Review TRENDS in Cell Biology
2 February 2005
Regulation of microtubules in cell
migration
Takashi Watanabe, Jun Noritake and Kozo Kaibuchi
Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya,
Aichi 466-8550, Japan
Directional cell migration is a fundamental process in all
organisms that is stringently regulated during tissue
development, chemotaxis and wound healing. Migrat-
ing cells have a polarized morphology with an asym-
metrical distribution of signaling molecules and the
cytoskeleton. Microtubules are indispensable for the
directional migration of certain cells. Recent studies
have shown that Rho family GTPases, which are key
regulators of cell migration, affect microtubules, in addi-
tion to the actin cytoskeleton and adhesion. Rho family
GTPases capture and stabilize microtubules through
their effectors at the cell cortex, leading to a polarized
microtubule array; in turn, microtubules modulate the
activities of Rho family GTPases. In this article, we dis-
cuss how a polarized microtubule array is established
and how microtubules facilitate cell migration.
Introduction
Cell migration is necessary for developmental morpho-
genesis, tissue repair and regeneration, and tumor meta-
stasis. Directional cell migration is initiated usually in
response to extracellular cues such as chemokines and
signals from the extracellular matrix. A migrating cell is
highly polarized and contains complex regulatory path-
ways downstream of various stimuli. Cell polarization
includes the asymmetrical distribution of signaling mol-
ecules and the cytoskeleton, in addition to directed mem-
brane trafficking. A polarized migrating cell has a single
leading edge and filopodia at the front end; the micro-
tubule-organizing center (MTOC) and the Golgi apparatus
are oriented towards the migrating direction, and tem-
poral capture and stabilization of some microtubule plus
ends occur near the leading edge, which are thought
principally to enable the cell to exert directed vesicular
transport (Figure 1). Cell migration requires the coordi-
nation of these processes, especially the reorganization of
the actin cytoskeleton and microtubules [1,2].
Recent advances in imaging technology have begun to
uncover when and how the cytoskeleton, signaling mol-
ecules and adhesion molecules are asymmetrically dis-
tributed during migration. Rho family GTPases, including
Rac1, Cdc42 and RhoA, have pivotal roles in cell polar-
ization and migration through the regulation of actin
cytoskeletal and adhesion rearrangements [3–9]. Recent
intensive analyses have revealed that Rho family
Corresponding author: Kaibuchi, K. (kaibuchi@med.nagoya-u.ac.jp).
Available online 5 January 2005
www.sciencedirect.com 0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.12.006
Vol.15 No.
GTPases also regulate the dynamics of microtubules
and, in turn, microtubules affect the activities of Rho
family GTPases. In this article, we focus on this
relationship and we discuss how Rho family GTPases
differentially or coordinately regulate microtubules.
Microtubules in cell migration
Microtubules are one of the major components of the
cytoskeleton and they are essential for cell division, cell
migration, vesicle transport and cell polarization. Micro-
tubules are hollow tubes composed of 13 protofilaments
of a- and b-tubulin dimers organized in a head-to-tail
fashion. Microtubules are nucleated from their minus
ends, which localize predominantly at the MTOC
(Figure 1a). The plus ends of microtubules alternate
between two phases of growth and shrinkage (a state
termed ‘dynamic instability’) to explore intracellular
spaces [10] (Figure 1b). Through this search process, the
plus ends are captured and stabilized at target desti-
nations such as kinetochores on the mitotic spindle and
cell cortex. The search-and-capture of microtubules is
important for generating an asymmetrical microtubule
array and maintaining cell shape [10–12]. During cell
migration, selective stabilization of the plus ends of micro-
tubules enables the MTOC to reorient towards the leading
edge, which results in a polarized microtubule array that
facilitates cell migration (Figure 1). Consistent with this,
when nanomolar concentrations of microtubule antagon-
ists are applied that decrease dynamic instability without
affecting microtubule mass, growth-cone motility and
fibroblast migration are diminished [13]. Thus, stabiliz-
ation of microtubule plus ends is necessary for cell
migration during the search-and-capture process.
Recent studies using green fluorescent protein (GFP) in
living cells have shown that some microtubule-associated
proteins (MAPs) accumulate specifically at the plus ends
of growing microtubules. These MAPs are termed CTIPs
and they include CLIP-170, CLASPs, EB1, APC, dynein,
dynactin and Lis1 [14,15]. The molecular mechanisms
underlying the accumulation of CTIPs only at the plus
ends of growing microtubules have been discussed in
previous studies [14–16]. Although microtubule tread-
milling probably accounts for their plus-end tracking,
Tip1p and Bik1p – the homologs of CLIP-170 in fission
yeast and budding yeast, respectively – seem to accumu-
late at microtubule plus ends after being transported
as complexes with kinesin, which suggests that this
 Review TRENDS in Cell Biology Vol.15 No.2 February 2005 77

