centrosome of the interphase cell duplicates (Bettencourt-Dias
and Glover, 2007). At the onset of mitosis, the duplicated
centrosomes recruit several components to the PCM, including ctubulin whose amount increases by more than three times
First observation of MTs
originating at the mitotic
chromosomes (Witt et al., 1980)
Mitotic spindle is a dynamic
structure made of filaments
called MTs (Inoue and Sato, 1967)
1967
1968
Identification of tubulin as
the component of MTs
(Mohri, 1968)
1984
Acentrosomal spindle
assembly in
mammalian cells
(Khodjakov et al., 2000)
DNA-coated beads
induce spindle assembly
(Heald et al., 1996)
MT dynamic instability
(Mitchison and
Kirschner, 1984)
1980
Discovery of the
Augmin pathway
(Goshima et al., 2008)
γ-Tubulin
nucleates MTs
(Wilde and Zheng, 1999)
1986
‘Search-and-capture’
model
(Kirschner and
Mitchison, 1986)
DNA triggers MT assembly
(Karsenti and Kirschner, 1984)
2807
1996
1999
2000
Discovery of the
Ran-GTP pathway
(Carazo-Salas et al., 1999;
Kalab et al., 1999;
Zhang et al., 1999;
Ohba et al., 1999)
2004
2008
Discovery of the
CPC pathway
(Sampath et al., 2004)
Fig. 2. Timeline of the key discoveries on spindle formation. The timeline highlights the key findings that have led to the current model of spindle formation
involving the different pathways of MT assembly discussed in the text. For previous historical discoveries, please see the excellent reviews by Paweletz
(Paweletz, 2001) and Mitchison and Salmon (Mitchison and Salmon, 2001).
2808
Journal of Cell Science 125 (12)
organization in M phase (Heald et al., 1996; Walczak et al.,
1998). Furthermore, MT nucleation away from the centrosome
has also been observed during mitosis in Drosophila and
mammalian cells (Khodjakov et al., 2000; Mahoney et al.,
2006; Tulu et al., 2006; Witt et al., 1980). Extensive studies over
the past 10 years have provided mechanistic insights into the noncentrosomal MT assembly during cell division and have shown
that two distinct chromosomal MT assembly pathways are
involved: one is triggered by GTP-bound Ran and the other is
controlled by the chromosomal passenger complex (CPC)
(Fig. 2, timeline).
Chromosomes as a source of MTs in dividing
cells
Journal of Cell Science
The Ran-GTP pathway
Ran is a small GTPase that drives nucleo-cytoplasmic transport
during interphase, whereby the high concentration of the GTPbound form of Ran (Ran-GTP) in the nucleus allows the release
of newly imported proteins from their binding to importins
(Clarke and Zhang, 2008). In the late 1990s, several groups
identified a mitosis-specific function for Ran in MT assembly.
Their studies defined a new pathway that turned out to be
essential for spindle assembly (Carazo-Salas et al., 2001; CarazoSalas et al., 1999; Kalab et al., 1999; Nachury et al., 2001; Ohba
et al., 1999; Zhang et al., 1999). Spatially, the system is defined
by the presence of the Ran guanine nucleotide exchange factor
RCC1 (regulator of chromosome condensation 1) on chromatin
(Karsenti and Vernos, 2001). This results in the formation of a
Ran-GTP gradient that is centered around the chromosomes,
which has been observed using fluorescence resonance energy
transfer (FRET) approaches in Xenopus egg extract as well as in
human somatic cells (Kaláb et al., 2006; Kalab et al., 2002). This
Ran-GTP gradient ensures the local nucleation of MTs close to
the chromatin and has a longer-range effect on MT stabilization
(Caudron et al., 2005). The fine-tuned regulation of this system in
space and time is still not fully understood and will require
further investigation (Arnaoutov et al., 2005; Joseph et al., 2002;
Torosantucci et al., 2008).
