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CHAPTER TWO
Microtubule Plus-End TrackingProteins and Their Roles in CellDivisionJorge G. Ferreira*,†, Ana L. Pereira*, Helder Maiato*,†,1*Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universityof Porto, Porto, Portugal†Cell Division Unit, Department of Experimental Biology, University of Porto, Porto, Portugal1Corresponding author: e-mail address: maiato@ibmc.up.pt
Contents
1. Introduction 602. Microtubules in Cell Division 64
2.1 Mitotic entry 642.2 Prometaphase–metaphase transition 652.3 Metaphase 662.4 Metaphase–anaphase transition 672.5 Mitotic exit and cytokinesis 67
3. Families of Microtubule Plus-End-Tracking Proteins (þTIPs) 693.1 CLIP family 693.2 EB family 733.3 CLASP family 773.4 APC family 813.5 Motor proteins 823.6 Lis1 843.7 Kinesin-13 family 853.8 TOG family 873.9 Other þTIPs 88
4. Recognition of Microtubule Plus Ends by þTIPs 894.1 Recognizing the microtubule plus end 894.2 Copolymerization 914.3 Diffusion versus motor-based transport 924.4 Hitchhiking 934.5 Turnover at microtubule plus end 95
5. þTIPs in Mitosis 975.1 þTIPs in mitotic spindle organization and positioning 975.2 þTIPs at mitotic centrosome 1025.3 þTIPs at kinetochore 1045.4 þTIPs regulation during mitosis 1095.5 þTIPs in mitotic exit and cytokinesis 113
International Review of Cell and Molecular Biology, Volume 309 # 2014 Elsevier Inc.ISSN 1937-6448 All rights reserved.http://dx.doi.org/10.1016/B978-0-12-800255-1.00002-8
59
6. Concluding Remarks 116Acknowledgments 117References 117
Abstract
Microtubules are cellular components that are required for a variety of essential pro-cesses such as cell motility, mitosis, and intracellular transport. This is possible becauseof the inherent dynamic properties of microtubules. Many of these properties are tightlyregulated by a number of microtubule plus-end-binding proteins or þTIPs. These pro-teins recognize the distal end of microtubules and are thus in the right context to con-trol microtubule dynamics. In this review, we address how microtubule dynamics areregulated by different þTIP families, focusing on how functionally diverse þTIPs spa-tially and temporally regulate microtubule dynamics during animal cell division.
1. INTRODUCTION
Division of one cell into two genetically identical daughter cells occurs
through two coordinated processes known as mitosis (division of the
nucleus) and cytokinesis (division of the cytoplasm). In order to do so, cells
have to assemble a dynamic array of MTs known as the mitotic spindle.
Differences in MT dynamic behavior are observed in vivo and can occur
via two distinct mechanisms. One involves the addition and loss of tubulin
subunits at the same end of MTs—a mechanism known as dynamic instabil-
ity (Mitchison and Kirschner, 1984; Sammak and Borisy, 1988; Schulze and
Kirschner, 1988). The other occurs through gain of tubulin at the plus
ends of MTs and loss of tubulin at the minus ends of MTs—a mechanism
known as treadmilling (Margolis and Wilson, 1978; Rodionov and
Borisy, 1997).
Dynamic instability is driven mainly by GTP hydrolysis (Hyman et al.,
1992). Tubulin subunits are incorporated into a protofilament when bound
to GTP (Fig. 2.1). After incorporation, GTP hydrolysis occurs very rapidly
in the b-tubulin subunit (Desai and Mitchison, 1997). This means that the
MT lattice is enriched in GDP-tubulin. As a consequence, MT plus ends are
less stable and tend to adopt a curved conformation, favoring depolymeri-
zation (Desai and Mitchison, 1997; Melki et al., 1989). Given this, how is it
then possible for MTs to stabilize and polymerize? Hydrolysis of GTP is
favored by the addition of new heterodimers and therefore does not occur
60 Jorge G. Ferreira et al.
in the last subunit added to the protofilament but in the one before last. For
this reason, it was proposed that MTs have a GTP b-tubulin cap that would
be sufficient to stabilize them (Mitchison and Kirschner, 1984). The exact
size of the GTP cap is still unclear and many studies have reached different
conclusions, with values ranging from 40 GTP subunits (Voter et al., 1991)
to a single GTP subunit on each protofilament (Caplow and Shanks, 1996;
Drechsel and Kirschner, 1994).
Four parameters are currently used to define dynamic instability: growth
velocity, shrinking velocity, rescue frequency, and catastrophe frequency
(Walker et al., 1988). MT growth velocity depends on soluble tubulin con-
centration and the rate of association of GTP-tubulin to the MT. On the
other hand, shrinking velocity is independent of tubulin concentration
but depends on the dissociation rate of GDP-tubulin. Therefore, increasing
tubulin concentration can increase growth rate which, in turn, leads to a
Polymerization
Growing microtubule
Shrinking microtubule
Catastrophe Rescue
Depolymerization
GDP
GTP
Figure 2.1 Microtubule dynamic instability. GTP-bound tubulin assembles at themicro-tubule plus end creating a stable GTP cap that prevents microtubules fromdepolymerizing. When GTP hydrolysis occurs, the microtubule becomes unstable anddepolymerizes by the outward curving of individual protofilaments, which leads to fur-ther destabilization of themicrotubule structure. When GDP is substituted for GTP in thedisassembled tubulin subunits, the cycle can begin again.
61+TIPs in Cell Division
decrease in MT shortening rate. Catastrophe/rescue frequency is defined as
the number of catastrophes/rescues undergone during the total growth time
of an MT, respectively. These dynamic parameters can be easily visualized
using kymographic tools (Pereira and Maiato, 2010), which provide a visual
representation of the MT plus end over time (Fig. 2.2).
Recent in vitro systems have made possible to recreate physiologic
dynamic instability with minimal components such as MTs, an MT stabi-
lizer, and an MT destabilizer (Kinoshita et al., 2001; Li et al., 2012;
Zanic et al., 2013). Typical growth velocities of MTs in vitro are around
1–5 mm/min, but these values can be much higher in living cells. MT short-
ening velocities are in the order of 10–50 mm/min and are normally 10 times
higher than growth velocities. Hydrolysis of GTP, which occurs at the MT
plus ends, also plays a crucial role in the transition between MT growth and
shrinkage (Hyman et al., 1992). In fact, GTP hydrolysis causes tubulin to
adopt a curved conformation, ultimately leading to destabilization of the lat-
tice (Melki et al., 1989). Because these GDP-tubulin subunits are not
allowed to completely curve while in the lattice, energy released from
hydrolysis is stored as mechanical strain within an MT (Caplow et al.,
1994). This means that when catastrophe events occur, protofilaments adopt
an outward curvature, leading to rapid depolymerization of an MT
(Fig. 2.1).
MT treadmilling was first proposed when it was observed that
isolated bovine brain tubulin continuously incorporated intoMTs at a con-
stant rate, while the MT length remained constant (Margolis and Wilson,
1978). This mechanism implies that (1) there has to be a unidirectional flow
of tubulin subunits with incorporation at the plus end and dissociation
at the minus end and (2) the rate of tubulin association has to be similar
to the rate of tubulin dissociation. The treadmilling model implies that
this mechanism could be bidirectional, depending on the available tubulin
concentration at each given moment. In fact, fluorescence speckle micro-
scopy techniques demonstrated a lack of polarity in treadmilling (Grego
et al., 2001).
It is now widely known that MT behavior is modulated by a number of
MT-associated proteins (MAPs), which can influence dynamic instability
parameters and consequently impact on mitotic progression and fidelity.
Many of these MAPs share the ability to recognize only the distal part of
a polymerizingMT, known as theMT plus end. For this reason, theseMAPs
are currently known as MT plus-end-tracking proteins (þTIPs)
(Akhmanova and Steinmetz, 2008; Schuyler and Pellman, 2001). In this
review, we will cover a range of topics related to the role and regulation
62 Jorge G. Ferreira et al.
of þTIPs in animal cell division which include (1) how þTIPs can specif-
ically recognize and bind to the plus ends of MTs, (2) howþTIPs are able to
modify MT behavior, and finally, (3) how different þTIPs interact with
each other to coordinate entry, progression, and exit from mitosis.
Space
Tim
e
Growth
Shrinkage
Figure 2.2 Typical kymograph (plot of distance vs. time) with changes in microtubulelength and transitions over time. Kymograph obtained from HeLa cell-expressing GFP-tubulin. Vertical scale bar is 10 s; horizontal scale bar 5 mm. Microtubules will normallyswitch stochastically between growth and shrinkage. Highlighted is one growth phaseand a subsequent shrinkage phase (dashed white lines). A rescue event corresponds toa transition from shrinkage to growth and a catastrophe corresponds to a transitionfrom growth to shrinkage.
63+TIPs in Cell Division
2. MICROTUBULES IN CELL DIVISION
2.1. Mitotic entryCell-cycle progression is accompanied by changes in MT dynamics at very
specific stages. In fact, there is an increase in MT dynamics which occurs
concomitantly with NEB that could be important for spindle morphogen-
esis (Piehl and Cassimeris, 2003; Zhai et al., 1996). These changes in MT
dynamics are accompanied by a decrease in tubulin polymer (Zhai and
Borisy, 1994; Zhai et al., 1996), leading to the hypothesis that
MT-stabilizing proteins would have to be inactivated upon mitotic entry
(Cassimeris, 1999). This was supported by the fact that addition of cyclins
or activated CDK1 toXenopus extracts was sufficient to induce a mitotic-like
catastrophe rate ofMTs (Belmont et al., 1990;Murray and Kirschner, 1989).
Curiously, inactivation of CDK1 upon anaphase onset was shown to require
intact MTs (Andreassen and Margolis, 1994) and inhibition of CDK1 pro-
motes MT growth (Moutinho-Pereira et al., 2009; Skoufias et al., 2007).
Why is it necessary for a cell to alter MT dynamics dramatically upon
mitotic entry? During the initial stages of mitosis, spindle poles nucleate
MTs that spatially search for kinetochores. This “search and capture” model
proposed that MTs randomly probe the entire cell volume until they contact
the kinetochore (Kirschner and Mitchison, 1986). However, it became
obvious, based on experimental and theoretical evidence, that this model
alone could not account for the typical mitotic timing (Magidson et al.,
2011; Paul et al., 2009; Wollman et al., 2005). In fact, it was demonstrated
that both the distribution of chromosomes in prometaphase and their move-
ments and rotations significantly reduce spindle assembly time without
compromising mitotic fidelity (Magidson et al., 2011; Paul et al., 2009).
Curiously, assembly or disassembly of MTs can also generate force with-
out direct contribution of motor proteins (Dogterom and Yurke, 1997;
Koshland et al., 1988), and these are sufficient to move subcellular structures
such as chromosomes and organelles, or assist in mitotic spindle positioning
(Dogterom et al., 2005; Inoue and Salmon, 1995;Mogilner andOster, 2003;
Tolic-Norrelykke, 2008). Accordingly, MT polymerization can generate
pushing forces. Addition of tubulin subunits to the MT plus end will induce
its compression when MT hits an object, and this leads to a movement of
MT in the opposite direction, unless MT is attached to some structure
(Dogterom and Yurke, 1997; Holy et al., 1997). These forces can only
64 Jorge G. Ferreira et al.
be exerted over very short distances because MTs have a tendency to buckle
when they grow too long (Dogterom and Yurke, 1997; Dogterom et al.,
2005). Because of this, in vivo evidence for MT pushing forces has been lim-
ited. However, it has been shown that MTs can contribute to the polar ejec-
tion forces that push chromosome arms away from the pole, thereby
contributing for chromosome alignment (Brouhard and Hunt, 2005;
Inoue and Salmon, 1995; Rieder and Salmon, 1994).
2.2. Prometaphase–metaphase transitionWhen an MT comes into contact with a kinetochore, it becomes stabilized
(Hayden et al., 1990), leading to a poleward movement of the chromosome,
which is dynein dependent (Echeverri et al., 1996; Rieder and Alexander,
1990; Yang et al., 2007). Afterward, CENP-E-mediated forces at the kinet-
ochore move the chromosome to the metaphase plate (Kapoor et al., 2006;
Wood et al., 1997). These traction forces are coordinated with polar ejection
forces, which act on chromosome arms and are driven by chromokinesins
and MT polymerization (Brouhard and Hunt, 2005; Cane et al., 2013;
Ke et al., 2009; Rieder and Salmon, 1994; Yajima et al., 2003). Altogether,
these forces facilitate chromosome alignment at the metaphase plate and help
stabilize kinetochore–MT attachments. However, in the initial stages of
mitosis, most kinetochores can become attached in an incorrect way as mon-
otelic (i.e., only one kinetochore attached), syntelic (i.e., both kinetochores
attached and oriented to the same spindle pole), or merotelic (i.e., one kinet-
ochore attached and oriented to both spindle poles). These need to be
corrected so that kinetochores become amphitelically attached (i.e., each
kinetochore attached to MTs oriented to a single spindle pole). The mech-
anisms involved in kinetochore–MT error correction have been extensively
studied and include the destabilization of kinetochore–MTs (k-fibers) by
Aurora-B-mediated activity (Biggins and Walczak, 2003; Cimini et al.,
2003, 2006; Kline-Smith and Walczak, 2004; Lampson et al., 2004; Liu
et al., 2009a; Loncarek et al., 2007; Magidson et al., 2011). Interestingly,
increasing kinetochore tension, such as happens when chromosomes
become bioriented, induces a spatial separation of Aurora-B from its kinet-
ochore substrates, leading to stabilization of k-fibers (Liu et al., 2009a).
In addition to the biochemical signals generated at the kinetochore, the
dynamic state of MTs is also important for mitotic fidelity. Accordingly, it
has been shown that the temporal regulation of MT dynamics during early
mitosis is essential for genomic stability (Bakhoum et al., 2009a,b).
65+TIPs in Cell Division
This regulation depends on the activity of kinesin-13 members Kif2B
and MCAK, which control MT turnover in prometaphase and metaphase,
respectively. Interestingly, it was shown that interaction of Kif2B with
CLASP1 during prometaphase promotes k-fiber turnover, whereas in
metaphase, CLASP1 associates with Astrin to promote k-fiber stability
(Manning et al., 2010). In agreement, increasing the stability of k-fibers pre-
maturely in prometaphase resulted in chromosome missegregation
(Bakhoum et al., 2009a).
Taken together, these results demonstrate that, during prometaphase,
k-fiber stability is reduced so that erroneous attachments can be efficiently
corrected. As cells progress to metaphase and chromosomes become bio-
riented, there is an increase in k-fiber stability which is essential for spindle
assembly checkpoint (SAC) satisfaction. In fact, the SAC constantly moni-
tors for unattached kinetochores so that the mitotic progression is delayed
until all kinetochores are stably attached to k-fibers (Rieder and Maiato,
2004; Rieder et al., 1995).
2.3. MetaphaseUpon establishment of the metaphase spindle, its length and shape appear
relatively stable. However, the spindle itself is quite heterogeneous and
dynamic. Experiments demonstrated that spindle MT turnover was mainly
derived from the high dynamic instability of nonkinetochore–MTs (Buster
et al., 2007; Gorbsky et al., 1990; Salmon et al., 1984; Zhai et al., 1995).
Similar measurements made in kinetochore–MTs showed that, although still
capable of turnover, they do so at much lower rates relative to non-
kinetochore–MTs (�10�) (Zhai et al., 1995). Interestingly, there is a strik-
ing reduction of MT turnover rates and MT flux at anaphase onset,
suggesting that kinetochore–MT attachment is stabilized at this stage
(Gorbsky and Borisy, 1989; Zhai et al., 1995). This further demonstrates that
MTs can also change their dynamic behavior during different stages of
mitosis.
In addition to dynamic instability, a second mechanism also ensures
proper spindle dynamics in metaphase, which is known asMT poleward flux
(Mitchison, 1989). This is a highly conserved feature of the mitotic spindle
in higher eukaryotes and is associated with the incorporation ofMT subunits
at the MT plus ends and disassembly of subunits at the MT minus ends
(Mitchison et al., 1986). Current models proposed to explain MT flux take
into account the following premises: active incorporation of tubulin sub-
units at the kinetochore, disassembly of tubulin subunits at the centrosome
66 Jorge G. Ferreira et al.
(Buster et al., 2007; Kwok and Kapoor, 2007; Mitchison, 1989), as well
as sliding of MTs through the action of plus-end-directed motors
(Brust-Mascher and Scholey, 2002; Matos et al., 2009; Pereira and
Maiato, 2012). The net result is the stabilization of the spindle size while
maintaining the structure highly dynamic.
2.4. Metaphase–anaphase transitionDifferentMT populations have distinct dynamic properties in the metaphase
spindle. Nonkinetochore–MTs have a higher turnover when compared to
k-fibers (Cassimeris et al., 1990; Mitchison et al., 1986; Saxton et al., 1984).
These differences will be reflected on MTs as cells enter anaphase. In fact,
turnover of k-fiber was shown to decrease as cells enter anaphase by as much
as fivefold when compared to the same population ofMTs in metaphase cells
(Gorbsky and Borisy, 1989; Zhai et al., 1995), whereas turnover of non-
kinetochore fibers is not affected during the transition from metaphase to
anaphase (Zhai et al., 1995).
Shortening of k-fibers during anaphase should occur either by activeMT
depolymerization at the pole region (known as the “Traction Fiber” model)
(Buster et al., 2007;Matos et al., 2009;Waters et al., 1996) orbydisassemblyof
MTs at the kinetochore (known as the “Pacman” model) (Cassimeris and
Salmon, 1991; Gorbsky et al., 1987, 1988; Maiato, 2010; Nicklas, 1989).