(a)
MTOC + Ruffling
Signal
+ + +
+ +
N -
+ +
- N

+ Reorientation +
+
+ + +
+ Filopodia
Microtubule

(b) Growing Shrinking

Rescue

Catastrophe

TRENDS in Cell Biology

Figure 1. Polarized microtubules in migrating cells. (a) In resting cells, the minus ends of microtubules (K) are anchored at the MTOC, which reorients towards the direction
of migration. The plus ends of microtubules (C) are stabilized at leading edges. Abbreviation: N, nucleus. (b) Microtubule plus ends alternate between two phases: growth
and shrinkage [boxed area in (a)] – a state termed ‘dynamic instability’. The growing microtubules are thought to form an open sheet of tubulin polymer that comprises GDP
a–b-tubulin heterodimers containing GTP b-tubulin at the tips (known as the GTP cap). During shrinking, microtubule plus ends have curved protofilaments that peel away
from the microtubule wall.

accumulation of CTIPs could be a consequence of multiple
mechanisms [17,18]. CTIPs are well conserved from yeast
to mammals and they have central roles in sensing cortical
sites at which a local signal activates microtubule-capping
structures. Similar to CLIP-170, the EB1 family is also
conserved from yeast to humans, and it includes Bim1p
from budding yeast and Mal3p from fission yeast. Genetic
analyses in yeast have revealed that these CTIPs are
essential for an organized microtubule array [12,14,16].
Linkage of microtubule plus ends with the cell cortex
is required for the polarized microtubule array that is
characteristic of migrating cells, although it remains
unclear how cortical molecules capture and stabilize the
plus ends of microtubules during cell migration.
Rho family GTPases: key regulators of cell migration
Rho family GTPases function as molecular switches
Rho family GTPases are well-conserved proteins, 20 of
which have been identified in mammals. They function as
molecular switches in cells by cycling between GDP-bound
inactive forms and GTP-bound active forms to transmit
intracellular signals. The activities of GTPases are con-
trolled both spatially and temporally by three kinds of
regulators: guanine-nucleotide-exchange factors (GEFs),
which promote the exchange of GDP for GTP; Rho
GDP-dissociation inhibitors (GDIs), which interact with
GDP-bound Rho GTPases and inhibit the exchange of GDP
for GTP; and GTPase-activating proteins (GAPs), which
enhance the intrinsic GTPase activities of Rho GTPases
(Figure 2a) (for review of the regulation of the activities of
Rho family GTPases, see Ref. [19]).
Activated Rho family GTPases interact with their
specific effectors and elicit diverse responses. In tissue
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culture studies using a variety of cells, it has been shown
clearly that Rho family GTPases regulate actin dynamics
and organization by interacting with their effectors. The
use of constitutively active mutants and dominant–
negative mutants has revealed the specific function of
each Rho family GTPase in the regulation of actin fila-
ments. RhoA regulates the assembly of contractile actin
and myosin, which seems to lead to contraction at the rear
of migrating cells; the polymerization of actin is regulated
by Rac1 and Cdc42 to form lamellipodia and filopodia,
respectively, at the front of migrating cells (Figure 2b).
Because Cdc42 is required for the establishment of cell
polarity in various cells and embryos, it is thought to
function as sensing machinery in determining the direc-
tion of cell migration (Figure 2b). Because the plus ends of
microtubules are captured and stabilized at leading edges
in migrating cells, Rac1 and Cdc42 might regulate the
dynamics of microtubule plus ends.
Several proteins have been identified as being effectors
of Cdc42, including p21-activated kinase (PAK), Wiskott–
Aldrich syndrome protein (WASP), neuronal WASP,
IQGAP1, Par6 and myotonic-dystrophy-kinase-related
Cdc42-binding kinase (MRCK). Effectors of Rac1 include
PAK, IQGAP1, p140Sra-1 and IRSp53. As can be seen,
PAK and IQGAP1 interact with both Cdc42 and Rac1.
Effectors of RhoA include Rho kinase (also known as ROK
or ROCK), the myosin-binding subunit of myosin phos-
phatase, protein kinase N (PKN), mDia, citron kinase
and rhotekin. Most of these effectors perform their physio-
logical functions during actin organization. Accumulating
evidence has shown that some of these effectors are also
involved in the regulation of microtubule dynamics and
organization.
78 Review TRENDS in Cell Biology Vol.15 No.2 February 2005