The basic mechanism by which the Ran system functions both
during interphase and in dividing cells is based on the differential
affinity of the GDP- and GTP-bound forms of Ran for
karyopherins (Clarke and Zhang, 2008). It has been postulated
that, during cell division, Ran-GTP can trigger the release of
putative spindle assembly factors (SAFs) from importins, thereby
enabling the SAFs to perform their function in spindle assembly.
The first protein that was shown to be regulated in this way is
TPX2 (targeting protein for XKLP2) (Gruss et al., 2001;
Wittmann et al., 2000). Once released from importin-a and
importin-b, TPX2 triggers MT assembly around the chromatin
and this activity is essential for spindle assembly (Gruss et al.,
2001; Gruss et al., 2002; Tulu et al., 2006) (Fig. 3). Over the last
decade, a number of Ran-GTP-regulated SAFs have been
identified, and most of them have been shown to be involved
in MT stabilization and/or organization (Table 1). In addition,
other proteins that are not directly regulated by RanGTP, like ctubulin and ch-TOG (also known as CKAP5) (Groen et al., 2009;
Wilde and Zheng, 1999), are also required for MT assembly
through this pathway. However, the precise mechanism by which
all these proteins promote MT assembly and organization is still
not clearly understood.
The CPC pathway
Using a ‘mitosis with unreplicated genomes’ (MUG) system in
which chromatin is, at least partially, excluded from the spindle,
O’Connell and collaborators have shown that MT assembly also
occurs at the kinetochores in a manner that is independent of
chromatin, RCC1 and the Ran-GTP gradient (O’Connell et al.,
2009). In fact, the molecular nature of this pathway has been
described by the Funabiki group, and it is now known to involve
the CPC, which comprises INCENP (inner centromere protein
antigens 135/155 kDa), survivin (also known as BIRC5), borealin
(also known as CDCA8) and the kinase Aurora B (Sampath et al.,
2004; Tseng et al., 2010). The role of the CPC in MT
stabilization and spindle assembly has been extensively
characterized in Xenopus egg extracts (Kelly et al., 2007;
Maresca et al., 2009; Sampath et al., 2004; Tseng et al., 2010).
Beads coated with reconstituted CPC have been shown to
promote MT assembly and the formation of bipolar spindles in
Xenopus egg extract in the absence of Ran-GTP (Kelly et al.,
2007; Maresca et al., 2009). The mechanism involves the local
B
A
TPX2
CPC
Ran-GTP
αβ
TPX2
RCC1
Ran-GDP
C
FAM29A
HICE1
Fig. 3. MT assembly pathways. (A) Schematic
prometaphase spindle, showing the three known sites of MT
assembly during spindle assembly. MTs that originate from
the centrosomes are in red, those originating from the
chromosomes in orange and those emanating directly from
pre-existing MTs are shown in green. (B,C) Representation
of the main components of the two pathways of
acentrosomal MT nucleation. (B) At the chromosomes, the
presence of RCC1 bound to chromatin creates a Ran-GTP
gradient around the chromosomes, allowing the release of
SAFs such as TPX2 from importins (which inhibit SAF
activity). TPX2, together with c-tubulin complexes (grey
rings), then triggers MT nucleation. Chromosomal MTs are
stabilized around the kinetochores by a mechanism that
depends on the CPC (see text for details). (C) During MT
amplification on pre-existing MTs, the Augmin complex
(purple) is recruited to a preexisting MT (red), through its
subunit HICE1. FAM29A is responsible for recruiting ctubulin (grey ring) to the complex and thereby triggering
MT assembly.
