Molecular Biology of the Cell
Vol. 20, 3660–3670, August 15, 2009
Interaction of CDK5RAP2 with EB1 to Track Growing
Microtubule Tips and to Regulate Microtubule Dynamics
Ka-Wing Fong, Shiu-Yeung Hau, Yik-Shing Kho, Yue Jia, Lisheng He,
and Robert Z. Qi
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay,
Kowloon, Hong Kong, China
Submitted January 6, 2009; Revised June 9, 2009; Accepted June 11, 2009
Monitoring Editor: Tim Stearns
Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function
at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips
in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule
plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif
is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting
a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes
EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression
inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2–EB1 complex regulates
microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of
microtubules. The CDK5RAP2–EB1 complex induces microtubule bundling and acetylation when expressed in cell
cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that
CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.
Microtubules (MTs) have highly dynamic and polarized
tubular structures, which are essential in controlling a vari-
ety of cellular events. A group of diverse proteins binds to
the plus ends of growing MTs and are therefore named
plus-end tracking proteins (?TIPs). These proteins include
EB1, the dynactin subunit p150glued, adenomatous polyposis
coli protein (APC), cytoplasmic linker proteins (CLIPs), and
CLIP-associating proteins (Carvalho et al., 2003; Galjart,
2005; Akhmanova and Steinmetz, 2008). Several mecha-
nisms have been proposed for ?TIPs to achieve their local-
ization to growing microtubule (MT) tips. EB1, a prototypic
?TIP, recognizes and accumulates at growing MT tips, per-
haps via an autonomous mechanism (Tirnauer et al., 2002;
Bieling et al., 2007, 2008). Other mechanisms known for plus-
end tracking include “hitchhiking” through interaction with a
?TIP such as EB1 and motor-driven plus-end–directed trans-
port (Carvalho et al., 2003; Galjart, 2005; Akhmanova and
Steinmetz, 2008). A ?TIP may adopt several mechanisms for
plus-end targeting. For example, APC moves along MTs in
three distinct ways: direct association with MTs, hitchhiking
on EB1, and kinesin-mediated transport (Askham et al., 2000;
Mimori-Kiyosue et al., 2000b; Jimbo et al., 2002; Kita et al.,
EB1 represents a highly conserved group of proteins that
localize to cytoplasmic MTs (Vaughan, 2005; Akhmanova
and Steinmetz, 2008). In mammalian cells, EB1 has two
homologues: EB2 and EB3. Among them, EB1 and EB3 but
not EB2 are enriched at growing MT tips. In addition, EB1 is
ubiquitously expressed and best characterized, whereas EB3
is primarily expressed in neurons and muscle cells. The
structure of EB1 comprises a calponin homology domain
responsible for MT binding, a coiled-coil region responsible
for its homodimerization, and a tail region for binding to
most known ?TIPs (Bu and Su, 2003; Hayashi and Ikura,
2003; Hayashi et al., 2005; Honnappa et al., 2005; Slep et al.,
2005). At the plus ends, EB1 promotes MT polymerization
and prevents MTs from pausing (Tirnauer et al., 2002; Rogers
et al., 2002; Busch and Brunner, 2004). EB1 has intrinsic
activity of promoting MT assembly, and such activity is
tightly controlled by its tail region (Ligon et al., 2003;
Hayashi et al., 2005). It is interesting that the autoinhibition
can be relieved by binding APC or p150gluedto the EB1 tail
(Nakamura et al., 2001; Hayashi et al., 2005; Honnappa et al.,
2005; Slep et al., 2005), implicating cooperation between EB1
and other ?TIPs in the regulation of plus-end dynamics.
CDK5RAP2 (also known as Cep215) is a protein whose
mutations associate with a neurogenic disorder, autosomal
recessive primary microcephaly (Ching et al., 2000; Andersen et
al., 2003; Bond et al., 2005). Recently, CDK5RAP2 has been
characterized for its function at centrosomes. In somatic cells,
CDK5RAP2 localizes to centrosomes throughout the cell
cycle (Bond et al., 2005; Graser et al., 2007; Fong et al., 2008).