(a) Plasma membrane

Rho
Rho GEF GTP Rho GAP
Effector

GDP Pi

GTP

Rho GDI Rho GDI

Rho GDI

Rho

GDP

(b) Direction (Cdc42)

Microtubule Adhesion

MTOC

Rear retraction
(Rho)
Adhesion maturation Protrusion and
(Rho) adhesion formation
(Cdc42 and Rac1)

TRENDS in Cell Biology

Figure 2. Rho family GTPases in cell migration. (a) Regulatory mechanisms of Rho family GTPases. In resting cells, Rho exists mostly in the GDP-bound form (GDP$Rho) and
in complexes with Rho GDI in the cytosol. Following stimulation by extracellular signals, Rho probably dissociates from Rho GDI and is targeted to specific membranes by its
C-terminal prenyl group. At the membrane, specific GEFs for Rho are activated and GDP$Rho is then converted to GTP$Rho, which interacts with its specific effectors to exert
its functions. GAPs enhance the GTPase activity of Rho and reconvert Rho to its inactive GDP-bound form. Rho GDI can then form a complex with GDP$Rho, extract it from the
membrane and return it to the cytosol. (b) Possible roles of Rho family GTPases in cell migration. Cdc42, together with Par proteins and aPKC, is involved in generating cell
polarity. At the front of the cell, Rac1 and Cdc42 regulate the formation of ruffles and filopodia, respectively, in concert with their effectors to promote cell migration.
Protrusions are stabilized by the formation of adhesions. This process requires integrin activation, clustering and the recruitment of structural and signaling components to
nascent adhesions, which results in the activation of Rac1 and Cdc42. RhoA is thought to be involved in retraction at the rear of the cell through the regulation of actin–myosin
contractility. Activation of RhoA also induces the maturation of focal adhesions behind leading edges.