MT depolymerization per se is sufficient to drive chromosome movement
in vitro (Coue et al., 1991; Koshland et al., 1988) and for generating force
(Grishchuk et al., 2005). This was first demonstrated in vitro when it was
shown that depolymerizingMTs alone could generate sufficient pulling force
to move chromosomes without the contribution of motors (Koshland et al.,
1988). Subsequent reports demonstrated that, in an in vitro system, chromo-
somes were being pulled at about 30 mm/min in an ATP-independent man-
ner and, thus,were relying only onMTdepolymerization (Coue et al., 1991).
However, there is evidence that this process might also require the assistance
ofmotor proteins tomove chromosomes (Desai andMitchison, 1997;Maiato
and Lince-Faria, 2010; Pfarr et al., 1990).
2.5. Mitotic exit and cytokinesisMTs are also necessary for changes in cell shape and size during anaphase and
telophase. Upon anaphase onset, depolymerization of spindle MTs has to be
compensated by an increase in astral MT polymerization/elongation
(Morrison and Askham, 2001; Strickland et al., 2005b). Elongation of astral
67+TIPs in Cell Division
MTs is necessary for their interaction with the cell cortex and definition of
the cytokinetic furrow, but apparently is not essential for anaphase progres-
sion itself, as the cytokinetic furrow can still be formed even in the absence of
astral MTs (Rankin and Wordeman, 2010; Strickland et al., 2005a,b;
Sullivan and Huffaker, 1992).
MT reorganization during mitotic exit is strictly associated with the inac-
tivation of the mitotic kinase CDK1, which triggers the formation of ana-
phase MTs and the midbody (Wheatley et al., 1997). A similar phenomenon
was also observed in Drosophila S2 cells and shown to involve acentriolar
MT-organizing centers (aMTOCs). These aMTOCs were able to nucleate
MTs de novo upon CDK1 inhibition at anaphase onset (Moutinho-Pereira
et al., 2009), and this was dependent on the activity of Msps/XMAP215
and KLP10A/kinesin-13. This reorganization also depends on the precise
regulation of MT dynamics and allows daughter cells to adhere simulta-
neously to the substrate (Ferreira et al., 2013).
Cytokinesis relies on MTs in several ways. First, definition of the cleav-
age plane is specifically determined by astral MTs (and not spindle MTs) as
furrowing still occurs in the presence of asters without any intervening spin-
dle (Rieder et al., 1997). However, successful completion of cleavage does
require interaction of midzone MT bundles with the cell cortex (Wheatley
and Wang, 1996). Moreover, if anaphase astral MT formation is suppressed
by interfering with the þTIP EB1 or with dynactin, cytokinesis is delayed
(Strickland et al., 2005b), which supports the necessity of MT interaction
with the cortex to define cleavage plane localization (Bement et al., 2005;
Strickland et al., 2005a). At this stage, regulation of MT dynamics seems
to be dispensable, as contact of MTs with the cortex is sufficient to trigger
the process. In contrast with earlier stages of cytokinesis, MTs are essential
for completion of the process (Savoian et al., 1999). MTs that establish the
midbody are acetylated, highly stable (Margolis et al., 1990), and resistant to
nocodazole treatment (Foe and von Dassow, 2008; Piperno et al., 1987).
Nevertheless, some midbody MTs are still able to exhibit a highly dynamic
behavior as can be seen by live imaging of MT plus ends with fluorescent-
tagged EB proteins, which show comets moving in and out of the midbody
(Rosa et al., 2006). Thus, it is not surprising that g-tubulin was found in themidbody during late cytokinesis (Julian et al., 1993), suggesting active MT
nucleation. Notably, g-tubulin interacts with the Augmin complex during
anaphase, and this is required for MT nucleation in the central spindle and
successful cytokinesis (Uehara et al., 2009). Final disassembly of the midbody
68 Jorge G. Ferreira et al.
requires that MTs are cut, which is accomplished by a mechanism that
involves the MT-severing enzyme spastin (Guizetti et al., 2011).
3. FAMILIES OF MICROTUBULE PLUS-END-TRACKINGPROTEINS (+TIPs)
Many proteins have the ability to associate with MTs. Among these, a
large number of MAPs specifically recognize the terminal portion of MT
(Table 2.1). These are collectively known as MT plus-end-tracking proteins
or þTIPs (Akhmanova and Steinmetz, 2008; Schuyler and Pellman, 2001).
When theseþTIPs are labeled with a fluorescent tag, they appear as comets
in the MT tip, moving throughout the cell as MT grows and disappearing
when MT shrinks (Howard and Hyman, 2003). In this section, we will
cover the structural features and function of the main families of þTIPs.
3.1. CLIP familyThe first descriptionof tip-tracking behavior came fromworkofKreis and col-
laborators, who demonstrated that cytoplasmic linker protein (CLIP) 170 was
able to specifically associate with the plus end of polymerizing MTs
(Diamantopoulos et al., 1999; Perez et al., 1999). The CLIP family of proteins
is comprisedof twomembers inmammalians:CLIP170 andCLIP115.The lat-
ter is a brain-specific CLIP that shares functional similarities with CLIP170
(DeZeeuwet al., 1997).These proteins have a characteristicCAP-Gly domain
(Fig. 2.3) which is necessary for interaction with tubulin and EB1 (Weisbrich
et al., 2007). TheseCAP-Gly domains are surrounded bybasic, serine-rich res-
idues that assist in the binding to MTs (Hoogenraad et al., 2000). In order to
perform its function, CLIP170 needs to form a parallel homodimer. Each
monomer is composed of an N-terminal domain required for MT binding
(with two CAP-Gly domains per monomer), a central coiled-coil domain
required for dimerization, and a C-terminal metal-binding domain (with
two zinc fingers per monomer; Fig. 2.3) (Gupta et al., 2009; Pierre et al.,
1994; Scheel et al., 1999). Both the CAP-Gly domains at the N-terminus
and the zinc fingers at the C-terminus are thought to play an important role
in the autoregulation of CLIP170 (Hayashi et al., 2007; Lansbergen et al.,
2004). In accordance, it was shown that they can interact with each other, cre-
ating a doughnut-shapedmolecule that no longer interacts withMTs. In addi-
tion, these autoinhibitory interactions use the same binding determinants as
CLIP170’s intermolecular interactions with p150glued, suggesting that
69+TIPs in Cell Division
Table 2.1 Main þTIP families and main functions during cell divisionþTIP Homologues Interacting þTIPs Main mitotic functions References
EB1 family (EB1,
EB2, EB3)
Mal3 (Sp)
Bim1 (Sc)
AtEB1 (At)
Most þTIPs (with
SxIP and CAP-Gly
domains)
Nucleation of astral microtubules;
loading of þTIPs to plus ends;
spindle positioning; cytokinesis
completion; postmitotic cell
adhesion
Tirnauer and Bierer (2000),
Toyoshima and Nishida (2007),
Rogers et al. (2002), Stout et al.
(2011), Ferreira et al. (2013)
CLIP family
(CLIP170,
CLIP115)
Tip1 (Sp)
Bim1 (Sc)
CLIP190
(Dm)
EB family
CLASPs p150glued
Cytoplasmic dynein
Microtubule rescue and
stabilization; targeting of dynein to
plus ends; required for mitotic
progression; microtubule
interaction with cell cortex and
kinetochores
Arnal et al. (2004), Dujardin et al.
(1998), Wieland et al. (2004),
Tanenbaum et al. (2006)
APC family (APC,
APC2/APC-L)
Kar9 (Sc)
APC1/2
(Dm)
APR-1 (Ce)
EB family Microtubule stabilization;
regulation of kinetochore–
microtubule interaction;
chromosome segregation; spindle
positioning
Kaplan et al. (2001), Fodde et al.
(2001), Green et al. (2005),
McCartney et al. (2001), Zhang
et al. (2007a)
CLASP family
(CLASP1,
CLASP2)
Peg1 (Sp)
Stu1 (Sc)
MAST/Orbit
(Dm)
Cls-2 (Ce)
CLASP (At)
EB family
CLIP170
CLIP115
Kinesin-7
Spindle microtubule dynamics;
mitotic spindle organization and
assembly; spindle pole integrity;
kinetochore–microtubule
attachment; cytokinesis completion
Mimori-Kiyosue et al. (2006),
Logarinho et al. (2012), Lemos
et al. (2000), Maiato et al. (2005),
Maiato et al. (2003a), Pereira et al.
(2006), Maffini et al. (2009)
Motor proteins
(kinesin-7, kinesin-
14, dynein)
Kinesin-7
Tea2 (Sp)
Klp2 (Sc)
Kinesin-14
Ncd (Dm)
KLP2 (Sp)
Kar3 (Sc)
Cytoplasmic
Dynein
EB family
Dynein
CLASPs;
EB family;
Dynactin
(p150glued)
LIS1
Spindle formation; chromosome
congression; microtubule plus-end
elongation; interpolar microtubule
sliding; metaphase chromosome
alignment; spindle pole focusing;
spindle positioning
Kapoor et al. (2006), Wood et al.
(1997), Kapitein et al. (2005),
Cooke et al. (1997), Sardar et al.
(2010), Goshima et al. (2005),
Kiyomitsu and Cheeseman (2012),
Maffini et al. (2009), O’Connell
and Wang (2000)
Kinesin-13 family
(Kif2C/MCAK)
XKCM1 (Xl)
KLP10A
(Dm)
AtKinesin-13
(At)
EB family
CLIP170
APC
Microtubule depolymerization;
spindle assembly; kinetochore–
microtubule turnover; error
correction
Ganem andCompton (2004), Ems-
McClung et al. (2007), Moore and
Wordeman (2004), Wordeman
et al. (2007), Bakhoum et al.
(2009b), Ganem et al. (2005)
TOG family (ch-
TOG)
XMAP215
(Xl)
Dis1
Alp14(Sp)
Stu2 (Sc)
Msps (Dm)
ZYG-9 (Ce)
EB family
Dynein
Microtubule stabilization; spindle
pole organization; centrosome
integrity; spindle assembly;
protecting kinetochore fiber
disassembly
Gergely et al. (2003), Cassimeris
and Morabito (2004), Barr and
Gergely (2008), Booth et al. (2011)
At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; An, Aspergillus nidulans; Ce, Caenorhabditis elegans; Xl,Xenopus laevis; Hs, Homo sapiens.
regulation of MT binding by þTIPs occurs through direct competition
between homologous binding interfaces (Hayashi et al., 2007).
CLIP proteins were described to impact on MT dynamics either directly
or by recruiting a rescue factor (Komarova et al., 2002). Although CLIP115
1 444 735
TOG/TOG-like domain Helical region Basic/Ser
CLASP-like protein
Helical region Helical region
1538
1 14 116 185
CH domain Coiled-coil EBH domain EEY/F
268255
EB-like protein
Zn fingerBas/Ser
1 78 120 232 274 350 1353
Bas/Ser CAP-Gly Bas/Ser CAP-Gly Coiled-coil Zn finger EEY/F
1438
CLIP-like protein
Basic/Ser
1 248 767
Coiled-coil Helical region
2843
APC protein
Coiled-coil
Armadillo repeats
Basic/Ser
453
Coiled-coil
1 181 1868
Dynein heavy chain protein
Helical region Coiled-coil Helical region Coiled-coilAAA ATPase AAA ATPase
444 1171 3189 3553 4646
1 255 658
Kinesin domainHelical region Basic/Ser Coiled-coil
725618518
Kinesin-like protein
1 159
TOG domain Basic/Ser Helical region
1399 2032
TOG domain TOG domain TOG domain TOG domain
ch-TOG protein
1 39
WD40 repeatLisH Coiled-coil
41085 96
Lis1 protein
Figure 2.3 Structural diagram of the main þTIP families. Cartoon depicting relevantdomains in the main þTIP families. Bas/Ser-basic and proline/serine-rich sequenceregions; CAP/Gly, cytoskeleton-associated protein/glycine-rich domain; Zn finger, zincfinger; CH, calponin homology domain; TOG, tumor overexpressed gene domain;EBH, end binding homology domain; LisH, Lis1-homology motif.
72 Jorge G. Ferreira et al.
lacks the C-terminal domain of CLIP170, they share a similar N-terminal
domain, which means they could regulate MT dynamics in a similar fashion
(Hoogenraad et al., 2000; Komarova et al., 2002). In fact, both in vivo and
in vitro studies using the N-terminus of CLIP170 demonstrated that this pro-
tein acts by preventing catastrophes or promoting rescue events (Arnal et al.,
2004; Komarova et al., 2002). The exact mechanism of CLIP170-mediated
MT rescue is still unclear but may involve stabilization of the curved
protofilaments by the N-terminus of CLIP170 or coassembly of CLIP170
with tubulin oligomers into MTs (Arnal et al., 2004; Diamantopoulos
et al., 1999). Interestingly, although CLIPs track only growing MT plus
ends, they also influence the behavior of depolymerizingMTs. This is a puz-
zling observation and indicates that CLIPs function is not totally understood.
3.2. EB familyEnd binding (EB) proteins are part of a highly conserved family which, in
mammalians, comprises three members encoded from three different genes:
EB1, EB2 (RP1), and EB3 (EB3F) (Su and Qi, 2001). EB1 was the first
member identified in a yeast two-hybrid screen as an interactor of the
C-terminus of the adenomatous polyposis coli (APC) tumor suppressor pro-
tein (Su et al., 1995). Both EB1 and EB3 seem to be ubiquitously expressed,
whereas EB2 expression is restricted to only certain cell types/tissues (Su and
Qi, 2001). Normally, EB1 is expressed in higher levels when compared to
other EBs. However, EB3 is also highly expressed in specific cell types. EB3
was originally reported in neurons, where it was shown to interact with a
brain-specific form of APC (APC2), but it is also highly abundant in muscle
cells (Nakagawa et al., 2000; Straube and Merdes, 2007).
EBs are relatively small, elongated proteins (around 32 kDa) with con-
served structural features (Fig. 2.3). All members have at the N-terminal
region an MT-binding portion containing a calponin homology (CH)
domain with a highly conserved fold (Akhmanova and Steinmetz, 2008).
The structural basis for EB1 binding to MTs has already been described
(Hayashi and Ikura, 2003; Slep and Vale, 2007). It was shown that this
CH domain is both required and sufficient for binding to MT plus ends
(Hayashi and Ikura, 2003; Komarova et al., 2009). The C-terminal portion
of EB1, on the other hand, contains a coiled-coil region which is necessary
for EB dimerization (Su and Qi, 2001). This is essential not only because
they need two CH domains to interact with MTs but also to form the func-
tional C-terminal domain (Buey et al., 2011; Honnappa et al., 2005).
73+TIPs in Cell Division
Recently, it was demonstrated that EB1 and EB3 prefer heterodimerization
to EB1/EB1 or EB3/EB3 homodimers (De Groot et al., 2009), while EB2
does not show preferential association with any other EB member. This
chain exchange between EBs can be suppressed by specific EB interaction
partners, which indicates an extra layer of regulation of EB function
(De Groot et al., 2009).
The coiled-coil region partially overlaps the end binding homology
(EBH) domain, which was shown to be required for efficient interaction
with EB-binding partners (Akhmanova and Steinmetz, 2008; Bjelic et al.,
2011). Solving the C-terminal structure of EB1 (EB1c) by X-ray crystallog-
raphy demonstrated that the coiled-coil terminates in a 4-helix bundle with
a hydrophobic cavity (Honnappa et al., 2005; Slep et al., 2005). In addition,
EB1c has an EEY/F motif that is very similar to the one found in a-tubulinand CLIP170 (Komarova et al., 2005; Mishima et al., 2007;Weisbrich et al.,
2007) and might be important to help in the regulation of EB1/CLIP170/
tubulin association (Bieling et al., 2008;Mishima et al., 2007). Both EB1 and
EB3 have very similar structures, which are highlighted by the fact that they
share some functional similarity (Komarova et al., 2005, 2009). On the other
hand, EB2 appears to have fewer similarities with the other two family
members. Not only the interaction partners are substantially different
between this and other EBs, but also EB2 does not promote persistent
MT growth or restore CLIP association to the MT plus ends (De Groot
et al., 2009; Komarova et al., 2005, 2009). In fact, EB2 does not interact
to the same extent with MCAK, APC, or CLIP170 (Bu and Su, 2003;
Komarova et al., 2005; Lee et al., 2008). This can be explained by the fact
that the C-terminal domain of EB2 is significantly different from EB1 and
EB3, with fewer acidic residues. Furthermore, EB2 has a longer N-terminal
region, containing approximately 40 amino acids in excess when compared
to EB1 and EB3 (Komarova et al., 2009). Interestingly, this difference in the
N-terminal domain is clustered around the sequence SRHD in the CH
domain, which is essential for MT binding and can explain the differences
observed between EB2 and the other family members in this aspect
(Komarova et al., 2009).
EB proteins are associated with MT plus ends in both interphase and
mitotic cells (Fig. 2.4; Berrueta et al., 1998; Mimori-Kiyosue et al.,
2000b; Morrison et al., 1998). The first report regarding the possible role
of EB proteins inMT dynamics came from the observation that, when over-
expressed, these proteins induced the formation of acetylated MT bundles
that were resistant to nocodazole treatment (Bu and Su, 2001). In addition,
74 Jorge G. Ferreira et al.
their ability to tip-track MTs led to the possibility that they might be
involved in MT dynamics regulation, particularly in promoting MT growth
(Nakamura et al., 2001; Tirnauer and Bierer, 2000). This was confirmed in
many independent studies using not only different model organisms such as
budding and fission yeast, Drosophila, and human cells, but also in vitro sys-
tems (Beinhauer et al., 1997; Coquelle et al., 2009; Komarova et al., 2009;
Nakamura et al., 2001; Rogers et al., 2002; Tirnauer et al., 1999).