Microtubule assembly in mitosis
2809
Table 1. Ran-GTP-regulated SAFs
Protein
MT nucleation
TPX2
MT stabilization
HURP
RAE1
ISWI1
CDK11
APC
TACC3
MCRS1
Journal of Cell Science
NUSAP1
Spindle organization
NUMA1
Kid
HSET
Transport receptors
Properties
Mice models (effect of loss-of-function)
Importin-a, importin-b (Gruss
et al., 2001; Wittmann et al.,
2000)
Interacts with and activates Aurora A
(Bayliss et al., 2003)
Early embryonic lethality (AguirrePortolés et al., 2012)
Importin-b (Silljé et al., 2006)
MT-bundling activity (Koffa et al., 2006; Silljé
et al., 2006; Wong and Fang, 2006)
Ribonuncleoprotein complex involved in spindle
assembly (Blower et al., 2005; Wong et al., 2006)
MT-bundling activity (Yokoyama et al., 2009)
Female infertility (Tsai et al., 2008)
Importin-b (Blower et al.,
2005; Wong et al., 2006)
Importin-a, importin-b
(Yokoyama et al., 2009)
Importin-b (Yokoyama et al.,
2008)
Importin-b (Dikovskaya et al.,
2010)
Importin-b
(Albee et al., 2006)
Importin-b (Meunier and Vernos,
2011)
Importin-a and/or importin-b,
importin 7 (Ribbeck et al.,
2006; Ribbeck et al., 2007)
Importin-b (Nachury et al.,
2001; Wiese et al., 2001)
Importin-a, importin-b (Tahara
et al., 2008)
Importin-a, importin-b
(Ems-McClung et al., 2004)
Necessary for Ran-GTP-dependent MT
stabilization(Yokoyama et al., 2008)
MT-bundling activity (Dikovskaya et al., 2010)
Phosphorylated by Aurora A
(Barros et al., 2005; Peset et al., 2005)
Binds and protects chromosomal MT minusends (Meunier and Vernos, 2011)
Interacts with chromosomes and MTs (Ribbeck
et al., 2006; Ribbeck et al., 2007)
Pole focusing (Haren et al., 2009; Merdes et al.,
2000; Merdes et al., 1996; Silk et al., 2009)
Chromokinesin; additional MT-binding site (ATP
independent) (Antonio et al., 2000; Funabiki and
Murray, 2000; Tokai et al., 1996)
Minus-end directed motor; additional
MT-binding site (ATP independent)
(Walczak et al., 1997)
Early embryonic lethality (Babu
et al., 2003)
Early embryonic lethality (Stopka
and Skoultchi, 2003)
Early embryonic lethality (Li
et al., 2004)
Model of colorectal cancer (Oshima
et al., 1996)
Malformations of axial skeleton
(Yao et al., 2007)
n.d.
Early embryonic lethality
(Vanden Bosch et al., 2010)
n.d.
50% embryonic lethality (Ohsugi
et al., 2008)
n.d.
TPX2, the first Ran-GTP-regulated SAF to be identified, is essential for MT nucleation, some SAFs are involved in MT stabilization or bundling and others are
more likely to be involved in spindle organization. n.d., not determined.
activation of the kinase Aurora B (Sampath et al., 2004), which
must be targeted to the MTs (through INCENP) in order to
promote spindle assembly (Tseng et al., 2010). Taken together,
these studies show that the CPC has a role in MT assembly that
might be independent of the Ran-GTP pathway.
It is not currently clear whether the CPC-dependent pathway is
directly involved in MT nucleation or stabilization. There are no
reports on the involvement of c-tubulin in the CPC-induced
mechanism. However, Aurora B has been shown to negatively
regulate the MT-catastrophe-promoting factors MCAK (for
mitotic centromere-associated kinesin, also known as KIF2C)
(Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004) and
stathmin 1 (STMN1, also known as OP18) (Gadea and
Ruderman, 2006). It is therefore probable that the CPC
pathway promotes MT stabilization, rather than nucleation itself.
The Ran-GTP- and CPC-dependent pathways probably work
together, first nucleating MTs near chromatin and then stabilizing
these nascent MTs preferentially near the kinetochores (Maresca
et al., 2009; Needleman et al., 2010; Tulu et al., 2006) (Fig. 3).