It binds to the ?-tubulin ring complex and is required for the
centrosomal assembly of ?-tubulin, playing a critical role in
the MT-organizing function of centrosomes (Fong et al.,
2008). CDK5RAP2 also has been shown to have an indirect
function in centrosome cohesion (Graser et al., 2007). There-
fore, CDK5RAP2 may play a multifunctional role at centro-
This article was published online ahead of print in MBC in Press
on June 24, 2009.
Address correspondence to: Robert Z. Qi (email@example.com).
Abbreviations used: MT, microtubule.
3660 © 2009 by The American Society for Cell Biology
In this report, we show that CDK5RAP2 tracks MT plus
ends via association with EB1. CDK5RAP2 contains the basic
and Ser-rich motif that has been found in several ?TIPs and
is responsible for EB1 interaction. It is interesting that the
EB1-binding motif is conserved in the chimpanzee, bovine,
and dog but not in the rat and mouse sequences of
CDK5RAP2, pointing to a function gained during evolution.
The suppression of CDK5RAP2 expression or the coexpres-
sion of CDK5RAP2 and EB1 impacts MTs. Furthermore, the
CDK5RAP2–EB1 complex promotes MT assembly in vitro.
Together, these results indicate that CDK5RAP2 forms a
plus-end complex with EB1 to regulate MT dynamics.
MATERIALS AND METHODS
DNA Clones and Reagents
DNA plasmids were constructed with standard molecular cloning techniques
and mutations were created by polymerase chain reaction (PCR) methods.
The constructs were all verified by DNA sequencing. The cDNA of mouse
CDK5RAP2 (GenBank accession AK129411) was obtained from the Kazusa
DNA Research Institute (Chiba, Japan). The EB1-green fluorescent protein
(GFP) plasmid (Mimori-Kiyosue et al., 2000a) was a gift from Dr. Yuko
Mimori-Kiyosue (KAN Research Institute, Kobe, Japan) and the mCherry-?-
tubulin plasmid (Shaner et al., 2004) was from Dr. Roger Y. Tsien (University
of California, San Diego, La Jolla, CA). Small interfering RNA (siRNA) du-
plexes against human EB1 (UUGCCUUGAAGAAAGUGAA; Louie et al.,
2004) and CDK5RAP2 (UGGAAGAUCUCCUAACUAA; Fong et al., 2008)
were used as reported previously. The siRNA-resistant construct of
CDK5RAP2 was prepared by introducing four silent substitutions into the
targeted site (i.e., the resulting sequence is TGGAGGAACTGCTAACGAA).
The antibody recognizing a carboxy-terminal region of CDK5RAP2 was
described previously (Fong et al., 2008). The following antibodies were pur-
chased: anti-acetylated-?-tubulin, anti-FLAG (monoclonal M2 and poly-
clonal) from Sigma-Aldrich (St. Louis, MO), anti-?-tubulin from Calbiochem
(San Diego, CA), anti-His6(H-15) and anti-GFP from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA), and anti-EB1 and anti-active caspase-3 from BD Bio-
sciences (San Jose, CA). A rabbit anti-pericentrin antibody (polyclonal M8)
was obtained from Dr. Stephen J. Doxsey (University of Massachusetts,
Amherst, MA). The monoclonal antibody GT335 was from Dr. Carsten Janke
(Centre de Recherches de Biochimie Macromole ´culaire, Montpellier, France).
Cell Culture, Transfection, and Stable Line Generation
Human embryonic kidney (HEK) 293T, HeLa, and U2OS were cultured in
DMEM plus 10% fetal bovine serum (Invitrogen, Carlsbad, CA). DNA plas-
mids and siRNA duplexes were transfected into cells using Lipofectamine
Plus and Lipofectamine 2000 (Invitrogen), respectively. To generate stable
transfectants of yellow fluorescent protein (YFP)-?-tubulin (Clontech, Moun-
tain View, CA), EB1-GFP, and GFP-CDK5RAP2, cells were selected with 2
mg/ml G418 (Invitrogen) 24 h after transfection. Resistant clones were picked
and cultured. Expression of the transfected genes was examined by immu-
Cells grown on coverglasses were fixed with 4% paraformaldehyde in phos-
phate-buffered saline (PBS) at room temperature for 20 min. Methanol fixation
also was used to evaluate the centrosomal localization of CDK5RAP2. Images
were acquired by confocal laser scanning microscopy (LSM510 META; Carl
Zeiss Microimaging, Thornwood, NY) or by wide-field microscopy using an
inverted microscope (Eclipse TE2000; Nikon, Tokyo, Japan) equipped with
the camera SPOT RT1200 (Diagnostic Instruments, Sterling Heights, MI).