The overall picture that has emerged confirms the role of EB proteins in
the regulation of MT dynamics, but their precise effect is still not fully
understood. In mouse fibroblasts, EB1 depletion leads to an increase in
MT pausing and a decrease in MT growth time (Kita et al., 2006). In addi-
tion, EB1 was also shown to induce MT stabilization by interacting with
mDia and APC (Wen et al., 2004) and to localize to stable Glu-MTs. In
these conditions, knockdown of EB1 leads to the appearance of more
dynamic MTs, as demonstrated by the concomitant decrease in Glu-
MTs. EB3 also interferes with MT dynamics. In fact, it was shown in myo-
blasts that EB3 depletion induced MT overgrowth near the cell cortex and a
significant decrease in MT shrinkage rate (Straube and Merdes, 2007). EB1
and EB3 also promote persistent growth of internal MTs by suppressingMT
catastrophes (Komarova et al., 2009).
The impact of EB proteins on interphase MT dynamics may also involve
their interaction with otherþTIPs. In fact, differences in the expression and
regulation of several þTIPs in different cell types may be responsible for
the observed differences in specific MT populations (Ligon et al., 2003).
A B
Figure 2.4 Localization of EB1 during (A) interphase and (B) mitosis. Immunolocalizationof EB1 (green) and a-tubulin (red) in fixed cells using specific antibodies. EB proteinsassociate with the growing ends of microtubules throughout the cell cycle. In addition,EB1 also associates with the centrosome. Scale bars, 5 mm.
75+TIPs in Cell Division
In addition, data derived from in vitro assays demonstrated that EB1 can act
cooperatively with other þTIPs such as CLIP170 in the regulation of MT
dynamics. Accordingly, it was demonstrated that both EB1 and CLIP170
can synergize to modulate MT dynamics, possibly by modifying the
MT-stabilizing cap (Lopus et al., 2012). Moreover, EB association with
CLASPs was also reported to affect MT dynamics at the cell cortex by
increasing MT rescue events (Mimori-Kiyosue et al., 2005). Interestingly,
EBs can also associate with and load MT depolymerizers such as MCAK
to the MT plus ends (Montenegro Gouveia et al., 2010; Moore et al.,
2005). This interaction is important for the localization ofMCAK to the plus
ends but also to enhance its catastrophe-inducing activity. Thus, by allowing
the accumulation of polymerizers and depolymerizers at the MT plus end,
EB proteins facilitate the rapid switching between MT growth and
shortening.
Interestingly, modulation of MT dynamics by EB proteins can also be
regulated by phosphorylation. In budding yeast, the single EB-like protein
was described to be phosphorylated by Ipl1p/Aurora-B and this is important
to regulate the association of EB to spindle MTs (Zimniak et al., 2009).
Moreover, a mutation in the fission yeast EB-like protein was sufficient
to increase MT binding, leading to their stabilization (Iimori et al., 2012).
In humans, less is known about the phosphoregulation of EB proteins.
Recent work demonstrated that EB3 is phosphorylated by Aurora kinases
on S176 during mitosis (Ban et al., 2009). This Aurora-mediated EB3
phosphorylation leads to a significant increase in MT growth, allowing
stabilization of the midbody (Ferreira et al., 2013). In this context, dephos-
phorylation of EB3 restricts cortical MT growth, allowing proper daughter
cell adhesion to the substrate. Inversely, phosphorylation of EB3 on S162 by
the Src-PLCg2 signaling pathway was shown to block MT growth, leading
to adherens junction stabilization in interphase cells (Komarova et al., 2012).
Taken together, these data demonstrate that EB protein association to the
MT plus ends can be regulated by phosphorylation, although it is still unclear
how different phosphorylation events integrate to control EB function, thus
regulating MT dynamics in different tissues.
Besides its plus end localization, EB proteins were also shown to bind
other subcellular structures either directly (centrosome) or indirectly
(F-actin and membranes). In fact, EB1 is a functional component of centro-
somes and binds to this structure independently of MTs through its
C-terminal domain (Louie et al., 2004). Curiously, the C-terminal domain
of EB1 is also required for the recruitment of g-tubulin to centrosomes and
76 Jorge G. Ferreira et al.
anchoring of MTs to this structure (Askham et al., 2002). Moreover, EB1
was shown to interact with the centrosomal protein FOP (Yan et al.,
2006). This interaction is essential for recruitment of EB1 to the centrosome
and its later association with CAP350, forming an MT-anchoring complex.
EB3 also localizes to the centrosome (Ban et al., 2009; Ferreira et al., 2013).
EBs can also interact indirectly with actin filaments or membrane structures.
EB1was shown to interactwith the spectraplakinACF7/MACF1, providing
a link between MT and actin cytoskeletons (Kodama et al., 2003). Overall,
EB proteins act, either directly or through interaction with a partner, as
mediators of cellular functions by regulating MT dynamics. More detailed
data on the physiological relevance of EB proteins still await the develop-
ment of mammalian knockout models.
3.3. CLASP familyCLIP-associating proteins (CLASPs) are highly conserved þTIPs involved
in the regulation and organization of cellular MT dynamics, motility, and
cell division. The CLASP protein family was first identified in a genetic
screen for mitotic mutants in Drosophila and was named as multiple asters
(MAST)/Orbit (Inoue et al., 2000; Lemos et al., 2000). In mammals, there
are two paralog genes encoding for CLASP1 and CLASP2 proteins, which
were found in a yeast two-hybrid screen as interacting proteins with
CLIP115 and CLIP170 (Akhmanova et al., 2001). While CLASP1 is more
ubiquitously expressed, CLASP2 is predominantly expressed in the brain
and reproductive organs (Akhmanova et al., 2001), as well as in the hema-
topoietic organs in mice (Drabek et al., 2012). All the data collected in dif-
ferent model organisms suggest a functional role of CLASPs starting at
embryogenesis (Inoue et al., 2000; Lemos et al., 2000; Park et al., 2012).
Both clasp1 and clasp2 genes can undergo alternative splicing events, origi-
nating several isoforms. So far, only one biologically active isoform has been
found for CLASP1, known as CLASP1a (�170 kDa). On the other hand,
three isoforms have been described for CLASP2, namely, CLASP2a(�170 kDa), CLASP2b (�140 kDa), and CLASP2g (�140 kDa), which
result from alternative splicing events (Akhmanova et al., 2001).
CLASPs display a conserved structure, sharing approximately 77%
sequence homology (Akhmanova et al., 2001), and contain two short
Ser-x-Ile-Pro (SxIP) polypeptide motifs embedded in an extensive central
sequence region enriched with positively charged serine and proline residues
(Fig. 2.4), which is highly conserved across species (Honnappa et al., 2009;
77+TIPs in Cell Division
Kumar et al., 2012; Mimori-Kiyosue et al., 2005). These motifs have been
shown to be essential for the interaction with the C-terminal domain of EB
proteins and are important for the plus-end-tracking activity of CLASPs
(Honnappa et al., 2009). The recurrence of the SxIPmotif found in CLASPs
also enables the intermolecular cooperation between them to significantly
improve the efficiency of MT tip-tracking (Honnappa et al., 2009).
Until recently, the general perception regarding the N-terminal domain
of CLASPs was that it contained only one TOG domain, accompanied by a
variable number of TOG-like regions that hold a weak sequence homology
to those found in proteins of the Dis1/TOG family (Lemos et al., 2000).
However, recent evidence gathered from X-ray crystallography determined
that human CLASP1 has, at least, two legitimate TOG domains: TOG1 and
TOG2 (previously classified as cryptic TOG-like 2) (Leano et al., 2013). In
yeast, the TOG domains of CLASP are capable of binding directly to soluble
tubulin dimers, but not to dimers that are already incorporated in the MT
lattice (Al-Bassam and Chang, 2011; Al-Bassam et al., 2010). The detailed
mechanism behind the interaction of TOG domains with soluble tubulin
is yet to be fully understood, but important new data may have shed light
on the precise mechanism that controls association of human CLASP1
with MTs. Accordingly, CLASP1 TOG2 domain has a distinctive bent
conformation, which is hypothesized to be a good fit to bind to the curved
conformation of tubulin dimers on depolymerizing MTs. This leads to their
stabilization, possibly leading to a rescue event (Leano et al., 2013). How-
ever, the authors suggest that this conformational variation in TOG2 may
only occur upon lattice binding. This particular domain also seems to be
important for the establishment of a CLASP-mediated bipolar spindle
(Leano et al., 2013).
Interestingly, the N-terminal region is different between the CLASP2
isoforms. Notably, the previously described CLASP2 TOG domain only
exists in the longer alpha isoform, while being absent from the shorter
isoforms (Akhmanova et al., 2001). In CLASP2b, it is replaced by a short
N-terminal palmitoylation motif, which gives CLASP2b the ability to
anchor membranes. On the other hand, CLASP2g contains the inconspic-
uous peptide—MAMGDD—in this region.
The central region of CLASPs contains six HEAT repeats embedded
between the TOG domains. These repeats were suggested to be involved
in intracellular transport, MT dynamics, and chromosome segregation,
but their exact function is still unknown (Neuwald and Hirano, 2000;
Tournebize et al., 2000). Within this central region, the SxIP motif and
78 Jorge G. Ferreira et al.
the innermost TOG domain of CLASP1 are responsible for its binding to
PRC1, an MT-bundler protein of the central spindle (Patel et al., 2012).
Of interest, CLASP2a association with actin stress fibers occurs through this
middle serine–arginine (SR)-rich motif and the N-terminal Dis1/TOG
domain in an MT-independent way, providing a direct cross-link between
MTs and the actin cytoskeleton, which is important for cell morphogenesis
(Tsvetkov et al., 2007).
TheC-terminal domain of CLASPs participates in the interactionwith the
Golgi apparatus by binding with the trans-Golgi network protein GCC185,
an interaction that contributes to an asymmetry of the MT array nucleated at
the Golgi (Efimov et al., 2007). The coiled-coil domain present in this region
is also important for the binding of CLASPs to interacting partner proteins,
such as CLIP170, CENP-E, and Plk1 (Akhmanova et al., 2001; Hannak
and Heald, 2006; Maffini et al., 2009; Maia et al., 2012), as well as kinesin-
10/Kid, a chromokinesin which is involved in chromosome congression by
generating polar ejection forces (Antonio et al., 2000; Levesque and
Compton, 2001; Patel et al., 2012; Wandke et al., 2012). The interaction
of CLIP170 with the C-terminal domain of CLASPs has been shown to
enhance CLASPs plus-end association (Mimori-Kiyosue et al., 2005). How-
ever, this CLIP170 interacting region does not seem to be required forCLASP
plus-end tracking or lattice binding (Wittmann andWaterman-Storer, 2005).
Finally, the C-terminal region also seems to be implicated in the
homodimerization of CLASPs (Al-Bassam et al., 2010; Patel et al., 2012).
In interphase,CLASPs canbe found associatedwith theplus endsof grow-
ing MTs, centrosomes, and perinuclear region, consistent with Golgi appa-
ratus localization (Akhmanova et al., 2001; Efimov et al., 2007). They were
also shown to be required for the stabilization of MTs at the leading edge of
motile fibroblasts (Akhmanova et al., 2001). It was demonstrated that
CLASP2 is necessary for the establishment of a stable, polarized MT array
in mouse embryonic fibroblasts, promoting persistent directional motility
in these cells (Drabek et al., 2006). Depletion of both CLASPs by RNAi
resulted in a decrease in the levels of acetylated tubulin, which was accompa-
nied by a reduction inMTdensity (Mimori-Kiyosue et al., 2005). This led to
the hypothesis that, when bound to the plus ends of MTs, CLASPs are
required for rescue events by reducing the number of long depolymerization
episodes (Akhmanova et al., 2001; Al-Bassam et al., 2010; Mimori-Kiyosue
et al., 2005; Sousa et al., 2007).Additional evidence further demonstrated that
CLASPs also increase MT longevity by promoting MT “pausing,” and con-
sequently their stability, without affecting overall MT polymerization rate.
79+TIPs in Cell Division
The accumulation of CLASPs at MT plus ends and their
MT-stabilization capacity are also important for the interaction of MTs with
the cell cortex through a complex with LL5b, a process that also seems to
require the spectraplakin MACF/ACF7 (Drabek et al., 2006; Lansbergen
et al., 2006). Notably, it was recently demonstrated, using a mouse knockout
model for Clasp2, that this protein is important for cell attachment and
proper organization of the MT network in hematopoietic stem cells
(Drabek et al., 2012). In this way, CLASP2 is an important player in the
homing and maintenance of hematopoietic stem cells in vivo.
The mechanisms by which CLASPs are able to interact with MTs are
only now being unraveled. It is now known that association of CLASPs
to MTs can be regulated by posttranslational modifications, such as phos-
phorylations. It was demonstrated that the interaction of CLASPs with
MTs is negatively regulated by glycogen synthase kinase (GSK)-3b, a
downstream target of phosphoinositide PI3-kinase (Akhmanova et al.,
2001). Initial observations implied a major increase in CLASP2 signal at dis-
tal MT ends upon GSK-3b inhibition in 3T3 fibroblasts (Akhmanova et al.,
2001). On the other hand, overexpression of a constitutively active GSK-3bform severely prevented CLASP2 localization to MT plus ends and strongly
disrupted CLASP2 MT lattice binding (Akhmanova et al., 2001; Wittmann
and Waterman-Storer, 2005), reinforcing the requirement of GSK-3bkinase activity for CLASP2 association to different subsets of MTs. On
the contrary, inhibition of the kinase stimulated ectopic MT lattice associ-
ation in the cell body. Based on these data, it was proposed that the
MT-binding domain of CLASP2 comprises different functions: it is required
for high affinity binding of CLASP2 to the MT lattice in the lamella, as well
as plus-end tracking. Later experiments identified the GSK-3b phosphory-
lation sites in the MT-binding domain that are involved in the transition
between plus-end tracking and lattice binding (Kumar et al., 2009), con-
firming that CLASP2 is spatially regulated in cells. The fact that these phos-
phorylations by GSK-3b affect the association between CLASP2 and EB1
may explain the alterations observed in CLASP2 tip-tracking ability
(Kumar et al., 2012). Importantly, a priming site phosphorylation of
GSK-3b by CDKs is necessary for GSK3b-mediated CLASP2 phosphory-
lation. Similarly, location and regulation of CLASPs in specific structures
during mitosis seems to be controlled through the phosphorylation activity
of CDK1 and Plk1 (Kumar et al., 2012; Maia et al., 2012), which will be
discussed in more detail in Section 5.4. Finally, the latest results obtained
with a Clasp2 knockout mouse model confirmed the importance of GSK3b
80 Jorge G. Ferreira et al.
in the regulation of CLASP2 activity, especially at the neuromuscular junc-
tions (Schmidt et al., 2012).
3.4. APC familyThe APC protein is a large protein of approximately 300 kDa encoded by
theApc gene (Smith et al., 1993). In mammalians, an additional form of APC
can be found, which is a product of theAPCL/APC2 gene (Nakagawa et al.,
1998). In structural terms, APC is composed of several domains (Fig. 2.3).
Close to the N-terminus, there is an oligomerization domain and an arma-
dillo repeat domain (ARD). In the middle of the protein, there are
b-catenin-binding motifs, Axin-binding motifs, and also a mutation cluster
region. In addition, there is a KKKK stretch, which is postulated as a putative
nuclear localization signal. In the C-terminus, there is an MT-binding
domain and an EB1-binding domain (Bienz, 2002). The interaction of
EB1 with APC was first mapped to a small region in the C-terminus of
APC which comprises amino acids 2559–2843 (Su et al., 1995). Subsequent
work narrowed this region to the last 170 amino acids of APC (Askham
et al., 2000), and finally, the interaction was attributed to a basic, serine-rich
sequence in the C-terminus of APC named APCp1 (Honnappa et al., 2005).
More specifically, interaction of APC with EB1 depends on the SxIP motif
(Ile2805 and Pro2806) of APC (Honnappa et al., 2005, 2009). Interestingly,
mutations within this region are sufficient to abolish EB1 interaction and
also the ability of APC to tip-track.
APC is involved in the regulation of MT function. In fact, APC directly
associates with MTs and promotes their polymerization and stabilization
in vitro (Munemitsu et al., 1994; Nakamura et al., 2001; Zumbrunn et al.,
2001). As was mentioned earlier, interaction of APC with EB1 seems to
be important for its ability to track MT plus ends (Mimori-Kiyosue et al.,
2000a). However, this might not be the only mechanism that APC uses
to localize to growing MT ends, as APC association to MTs can occur even
in the absence of EB1 (Kita et al., 2006). Moreover, APC can also accumu-
late at the MT plus ends by interacting with Kif3A/Kif3B (Jimbo et al.,
2002). Nevertheless, it seems that APC is mainly loaded onto plus ends
by hitchhiking on EB1 (Honnappa et al., 2009). This interaction is impor-
tant because it was shown that it can help regulate MT stability and promote
cell migration (Wen et al., 2004), although another study with mouse
embryonic fibroblasts derived from mice carrying a truncated Apc allele
demonstrated that the APC–EB1 interaction is not essential for MT
81+TIPs in Cell Division
stabilization (Drabek et al., 2006). APC is also involved in the interaction
between MT and actin cytoskeletons (Moseley et al., 2007) and in the
regulation of cell polarity (Etienne-Manneville and Hall, 2003).
3.5. Motor proteinsMany organisms have a set of motor proteins that can travel along MTs
toward their plus- or the minus ends. Recently, many of these motor pro-
teins have also been identified asþTIPs (Wu et al., 2006). These include the
plus-end directed, kinesin-7 family member, CENP-E (Cooke et al., 1997;
Sardar et al., 2010) as well as kinesin-5 Eg5 (Jiang et al., 2012) and theminus-
end-directed dynein (Kobayashi and Murayama, 2009). In this section, we
will focus on the functional relevance and mechanisms involved in motor
protein accumulation at MT plus ends.