Rho family GTPases are activated at specific sites
Following stimulation by growth factors and cell-adhesion
molecules, Rho GTPases are thought to shuttle between
the cytosol and specific membrane sites or the actin
cytoskeleton. In a migrating cell, Cdc42 and Rac1 localize
at the leading edge. Cdc42 also localizes to the Golgi
apparatus, at which it is thought to regulate the establish-
ment of cell polarity through the control of secretory and
endocytic transport. RhoA is thought to localize mostly
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in the cytosol, although there is evidence that it also
accumulates in stress fibers [20].
Researchers have examined the activation sites of
Rho family GTPases, and their activities have been
measured biochemically by affinity precipitation using
the Rho-GTPase-binding region of specific effectors such
as WASP, PAK and rhotekin. Recently developed tech-
nology based on fluorescence resonance energy transfer
(FRET) enables the nucleotide state of Rac1 and Cdc42 to
 Review TRENDS in Cell Biology
be visualized in living cells. Fluorescence activation indi-
cator for Rho proteins (FLAIR) reveals that, in wound-
induced polarized fibroblasts, the activity of Rac1 shows a
gradient, with the highest levels occurring at leading
edges [21]. The localization of Rac1 activity seems to be
distinct from that of Cdc42 [22]. Levels of Rac1 activity are
highest immediately behind leading edges, whereas levels
of Cdc42 activity are highest at the tip of leading edges.
Although RhoA is activated at the membrane in inter-
phase cells, visualization of its activity in migrating cells
has not yet been reported [23]. Thus, Rac1 and Cdc42 are
activated at leading edges, at which they can regulate
microtubule plus ends.
Regulation of microtubules by Rho family GTPases
RhoA induces microtubule stabilization
Rho family GTPases regulate the dynamics and organiz-
ation of microtubules during cell migration. Studies have
been performed using an antibody against detyrosinated
tubulin – Glu-tubulin – which is a posttranslationally
modified tubulin that accumulates in stable microtubules
(termed Glu-MTs) and has a half-life of 1 h [24]. According
to these studies, RhoA, but not Rac1 and Cdc42, is neces-
sary and sufficient for lysophosphatidic acid (LPA)-
induced Glu-MT formation [25]. The RhoA effector mDia
induces the formation of Glu-MTs, whereas an mDia2
mutant that lacks the Rho-binding region and is consti-
tutively active can interact with microtubules and localize
at Glu-MTs, which suggests that mDia caps and stabilizes
microtubules [26,27].
Although stabilized microtubules orient towards the
direction of cell migration, the distribution of RhoA and
Cdc42
Par6 IQGAP1
aPKC
GSK-3β
APC APC
EB1 CLIP-170
Capture
MTOC polarization
Figure 3. Signaling network from Rho family GTPases to microtubules. Rho family GTPases regulate microtubules through their effectors (e.g. Par6, IQGAP1, PAK and mDia).
These effectors interact with CTIPs (e.g. APC, EB1 and CLIP-170) or modify the activities of CTIPs and MAPs (e.g. APC and stathmin) through their phosphorylation, leading
to the corresponding effects on microtubules. Rac1 and Cdc42 affect microtubule dynamics, using some of the signaling pathways that are also involved in the polarization of
the MTOC. RhoA induces the stabilization of microtubules (the formation of Glu-MTs).
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Vol.15 No.2 February 2005 79
mDia is not restricted to leading edges in migrating cells.
Where do RhoA and mDia regulate the formation of stab-
ilized microtubules? Cell adhesion to substrate also affects
the formation of Glu-MTs. Integrin-mediated activation of
focal adhesion kinase (FAK), a crucial mediator of integrin
signaling, is required for the formation of Glu-MTs through
regulation of the local coupling of RhoA to mDia at lipid
rafts in the leading edges of migrating cells [28]. Further-
more, because mDia1 and mDia2 interact with the tumor
suppressor adenomatous polyposis coli (APC) and EB1
(an APC-interacting CTIP) and because mDia1 localizes
at the tips of Glu-MTs together with these proteins [29],
the formation of Glu-MTs can be regulated in the