Nuclear DNA was labeled with 1 ?M Hoechst 33528 (Sigma-Aldrich). Anti-
gen blocking was performed by incubating the CDK5RAP2 antibody with
antigen protein in excess before immunostaining.
Cells grown on 35-mm glass-bottomed dishes were changed to the phenol
red-free medium before being imaged on the Nikon microscope equipped
with an incubator to maintain the culture conditions (e.g., 37°C and 5% CO2).
Live-cell imaging was performed using an EMCCD camera (SPOT BOOST
BT2100; Diagnostic Instruments). TIF image stacks were exported as MOV
files using MetaMorph (Molecular Devices, Sunnyvale, CA). The play-back
rate is 5 frames/s. To determine the dynamic behaviors of MTs, data were
collected by tracking MT ends using the “track points” function of Meta-
Morph and were transferred to Excel (Microsoft, Redmond, WA) to plot the
life history (Rusan et al., 2001). The phases of growth, shrinkage, and pause
were then identified, and the time spent in each phase was determined. To
derive rescue frequency, the number of transitions from shrinkage to growth
or pause was divided by the duration of shrinkage. To calculate catastrophe
frequency, the number of transitions from growth to shrinkage and pause to
shrinkage was divided by the duration of growth and pause.
To determine the amount of CDK5RAP2 at MT plus ends, the fluorescence
intensity was measured from a rectangle covering the entire tip area (8 ? 30
pixels, width ? height). The intensity was also measured from a cytoplasmic
area of same size. After background subtraction, the intensity ratios of MT
tips to cytoplasm were determined and presented as a histogram.
Preparation of Recombinant Proteins
Escherichia coli BL21(DE3) was used to express glutathione transferase (GST)
fusion and His6-tagged proteins from pGEX and pET21b constructs, respec-
tively. The expressed proteins were purified by binding His6to Ni2?-nitrilo-
triacetic acid resin (QIAGEN, Valencia, CA) or GST to glutathione (GSH)-
Sepharose (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United
Kingdom) as described in previous reports (Lim et al., 2004; Fu et al., 2006).
Immunoprecipitation and In Vitro Protein Binding
Cell extracts were prepared in the buffer of 25 mM Tris-HCl, pH 7.4, 0.5%
NP-40, 100 mM NaCl, 5 mM MgCl2, 5 mM NaF, 20 mM ?-glycerophosphate,
1 mM dithiothreitol, 1 mM EDTA, and the Compete Protease Inhibitor Cock-
tail (Roche Diagnostics, Indianapolis, IN). After the extracts were clarified by
centrifugation (13,000 ? g for 15 min), proteins were immunoprecipitated
using anti-FLAG M2-coupled beads (Sigma-Aldrich) or otherwise antibodies
coupled to protein A/G-Agarose (Invitrogen) as indicated. Immunoprecipi-
tated products were detected by immunoblotting. To test protein binding in
vitro, the CDK5RAP2 fragment 926-1208 and its L938A/P939A mutant pre-
pared as recombinant GST proteins were incubated at 4°C with His6-EB1 in
the buffer described above containing 2 mg/ml bovine serum albumin. In a
control, GST was used instead of the CDK5RAP2 fragment. After the incu-
bation, the GST proteins were bound to GSH-Sepharose and retrieved for
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.
The light scattering assay was carried out with 18 ?M tubulin purified from
porcine brain and free of MT-associated proteins as described in a previous
report (Hou et al., 2007). To test MT-elongating activity, MT seeds, prepared
by shearing Taxol-stabilized MTs with a 26-gauge needle, were included in
assembly assays. MT assembly also was examined by polymerization of
rhodamine-labeled (Cytoskeleton, Denver, CO) and unlabeled tubulin (1:20)
for epifluorescence microscopy (He et al., 2008).