3.5.1 KinesinsMost kinesins show plus-end-directedmotility. Kinesins have ATPase activ-
ity, generate movement through the motor domain (Vale and Fletterick,
1997), and are classified according to its position within the proteins
(Miki et al., 2005). These structural features led to the separation of kinesins
into 15 different families (Hirokawa et al., 2009). In addition to the motor
domain, all kinesins have one or more coiled-coil domains. Depending on
the kinesin family, they can also have a CAP-Gly domain, a pleckstrin
homology (PH) domain, a Phox homology (PX) domain, and WD40
repeats (Hirokawa et al., 2009). Any kinesin that does not have a dis-
tinguishing feature falls into the orphan kinesin group (Fig. 2.3; Miki
et al., 2005). So far, kinesins have been involved in many cellular functions
such as organization of the interphase MT cytoskeleton, axonal transport,
organelle movement, and mitosis.
Some kinesin-like proteins have already been described to tip-track
MTs. CENP-E was described to localize to the plus ends of MTs, where
it promotes their elongation, possibly by stabilizing a straight-end conforma-
tion, which favors tubulin addition to the plus end (Sardar et al., 2010). In
theory, all plus-end-directed motors could concentrate on MT plus ends
due to their function, but most of them do not. This probably happens
because they have to interact with otherþTIPs or, in alternative, must show
some specificity for the MT plus end to do so (Bieling et al., 2007; Busch
et al., 2004). In fact, the yeast kinesin Tea2 needs to interact with Mal3
(the EB-like homologue) to track MT plus ends and to stimulate its ATPase
activity (Bieling et al., 2007; Browning and Hackney, 2005; Busch and
82 Jorge G. Ferreira et al.
Brunner, 2004). The same behavior was described for kinesin-5 Eg5, which
interacts with EB proteins through a classical SxIP motif (Jiang et al., 2012)
and for kinesin-8 Kif18B which does not contain a canonical SxIP motif but
has similar sequences (Stout et al., 2011).
3.5.2 DyneinDynein is a large macromolecular complex with a molecular weight of
approximately 1.2 MDa. It is composed of heavy intermediate, light inter-
mediate, and light chains. The heavy chains contain the motor domains with
six AAA ATPase domains and an MT-binding stalk (Fig. 2.3; Oiwa and
Sakakibara, 2005). Dynein is a minus-end-directed motor that uses ATP
hydrolysis to power its movement and requires interaction with the dynactin
complex. One of the subunits of the dynactin complex is p150glued. This
protein is a þTIP that has a CAP-Gly domain and two coiled-coil regions
which are required for dimerization and interaction with the dynein inter-
mediate chain (King et al., 2003). Early reports of dynein accumulation on
MT plus ends came from work with the filamentous fungus Aspergillus. In
this organism, dynein exhibits plus-end-directed movement at velocities
similar to MT polymerization rates, which suggests that dynein is associated
to, and moving with, the polymerizing ends of MTs (Xiang et al., 2000).
Subsequent reports described the accumulation of both dynein and NUDF
(the homologue of Lis1) at MT plus ends in a comet-like structure (Zhang
et al., 2003). In the same system, dynein and dynactin required each other for
plus-end accumulation but NUDF specifically required dynein to tip-track.
After arriving at the plus ends, dynein also exhibits some retrograde move-
ment and this movement is also MT dependent (Xiang et al., 2000). The
interaction of dynein with LIS1 is important for dynein-mediated retrograde
transport because it allows the release of the dynein–dynactin complex from
CLIP170-decorated MT plus ends (Lansbergen et al., 2004). In vitro work
estimated that the dynein comet consists of approximately 55 dyneinmotors.
About half of the motors show a slow turnover and are actively kept at the
plus ends by a retention mechanism that requires interaction with dynactin
and EB1 (Schuster et al., 2011). Therefore, dynein retention at the plus ends
involves a combination of both stochastic accumulation and active retention
to allow formation of the dynein comet and ensure capturing of organelles
by minus-end-directed motors (Schuster et al., 2011).
During mitosis, dynein localizes at the cell cortex (Kiyomitsu and
Cheeseman, 2012; O’Connell and Wang, 2000). In yeast, it was proposed
that dynein offloads directly from the MT plus ends to the cell cortex by an
83+TIPs in Cell Division
active MT-mediated delivery (Markus and Lee, 2011), a process which
requires the neck region of dynein. Longer neck regions allow enhanced
off-loading without affecting motor activity, while shorter necks block
delivery to the cortex. This led to the proposal that a conformational change
in dynein could be regulated by a masking/unmasking event that controls
dynein off-loading from MTs. Moreover, the N-terminal tail domain is
essential for targeting dynein to cortical receptor sites, whereas the
C-terminal domain is required for plus-end targeting in a Bik1/CLIP170-
and Pac1/LIS1-dependent manner (Markus et al., 2009). Curiously, expres-
sion of the motor domain alone blocks the MT plus-end accumulation of
dynein, and this can be rescued by overexpression of LIS1.
Additional dynein functions include centrosome separation and nuclear
translocation (Tsai et al., 2007). Dynein and Lis1 appear to generate tension
between the nucleus and the centrosome (Tanaka et al., 2004) and also at the
interface between MT tips and the cell cortex (Dujardin et al., 2003). Inter-
estingly, the role of dynein in nuclear movement appears to be conserved in
different cell types. Both dynein and kinesin seem to be required for the
bidirectional movement of the nucleus by interacting with the nuclear pore
complex. Interaction of dynein or kinesin-1 with Bicaudal D2 is essential for
nuclear and centrosomal position during mitotic entry (Splinter et al., 2010).
This may also involve the interaction of dynein/dynactin with a CENP-F–
NudE/EL–Nup133 complex (Bolhy et al., 2011).
3.6. Lis1Lissencephaly 1 (Lis1) proteins were first described as the result of a mutation
that leads to severe defects in brain development in humans (Dobyns et al.,
1993; Vallee et al., 2001). So far, many orthologs have been identified from
yeast (Geiser et al., 1997) to Caenorhabditis elegans (Dawe et al., 2001) and
Drosophila (Sheffield et al., 2000). Sequences from all orthologs are highly
conserved, suggesting a functional conservation. In structural terms, Lis1
proteins have three distinct regions (Fig. 2.3). The N-terminal region is
called LIS1-homology motif (LisH), which ranges between residues 1–39
and has been recently recognized as an ubiquitous motif, found in another
114 eukaryotic proteins (Emes and Ponting, 2001; Kim et al., 2004). The
region between amino acids 40–85 is predicted to be a coiled-coil region
which, together with the LisH domain, is involved in dimerization (Tai
et al., 2002). Near the C-terminal region, there are seven WD40 repeats
which range from amino acids 96–410 containing a b propeller domain,
84 Jorge G. Ferreira et al.
which are important for lateral interactions with other proteins (Tarricone
et al., 2004).
Although it can act as a þTIP, Lis1 seems to target MTs by WD40-
mediated binding to CLIP170, dynein, and dynactin, rather than binding
the plus ends directly (Coquelle et al., 2002; Tai et al., 2002). The interac-
tion of Lis1 with CLIP170 is positively regulated by phosphorylation
(Coquelle et al., 2002). During mitosis Lis1 is recruited to the cell cortex
and kinetochores in a dynein/dynactin-dependent manner (Coquelle
et al., 2002; Faulkner et al., 2000). The C-terminal WD40 repeat region
of Lis1 seems to be sufficient for kinetochore targeting (Tai et al., 2002).
When overexpressed, Lis1 induces a displacement of CLIP170 from the
kinetochores but also interferes with spindle orientation and mitotic pro-
gression (Faulkner et al., 2000; Tai et al., 2002; Vallee et al., 2001).
3.7. Kinesin-13 familyMembers of the kinesin-13 family were named so because of the position of
the motor domain in the middle of the protein. The first 12 families (from
kinesin-1 to -12) have the motor domain close to the N-terminal region,
and kinesin-14 has the motor domain in the C-terminal region (Lawrence
et al., 2004; Miki et al., 2005). These kinesin-13 members were also initially
named M kinesin family (for “Middle Type Motor”) or KinI family (for
“InternalTypeMotor”).Within thekinesin-13 family, there are two subfam-
ilies: the ubiquitous KIF24 subfamily and the mammalian-specific KIF2 sub-
family. This last subfamily is comprised of three members: Kif2A, Kif2B, and
Kif2C/MCAK. All members of the family have an N-terminal globular
domain, followed by a positively charged neck upstream of the centrally
located catalytic core, and a C-terminal dimerization domain (Fig. 2.3;
Ogawa et al., 2004;Wordeman, 2005). TheKIF24 subfamily has the catalytic
core close to the N-terminal region, whereas the KIF2 subfamily has the cat-
alytic core closer to the center of the molecule (Miki et al., 2005). Interest-
ingly, it was demonstrated that MCAK requires dimerization through the
coiled-coil domain in the C-terminal region, and this has a role in regulating
the ATPase activity of the protein (Ems-McClung et al., 2007).
Members of the kinesin-13 family have been implicated in vesicle trans-
port (Noda et al., 1995) and, more importantly, in MT depolymerization
(Desai et al., 1999; Manning et al., 2007; Mennella et al., 2005; Walczak,
2003). Upon binding to the MT end, they induce a conformational change
in its structure that leads to a catastrophe event (Desai et al., 1999). The
MT-destabilizing properties of kinesin-13 members are unique because they
85+TIPs in Cell Division
use ATP hydrolysis to induce depolymerization of MTs from both ends,
instead of using it to walk along MTs (Desai et al., 1999; Helenius et al.,
2006; Hunter et al., 2003). The best studiedmember of the family isMCAK.
This þTIP was shown to target the plus ends of MTs and, once there,
14 MCAK dimers form an ATP-hydrolyzing complex that processively
depolymerizes MTs (Hunter et al., 2003). For this reason, MCAK was
described as a major MT remodeler by preventing MT aging and inducing
random catastrophes (Gardner et al., 2012).
One puzzling observation comes from the fact that these proteins, while
having potent MT depolymerization activity, are still able to accumulate in
the plus ends of MTs (Moore et al., 2005). This suggests that the
MT-depolymerizing activity must be inhibited or controlled at this location
and raises the question of how does MCAK reach the MT tip. Microscopy
studies using single molecules demonstrated that MCAK rapidly moves
along the MT lattice in a random walk (Helenius et al., 2006). Contrary
to its requirement for the MT-depolymerizing effect, this diffusion does
not require ATP hydrolysis and is more rapid than direct binding to the plus
end from solution (Helenius et al., 2006). In addition to this, MCAK also
associates with EB proteins. In fact, MCAK associates with the
C-terminal region of both EB1 and EB3 and colocalizes with EB1 at MT
plus ends (Lee et al., 2008; Montenegro Gouveia et al., 2010). This raises
the possibility that MCAK could also use an EB-hitchhiking mechanism
to bind MT plus ends, in addition to lattice diffusion. These were proposed
as complementary mechanisms that would allow MCAK to remain associ-
ated with MT even after EB displacement from the plus end. Recent work
demonstrated that MCAK contains an SxIP motif near its C-terminal
domain that is crucial for associating with EB1 (Honnappa et al., 2009). This
property of MCAK seems to be conserved with other kinesin-13 proteins in
Drosophila such as KLP10A, which associates with EB1 and is necessary for
KLP10A targeting to MT plus ends (Mennella et al., 2005). The association
of MCAK with MTs can also be regulated in a posttranslational manner.
Indeed, Aurora-B was shown to phosphorylate MCAK and this is crucial
for its function (Andrews et al., 2004; Lan et al., 2004). In addition, most
of these phosphorylation sites seem to cluster in a region close to the SxIP
motif, which alters the ability of MCAK to interact with EB1 and tip-track
(Honnappa et al., 2009; Moore et al., 2005). Curiously, the other family
members Kif2A and Kif2B do not accumulate at MT plus ends, and this
is explained by the fact that they do not have an SxIP motif.
86 Jorge G. Ferreira et al.
3.8. TOG familyThe tumor overexpressed gene (TOG) proteins belong to a highly con-
served family involved in MT dynamics regulation (Al-Bassam and
Chang, 2011; Slep, 2009). The founding member of this family, XMAP215,
was identified in Xenopus as a protein that promotes rapid MT growth (Gard
and Kirschner, 1987). Many orthologues have been described, including
ch-TOG in humans (Charrasse et al., 1998). In structural terms, these pro-
teins are characterized by the presence of a variable number of TOG
domains near the N-terminal region (Fig. 2.3). These domains have approx-
imately 200 amino acids and comprise between 2–5 units, depending on the
organism (Ohkura et al., 2001). Interestingly, each of these domains contains
several HEAT repeats, which are thought to mediate protein–protein inter-
actions (Cassimeris et al., 2001). The human ch-TOG contains five TOG
domains near theN-terminus, regions with sequences rich in serine, glycine,
and lysine (SK-rich domains) and a conserved C-terminal nonrepeat domain
(Al-Bassam and Chang, 2011). Interestingly, CLASPs also have TOG
domains and SR-rich regions, which provide a structural link between
the possible functions of both classes of proteins (Lemos et al., 2000; Slep,
2010). Detailed studies revealed that the N-terminal domain contains
an MT-stabilizing region, whereas the C-terminal domain is necessary for
centrosome and MT targeting (Popov et al., 2001).
TOG proteins not only localize to MT plus ends but can also bind the
MT lattice and soluble tubulin. They have an intrinsic ability to promote
MT elongation from both ends although they do so more efficiently on
the plus ends (Gard and Kirschner, 1987; Vasquez et al., 1994). In vitro stud-
ies with recombinant XMAP215 confirmed that these molecules can asso-
ciate directly to MT plus ends, stimulating their growth (Brouhard et al.,
2008; Kinoshita et al., 2001). These studies further demonstrated that
XMAP215 transiently binds the MT plus end and adds 25 tubulin dimers
to MT before dissociating (Brouhard et al., 2008). The initial hypothesis
for XMAP215 action involved the binding and recruitment of tubulin olig-
omers to MT ends (Cassimeris et al., 2001). However, later it became clear
that TOG proteins can only bind one tubulin dimer at a time (Al-Bassam
et al., 2006; Brouhard et al., 2008). Curiously, in Xenopus egg extracts,
the N-terminal region is able to stimulate MT growth at the plus ends by
inhibiting catastrophes, while the C-terminal region suppresses MT growth
by promoting catastrophes (Popov et al., 2001). Additional studies in differ-
ent systems further confirmed the role of TOG proteins in MT growth and
87+TIPs in Cell Division
stabilization (Charrasse et al., 1998; Dionne et al., 2000; Tournebize et al.,
2000). ch-TOG has been shown to promote MT assembly both in solution
and from nucleation centers (Charrasse et al., 1998), and to be essential for
the formation of taxol-induced asters in human mitotic extracts (Dionne
et al., 2000). In vivo, these proteins increase MT growth and knockdown
of the proteins is associated with short interphase MTs, reduced growth
rates, and increased catastrophes and pauses (Brittle and Ohkura, 2005;
Cullen et al., 1999; Tournebize et al., 2000; Wang and Huffaker, 1997).
It was proposed that the stabilizing effect of these proteins might be due
to their interaction with MT-destabilizing proteins. In fact, XMAP215
seems to stabilize MTs by opposing the action of destabilizers such as
XKCM1 (the Xenopus homologue of MCAK).
3.9. Other þTIPsIt is well known that many þTIPs require interaction with EB proteins in
order to localize to the MT plus end (Akhmanova and Steinmetz, 2008).
The discovery, that conserved SxIP motifs are sufficient to target these pro-
teins to the plus ends (Honnappa et al., 2009), has allowed for the screening
and identification of an ever increasing number ofþTIPs (Jiang et al., 2012).
Examples of some of theseþTIPs include the stromal interactionmolecule 1,
which exhibits EB1-dependent tip-tracking behavior (Grigoriev et al.,
2008) and is involved in ER remodeling. Similarly, navigators were
described to associate withMT plus ends and to be important for cytoskeletal
reorganization (Martinez-Lopez et al., 2005; van Haren et al., 2009). In
addition, þTIPs which are involved in MT organization such as tastin
and DDA3 also have SxIP motifs (Jiang et al., 2012; Zhang et al., 2013).
Both tastin and DDA3, unlike the majority of other þTIPs, also have the
ability to track depolymerizing MTs. Surprisingly, among the new SxIP-
containing proteins, there were also membrane-associated þTIPs such as
AMER2/FAM123A, which was originally described as an APC-binding
protein (Grohmann et al., 2007), and kinases such as TTBK1 and TTBK2,
which are involved in the phosphorylation of MT-associated tau (Houlden
et al., 2007; Sato et al., 2006).
On the other hand, there areþTIPs that do not seem to interact directly
with EB proteins but are able to tip-track nonetheless. Two of such þTIPs
are Astrin and Kinastrin. Astrin was originally identified as a mitotic,
MAP (Mack and Compton, 2001). Recently, it was shown that Astrin
can bind to MT plus ends by associating with its interactor Kinastrin
88 Jorge G. Ferreira et al.
(Dunsch et al., 2011). Once at the plus ends, this Astrin/Kinastrin complex
can induce MT polymerization, possibly by stabilizing the plus ends.
4. RECOGNITION OF MICROTUBULE PLUSENDS BY +TIPs
Accumulation at the MT plus ends is what defines a þTIP. Although
their localization can be confined to a small terminal region of the MT, they
employ different mechanisms to recognize and move along MTs. This sec-
tion will focus on howþTIPs recognize the plus end and how they are able
to move along the MT lattice. Tip-tracking behavior implies that þTIPs
must either have the ability to directly bind tubulin orMTs or, in alternative,
be recruited indirectly through binding to other factors. The fact that many
different classes of proteins can exhibit tip-tracking (Table 2.1) led to the
proposal of four models to account for this behavior: end binding, copoly-
merization, directed transport, and hitchhiking (Fig. 2.5). Curiously, it
seems that the same þTIP can exhibit different behaviors depending on
the conditions or organism: for example, in mammalian cells, APC can
be loaded to the plus ends in an EB1-dependent manner (Slep et al.,
2005), can tip-track autonomously (Kita et al., 2006), or can do so by asso-
ciating with kinesin-2 (Jimbo et al., 2002). On the other hand, loading of
CLIP170 to plus ends can be mediated by motors in yeast (Busch et al.,
2004; Carvalho et al., 2004; Maekawa and Schiebel, 2004), whereas in
mammalian cells, it involves direct binding and treadmilling on MT plus
ends (Perez et al., 1999).