MT branching and amplification by the
Augmin complex
The observations reporting the localization of c-tubulin to spindle
MTs (Khodjakov and Rieder, 1999; Lajoie-Mazenc et al., 1994)
and the distribution of MT minus-ends throughout the whole
spindle in Xenopus egg extracts (Burbank et al., 2006), could be
explained by the transport of the MTs nucleated on chromosomes
towards the poles. However, a mechanism of MT nucleation on
preexisting MTs has been described in higher plants and fission
yeast (Bratman and Chang, 2008), and the identification the Dgt
(for dim c-tubulin) protein family (Goshima et al., 2007) has
revealed a similar mechanism for MT amplification in the
spindles of animal cells. These proteins define a complex called
Augmin in Drosophila (Goshima et al., 2008) and HAUS
(homologous to Augmin subunits) in human cells (Lawo et al.,
2009; Uehara et al., 2009). The Augmin complex has been shown
to recruit the c-TuRC to preexisting MTs and to promote MT
nucleation, amplification or branching (Zhu et al., 2008; Zhu
et al., 2009) (Fig. 3). The role of the different Augmin complex
subunits has not been entirely resolved, but FAM29A (also
known as Dgt6 and HAUS6) binds the c-TuRC, and another
component, HICE1 (also known as HAUS8), associates with
MTs (Tsai et al., 2011; Zhu et al., 2008). These two activities
might account for targeting of the c-TuRC to a pre-existing MT.
It is currently unknown whether this targeting can happen
anywhere along a MT or whether there are some specific sites for
MT branching and/or amplification. Moreover, it is also unknown
whether all spindle MTs can act as docking sites with the same
efficiency.
The severity of the phenotypes observed when the function of
this complex is impaired seem to be largely dependent on the
system and more specifically on the presence or absence of
centrosomes (Petry et al., 2011; Uehara et al., 2009; Wainman
et al., 2009). In the absence of centrosomes, Augmin-dependent
2810
Journal of Cell Science 125 (12)
Journal of Cell Science
MT amplification is required, together with the chromosomal
pathway, to build a bipolar spindle (Wainman et al., 2009).
During anaphase, the Augmin complex is essential for central
spindle assembly, for promoting MT nucleation and possibly for
the very specific organization of the interpolar MTs composing
the structure (Uehara and Goshima, 2010; Uehara et al., 2009).
A unifying principle for MT nucleation in
dividing cells
A common requirement for the assembly of MTs through any of the
three pathways – centrosomal, chromosomal and MT-dependent –
is c-tubulin, which acts as a template for MT nucleation (Goshima
et al., 2008; Mishra et al., 2010; Wilde and Zheng, 1999). The
mitotic kinases cyclin-dependent kinase 1 (CDK1), polo-like
kinase 1 (PLK1) and Aurora A have been shown to be involved in
c-tubulin recruitment to the centrosome (Blagden and Glover,
2003). PLK1 and Aurora A activities are involved in c-TuRC
recruitment by the Augmin complex (Johmura et al., 2011; Tsai
et al., 2011) (Tables 2, 3). Although the mechanism of c-tubulin
targeting in the chromosomal pathway remains poorly understood,
Aurora A kinase is also required for MT nucleation and
stabilization around chromatin (Bayliss et al., 2003; Sardon
et al., 2008) (Table 3). The targeting of c-tubulin to each MT
nucleation site is, at least in part, directed by the c-TuRCassociated protein NEDD1 (neural precursor cell expressed,
developmentally down-regulated 1, also known as GCP7)
(Lüders et al., 2006; Zhu et al., 2008) (Table 2). Interestingly,
NEDD1 is hyperphosphorylated in mitosis, and its activity is
differentially regulated by mitotic kinases at the centrosomes and
in the acentrosomal MT amplification pathway (Johmura et al.,
2011; Zhang et al., 2009).
The selective targeting and activation of c-TuRCs by factors
specific for each pathway might control MT assembly at different
sites in the mitotic cell (Tables 2, 3). It is an interesting
possibility that these different targeting complexes compete for a
limited pool of c-TuSCs and c-TuRCs in the cell. The efficiency
and/or regulation of c-tubulin targeting would therefore explain
the relative contribution of the different MT assembly pathways,
which varies dramatically depending on the organism and/or the
cell type.
In addition to the distinct mechanisms of c-tubulin targeting,
the type of c-tubulin-containing complex involved in MT
nucleation in each MT assembly pathway might be different.