RhoA–mDia–APC–EB1–microtubule pathway (Figure 3),
which is facilitated by integrin–FAK signaling through
the targeting of RhoA to lipid rafts (Table 1). Because
members of the formin family, including mDia, have been
shown to nucleate unbranched actin filaments, it will be
important to examine whether mDia coordinately regu-
lates both actin and microtubules downstream of RhoA
during cell migration.
Constitutively active mDia1 induces the longitudinal
alignment of microtubules parallel to actin bundles along
the long axes of cells, in addition to the formation of
Glu-MTs [29,30], whereas Rho-kinase, which is another
RhoA effector, is reported to interact with and phosphory-
late MAP-2 and Tau. In one study, a Tau mutant in which
the Rho kinase phosphorylation sites were substituted
with Asp diminished Tau-induced tubulin polymerization
[31]. Although Rho kinase activity is not required for
the induction of Glu-MT formation [26], mDia and Rho
kinase seem to regulate the dynamics and organization of
Rac1 RhoA
PAK mDia
APC
Stathmin EB1
Anti-catastrophe Glu-MT formation
TRENDS in Cell Biology
80 Review TRENDS in Cell Biology
Table 1. Molecular mechanisms underlying Rho family GTPase regulation of microtubule dynamicsa
Extracellular signals Cells Rho family GTPases
LPA, integrin Fibroblasts RhoA
GF, HGF Fibroblasts Rac1, Cdc42
LPA Fibroblasts Cdc42
Fibroblasts Rac1, Cdc42
Wound Fibroblasts Rac1, Cdc42
Wound Astrocytes Cdc42
Shear stress Endothelial cells Cdc42
Cell–cell contact T cells Cdc42
a
Abbreviations: EGF, epidermal growth factor; HGF, hepatocyte growth factor; LPA, lysophosphatidic acid; MT, microtubule; MTOC, microtubule-organizing center.
microtubules under the control of RhoA. However, it is
largely unknown how such effectors regulate microtubules
cooperatively, and further analyses are necessary to
address these issues. The formation of Glu-MTs seems
to be regulated by RhoA, although mDia2 and mDia3
have been shown to be effectors for both Cdc42 and RhoA
[32,33]. It will be worthwhile to examine whether mDia
is also involved in the regulation of microtubules down-
stream of Cdc42.
Cdc42 and Rac1 regulate microtubule dynamics
PAK, which is an effector of Rac1 and Cdc42, directly
phosphorylates stathmin (Op18) [34,35]. Although stath-
min is known to destabilize microtubules, its mechanism
is currently the topic of much debate [36]. Stathmin is
phosphorylated in response to several signals, including
those for cell migration and cell proliferation. Some
kinases such as Ca2C/calmodulin-dependent protein
kinase (CaMK), cyclin-dependent kinase (CDK), cAMP-
dependent kinase (PKA) and PAK have been shown to be
responsible for the phosphorylation of stathmin. Phos-
phorylation at Ser16 (where PAK can phosphorylate)
turns off the activity of stathmin and, consequently, PAK
phosphorylation here can induce microtubule stabiliz-
ation. In fact, constitutively active Rac1 affects the
dynamics of microtubules at the leading edges of cells
through PAK. These microtubules are termed ‘pioneer
microtubules’ and they exhibit a decreased catastrophe
[i.e. the event by which microtubules switch from growth
to shrinkage (Figure 1b)] frequency and an increased
growth time [35,37,38]. Thus, Rac1 and Cdc42 stabilize
microtubules through PAK phosphorylation of stathmin at
leading edges (Table 1 and Figure 3).
Cdc42 regulates MTOC polarization by affecting Par6,
atypical protein kinase C (aPKC), glycogen synthase
kinase-3b (GSK-3b) and APC in migrating primary
astrocytes during wound healing. In this pathway, Cdc42
is activated by integrin-mediated adhesion to the sub-
strate [39,40]. The Par6–aPKC complex is an evolution-
arily conserved cassette in cell polarity, and Cdc42
activates aPKC through Par6 [41–43]. APC and GSK-3b
are involved not only in mediating the degradation of
b-catenin under the control of Wnt signaling but also in
the stabilization of microtubules under the control of
Cdc42. In fact, APC accumulates at microtubule ends that
extend into actively migrating regions [44,45]. APC also
stabilizes microtubules, and phosphorylation by GSK-3b
decreases APC-induced microtubule bundling [46]. Cdc42
activates aPKC through Par6, leading to phosphorylation