4.1. Recognizing the microtubule plus endHow is it that some þTIPs such as EB proteins are able to directly associate
to the growing end ofMTs? This question is of great importance because EB
proteins are responsible for loading the majority of other þTIPs, including
CLIPs, CLASP, and APC (Lansbergen and Akhmanova, 2006), and can
influence drastic changes in MT dynamics. This means that they must rec-
ognize specific features on plus ends that are different from the lattice
(Fig. 2.5). The first obvious hypothesis is the GTP cap itself. Recently, it
was reported that introducing GTPgS (a slowly hydrolysable form of GTP)
on plus ends mimicked the EB-binding site (Maurer et al., 2011). This is in
line with the finding that EB1 can recognize the nucleotide state of tubulin
independently of its location. Under these conditions, EB1 recognizes the
GMPCPP MT lattice as opposed to the GDP lattice (Zanic et al., 2009).
89+TIPs in Cell Division
A different study revealed that EBs can recognize the nucleotide state of the
plus end and this is crucial both for EB binding and for stabilizing a structural
cap that protects MT from depolymerization (Maurer et al., 2012).
Although it is tempting to assume that the nucleotide state of tubulin alone
is sufficient to determine plus-end binding, there is evidence that argues
against such a simple model. In fact, the GTP cap size is thought to be very
small when compared to the region decorated by the EB comet. Typical
comets can vary between 0.5 and 3 mm in length, depending on the growth
rate but not þTIP concentration (Bieling et al., 2007). This means that
comets have to encompass several hundreds or thousands of tubulin sub-
units, which is much bigger than the presumable GTP cap size (Caplow
and Shanks, 1996; Seetapun et al., 2012; Walker et al., 1991). It should
be noted, however, that recent studies propose the existence of longer
GTP caps that exhibit dynamic behavior and could partly account for this
discrepancy (Schek et al., 2007).
Additionally, it was suggested that EB1, instead of binding the
protofilaments themselves (Maurer et al., 2012), could bind to tubulin while
still in the sheet conformation (Vitre et al., 2008). Thismeans that EB1would
promote sheet closure and bind to theMT seam instead of the protofilaments
(Vitre et al., 2008). In fact,Mal3, the EB1homologue,was reported to act as a
Kinesin-mediatedtransport
Lateral diffusion
Direct recognition of plus-end-specific structure
Copolymerizationwith tubulin
Hitchhiking
Plus-end-directed kinesin
Autonomous +TIP
+TIP with partner-binding plus-end tracking
Tubulin dimer
Figure 2.5 Mechanisms of plus-end recognition by þTIPs. þTIPs can arrive at the plusend by lateral diffusion along the microtubule lattice or diffusion from the cytoplasm. Inalternative, they can be transported by kinesins or associate with the growing end ofmicrotubules by attaching to another þTIP (hitchhiking). Some þTIPs can recognizespecial structural features of the plus ends of microtubules, or they may copolymerizewith tubulin dimers or oligomers. Adapted with permission fromMacmillan Publishers Ltd:Nature Reviews Molecular Cell Biology (A Akhmanova and MO Steinmetz; Tracking theends: a dynamic protein network controls the fate of microtubule tips), copyright (2008).
90 Jorge G. Ferreira et al.
molecular zipper by binding to the seam and leading to changes inMT struc-
ture (desGeorges et al., 2008; Sandblad et al., 2006).Overall these results indi-
cate that the nucleotide state of tubulin plays an important role in plus-end
binding, but there may be additional mechanisms that contribute to þTIP-
MT association. An alternative explanation for specific EB association to the
plus end could depend on the electrostatic interactions between the
C-terminal portion of EB1 and theMT lattice (Buey et al., 2011). In this case,
long-range electrostatic repulsive interactions between the C-terminus of
EB1 and theMT latticemay be able to drive accumulation of EBs on growing
MT ends. In fact, replacing the negatively charged C-terminal portion for a
neutral coiled-coil increased the dwell time of EB1 onMTwithout affecting
interaction with the plus end. Other possible mechanisms may involve the
posttranslational modification of EB proteins themselves. Actually, recent
reports demonstrated that phosphorylation of EB proteins might have an
important role in their association to the plus end. One study described a
mutation on the linker region of Mal3 that is sufficient to reduce the affinity
of the protein forMTs (Iimori et al., 2012), while another demonstrated that
phosphorylation of Bim1p by Aurora/Ipl1p was sufficient to remove Bim1p
from static and dynamic MTs (Zimniak et al., 2009). Curiously, the same
study indicates that both dimerization of Bim1p and the presence of the linker
domain are required for efficient tip-tracking.
4.2. CopolymerizationIn addition to recognizing MT plus ends, someþTIPs such as CLIP170 also
have the ability to directly bind tubulin subunits (Fig. 2.5) (Arnal et al., 2004;
Folker et al., 2005). This suggests that, in order to tip-track, these proteins
copolymerize with tubulin into MT and then quickly dissociate from the
“older” part of MTs as it grows (Akhmanova and Hoogenraad, 2005).
Moreover, these þTIPs must have a higher affinity for free GTP-tubulin
subunits than the GTP or GDP polymer itself. Association of CLIP170 with
free GTP-tubulin subunits is thought to occur through its CAP-Gly domain
which is able to bind directly the EEY/F motif on the C-terminal a-tubulintail (Mishima et al., 2007). Interestingly, the CAP-Gly domain of CLIP170
also interacts with EB1 and explains how it recognizes a composite binding
site on MTs plus ends composed of EB1 (including its C-terminal tyrosine)
and tyrosinated a-tubulin (Bieling et al., 2008; Mishima et al., 2007). Taken
together, these results provide a model for copolymerization of CLIP170
with tubulin, but they do not explain how CLIP170 dissociates from the
growing MT.
91+TIPs in Cell Division
While copolymerization seems to explain CLIP170 behavior, it falls
short of explaining general þTIP behavior. First, other þTIPs such as
EB1 do not use the copolymerizationmechanism. In fact, EB1 seems to bind
the tubulin polymer but not the individual subunits (Gache et al., 2005).
Moreover, using reconstituted in vitro systems, it was possible to demonstrate
that the yeast EB-like protein Mal3 did not bind tubulin subunits and that
accumulation of Mal3 on plus ends was independent of tubulin concentra-
tion (Bieling et al., 2007). Furthermore, in vitro systems were able to recreate
plus-end tracking without the presence of exogenous enzymes, which
means that tip-tracking is independent of GTP or GDP and argues against
its role in MT recognition (Bieling et al., 2007, 2008; Maurer et al., 2011).
Recent experiments using FRAP demonstrated that þTIPs associate
very transiently with the plus end of MTs (Dragestein et al., 2008;
Wittmann and Waterman-Storer, 2005). Interestingly, turnover measure-
ments of CLIP170 and EB3 demonstrated that they show diffusion at both
the plus and the minus ends of MTs, which is inconsistent with the copo-
lymerization model (Dragestein et al., 2008). Taken together, these results
argue against the role of copolymerization as the major contributor to plus-
end tracking.
4.3. Diffusion versus motor-based transportAccumulation of þTIPs does not necessarily involve direct binding to the
plus end in all situations. SometimesþTIPs will bind to the lattice and move
toward the plus end ofMTswhere they accumulate. To do so, these proteins
use two different mechanisms: diffusion and motor-based transport
(Fig. 2.5).
Diffusional motility is defined as a one-dimensional walk along the MT
lattice driven solely by thermal energy (Cooper and Wordeman, 2009).
Simple diffusion of molecules along an MT is a simple, low-energy mech-
anism that also has the advantage of allowing bidirectional movement. This
mechanism is represented by the same mathematical equations that define
Brownian motion although diffusion coefficients tend to be smaller (Ali
et al., 2007; Gestaut et al., 2008; Helenius et al., 2006). The first observations
of single-molecule diffusional motility on MTs were performed using non-
processive kinesin motors (Inoue et al., 2001; Okada and Hirokawa, 1999).
While kinesin motor proteins usually “walk” along MTs using ATP hydro-
lysis, they can sometimes show a “biased diffusion.” This has already been
demonstrated for a number of kinesins which include KIF1A, CENP-E,
92 Jorge G. Ferreira et al.
Ncd, and Eg5 (Furuta and Toyoshima, 2008; Kim et al., 2008; Kwok et al.,
2006; Okada and Hirokawa, 1999). It has been proposed that this type of
motor protein motility occurs when the motor domain is not so tightly
bound to the MT. In accordance, in experiments where ADP is added
instead of ATP, these proteins exhibit a pure diffusional movement presum-
ably because of weaker binding to MT (Kwok et al., 2006; Okada and
Hirokawa, 1999). This type of diffusion appears to occur ubiquitously
and provides some advantages over motor-based movement. First, it makes
the system more flexible by allowing unbiased binding of proteins at both
MT ends. Interestingly, many MAPs (such as MCAK) that require localiza-
tion at both the plus and minus end ofMTs also use this mechanism (Oguchi
et al., 2011). Second, by making weaker attachments to the lattice, in theory
it could allow these proteins to overcome obstacles that may exist along MT
by jumping between protofilaments in a side-step manner (Wang et al.,
1995). Third, diffusion does not require ATP consumption to move pro-
teins. Finally, over short distances (<1 mm), diffusional motility is faster than
directed motility which may allow quicker delivery of molecules to the plus
ends (Cooper andWordeman, 2009). However, because it is a random pro-
cess, it is very ineffective over longer distances.
Contrary to unbiased diffusional motility, molecules can exhibit a direc-
tional movement on MTs that is dependent on motor proteins. This mech-
anism requires the action of kinesin motor proteins which can contribute to
plus end accumulation. However, kinesin action alone is not sufficient to
induce the formation of a comet, as theremust be some retentionmechanism
that allows the þTIP to remain associated with the plus end (Galjart and
Perez, 2003). In yeast, CLIP170 homologues Tip1 and Bik1 are transported
to the plus end by the action of kinesins Tea2 and Kip2, respectively (Busch
et al., 2004; Carvalho et al., 2004). Although not so common in mammals,
motor-mediated transport can also be observed for APC, which requires
kinesin-2 for plus-end accumulation (Jimbo et al., 2002). This accumulation
occurs if the motor transport velocity is higher than MT polymerization/
depolymerization velocity (Busch et al., 2004; Carvalho et al., 2004). Inter-
estingly,þTIPs that are transported bymotors can also track depolymerizing
MTs in a mechanism known as backtracking (Carvalho et al., 2004).
4.4. HitchhikingLoading þTIPs via a motor-based transport requires that they hitchhike on
motor proteins. However, mostþTIPs accumulate at plus ends indirectly by
93+TIPs in Cell Division
hitchhiking on other þTIPs (Lansbergen and Akhmanova, 2006). This
implies that a core component of þTIPs must exist that is able to associate
to MTs independently of any other factor (Fig. 2.5). EB proteins and also
XMAP215/ch-TOG can perform this function because it has been shown
that they can autonomously track MT plus ends (Al-Bassam and Chang,
2011; Bieling et al., 2007, 2008; Brouhard et al., 2008; Gard and
Kirschner, 1987). Furthermore, in the case of EB proteins, they are known
to interact with most other plus-end-associated proteins (Akhmanova and
Steinmetz, 2008; Jiang et al., 2012).
The interaction of EBs with other þTIPs occurs through the EB
C-terminal domain, also known as EBH domain (Bu and Su, 2003).
Although all these EB-interacting proteins share the ability to tip-track,
there is no apparent functional similarity between them, raising the question
of whether there is any common feature that accounts for their behavior. It is
now known that these EB partner proteins rely on the SxIP motif, which is
embedded within basic and proline/serine-rich sequence regions, to bind to
EB proteins and hitchhike onMTs. The first report on the role of an Ile-Pro
peptide in þTIP interaction came from structural work on the EB1–APC
interaction (Honnappa et al., 2005). The authors demonstrated that this Ile-
Pro peptide was part of the APC region that bound EB1 and that mutating it
was sufficient to impair the interaction. Further studies revealed that many
þTIPs such as MCAK, CLASPs, APC, and ACF7/MACF1 have a similar
SxIP motif (Honnappa et al., 2009; Jiang et al., 2012). In addition, it was also
demonstrated that this SxIPmotif is sufficient to load theseþTIPs to theMT
plus ends through interaction with EB1. Two interesting observations were
derived from this study: it is possible to abolish the interaction between these
þTIPs and EB1 and, as a consequence, inhibit tip-tracking by simply mutat-
ing the SxIP motif (for instance, by substituting the Ile-Pro with Asp); it also
became possible to “transform” a protein into a þTIP by introducing the
SxIP motif in its amino acid sequence. Taken together, these findings allow
the establishment of a generalMT tip localization signal and create a unifying
mechanism for plus-end targeting.
In addition to SxIP motifs, proteins can also use the CAP-Gly domain to
interact with the plus ends of MTs. Proteins that have a CAP-Gly domain
were the first to be identified that exhibit tip-tracking behavior (Perez et al.,
1999), and these include CLIP170, CLIP115, and p150glued, among others
(Steinmetz and Akhmanova, 2008). The CAP-Gly domain is highly con-
served in eukaryotes, can exist in either single or multiple copies, and is
involved in the regulation of protein interactions and formation of protein
94 Jorge G. Ferreira et al.
networks (Akhmanova and Steinmetz, 2008; Galjart, 2005). Structural data
derived from X-ray crystallographic analyses of these domains demonstrated
that the conserved motif GKNDG is essential for interaction with the
EEY/F motif of a-tubulin, EB1, and CLIP170 (Steinmetz and
Akhmanova, 2008; Weisbrich et al., 2007). CAP-Gly proteins are unable
to bind tubulin dimers that lack the C-terminal tyrosine of EEY/F (Peris
et al., 2006), and moreover, mutations in the Lys-Asn of the GKNDG or
the EEY/F motives are sufficient to abolish this interaction (Steinmetz
and Akhmanova, 2008; Weisbrich et al., 2007). These interactions with
CAP-Gly proteins have dissociation constants in the micromolar range,
which is similar to what is observed for the interaction of the C-terminal
of EB1with an APCC-terminal peptide and indicates they are very dynamic
(Honnappa et al., 2005, 2006; Mishima et al., 2007; Weisbrich et al., 2007).
Conceptually, the hitchhiking mechanism implies that þTIPs that use
this mechanism are not able to interact efficiently with tubulin or MTs,
but this is not always the case. Notably, proteins that contain CAP-Gly
domains can efficiently associate with tubulin (Dixit et al., 2009; Folker
et al., 2005; Mishima et al., 2007). In addition, although some proteins with
SxIP domains such as RhoGEF2 and melanophilin do not bind tubulin
directly, many others such as MCAK and CLASPs are able to do so
(Al-Bassam et al., 2010; Helenius et al., 2006; Rogers et al., 2004; Wu
et al., 2005). Although direct binding to tubulin or MTs circumvents the
necessity for hitchhiking, it seems that plus-end accumulation mainly
depends on the hitchhiking mechanism. In fact, CLIP170 requires EB1
to tip-track (Dixit et al., 2009) and both EB1 and EB3 enhance the binding
of CLIPs to the MT plus ends (Komarova et al., 2005). Interestingly,
hitchhiking seems to be necessary for the loading but not dissociation of
þTIPs. These results were based on in vitro observations that CLIP170
(which hitchhikes on EB1) remains associated with MT longer than EB1
itself (Dixit et al., 2009). This probably happens because CLIP170, besides
binding to EB1, is able to bind directly to the C-terminal tails of tubulin
(Mishima et al., 2007).
4.5. Turnover at microtubule plus endThe balance betweenMT association–dissociation must be tightly regulated
so thatþTIPs remain confined to the plus end. This is clearly observedwhen
þTIPs are overexpressed and label the entire MT lattice (Schwartz et al.,
1997; Tirnauer and Bierer, 2000). Dissociation of þTIPs from MT may
involve changes in the MT lattice (such as GTP hydrolysis) or a structural
95+TIPs in Cell Division
change in the þTIP itself (Akhmanova and Hoogenraad, 2005). Another
convenient mechanism would be þTIP phosphorylation. In fact, CLIP170
association to MT is negatively regulated by phosphorylation (Rickard and
Kreis, 1991). So far, several kinases have been described to affect CLIP170
phosphorylation, including mTOR (Choi et al., 2002), Plk1 and CK2
(Li et al., 2010), and AMPK (Nakano et al., 2010). Surprisingly, inhibition
of mTOR decreases the binding of CLIP170 to MTs, whereas inhibition of
AMPK increases the binding of CLIP170 toMTs. These data emphasize that
multiple layers of regulation must exist that control association of CLIP170
to MT. Experiments with the EB-like protein in yeast cells have also
suggested phosphorylation as a possible mechanism for EB binding to
MTs in a cell-cycle-dependent manner (Iimori et al., 2012; Zimniak
et al., 2009). However, there is no evidence so far for a phosphoregulatory
mechanism that specifically controls association/dissociation of individual
molecules to the MT plus ends.