Although the c-TuRC is an active MT nucleator in higher
eukaryotes, work in Drosophila has shown that the c-TuSC alone
is essential and sufficient for MT nucleation at the centrosome.
Indeed, none of the specific c-TuRC proteins (i.e. GCP4, GCP5
and GCP6) are required for c-tubulin recruitment to the
centrosome or for MT nucleation in Drosophila cells (Vérollet
et al., 2006) (Table 2). Interestingly, it has recently been shown
that c-TuSCs are able to form a ring structure without the need of
the other GCPs (Kollman et al., 2010). It is therefore tempting to
speculate that the c-TuSCs could be sufficient to promote MT
nucleation in metazoans, as it is the case in budding yeast
(Kollman et al., 2011; Vinh et al., 2002). Taken together, these
data suggest the possibility that different c-tubulin nucleation
complexes might exist in the eukaryotic cell. If this is the case, it
will be important to determine with precision which complexes
function in the different MT assembly pathways and whether
there are functional implications.
Could different origins confer specificity to
distinct spindle MTs?
Having identified different means by which spindle MTs are
assembled, the question remains as to what the relative
contribution of the different MTs assembly pathways is to
spindle assembly and function. The current view is that MTs that
originate from the different pathways cooperate to form the
spindle: the centrosome asters define the position of the two
spindle poles and the axis of cell division; the chromosomal MTs
facilitate the capture of the kinetochores and the formation of the
K-fibers; and MT amplification by the Augmin complex
contributes to the strengthening of the whole structure (Figs 1,
3). The combination of MTs from the three pathways could
therefore allow the timely assembly of the bipolar spindle and
allow mitosis to progress successfully. The recent identification
of microspherule protein 1 (MCRS1) as a SAF (Meunier and
Vernos, 2011) suggests that there is a possibility that the different
MTs are not equivalent but differ in terms of dynamics and
molecular composition.
Table 2. MT nucleation complexes
Protein
Centrosomal pathway
c-TuRC proteins
c-tubulin (c-TuSC component)
GCP2 (c-TuSC component)
GCP3 (c-TuSC component)
GCP4
GCP5
GCP6
3
3
3
7 (Vérollet et al., 2006)
7 (Vérollet et al., 2006)
7 (Vérollet et al., 2006)
3
?
?
?
?
?
3 (Mishra et al., 2010)
3 (Mishra et al., 2010)
3 (Mishra et al., 2010)
?
?
?
?
?
?
7
7
7
7
7
7
3 (Lüders et al., 2006)
3 (Zhu et al., 2008)
3 (Lüders et al., 2006)
c-TuRC-associated proteins
MOZART1
(Hutchins et al., 2010)
MOZART2A
(Teixidó-Travesa et al., 2010)
MOZART2B
(Teixidó-Travesa et al., 2010)
NEDD1
MT amplification pathway
Chromosomal pathway
The table shows the requirements of the components of the c-TuSC, c-TuRC and c-TuRC-associated proteins in the three MT assembly pathways (centrosomes,
MTs and chromosomes). 7, not required for the assembly pathway; 3, required for the assembly pathway; ?, involvement not known.
Microtubule assembly in mitosis
2811
Table 3. c-Tubulin-targeting mechanisms
Centrosomes
Targeting factors
CEP192, ninein, centrosomin (Gomez-Ferreria
et al., 2007; Raynaud-Messina and Merdes, 2007)
Targeting regulators
PLK1, Aurora A, CDK1 (Barr and Gergely, 2007)
MT amplification
Chromosomes
Augmin complex, FAM29A
(Bucciarelli et al., 2009)
TPX2, RHAMM (also
known as HMMR)
(Groen et al., 2004)
PLK1, Aurora A (Johmura et al., 2011;
Tsai et al., 2011)
Ran-GTP, Aurora A
(Gruss et al., 2001; Tsai et al., 2003)
Journal of Cell Science
In the upper part of the table, the proteins involved specifically in c-tubulin targeting to the MT nucleation sites are indicated for each of the three pathways. In
the lower part of the table, the main regulators of c-tubulin recruitment for each MT assembly pathway are shown.