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Vol.15 No.2 February 2005
Effectors MAPs Effects on microtubules Refs
mDia1, mDia2 APC, EB1 Glu-MT formation [25–29]
IQGAP1 CLIP-170 Capture [47]
Dynein, dynactin MTOC reorientation [51]
PAK Stathmin Anti-catastrophe [34,35,38]
IQGAP1 APC, CLIP-170 MTOC reorientation [48]
Par6 APC, EB1, dynein, dynactin MTOC reorientation [39,40]
Par6 MTOC reorientation [52]
MTOC reorientation [53]
and inactivation of GSK-3b at the leading edges of migrat-
ing astrocytes; this suggests that inactivated GSK-3b
enables APC to stabilize microtubules at leading edges
[39,40] (Figure 3). However, how and where APC is stab-
ilized at the specialized cortical region remain to be clarified.
The actin-binding protein IQGAP1, which is an effector
of Rac1 and Cdc42, is reported to interact with CLIP-170
[47]; IQGAP1 associates with microtubules through
CLIP-170 in vitro. In fibroblasts, IQGAP1 colocalizes
with actin filaments at polarized leading edges, to which
CLIP-170-decorated microtubules are targeted. Activated
Rac1 and Cdc42 enhance the interaction of IQGAP1 with
CLIP-170, and capture GFP–CLIP-170 at the base of the
leading edge and the filopodia, respectively [47]. Thus, it is
likely that activated Rac1 and Cdc42 can provide the
docking sites for microtubule plus ends near the cortex
through IQGAP1 and CLIP-170 (Table 1).
Recently, we found that IQGAP1 interacts with APC
[48]; both localize to leading edges in migrating fibro-
blasts, and the depletion of IQGAP1 or APC impairs actin-
meshwork formation, the immobilization of microtubule
plus ends and the polarization of the MTOC. Furthermore,
the depletion of IQGAP1 or APC decreases the accumu-
lation of APC or IQGAP1, respectively, at leading edges.
These results suggest that IQGAP1 anchors APC to actin
filaments in specialized cortical regions. In addition, APC
can stabilize microtubules directly through its microtubule-
binding domain. Thus, Rac1 and Cdc42 seem to mark
specialized cortical spots to which the IQGAP1–APC com-
plex is targeted, leading to the recruitment and immobil-
ization of microtubule plus ends in concert with the
interaction with CLIP-170 (Table 1 and Figure 3). Because
APC binds to Asef, thus activating its Rac1 GEF activity
[49], APC–Asef-recruited and -activated Rac1 might affect
not only microtubules but also actin filaments through its
effectors. This feedback loop might be composed locally
and positively for cell migration.
CLIP-115- and CLIP-170-associating proteins (CLASPs)
are key molecules involved in specifying polarized micro-
tubule arrays [50]. Unlike CLIP-170, CLASPs localize at
the plus ends of growing microtubules that are polarized
towards the leading edges of motile fibroblasts. This asym-
metrical CLASP distribution is mediated by phosphatidyl-
inositol 3-kinase (PI3K) and GSK-3b, which suggests that
the CLIP-170–CLASP complex assists polarized micro-
tubule growth in response to extracellular signals.
Because Cdc42 regulates GSK-3b and because PI3K is
part of a positive-feedback loop in the activation of Cdc42
and Rac1 [8], it is possible that Rac1 and Cdc42 regulate
 Review TRENDS in Cell Biology
the localization of CLASPs and that the CLIP-170–CLASP
complex is related to the CLIP-170–IQGAP1 complex and
other CTIPs. Further research is necessary to address
these issues.
Stabilization of microtubules by Rho family GTPases
Activated Rac1 and Cdc42 capture CLIP-170 for a rela-
tively short period at cortical regions (w2 min) [47]. The
immobilization of CLIP-170 is observed in most micro-
tubules that target to the periphery, towards the wound
[48]. Activated Rac1 also induces the formation of stab-
ilized pioneer microtubules, partially through the inacti-
vation of stathmin, at the leading edge of most cells
[35,38]. Glu-MTs, which are induced by the activation of
RhoA and which orient to the wound, have a longer half-
life of 1 h, and only 10% of microtubules in cells show the
accumulation of Glu-tubulin [13]. Although RhoA-induced
stabilization is a consequence of the accumulation of post-
translationally modified tubulin, such modifications are
not required for the capture and stabilization of micro-