While the mechanisms that regulate þTIP association to MT are still
elusive, considerable progress has been made in defining how these proteins
turnover at the plus end. The first model proposed that þTIPs bind only
once to the MT plus end and then dissociate when the MT lattice becomes
“mature” (Carvalho et al., 2003; Galjart, 2005). This process is called
treadmilling because of the similarities with the behavior of tubulin subunits
within MT (Fig. 2.6). While it seems that þTIPs are moving along as MT
Microtubulegrowth
Rapid exchange betweenplus-end and cytoplasm
Association
Dissociation
Association+TIP treadmillingalong microtubule(1A) (1B)
(2)
Figure 2.6 Mechanisms of þTIP dissociation from the microtubule. A þTIP associateswith the microtubule (1A) and remains attached to the structure until the plus-end isconverted into a regular lattice and then dissociates (1B). A new þTIP can then bindto the new microtubule plus-end (2). This mechanism is known as treadmilling. Onthe other hand, þTIPs may exchange rapidly with the cytoplasmic pool at their bindingsites in the plus end, while these sites decay exponentially over time. This mechanism isknown as rapid exchange. Adapted with permission fromMacmillan Publishers Ltd: NatureReviews Molecular Cell Biology (A Akhmanova and MO Steinmetz; Tracking the ends: adynamic protein network controls the fate of microtubule tips), copyright (2008).
96 Jorge G. Ferreira et al.
grows, they are, in fact, stationary and it is the addition of new þTIPs and
tubulin at the plus end that creates this optical illusion (Carvalho et al., 2003).
The treadmilling model also implies that fluorescence decay observed in the
comet’s tail is due to the dissociation of þTIPs as the MT matures. Initial
approaches using fluorescent speckle microscopy techniques proposed that
EB1 and CLIP170 probably used treadmilling onMT plus ends (Perez et al.,
1999; Tirnauer et al., 2002b; Waterman-Storer et al., 1998). However,
recent studies demonstrated that this is not the case. Based on single-
molecule studies and FRAP experiments on MT plus ends (Bieling et al.,
2007; Dragestein et al., 2008; Kumar et al., 2009), a model of fast exchange
was proposed (Fig. 2.6). This is supported by the observation that the MT
decoration time is much longer than the dwell time of single molecules of
Mal3 (Bieling et al., 2007), meaning that Mal3 molecules must continuously
turnover on the plus end. In addition, it was demonstrated that individual
þTIPs are very dynamic and can repeatedly bind to the same plus end with
low affinity. Accordingly, both CLIP170 and EB3 molecules exhibit rapid
turnover behavior on plus ends (Dragestein et al., 2008). This turnover
means that several molecules can attach to the same binding site on MT
and continuously exchange with the cytoplasmic pool, as was shown for
EB3 (Dragestein et al., 2008). Further studies confirmed that EB1 also
exhibited the same behavior (Dixit et al., 2009). Taken together, this means
that þTIP turnover is much higher than binding site turnover, which is in
disagreement with the treadmilling model. As a consequence, these exper-
iments show that accumulation of þTIPs in a comet-like structure depends
on the exponential decay of EB-binding sites in the MT. EB proteins bind
and dissociate very rapidly, which creates a large number of binding sites for
other þTIPs. This is in agreement with studies that demonstrate a necessity
of CLIP170 to bind simultaneously to EB1 and tubulin composite sites
(Bieling et al., 2008). In addition, other þTIPs also show a slower dissoci-
ation rate from theMT, when compared to EB proteins (Bieling et al., 2007;
Dragestein et al., 2008), which further supports the role of EBs in facilitating
the binding of þTIPs to MTs.
5. +TIPs IN MITOSIS
5.1. +TIPs in mitotic spindle organization and positioningThe transition from interphase to mitosis involves a dramatic reorganization
of the MT cytoskeleton. This is accompanied by an increase in MT dynam-
ics and an abrupt decrease inMT polymer level which tightly correlates with
NEB (Zhai et al., 1996). Moreover, mitotic MTs show increased
97+TIPs in Cell Division
catastrophe frequencies and spend less time in the “paused” state (Belmont
et al., 1990; Rusan et al., 2001). Many of these changes appear to be con-
trolled by phosphorylation-dependent regulatory mechanisms. Accord-
ingly, CDK1 was shown to play a role in remodeling the MT
cytoskeleton, as adding active CDK1 to Xenopus extracts increases MT
dynamics to mitosis-like levels (Verde et al., 1990). Furthermore, CDK1
induces the depolymerization of interphase MTs when injected into mam-
malian cells and also leads to the destabilization ofMTs when added tomam-
malian cell lysates (Lamb et al., 1990; Lieuvin et al., 1994). Furthermore,
protein phosphatases PP1 and PP2A have been shown to differentially reg-
ulate MT dynamics (Tournebize et al., 1997). While PP1 is required for
transitions into and out of mitosis, PP2A is required to maintain a steady-
state spindle length by controlling the level of catastrophes.
Many different classes of þTIPs have been involved in mitotic spindle
organization, some of which are regulated by phosphorylation. Notably,
CLIP170 is necessary for establishment of spindle bipolarity by interacting
with dynein (Tanenbaum et al., 2008). Interestingly, CLIP170 association
to the MTs is regulated by phosphorylation (Choi et al., 2002; Rickard
and Kreis, 1991), although it is not known whether this has an impact on
spindle organization.
EB proteins have also been implicated in spindle organization. Both
immunofluorescence analyses and live imaging using GFP tagging showed
that EB1 is able to localize to the growing ends of MTs throughout mitosis
(Berrueta et al., 1998; Morrison et al., 1998; Piehl and Cassimeris, 2003).
More in-depth observations demonstrated that EB1 can target to kineto-
chores with attached growingMTs (Tirnauer et al., 2002a). The first reports
indicated that depletion of EB1 in Drosophila by RNAi leads to the forma-
tion of short spindles and short astral MTs (Rogers et al., 2002), but in mam-
malian cells, depletion of EB1 by RNAi does not seem to interfere with
spindle assembly (Bruning-Richardson et al., 2012; Draviam et al., 2006;
Ferreira et al., 2013). In Xenopus egg extracts, EB1 was involved in spindle
organization and chromosome segregation by interacting with XMAP215
(Kronja et al., 2009). In addition, EB1 is also involved in astral MT nucle-
ation/stabilization possibly by interacting with Kif18B (Stout et al., 2011;
Toyoshima and Nishida, 2007). Interestingly, the EB1-interactor APC is
hyperphosphorylated during mitosis, which suggests that its binding to
MTs is regulated by phosphorylation (Bhattacharjee et al., 1996). Moreover,
depletion of APC has been shown to compromise the formation of spindles
in Xenopus extracts (Dikovskaya et al., 2004), although direct evidence for
98 Jorge G. Ferreira et al.
the role of APC in spindle assembly in animal somatic cells is still lacking.
APC is also involved in mitotic spindle positioning. When a mutated dom-
inant form of APC is expressed in cells or when APC is disrupted, the spindle
is displaced from the cell center (Draviam et al., 2006; Green and Kaplan,
2003). This was also observed when EB1, a known APC interactor, is
depleted from cells, and this correlates with a marked loss of astral MTs
(Draviam et al., 2006; Ferreira et al., 2013; Green et al., 2005;
Toyoshima and Nishida, 2007). Therefore, by stabilizing astral MTs, EB1
helps orient the spindle parallel to the cell-substrate and provides an addi-
tional link between the spindle and the cell cortex. This may be accom-
plished through the interaction between EB1 and the motor protein
Kif18B, a plus-end-directed kinesin that can modulate MT dynamics and
has been shown to regulate astral MT length in early mitosis (Stout et al.,
2011). Recently, EB1 was also shown to be required for spindle symmetry.
Upon injection of specific antibodies or a dominant-negative form of EB1 in
mitotic cells, the resulting daughter cells displayed unequal MT content, and
this correlated with an asymmetric spindle pole movement (Bruning-
Richardson et al., 2012). However, in this study, there was no significant
displacement of the spindle from the cell center.
Several studies have confirmed that both the localization pattern of
CLASPs and their role in mitotic spindle organization are conserved
between species (Inoue et al., 2000; Lemos et al., 2000; Maiato et al.,
2003a; Mimori-Kiyosue et al., 2006; Pereira et al., 2006). Studies in mam-
malian cells revealed that simultaneous depletion of both CLASPs resulted in
an increased mitotic index and a plethora of mitotic abnormalities including
misaligned chromosomes, shorter or collapsed bipolar spindles, as well as
multipolar and disorganized spindles (Logarinho et al., 2012; Maiato
et al., 2003a; Mimori-Kiyosue et al., 2006; Pereira et al., 2006). Further-
more, studies with cells derived from Clasp2 knockout mice demonstrated
that the absence of this protein per se results in a significant number of mitotic
abnormalities, enhancing the susceptibility for aneuploidy and chromosomal
instability (Pereira et al., 2006). These cells exhibit numerous spindle defects
that can be partially rescued by ectopic expression of CLASP1. Curiously,
when individual CLASPs are depleted from human cells by RNAi, mitotic
progression does not seem to be affected (Mimori-Kiyosue et al., 2005). This
is in agreement with the observation that removal of one of the CLASP par-
alogues does not affect localization of the other (Pereira et al., 2006). Taken
together, these observations suggest that CLASPs play, at least, partially
redundant roles in mitosis.
99+TIPs in Cell Division
However, some specific mitotic roles can still be assigned to individual
CLASPs. In fact, association of CLASP1 (but not CLASP2) with the tau-
related protein MAP4 was described as important for maintaining spindle
position and defining the division axis in human cells (Samora et al.,
2011). This association serves two purposes: whereas CLASP1 is required
for astral MT capture at the cortex, MAP4 is necessary to prevent engage-
ment of excess dynein motors creating an equilibrium situation. Impor-
tantly, under these conditions, depletion of MAP4 specifically induces
spindle misorientation relative to the substrate without affecting astral
MT nucleation, suggesting that the presence of astral MTs per se might
not be sufficient for accurate spindle positioning. InC. elegans, CLASPs have
a partial redundant role in spindle positioning and astral MT regulation dur-
ing asymmetric cell division (Espiritu et al., 2012). In this system, simulta-
neous depletion of the CLASP homologue CLS-1 together with CLS-2 or
CLS3 induces displacement of the spindle, together with changes in spindle
length. These cells also have a reduced complement of astral MTs, which
accounts for the positioning phenotype. However, depletion of CLS-2
alone in C. elegans embryos leads to defects in chromosome biorientation
without inducing spindle displacement (Cheeseman et al., 2005). Notably,
under these conditions, chromosome biorientation could be rescued by
inhibiting astral MT pulling forces. Overall, these results strengthen the
importance of CLASPs for proper mitotic progression and promotion of
mitotic fidelity.
TheTOG family of proteins also plays an important role in spindle assem-
bly. In fact, XMAP215 is required for this process inXenopus extracts, and its
immunodepletion results in either absence of spindle formation or very short
spindles (Tournebize et al., 2000). In mammalian cells, ch-TOG seems to be
required for the organization of spindle poles but has only a minor role in the
stabilization of spindle MTs (Gergely et al., 2003). The mechanism of
ch-TOG-mediated MT stabilization is partly regulated by its interaction
with TACC3 (Gergely et al., 2003). In addition, it can also protect kineto-
chore–MTs from depolymerization by MCAK (Barr and Gergely, 2008).
Overall, ch-TOG contributes to spindle bipolarity by increasing MT length
and density, focusing MT minus ends at the spindle poles and maintaining
centrosome integrity (Cassimeris and Morabito, 2004).
Many motor proteins that act asþTIPs also have an essential role in spin-
dle organization. Dynein is a minus-end-directed motor that shows tip-
tracking behavior (Vaughan et al., 1999). Dynein can bind to MTs and
induce their stabilization by tethering the plus ends (Hendricks et al., 2012;
100 Jorge G. Ferreira et al.
Steuer et al., 1990;Yoshida et al., 1985). Furthermore, cytoplasmic dynein also
localizes to the cell cortex and serves as an anchor for astralMTs (Busson et al.,
1998). This localization led to the hypothesis that dynein could be involved in
spindle positioning. In yeast, mutations in the dynein gene affect the move-
ment of the spindle into the budding daughter cell without affecting spindle
assembly or chromosome segregation (Li et al., 1993). Later work done in
mammalian cells showed that when the shape of epithelial cells is mechani-
cally manipulated during mitosis, the mitotic spindle will always align with
the longer cell axis (O’Connell and Wang, 2000) and this can be blocked
by inhibiting dynein. This led to the hypothesis that longer astral MTs, by
having a higher number of dynein motors, would be able to generate
increased forces on the spindle and align it with the long cell axis. This model
implies that either astral MTs are in contact with the actin cortex along their
entire length or that dyneinmotors can anchor to cytoplasmic complexes and
exert a pulling force on MTs, as was later also suggested for interphase cells
(Brodsky et al., 2007). Interestingly, dynein localization at the cortex seems to
depend on both spindle pole and chromosome-derived signals which affects
cortical force generation (Kiyomitsu and Cheeseman, 2012). Proximity of
spindle poles with the cortex displaces dynein to the opposite pole, which
results in spindle centering. Activity of Plk1 at the spindle poles is necessary
because it regulates the interaction between dynein–dynactin and the cortical
factors NuMA and LGN (Kiyomitsu andCheeseman, 2012). Furthermore, a
chromosome-derivedRanGTPgradient restricts the localization ofNuMA–
LGN to the lateral cortex which enforces the spindle orientation axis.
In addition, by using their minus-end-directedmotion, thesemotors exert
pulling forces that maintain spindle pole separation during mitosis (Laan et al.,
2012; Vaisberg et al., 1993) and transport different cargo to the centrosome
where they help maintain spindle pole integrity (Purohit et al., 1999;
Young et al., 2000). Interestingly, minus-end-directed motors can also bind
toMT ends such as theDrosophila kinesin-14Ncd (Goshima et al., 2005). This
accumulation occurs through interaction with EB1 and is thought to play a
role in the capture and transport of k-fibers along centrosomal MTs and help
to form a tightly focused bipolar spindle (Goshima et al., 2005).
Astrin has also been involved in mitotic progression and spindle assem-
bly. Its association to spindle MTs and kinetochores was shown to depend
on GSK3b-mediated phosphorylation (Cheng et al., 2008), as inhibition of
the kinase impairs Astrin accumulation and spindle formation. Additionally,
depletion of Astrin by RNAi in human cells also leads to the formation of
disordered spindles (Gruber et al., 2002). Overall, these results highlight the
101+TIPs in Cell Division
importance of Astrin for the formation of a bipolar spindle, which could be
due to the ability of Astrin to regulate spindle MT dynamics (Dunsch
et al., 2011).
There are other proteins such as kinesin-13 family member MCAK that
also plays a role in spindle assembly. MCAK localizes to spindle poles, cen-
tromeres, kinetochores, plus ends of MTs, and also the cytoplasm during
mitosis (Ems-McClung and Walczak, 2010; Moore et al., 2005;
Wordeman and Mitchison, 1995). During early mitosis, MCAK is required
for bipolar spindle assembly. How does MCAK regulate spindle bipolarity?
It has been reported that knockdown of another kinesin-13 member Kif2A
by RNAi leads to a dramatic increase in the number of monopolar spindles
(Ganem and Compton, 2004). When cells depleted of Kif2A are codepleted
for MCAK or treated with low doses of nocodazole, spindle bipolarity is
restored (Ganem and Compton, 2004). This means that Kif2A and MCAK
must be acting on spindle bipolarity through their ability to regulate MT
dynamics. In fact, in extracts depleted of XKCM1 (the Xenopus homologue
of MCAK), there is a fourfold decrease in catastrophe frequencies, which
leads to the formation of very longMTs and assembly of a monopolar spindle
(Walczak et al., 1996). In addition, the EB1-associated pool of MCAK was
proposed to limit the length of MTs in the assembling mitotic spindle, thus
favoring the formation of robust kinetochore–MT attachments (Domnitz
et al., 2012).
Interestingly, excessive nucleation can also induce defects in spindle
positioning. In fact, when MCAK is depleted from HeLa cells, very long
astral MTs are produced (Rankin and Wordeman, 2010). This same effect
can be accomplished by treating cells with the MT-stabilizing drug taxol. As
a consequence, the spindle shows dramatic rocking inside the cell, which is
dependent on Myosin II (Rankin and Wordeman, 2010). During mitosis,
other kinesins are involved in spindle formation (Haraguchi et al., 2006),
chromosome congression (Kapoor et al., 2006;Wood et al., 1997), and inte-
rpolar MT sliding (Kapitein et al., 2005). Interpolar MT sliding is achieved
by kinesin-5/Eg5, which is a plus-end-directed motor that can also tether
MT plus ends (Jiang et al., 2012; Kapitein et al., 2005).
5.2. +TIPs at mitotic centrosomeIn addition to their tip-tracking ability, many þTIPs are also capable of
binding to or contribute to centrosome function. EB1 was first reported
to localize to centrosomes in Dictyostelium. In this system, EB1 localized
102 Jorge G. Ferreira et al.
to MT-free isolated centrosomes. Moreover, EB1 is thought to be required
for initiation of spindle MT growth (Rehberg and Graf, 2002). In mamma-
lian cells, EB1 interacts with centrosomes independently ofMTs, through its
C-terminal domain (Louie et al., 2004). In addition, EB1 depletion leads to a
reduction in MT minus-end anchoring and delays MT regrowth from cen-
trosomes. More recently, it was shown that FOP (FGFR1 oncogene part-
ner) interacts with the C-terminal region of CAP350 and forms a
centrosomal complex necessary for MT anchoring. Interestingly, FOP is
required for EB1 centrosomal localization (Yan et al., 2006). This localiza-
tion could also be mediated by an interaction with CDK5RAP2. In fact, in
addition to its centrosomal localization, CDK5RAP2 exhibits a tip-tracking
behavior that depends on EB1 binding, through a basic and Ser-rich motif
(Fong et al., 2009). Moreover, CDK5RAP2 contains a centrosome-
targeting domain that has a high homology to the Motif 2 of Centrosomin
(CM2) and mediates the association with Pericentrin and AKAP450 (Fong
et al., 2009; Wang et al., 2010). Similar to EB1, APC also associates with
centrosomes (Louie et al., 2004). This interaction is mediated by the
N-terminal domain of APC (Louie et al., 2004; Tighe et al., 2001), although
the exact interaction sequence is not known.