MCRS1 is a nuclear protein that associates specifically with
the chromosomal Ran-GTP-dependent MTs. As such it is the first
example of a protein showing specificity for this class of MTs.
Interestingly, MCRS1 also shows full specificity for the minusends of the K-fibers. These data provide a molecular link between
the chromosomal MTs and the K-fibers that goes beyond
previous observations of the formation of acentrosomal MTs at
the kinetochores (Khodjakov et al., 2003; Maiato et al., 2004).
Interestingly, the distinct spindle MT subclasses have distinct
dynamic properties, (Rizk et al., 2009), and functional studies
have shown that MCRS1 could have a role in the regulation of Kfiber minus-end stability (Meunier and Vernos, 2011).
The localization and function of MCRS1 therefore suggests
that some specific proteins can be ‘loaded’ on the K-fibers by the
Ran-GTP pathway to confer specific properties in terms of
organization and dynamics to the K-fibers. Interestingly, other
Ran-GTP-regulated proteins show activities consistent with this
idea (Table 1). For instance, HURP (also known as DLGAP5),
nucleolar and spindle associated protein 1 (NUSAP1) and ISWI
(also known as SMRCA) have MT-bundling activity (Ribbeck
et al., 2006; Silljé et al., 2006; Yokoyama et al., 2009) and
preferentially bind to K-fibers (Silljé et al., 2006). Moreover,
NUMA1 is involved in K-fiber minus-end transport towards the
poles and is involved in the regulation of K-fiber stability (Haren
et al., 2009; Khodjakov et al., 2003; Silk et al., 2009). TACC3
has also recently been shown to have a role in MT bridging in Kfibers (Booth et al., 2011; Cheeseman et al., 2011). Taken
together, these data suggest a role for the Ran-GTP-dependent
MT assembly pathway in promoting and regulating K-fiber
formation.
It is however important to note that the MTs nucleated by the
chromosomal pathway are not all organized in K-fibers, as they
can also form interpolar MTs. It is therefore probable that both
the interaction of these MTs with the kinetochore and their plusend stabilization through the CPC also have a contribution in
conferring specific properties to K-fibers.
Perspectives
Although three pathways for MT assembly in mitotic cells have
been identified so far, it is possible that others exist. For instance,
remnants of the nuclear envelope have been shown to function as
MT nucleation sites in dividing Drosophila spermatocytes
(Rebollo et al., 2004) but nothing is known about this process
in mammalian cells. Golgi stacks have also been reported to
efficiently promote MT assembly in interphase cells (Efimov
et al., 2007; Rı́os et al., 2004; Rivero et al., 2009). Are these
membrane-dependent mechanisms also involved in mitotic
spindle assembly and how do they participate in successful cell
division? Further studies will certainly clarify these questions and
help us to obtain a global view of spindle assembly and function.
As a result of decades of extensive research in the field of MT
assembly during mitosis, we are now starting to reach a good
understanding of spindle formation. However, an exciting
challenge for the future will not only be to elucidate the exact
role of each MT assembly pathway and the mechanism by which
they work together to build the mitotic bipolar spindle but also to
identify new putative pathways of MT assembly during mitosis.
This is fundamental for our understanding of how, in only a few
minutes, the cell is able to build a machinery as complex as the
mitotic spindle.
Acknowledgements
We would like to thank M. Mendoza, R. Pinyol, T. Cavazza, M.
Schütz and J. Scrofani for critical reading of the manuscript, and all
the members of the Vernos lab for helpful discussions. We apologize
to authors whose works could not be cited owing to space limitation.
We thank the reviewers, whose comments greatly improved the
manuscript.
Funding
The work of our laboratory is supported by grants from the Spanish
Ministerio de Educación y Ciencia; the Ministerio de Ciencia e
Innovaciœn [grant numbers BFU2006-04694, BFU2009-10202,
CSD2006-00023]; and institutional funds.
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