CDK5RAP2 Localizes to MTs and Concentrates at the
In a previous report, we showed CDK5RAP2 association
with MTs in an MT-pelleting assay (Fong et al., 2008). We
extended this observation and further examined the cyto-
plasmic distribution of CDK5RAP2. Cell cultures were fixed
in paraformaldehyde and then examined by immunofluo-
rescence confocal microscopy. CDK5RAP2 staining dis-
played diffuse and punctuate patterns in the cytoplasm, in
addition to the centrosomal localization (Figure 1A). In the
cell lamellae, CDK5RAP2 signals showed filamentous struc-
tures coaligned with MTs (Figure 1A). The filamentous pat-
terns were most evident near the extending edge of cell
protrusions, where CDK5RAP2 labeling was concentrated at
the MT distal tips (Figure 1A). The staining of CDK5RAP2 to
MTs and the distal tips are specific, because the filamentous
patterns were eliminated upon CDK5RAP2 depletion by
using RNA interference (RNAi) or by preincubation of the
antibody with antigen (Figure 1, B and C). The CDK5RAP2
tracking of growing MT tips also was demonstrated by
time-lapse microscopy (see below).
CDK5RAP2 Is an EB1-binding Protein
Because EB1 is a core component of protein complexes at MT
plus ends, we tested the potential interaction between
CDK5RAP2 and EB1. HEK293T was transiently transfected
with CDK5RAP2 and EB1-GFP or the vector for coimmuno-
precipitation. Immunoblots revealed the presence of EB1-GFP
but not GFP by itself in the immunoprecipitate of ectopically
CDK5RAP2 Associates with EB1
Vol. 20, August 15, 20093661
expressed CDK5RAP2 (Figure 2A). We further probed the
interaction using EB1 fragments. The tail fragment EB1(185-
268) was readily detected to coimmunoprecipitate with
CDK5RAP2 (Figure 2A). In contrast, EB1(1-241), the con-
struct lacking the tail, did not show any binding activity
(Figure 2A). Therefore, CDK5RAP2 binds to the EB1 tail,
which is similar to several ?TIPs such as APC and p150glued
(Askham et al., 2002). We next performed coimmunoprecipi-
tation of the endogenous proteins. Immunoprecipitation of
endogenous EB1 specifically coprecipitated endogenous
CDK5RAP2 (Figure 2B), confirming the interaction under
To delineate the EB1-binding domain, CDK5RAP2 con-
structs expressing various regions were used to test EB1
binding by coimmunoprecipitation. The fragment 926-1208
but not the other regions were found to be responsible for
EB1 interaction (Figure 2C). The coimmunoprecipitation ex-
periments described above revealed the association of
CDK5RAP2 with EB1 as well as the binding regions but did
not show whether the binding is direct or indirect. To ad-
dress this, we prepared recombinant proteins of 926-1208
and EB1 by bacterial expression for testing the binding in
vitro. We also prepared the L938A/P939A mutant of 926-
1208, which lacks EB1-binding activity (see below). EB1
readily bound to 926-1208 but not to the L938A/P939A
mutant or the tag of the recombinant protein (Figure 2D),
indicating direct association of EB1 with the middle region
CDK5RAP2 Contains the Basic and Ser-rich Motif
The EB1-binding sites of APC and several other ?TIPs have
been defined as a small region rich in Ser and basic residues
and containing conserved motifs (Galjart, 2005; Honnappa et
al., 2005; Mimori-Kiyosue et al., 2005; Slep et al., 2005). Scan-
ning the EB1-binding region of CDK5RAP2 revealed a short
segment, 926–956, exhibiting features of the EB1-binding
domains found in the above-mentioned ?TIPs (Figure 3A).
Within the EB1-binding domain of APC, Ile2805-Pro2806 is
critically involved in the interaction with EB1 (Honnappa et
al., 2005). This dipeptide motif is conservatively substituted
in CDK5RAP2 as Leu-Pro (i.e., Leu938-Pro939; Figure 3A).
We double mutated Leu938-Pro939 to Ala and tested the
mutational effect on EB1 binding. The binding assays
showed that the mutation abolished the EB1-binding activity
of 926-1208 as well as that of the full-length protein (Figure
3B). Together, CDK5RAP2 harbors a basic and Ser-rich se-
quence for EB1 interaction.
In a search of GenBank databases, we found putative
CDK5RAP2 sequences from several lower mammalian spe-
cies, including mouse, rat, dog, bovine, and chimpanzee.