The observation that CLASPs can accumulate at the centrosome suggests
a function at this level, but little is known about their role in this structure
(Maiato et al., 2003a; Pereira et al., 2006). In both HeLa and Drosophila S2
cells, following colchicine treatment, CLASPs were found to colocalize
with g-tubulin in an MT-independent manner (Lemos et al., 2000;
Maiato et al., 2003a). Drosophila CLASP hypomorphic mutants displayed
atypical MT morphology that correlated with an abnormal pattern of cen-
trosome separation (Lemos et al., 2000), in spite of the fact that these cen-
trosomes were still capable of MT nucleation. Recently, CLASPs were
shown to be required for spindle pole integrity after bipolarization in
response to traction forces exerted by the motor proteins CENP-E and
Kid during chromosome alignment, by recruiting ninein to the centrosome
(Logarinho et al., 2012). This mechanism explains why suppression of
CLASPs leads to an increase in the number of multipolar spindles.
The TOG family of proteins also plays a relevant role at the centrosome.
InDrosophila embryos, the centrosomal protein D-TACC is required to effi-
ciently recruit ch-TOG/Msps to centrosomes (Lee et al., 2001). The role of
ch-TOG in spindle organization was proposed to occur in multiple ways. In
human somatic cells, ch-TOG is thought to play a major role in organizing
spindle poles and a more minor role in stabilizing spindle MTs via an
103+TIPs in Cell Division
interaction with TACC3 (Gergely et al., 2003). ch-TOG seems to be
required for centrosomal MT nucleation or stabilization, as absence of the
protein leads to both diminished assembly and less dynamic MTs (Barr
and Gergely, 2008). In addition, ch-TOG also acts by focusing MT minus
ends at the spindle poles ensuring centrosome integrity (Cassimeris and
Morabito, 2004) but also protects spindle MTs from MCAK activity at
the centrosome, which could lead to multipolar spindles (Holmfeldt
et al., 2004). The joint localization of ch-TOG with MCAK at the centro-
some, and subsequent centrosome stabilization, is regulated by Aurora-A
(De Luca et al., 2008). In fact, depletion of Aurora-A leads to an accumu-
lation of ch-TOG at spindle poles with a concomitant delocalization of
MCAK (De Luca et al., 2008).
Like other þTIPs, Astrin was also shown to localize to spindle poles
(Mack and Compton, 2001). Subsequently, it was reported that targeting
of Astrin to the centrosome during S and G2 phases of the cell cycle requires
its interaction with the centrosomal protein ninein (Cheng et al., 2007).
Interestingly, depleting Astrin by RNAi or inducing its mislocalization leads
to loss of spindle pole integrity and centriole disengagement (Cheng et al.,
2007; Thein et al., 2007).
5.3. +TIPs at kinetochoreManyþTIPs are also involved in the regulation of MT–kinetochore attach-
ments. Initial experiments with CLIP170 described its transient association
with prometaphase kinetochores, even before CLIP170 was shown to tip-
track (Dujardin et al., 1998). CLIP170 colocalizes with dynein and dynactin
at kinetochores and is required for the formation of robust k-fibers (Dujardin
et al., 1998). Subsequent studies determined that CLIP170 is necessary for
mitotic progression (Wieland et al., 2004) and that interfering with CLIP170
expression leads to defects in chromosome congression and a decrease in
the number of kinetochore–MT attachments (Tanenbaum et al., 2006).
However, this does not seem to affect MT dynamics or the stability of
kinetochore–MT attachments. These observations indicate that CLIP170
may help in the formation of kinetochore–MT attachments by mediating
the direct capture of MTs at the kinetochore (Tanenbaum et al., 2006).
Interestingly, this kinetochore–MT attachment mechanism may involve
phosphoregulation of CLIP170. Indeed, it was proposed that CK2-
mediated phosphorylation of CLIP170 is involved in its kinetochore local-
ization (Li et al., 2010). Moreover, Plk1 is necessary to enhance this
104 Jorge G. Ferreira et al.
association with CK2. Overall, Plk1- and CK2-associated phosphorylations
of CLIP170 are necessary for the timely formation of kinetochore–MT
attachments during mitosis. However, the mitotic defects that were attrib-
uted to loss of CLIP170 in cultured cells were not confirmed in mouse
models of CLIP170 deficiency, which raises the possibility that loss of
CLIP170 per se is not essential for establishing kinetochore–MT attachments
(Akhmanova et al., 2005). Interestingly CLIP190, theDrosophila orthologue
of CLIP170, was also reported to localize to unattached kinetochores and
this was shown to be dynein/dynactin-dependent (Dzhindzhev et al., 2005).
Given the localization of CLASPs at the kinetochore, one can assume a
functional role in this structure. Kinetochore localization of CLASPs relies
on CENP-E, independently of its motor activity (Maffini et al., 2009;
Maiato and Logarinho, 2011). This kinetochore targeting requires the
C-terminal domain of CLASP1 and CLASP2 but is independent of MTs
or CLIP170 association (Maia et al., 2012; Maiato et al., 2003a; Mimori-
Kiyosue et al., 2006).
Evidence to support the critical role of CLASPs in the regulation of spin-
dleMT dynamics in mammalian cells initially surfaced after injection of anti-
CLASP1 antibodies in HeLa cells stably expressing GFP-a-tubulin (Maiato
et al., 2003a,b). In this situation, injection of anti-CLASP1 antibodies sig-
nificantly reduced or suppressed the typical oscillatory dynamic behavior
of k-fibers, resulting in spindle collapse. Additionally, the fact that CLASP1
accumulates at the outer corona region strongly argued for a role in the reg-
ulation of the kinetochore–MT interface. Amore detailed analysis of mature
k-fibers using FRAP revealed that, in cells depleted for Drosophila CLASP,
k-fibers were not able to flux (Maiato et al., 2005). When severed with a
laser, the fraction of the k-fiber that remained attached to the kinetochore
was unable to regrow, contrary to what happens in the wild-type control
cells. This provided conclusive evidence regarding the essential role of
Drosophila CLASP in the incorporation of tubulin subunits at the kineto-
chore level. This also explains why its absence results in progressively short
k-fibers through tubulin depolymerization at the minus end, leading to
bipolar spindle collapse (Maiato et al., 2005). Interestingly, spindle collapse
could be reverted by depleting KLP10A (a Drosophila kinesin-13 MT
depolymerizer), which prevents MT minus-end depolymerization (Buster
et al., 2007; Laycock et al., 2006; Matos et al., 2009).
In mammalian cells, depletion of CLASPs at the kinetochores caused a
considerable decrease of k-fiber poleward flux and turnover rates, increasing
their stability (Maffini et al., 2009; Manning et al., 2010). Thus, the short
105+TIPs in Cell Division
spindles detected in mammalian cells upon CLASPs depletion can be related
to their particular function at the MT–kinetochore interface. In this way,
CENP-E-mediated recruitment of CLASPs to kinetochores is critical for
the rapid exchange of attached MTs, contributing to their instability. This
view is consistent with the decreased accumulation of CLASPs at kineto-
chores during anaphase and concomitant with the reduction of k-fiber
dynamics at anaphase onset when compared to prometaphase (Bakhoum
et al., 2009b; Gorbsky and Borisy, 1989; Gorbsky et al., 1987). Therefore,
it was proposed that during early mitosis an increase in kinetochore–MT
turnover would allow the correction of erroneous attachments. This balance
is achieved through a functional interaction between CLASPs and the MT
depolymerizer Kif2B, which localize to kinetochores during early mitosis
(Bakhoum et al., 2009b; Maffini et al., 2009; Manning et al., 2010). How-
ever, as cells go into metaphase, this complex is replaced by a CLASP1–
Astrin complex, which promotes k-fiber stability, chromosome alignment,
and SAC silencing. These different complexes appear to be mutually exclu-
sive, suggesting that their recruitment to kinetochores is sufficient to change
the dynamics of attached MTs. At the transition from metaphase to ana-
phase, CLASP levels at kinetochores are reduced via a dynein-dependent
minus-end-directed removal (Reis et al., 2009). It should be noted that
depletion of CLASPs does not seem to interfere with the targeting of other
proteins that might be involved in kinetochore–MT attachment, such as
CLIP170 or dynein (Maiato et al., 2002, 2003a).
EB1 localizes to the plus ends of polymerizing MTs, suggesting that it
may regulateMT dynamics duringmitosis (Tirnauer et al., 2002a). Although
EB1 was originally identified as an APC-interacting protein, its localization
is independent of APC (Berrueta et al., 1998; Morrison et al., 1998). Curi-
ously, the inverse is not true, as APC localization to the plus ends requires an
interaction with EB1 (Askham et al., 2000; Mimori-Kiyosue et al., 2000b).
Moreover, the interaction between APC and EB1 does not seem to be rel-
evant for EB1 mitotic localization, as immunoprecipitation studies demon-
strated that the EB1–APC interaction does not occur or is not detectable
during mitosis, possibly because of APC hyperphosphorylation (Askham
et al., 2000; Bhattacharjee et al., 1996; Nakamura et al., 2001). In Xenopus
meiotic extracts, both EB1 and APC interact with kinetochore-associated
BubR1 (Zhang et al., 2007a). In this system, BubR1 directly interacts with
APC and this is essential for chromosome positioning in the metaphase plate.
Curiously, earlier reports had already identified interaction of APC with
checkpoint proteins Bub1 and Bub3 at the kinetochore (Kaplan et al.,
106 Jorge G. Ferreira et al.
2001). This may explain why cells carrying a truncated APC gene (Min)
show defects in chromosome segregation. Furthermore, loss of APC leads
to changes in mitotic progression associated with a decrease in metaphase
interkinetochore tension (Dikovskaya et al., 2007; Draviam et al., 2006).
This was accompanied by a decrease in the association of Bub1 and BubR1
with kinetochores, which suggests that APC may be, directly or indirectly,
involved in the loading of these proteins.
The minus-end-directed motor dynein can also be found at kineto-
chores. This localization is regulated by MT attachment to the kinetochores
but does not depend on tension (King et al., 2000). In fact, dynein binding to
the kinetochore is very sensitive, as “fewer than half the normal number of
kinetochore–MTs leads to the loss of most kinetochore–dynein” (King
et al., 2000). Furthermore, the association of dynein to kinetochores was
reported to depend on Spindly (Barisic et al., 2010; Chan et al., 2009;
Gassmann et al., 2010). A significant pool of kinetochore–dynein is regu-
lated by Plk1-mediated phosphorylation, as inhibiting Plk1 severely affects
dynein localization to the kinetochore without affecting dynactin or Zw10
(Bader et al., 2011). What could be the role of dynein at the kinetochore?
Dynein associates with kinetochores during prometaphase and, as a minus-
end-directed motor, generates a pulling force on MTs. By interfering with
dynein localization at the kinetochore, cells fail to achieve efficient chromo-
some alignment and exhibit problems in MT capture (Li et al., 2007; Yang
et al., 2007). For this reason, kinetochore–dynein was proposed to produce a
poleward force that brings monooriented kinetochores close to the pole,
which facilitates MT capture by the kinetochore and promotes chromosome
congression. Accordingly, depleting or inhibiting kinetochore–dynein pre-
vents the rapid poleward motion of attached kinetochores but does not
interfere with kinetochore fiber formation (Yang et al., 2007). In addition,
dynein also plays a role in stableMT attachment and kinetochore orientation
during metaphase, although its kinetochore levels are reduced at that stage
(Varma et al., 2008; Yang et al., 2007). This effect may be related to the abil-
ity of dynein to remove some kinetochore components during mitosis to
ensure MT stability as was shown for CLASPs (Reis et al., 2009). Interest-
ingly, kinetochore–dynein is also required for normal anaphase chromo-
some movement, but it remains unknown whether this is directly due to
its ATPase activity (Yang et al., 2007).
Kinesin-7/CENP-E is a plus-end-directed motor required for meta-
phase chromosome alignment (Kapoor et al., 2006; Wood et al., 1997).
Although CENP-E is not a conventionalþTIP, due to its plus-end-directed
107+TIPs in Cell Division
movement, it appears to accumulate in the distal end of MTs in vitro (Sardar
et al., 2010) and in vivo (Cooke et al., 1997). In addition, CENP-E has crit-
ical roles during mitosis including kinetochore–MT attachment and move-
ment of chromosomes to the metaphase plate (Cooke et al., 1997; Kapoor
et al., 2006; Schaar et al., 1997; Wood et al., 1997).
In prophase, kinesin-13 MCAK localizes to the inner kinetochore and,
during chromosome congression, MCAK specifically associates with the
leading kinetochore (Kline-Smith et al., 2004). Depletion or disruption of
MCAK leads to defects in alignment and segregation of chromosomes
(Kline-Smith et al., 2004; Wordeman et al., 2007; Zhu et al., 2005). These
defects may be the consequence of improper kinetochore–MT attachments
that arise whenMCAK is disrupted and lead to the formation of merotelic or
syntelic attachments. For this reason, MCAK was proposed to play a role in
the prevention and/or correction of kinetochore–MT attachments (Kline-
Smith et al., 2004). Additional work demonstrated that MCAK is required
for the turnover of k-fibers, contributing to the directional switching
between sister centromeres (Rizk et al., 2009; Wordeman et al., 2007).
The pool of MCAK that is associated with EB1 at the MT tips was proposed
to be important for the promotion of stable kinetochore–MT attachments in
an indirect way, by limiting nonkinetochore–MT length (Domnitz et al.,
2012). Overall, MCAK’s role would be to contribute to error correction
either by allowing the release of MTs from the kinetochore or by promoting
MT turnover. This process seems to be regulated by Aurora-B-mediated
phosphorylation of MCAK at serine 196, which inhibits MCAK
MT-depolymerizing activity (Andrews et al., 2004; Lan et al., 2004). Given
their role in controlling k-fiber turnover, it is not surprising that these proteins
can influence chromosome segregation. In fact,MCAKwas first shown to be
important for chromosome segregation, as introduction of a motorless,
dominant-negative version of the protein leads to lagging chromosomes
(Maney et al., 1998). Simultaneous knockdownofMCAKandKif2A induces
an even higher number of lagging chromosomes (Ganem et al., 2005). Inter-
estingly,MCAK activitymay also be regulated by interactionwith ch-TOG.
By forming a complex with TACC3 and clathrin, ch-TOG physically cross-
links k-fibers and reduces MT catastrophes (Barr and Gergely, 2008; Booth
et al., 2011).Moreover, these TACC3/ch-TOG/clathrin k-fiber bridges are
regulated by Aurora-A (Cheeseman et al., 2011).
In addition to its centrosomal localization, Astrin and its interactor
Kinastrin also localize at the kinetochores (Dunsch et al., 2011; Manning
et al., 2010; Schmidt et al., 2010). This localization is negatively regulated
108 Jorge G. Ferreira et al.
by an Aurora-B-mediated phosphorylation so that Astrin only localizes to
bioriented kinetochores (Manning et al., 2010; Schmidt et al., 2010).
Accordingly, Astrin was shown to help stabilize kinetochore–MT attach-
ments and to promote mitotic progression (Dunsch et al., 2011; Gruber
et al., 2002; Manning et al., 2010; Schmidt et al., 2010). Importantly, the
plus-end localization of Astrin was reported to facilitate chromosome align-
ment (Dunsch et al., 2011). Moreover, depletion of Kinastrin induces the
same mitotic defects observed in Astrin-depleted cells.
5.4. +TIPs regulation during mitosisMany þTIPs exhibit different localization or behavior during mitosis. This
raises the question of how the different þTIPs are regulated in space and
time. CLIP170 was one of the first þTIPs described to be phosphorylated
in vivo at multiple sites (Choi et al., 2002). In this report, the authors iden-
tify an interaction of CLIP170 with FRAP kinase and treatment with
rapamycin interferes with the ability of CLIP170 to associate with MTs.
However, the same report describes several rapamycin-sensitive and -
insensitive phosphorylation sites, indicating there must be other kinases
regulating CLIP170 function. Accordingly, both Plk1 and CK2 have been
recently identified as CLIP170 kinases (Li et al., 2010). The CK2-mediated
phosphorylation is essential for kinetochore targeting of CLIP170 in a
dynactin-dependent manner. In this context, Plk1 seems to act as a priming
kinase, which enhances the ability of CLIP170 to bind to CK2. Expression
of phospho-null mutants of CLIP170 is sufficient to displace the protein
from the kinetochore and induce defects in the formation of k-fibers,
which further highlights the importance of CLIP170 phosphoregulation
(Li et al., 2010). This phosphoregulatory mechanism may also be relevant
to control CLIP170 association to the plus ends by inducing conforma-
tional changes in the protein. CLIP170 switches between two conforma-
tional states that alter its affinity for MTs (Lansbergen et al., 2004). In
its phosphorylated state, CLIP170 shows enhanced binding between the
N- and C-terminal domains and remains in a “closed” conformation
(Lee et al., 2010). This phosphorylated form of CLIP170 has lower affinity
for MTs and does not interact with p150glued. The phospho-null mutant
of CLIP170 is in an “open” conformation and has higher affinity for the
plus ends of MTs and p150glued (Lee et al., 2010). This leads to an auto-
inhibitory mechanism that confers tighter control of CLIP170 association
to the MT.