tubules by Rac1 or Cdc42. These Rho family GTPases
might affect the dynamics of microtubules temporally in
response to signals that induce reorganization of the cyto-
skeleton. These pathways might regulate microtubules
separately and have different physiological importance;
more-detailed analyses are required to address these pos-
sibilities. In this regard, the activation of RhoA (which is
necessary for the formation of Glu-MTs) is dispensable,
but Cdc42 signaling is necessary, for the reorientation of
the MTOC towards the stimuli [39,40,51–53]. Dynein and
dynactin, in addition to the effectors of Cdc42, also have
essential roles in this reorientation [40,51]. Dynein and
dynactin form a motor complex that directs the minus end
of microtubules. They can generate the directional force
from the plus end to the minus end of microtubules at the
specialized cortical region. They might pull microtubules
that are captured and stabilized by the activation of
Cdc42, although the mechanism by which the MTOC
orients towards the direction of the stimuli needs to be
characterized in more detail. Because CTIPs other than
CLIP-170 and EB1 also accumulate at the plus ends
of microtubules, it is important to identify the CTIP-
interacting molecules and to investigate their association
with Rho family GTPases and their effectors. Although
APC has pivotal roles in the stabilization of microtubules,
the detailed modes of this process are largely unknown.
The stabilization of microtubules by APC is under the
control of Cdc42, Rac1 and RhoA, but how these Rho
family GTPases control the functions of APC and the
specific stabilization effects of this CTIP on microtubules
need to be clarified.
Regulation of Rho family GTPases by microtubules
Microtubules influence cell migration by regulating
adhesion and protrusion through the modulation of Rho
family GTPase activity. Membrane ruffling is induced
during recovery from treatment with microtubule-
disrupting agent, leading to the activation of Rac1 [54].
During the recovery, microtubules extend towards and
into the membrane ruffle. It has been suggested that
microtubule outgrowth activates Rac1 at the cell front to
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Vol.15 No.2 February 2005 81
promote cell migration. Because Rac1 stabilizes micro-
tubules through its effectors and because microtubule
growth stimulates the activation of Rac1, microtubules
and Rac1 seem to be part of a feedback loop that maintains
the activity of Rac1 for cell migration. The formation of
protrusions, however, is not dependent on microtubules.
In fast-moving cells such as neutrophils, microtubules
concentrate in the rear. The positive-feedback loop
between microtubules and Rac1 might exist only in slow-
moving cells such as fibroblasts.
The disruption of microtubules also affects the activi-
ties of Rho family GTPases. The depolymerization of
microtubules results in the formation of contractile actin
bundles and focal adhesions [55] and, under this con-
dition, RhoA is activated. Although microtubules seem to
modulate the activities of Rho family GTPases, the
molecular mechanisms underlying how microtubule
growth activates Rac1 and how microtubule disruption
induces the activation of RhoA are largely unknown. Micro-
tubules might modulate the activity of GEFs towards Rho
family GTPases such as Asef and RhoGEF-H1 (for review,
see Ref. [56]).
Targeting of microtubules to adhesion complexes
In living cells, many microtubules seem to follow similar
tracks during polymerization, and the coordinated reor-
ganization of the cytoskeleton is required for cell polariz-
ation and migration, which suggests that microtubules are
guided along common cytoskeletal elements and targeted
to specific sites in cells; for example, microtubules speci-
fically target the adhesion complex [57]. Using total
internal reflection fluorescence microscopy (TIRFM), it
has been shown that the plus ends of growing micro-
tubules track close to the dorsal surface (on a nanometer
scale) and consistently target the substrate-adhesion
complex [58] (Figure 4).
How are microtubules guided along the cytoskeleton?
Based on the observation that guidance occurs in cells