Across this range of species, CDK5RAP2 shows a high de-
gree of sequence homology ranging from 70 to 99%. We then
examined EB1-binding sequences. The segment 926–956 of
human CDK5RAP2 shares significant overall homologies
with the corresponding regions from these sequences found
in lower mammals (Figure 3C). The EB1-binding motif pre-
dicted from the alignment of the basic and Ser-rich se-
quences is well conserved in the sequences from chimpan-
zee, bovine, and dog but not in the murine sequences
(Figure 3C). In particular, two residues including Pro of the
critical Ile/Leu-Pro motif are substituted in the rat and
mouse sequences. We expressed mouse CDK5RAP2 for test-
ing in an EB1-binding assay. Immunoprecipitation of EB1
failed to pull down mouse CDK5RAP2, whereas the immu-
noprecipitation specifically coprecipitated the human pro-
tein in a control assay (Figure 3D). In addition, the mouse
protein did not show plus-end tracking property when ex-
pressed, even though it localized to the centrosomes (data
not shown). These analyses indicate that the MT tip-binding
function of CDK5RAP2 is not conserved in the rat and
CDK5RAP2 Tracks Growing MT Tips in an
We proceeded to investigate CDK5RAP2 association with
MT plus ends and whether the plus-end association requires
EB1 binding. To this end, U2OS sublines were generated to
stably express CDK5RAP2 or the EB1-binding–deficient mu-
tant L938A/P939A. The proteins were expressed in fusion
with GFP at the amino terminus. Several stable transfectants
were selected with the proteins expressed at levels close to the
endogenous amount. The results were shown below using
the clones W1 and M3, which expressed the CDK5RAP2
wild type and the mutant, respectively. In these clones, the
amount of ectopically expressed CDK5RAP2 was ?1.4-fold
of the endogenous level (Figure 4A). All experiments were
repeated with at least two other selected clones of similar
The distribution patterns of CDK5RAP2 stably expressed
in U2OS cells were first examined by immunofluorescence
microscopy. The wild-type protein decorated along the shaft
of MTs and concentrated at the distal end regions in addi-
tion to centrosomal localization (Figure 4B). The L938A/
P939A mutant displayed a diffuse pattern without obvious
localization to MTs and the distal tips despite its retained
HeLa cells were stained with anti-CDK5RAP2 and anti-?-tubulin
antibodies for confocal microscopy. The boxed areas are enlarged in
insets. (B) HeLa cells depleted of CDK5RAP2 by RNAi were immu-
nostained as described in A. (C) The CDK5RAP2 antibody was
blocked with antigen protein and then subjected to immunostaining
of HeLa cells. Bars, 10 ?m.
CDK5RAP2 localization to MTs and the distal tips. (A)
K.-W. Fong et al.
Molecular Biology of the Cell3662
centrosomal localization (Figure 4B). To conduct time-lapse
microscopy, mCherry-?-tubulin was expressed at low levels
to allow imaging of MTs. In live cells, CDK5RAP2 wild type
displayed highly dynamic comet-like fluorescence patterns
that highlighted the growing MT tips and moved toward the
cell periphery (Figure 4C and Supplemental Movie 1), dem-
onstrating the plus-end tracking behavior. We then pro-
ceeded to analyze the stable line of the L938A/P939A mu-
tant. The mutant protein did not show MT plus-end tracking
behavior (Figure 4C and Supplemental Movie 2). Together,
the association of EB1 targets CDK5RAP2 to MTs and to the
We tested whether the plus-end tracking of CDK5RAP2
depends on MT assembly/disassembly dynamics. The sta-
ble cells of GFP-CDK5RAP2 were treated with low-dosage
nocodazole (0.1 ?M), which perturbs MT dynamics but does
not disrupt the array organization (Vasquez et al., 1997). The
treatment quickly eliminated the dynamic signals of
CDK5RAP2 (Supplemental Movie 3), which is similar to the
effect observed for EB1 (Mimori-Kiyosue et al., 2000a). These
data revealed the attachment of CDK5RAP2 with dynamic
MT plus ends.
Using the stable cells of GFP-CDK5RAP2, we also exam-
ined CDK5RAP2 dynamic movement under the condition of
EB1 depletion. The transfection of an eb1-targeting siRNA
effectively reduced the EB1 level (by ?90%) but did not
affect the expression of CDK5RAP2 and tubulin (Figure 5A).