109+TIPs in Cell Division
In a similar fashion, CLASPs localization during mitosis is also regulated
by phosphorylation, both in a direct and in an indirect manner. Astrin and
SKAP both bind MTs directly and are required for CLASP1 kinetochore
localization (Schmidt et al., 2010). However, the Astrin/SKAP complex tar-
gets only to bioriented kinetochores due to an Aurora-B-mediated regula-
tory mechanism. This provides a spatiotemporal control of kinetochore
composition through Aurora-B. In addition to this indirect regulation,
CLASP2 can also be directly phosphorylated during mitosis. Although
CLASP2 shows tip-tracking behavior in interphase, it is not easily detectable
at MT plus ends during metaphase. This raised the possibility that plus-end
localization could be under the regulation of specific mitotic kinases. Indeed,
priming phosphorylations of CLASP2 by CDK1 and subsequent GSK3bphosphorylation are required to induce CLASP2 displacement from the plus
ends of MTs during mitosis (Kumar et al., 2012). Interestingly, this tip-
tracking behavior depends on the interaction of CLASP2 with EB1, as
imposing the phosphorylations induced a disruption in the interaction
between the two proteins, leading to the displacement of CLASP2 from
the plus ends. Conversely, introducing phospho-null mutations on these
specific sites was sufficient to restore EB1 association and binding to the
plus end. More recently, it was shown that during mitosis CLASP2 is
predominantly phosphorylated at its C-terminal domain, close to the
kinetochore-associated and dimerization region (Maia et al., 2012). These
phosphorylations were mediated by CDK1 and Plk1 kinases at different sites
and were specific for CLASP2. Noteworthy, CLASP2 phosphorylation by
CDK1 acts as a priming event for further association of CLASP2 with Plk1.
In accordance, colocalization of CLASP2 and Plk1 was reported in the Golgi
apparatus, centrosomes, kinetochores (in an MT-independent way), spindle
midzone, and midbody. CDK1 phosphorylation of CLASP2 was required
not only to increase Plk1 levels at the kinetochore but was also necessary
to maintain spindle bipolarity. More specifically, CLASP2 phosphorylation
on serine 1234 by CDK1 was shown to stabilize kinetochore–MTs, as
expression of the respective phospho-null mutant of CLASP2 leads to the
formation of monopolar spindles due to k-fiber instability (Maia et al.,
2012). In this way, phosphorylation of CLASP2 by both CDK1 and Plk1
were shown to be important for proper kinetochore–MT attachment and
chromosome alignment.
Given their crucial role in the regulation of MT dynamics, surprisingly
little is known on how EB function is regulated. Much of the recent work
has focused on the budding or fission yeast homologues of EB1. The
110 Jorge G. Ferreira et al.
budding yeast EB-like protein Bim1p is regulated by multisite phosphory-
lation by the Aurora-B homologue Ipl1p (Zimniak et al., 2009). This
EB-like protein forms a stable complex with Aurora-B, which then phos-
phorylates a serine cluster in the linker region of EB, and this phosphoryla-
tion is sufficient to reduce the affinity of the protein for MTs. On the other
hand, a mutation of the fission yeast EB-like protein Mal3 was reported to
increase the affinity of the protein to MTs (Iimori et al., 2012). When the
glutamine on position 89 in the CH domain was replaced with an arginine,
this EB no longer behaved as a þTIP but associated with the entire MT
lattice. This also prevented EB dissociation from MTs even when it was
not growing, leading to a reduction in the shrinkage rate. How do these
phosphorylations regulate the affinity of EB proteins for MTs? One may
consider that they affect the interaction of EBs with partner proteins such
as CLASP2, and this could impact on the overall affinity of EBs for the plus
end. On the other hand, phosphorylation of EB proteins could introduce
negative charges in the protein which would disrupt the association with
MTs through electrostatic repulsive interactions as was recently proposed
(Buey et al., 2011). Nevertheless, it seems plausible that phosphorylation
of EBs could lead to an overall decrease of interaction with the MT. In fact,
phosphorylation of EB3 was recently reported in human cells. During mito-
sis, EB3 was reported to be phosphorylated by Aurora kinases on serine 176
(Ban et al., 2009). This phosphorylation induces a stabilization of the protein
because it prevents its polyubiquitination and proteasome-mediated degra-
dation. Furthermore, EB3 phosphorylation by Aurora-B during mitosis was
shown to spatially regulate MT dynamics (Ferreira et al., 2013). Such a spa-
tial phosphorylation pattern allows distinct pools of EB3 to fine-tune MT
dynamics at different cellular locations.
Motor proteins are also subject to regulation during mitosis. Dynein was
shown to be phosphorylated during meiosis when added to Xenopus egg
extracts. This was dependent on CDK1 and occurred specifically after incu-
bation with metaphase but not interphase extracts (Dell et al., 2000). At least
partially, dynein phosphorylation could direct its binding to specific partners
or structures. Phosphorylation of dynein intermediate chain favors its asso-
ciation to Zw10 instead of dynactin, and this triggers dynein accumulation at
the kinetochore (Whyte et al., 2008). Interestingly, this association persists
until chromosomes become bioriented, which results in dynein dephos-
phorylation. Dephosphorylated dynein then associates preferentially with
dynactin and exhibits poleward streaming, which removes it from the kinet-
ochore (Whyte et al., 2008). In addition to CDK1, kinetochore–dynein is
111+TIPs in Cell Division
also phosphorylated by Plk1 (Bader et al., 2011). Interfering with Plk1
induces mislocalization of dynein without significantly affecting dynactin
or Zw10, and this leads to defects in MT capture at the kinetochore.
MCAK is regulated by phosphorylation through several kinases, of
which Aurora-B seems to be the most significant (Tanenbaum and
Medema, 2011). Addition of phosphates in the N-terminal region of
MCAK, near the SxIP motif, seems to affect the tip-tracking behavior of
MCAK. Namely, Aurora-B-mediated phosphorylation of MCAK was pro-
posed to disrupt its interaction with theþTIPs TIP150 and EB1, decreasing
MCAK affinity for MT plus ends (Honnappa et al., 2009; Jiang et al., 2009;
Moore et al., 2005). Overall, these phosphorylations may contribute to a
decrease in the recruitment of MCAK to the plus ends of MTs, favoring
MT growth. In agreement, in vivo phosphorylation of the neck region of
MCAK varies according to the mitotic stage: it is high in early mitosis
but decreases when chromosomes become aligned and kinetochore–MT
attachments have to be stabilized (Lan et al., 2004). Many phosphorylation
sites regulate MCAK binding to spindle poles, kinetochores, centromeres,
and chromosome arms (Andrews et al., 2004; Lan et al., 2004; Zhang
et al., 2007b, 2008). Moreover, these phosphorylation events seem to affect
specific pools of MCAK. Accordingly, MCAK neck phosphorylation can be
found mainly at the centromere, whereas Aurora-B-mediated phosphoryla-
tion at serine 95 inhibits this localization. Strikingly, phosphorylation at ser-
ine 110 by Aurora-B increases centromere binding (Zhang et al., 2007b).
Taken together, these observations suggest multiple layers of regulation
depending on spatiotemporal constraints. A mitotic-specific phosphoryla-
tion of MCAK by CDK1 has also been reported (Sanhaji et al., 2010). This
modification inhibits MCAK’s MT-depolymerizing activity and can be
reproduced by expressing a phosphomimetic mutant. However, it is not
yet clear what the functional relevance of this modification is. In fact, if
MTs need to be more dynamic during mitosis, why should MCAK activity
be impaired at this stage? It may be that this phosphorylation (such as hap-
pens with Aurora-B-mediated phosphorylations) affects only a small pool of
MCAK, suggesting that there must be a local regulation of MT dynamics
during mitosis (Tanenbaum et al., 2011). In this regard, it is possible that
impairment of MCAK MT-depolymerizing activity on k-fibers alone
would allow them to stabilize, while active MCAK would still be acting
on nonkinetochore–MTs, allowing their faster turnover. Finally, the
C-terminal domain of MCAK, which affects its own MT depolymerase
activity, is phosphorylated by Plk1 (Moore and Wordeman, 2004; Zhang
112 Jorge G. Ferreira et al.
et al., 2010). Unlike CDK1- and Aurora-B-mediated phosphorylations,
Plk1 phosphorylation promotes the MT-destabilizing activity of MCAK.
Thus, many layers of regulation ensure that the localization and activity
ofþTIPs during mitosis are tightly regulated to allow successful completion
of the process. While phosphorylation emerges as the major regulatory
mechanism to control þTIPs interactions, it remains unclear how all these
can be integrated to form a coherent picture.
5.5. +TIPs in mitotic exit and cytokinesisThe completion of mitosis involves the final separation of sister chromatids
into two daughter cells and partitioning of the cytoplasm. This last step
requires the formation of an actomyosin ring that will constrict MTs in
the midzone region (Fujiwara and Pollard, 1976; Schroeder, 1972, 1973).
Myosin function in the cytokinetic ring requires astral MTs to interact with
the cortex (Foe and von Dassow, 2008), but this does not seem to depend on
the precise regulation of MT dynamics. In fact, both MT stabilization and
increases in MT dynamics are able to induce furrow formation (Strickland
et al., 2005a). Changing of the midzone to midbody correlates with furrow
ingression, and when this is prevented, cells accumulate midzone-like MT
structures (Straight et al., 2003). Most MTs that compose the spindle mid-
body are antiparallel MTs that derive from the spindle midzone (Elad et al.,
2010; Euteneuer and McIntosh, 1980; Mullins and Biesele, 1977). As
opposed to earlier stages of cytokinesis, MTs are essential for completion
of the process (Savoian et al., 1999). Although midbody MTs are relatively
stable (Margolis et al., 1990), they can also show de novo nucleation. This
process may involve g-tubulin, which is also required for successful comple-
tion of cytokinesis (Julian et al., 1993; Shu et al., 1995). Live imaging of MT
plus ends with EB proteins also indicated that some midbody MTs are still
able to exhibit a highly dynamic behavior (Rosa et al., 2006).
Exit from mitosis requires the inactivation of CDK1. This inactivation
induces a reorganization of the MT cytoskeleton that includes increased
astral MT nucleation and midbody formation (Wheatley et al., 1997). In
addition to this more general role in MT organization, yeast CDK1 was
shown to control Aurora kinase by phosphorylating its N-terminal domain
(Zimniak et al., 2012). Interestingly, this phosphorylation blocks association
of Aurora with the yeast EB-like protein until anaphase onset. Association
between Aurora and Bim1p is required for Bim1p phosphorylation on its
linker region (Zimniak et al., 2009). This phosphorylation is necessary for
113+TIPs in Cell Division
efficient EB tip-tracking and occurs specifically during anaphase as a means
to ensure normal spindle elongation and disassembly of the spindle midzone.
Therefore, if CDK1 fails to phosphorylate Aurora kinase, this leads to pre-
mature targeting of Aurora kinase to the spindle and constitutive EB phos-
phorylation, resulting in problems duringmitotic exit (Zimniak et al., 2012).
In human cells, EB proteins are tightly associated with midzone and mid-
body MTs (Berrueta et al., 1998; Morrison et al., 1998). Moreover, EB1
and Aurora-B were shown to colocalize in these same structures (Sun
et al., 2008). However, unlike in yeast cells, human EB1 is not a substrate
of Aurora-B but is required to enhance the kinase activity of Aurora-B. It
does so by preventing association of PP2A with Aurora-B and protecting it
from dephosphorylation (Sun et al., 2008). This is in apparent contradiction
with the studies performed in yeast but one must bear in mind that human
cells have more than one EB protein. Accordingly, in human cells, both
Aurora-A and Aurora-B were shown to phosphorylate EB3 during mitosis,
leading to EB3 stabilization (Ban et al., 2009). Interestingly, EB3 seems to be
a target of the recently described Aurora-B-mediated phosphorylation gra-
dient in late mitosis (Ferreira et al., 2013; Fuller et al., 2008). Accordingly,
Aurora-B-mediated phosphorylation of EB3 on serine 176 promotes MT
growth, stabilizing the midbody and allowing completion of cytokinesis
(Ferreira et al., 2013). Importantly, EB3 dephosphorylation near the cell
cortex restricts MT growth, which allows stabilization of focal adhesions
and daughter cell adhesion.
Taken together, the interactions between EB proteins and other mitotic
exit-related proteins highlight the importance of þTIPs in this context.
Accordingly, if formation of astral MTs is suppressed during anaphase by
interfering with either EB1 or dynactin, there is a significant delay in cyto-
kinesis (Strickland et al., 2005b). During anaphase, phosphorylated MCAK
localizes to the spindle midzone, and this is important because it helps reg-
ulate its MT depolymerization activity (Lan et al., 2004). The phosphory-
lation of MCAK is also carried out by Aurora-B (Fuller et al., 2008; Lan
et al., 2004). Recently, a new þTIP termed TIP150 was shown to localize
to the plus ends until anaphase B, in an EB1-dependent manner. TIP150 also
interacts with the MT depolymerase MCAK and appears to assist in the
EB1-mediated recruitment of MCAK to the plus ends (Jiang et al.,
2009). Interestingly, MCAK shares common cellular localizations with
EB1 and Aurora-B. Taken together, this means that, either directly or indi-
rectly, Aurora-B and CDK1 seem to regulate the localization or activity of
many EB1-associated proteins after anaphase onset and until cytokinesis.
114 Jorge G. Ferreira et al.
Other EB-associated proteins such as APC or CLASPs were also shown
to independently regulate mitotic exit. During anaphase and telophase, both
CLASP1 and CLASP2 accumulate in the spindle midzone and midbody
(Maiato et al., 2003a; Mimori-Kiyosue et al., 2006). Given the localization
of CLASPs in the spindle midzone and midbody, one can predict a role for
these proteins during mitotic exit. In accordance, previous studies have
implicated CLASPs in cytokinesis in C. elegans, Drosophila weak hypo-
morphic mutants, and human cells (Inoue et al., 2004; Mimori-Kiyosue
et al., 2006; Pereira et al., 2006; Skop et al., 2004). More recently, CLASP1
recruitment to the central spindle was shown to be dependent on its asso-
ciation with PRC1 at anaphase B (Liu et al., 2009b). Furthermore, reduc-
tion in CLASP1 levels or interference with the CLASP1–PRC1 interaction
leads to a disorganization of the central spindle, due to a reduction in the
amount of antiparallel MTs, as well as a chromatin-bridge phenotype and
failures in accurate chromosome segregation (Liu et al., 2009b). These
observations suggest a role for the CLASP1–PRC1 complex in antiparallel
MT elongation and central spindle stabilization. Interestingly, PRC1 itself is
directly regulated by phosphorylation by CDK1 and Plk1, which puts it in
the right context for regulating mitotic exit (Hu et al., 2012; Jiang et al.,
1998). Due to the partial redundancy of CLASPs during mitosis, it is not
surprising that cells derived fromCLASP2 knockout mice also present a mild
cytokinetic phenotype (Pereira et al., 2006). The presence of chromatin
bridges in CLASP2-depleted cells can be pointed out as a cause of cytoki-
nesis failure, leading to the generation of polyploid cells with multiple
centrosomes (Pereira et al., 2006).
APC, one of the main interactors of EB1, has been extensively impli-
cated in cytokinesis completion. Reports on APC mutants demonstrated
that these cells become polyploid over time (Fodde et al., 2001; Kaplan
et al., 2001; Tighe et al., 2004), which suggests that APC plays a role in cyto-
kinesis. Although the different APC alleles behave in a distinct manner, it is
believed that they may interfere with anchoring of the mitotic spindle
(Caldwell et al., 2007). In fact, in a C-terminal-truncated mutant of
APC, MTs make fewer contacts with the cell cortex. For this reason, spin-
dles rotate excessively and this leads to cytokinetic failures (Caldwell et al.,
2007). Interestingly, both inMin mice and APC knockout mice, there is an
increase in the number of tetraploid cells, which is a hallmark of cytokinesis
failure (Caldwell et al., 2007; Dikovskaya et al., 2007).
Other non-EB1-associated proteins play important roles in postanaphase
cells. The kinesin CENP-E localizes to the midbody where it uses its
115+TIPs in Cell Division
coiled-coil domain, to interact with Skp1 (Liu et al., 2006). These proteins
show an inverse correlation at the midbody, with CENP-E levels decreasing
as Skp1 associates with this structure. In fact, there is a tight spatiotemporal
regulation of CENP-E at the midbody that is essential for completion of
cytokinesis. In this context, the Skp1 interaction may be essential for
CENP-E proteolysis. Dynein may also play a role in cytokinesis, although
the mechanism is not completely clear. Dynein light intermediate chain 1
(LIC1) is concentrated at the midbody during abscission (Horgan et al.,
2010). Moreover, it was recently shown that dynein is necessary for
transport of Rab8-positive vesicles to the midbody, and this is required
for completion of cytokinesis (Kaplan and Reiner, 2011).
In conclusion, þTIPs can impact mitotic exit at many different levels.
They interact with the major kinases regulating transition from mitosis to
G1 such as CDK1, Plk1, and Aurora-B. Moreover, they are prominently
localized to the spindle midzone and midbody which are crucial in the out-
come of mitosis. Although þTIPs have a significant role in the direct reg-
ulation of MT function, there is a network of reciprocal interactions at the
midzone and midbody which is regulated by Aurora-B or CDK1-mediated
phosphorylations and involves many different families of þTIPs.
6. CONCLUDING REMARKS
In this review, we aimed to provide an up-to-date, systematic orga-
nization of MT functions as well as the role that the major þTIP families
play in this context. More specifically, we addressed the significance that
þTIPs have on the regulation of MT dynamics and stability, which makes
them important players during cell division. It is interesting that many of the
þTIPs share either a functional or spatial overlap, highlighting the complex
role that MT plus ends play throughout mitosis. While many of the features
that define a þTIP are already known, it is still unclear how they interact
with each other in specific contexts to regulate mitotic progression. The
identification of the main structural features that regulate association of pro-
teins to the MT plus ends has led to an overwhelming increase in the num-
ber of potential þTIPs. So far, around 800 proteins were defined as
containing SxIP motifs and many have been confirmed to track on MT plus
ends ( Jiang et al., 2012). Strikingly, little is known about the mitotic role of
most of these proteins, providing a fruitful ground for study in the
coming years.
116 Jorge G. Ferreira et al.
ACKNOWLEDGMENTSAna L. Pereira is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a
Ciencia e Tecnologia (FCT, Portugal). Work in the laboratory of H. M. is funded by Grants
PTDC/SAU-ONC/112917/2009 from FCT (COMPETE-FEDER), the Human Frontier
Science Program and the 7th framework program Grant PRECISE from the European
Research Council.
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