lacking intermediate filaments and that a myosin V homo-
log in budding yeast is involved in the directed polymer-
ization of microtubules [13,57], the most likely candidate
for assisting microtubule guidance is the actin cytoskele-
ton. Myosin VI was identified as being a molecule that
interacts with CLIP-190, the homolog of CLIP-170 in
Drosophila, and some actin-binding proteins have been
shown to interact with CTIPs such as IQGAP1 [47,59].
However, the clearest evidence of how microtubules are
guided along actin filaments was reported recently; ACF7
is a member of the spectraplakin family of cytoskeletal
cross-linking proteins that interacts with both actin and
microtubules. In ACF7-null endodermal cells, micro-
tubules still contain EB1 and CLIP-170 at the tips but
they do not grow along with actin filaments [60].
Furthermore, in the absence of ACF7, microtubules do
not pause and tether to actin-rich cortical sites; instead,
they form long and less-stable microtubules with skewed
cytoplasmic trajectories [60]. Thus, microtubules are
guided along actin filaments through multiple mechan-
isms (Figure 4). In addition, it should be noted that ACF7
is also involved in the formation of Glu-MTs downstream
of the RhoA–mDia pathway [60].
82 Review TRENDS in Cell Biology
Microtubule
Crosslinker
Actin
Extracellular matrix
Figure 4. Targeting of microtubules to focal adhesions. Microtubules are guided along actin filaments and targeted to focal adhesions within the basal region of migrating
cells. Several molecules seem to be involved in this guidance, including cross-linker proteins (which possess both actin- and microtubule-binding domains), CTIPs and their
interacting molecules, and actin-based motor protein myosins. The local targeting of microtubules modulates the activities of Rho family GTPases, resulting in the regulation
of focal adhesion turnover. Thus, the targeting of microtubules to focal adhesions contributes to cell migration.
Microtubule targeting and the physiological implications
for adhesion
Multiple microtubule-targeting events induce the dissoci-
ation of adhesion sites or their release from the substrate
to promote cell migration. The effects of microtubules on
cell migration might be attributable to their ability to
regulate the turnover of adhesion, to enable protrusion at
the front of cells and to promote retraction at the rear of
cells. At leading edges, focal adhesions show turnover,
which is required for the reorganization of actin filaments
and the recycling of components. Not surprisingly, Rho
family GTPases regulate substrate adhesion. Rac1 and
Cdc42 regulate the formation of focal adhesion at the
front of migrating cells, whereas RhoA induces the
maturation of focal adhesion behind leading edges and
the dissociation of focal adhesion at the rear [61]. During
adhesion, microtubules seem to modulate the activities of
Rho family GTPases locally and, in turn, Rho family
GTPases seem to regulate the guidance and the targeting
of microtubules to focal adhesion.
Concluding remarks
Cortical regulation of microtubules is thought to have a
pivotal role in the establishment of the polarized micro-
tubule array that is necessary for both morphogenesis
and cell migration. Recent evidence seems to show that
microtubule dynamics are regulated locally by extracellu-
lar signals through Rho family GTPases at specialized
cortical regions (Figure 3). Rho family GTPases and
CTIPs are also involved in the capture and stabilization
of kinetochore microtubules and neuronal microtubules.
Furthermore, under the control of Cdc42, mDia3 regulates
microtubule attachment to kinetochores [33]. CTIPs such
as CLASP and APC have essential roles in microtubule
regulation in mitotic cells and in the guidance of growth
cones [62–71]. The mechanisms of capture and stabiliz-
ation are well conserved from yeast to mammals, and
among motile and mitotic cells. Further studies are
required to elucidate exactly how microtubules are
regulated during cell migration and cell mitosis.
www.sciencedirect.com
Vol.15 No.2 February 2005
+TIPs
Focal adhesion
Myosin Integrin
Plasma membrane
TRENDS in Cell Biology
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