In cells transfected with eb1 siRNA, GFP-CDK5RAP2
showed little MT localization and plus-end tracking (Figure
5, B and C, and Supplemental Movie 4). To assess more
precisely the plus-end tracking activities, we determined the
ratio of GFP-CDK5RAP2 intensities between MT tips and
cytoplasm. The suppression of EB1 expression dramati-
cally reduced CDK5RAP2 at the plus ends to the levels
close to that of the cytoplasm (Figure 5C). This result
supports the principal role of EB1 in CDK5RAP2 attach-
ment to the plus ends.
The CDK5RAP2–EB1 Complex Regulates MT Dynamics
To probe CDK5RAP2 function in the control of MT dynam-
ics, we performed RNAi-mediated CDK5RAP2 depletion
and imaged MTs in live cells. The suppression of
CDK5RAP2 expression did not significantly affect EB1 track-
ing of MT plus ends. We derived the parameters of MT
dynamic instability from the time-lapse microscopic data of
interphase cells expressing YFP-?-tubulin. The dynamic
properties measured from control cells (Table 1) were simi-
lar to those reported previously (Rusan et al., 2001). MTs in
cells depleted of CDK5RAP2 were less dynamic than those
in the control cells. The most notable effects of the depletion
were shortened growth and prolonged pause of MTs (Table
1). CDK5RAP2 seems to have growth-promoting and pause-
suppressing effects on MTs.
Because CDK5RAP2 is required for ?-tubulin assembly
onto centrosomes, CDK5RAP2 depletion delocalizes centro-
somal ?-tubulin and disorganizes microtubules (Fong et al.,
2008). To investigate specifically the plus-end–associated
function, we carried out rescue experiments of CDK5RAP2
knockdown by using the EB1-binding–deficient mutant
CDK5RAP2(L938A/P939A). The construct was engineered
to contain silent mutations within the siRNA-targeted se-
quence, making it unaffected by the siRNA used (Supple-
mental Figure 1A). The expression of CDK5RAP2(L938A/
P939A) maintained the radial array of MTs in cells depleted
of endogenous CDK5RAP2 (Supplemental Figure 1B). Al-
though the mutant expression showed small rescuing effects
on MT dynamics, MTs in the cells still displayed a signifi-
cantly longer time in pause and a shorter time in growth
than those in the control cells (Table 1). Therefore, EB1
binding and thus plus-end tracking are required for
CDK5RAP2 to regulate MT dynamics.
tracts coexpressing Flag-CDK5RAP2 and either GFP or
GFP-tagged EB1 proteins were subjected to anti-FLAG
(50%) and the cell extracts (5%) were analyzed by im-
munoblotting (IB). EB1 constructs used were the full-
length protein (i.e., 1–268) and two fragments. (B) En-
dogenous EB1 was immunoprecipitated from HEK293T
extracts to detect coimmunoprecipitation of endoge-
nous CDK5RAP2. The immunoprecipitates (100%) and
the cell extract (10%) were analyzed. (C) CDK5RAP2
fragments (FLAG-tagged) were coexpressed with EB1-
GFP. The anti-FLAG immunoprecipitates (50%) and the
cell extracts (5%) were probed on immunoblots. (D) In a
GST pull-down assay, His6-EB1 was tested for binding
to GST or the CDK5RAP2 fragment in fusion with GST.
938/939A, 926-1208(L938A/P939A). The pull-downs
(20%) and the His6-EB1 input (10%) were resolved by
SDS-PAGE and transferred to membranes. Top, anti-
His6immunoblots. Bottom, membrane stained with
CDK5RAP2 binds to EB1. (A) HEK293T ex-
CDK5RAP2 Associates with EB1
Vol. 20, August 15, 20093663
Next, we generated U2OS sublines stably expressing EB1-
GFP at low levels; one of the selected clones, E2, expressed
the protein at ?1.5-fold of endogenous EB1 (Figure 6A). At
this level, EB1-GFP decorated the entire network of MTs
with enrichment at the plus ends (Figure 6B), which is in
agreement with the previous reports (Mimori-Kiyosue et al.,
2000a; Bu and Su, 2001; Ligon et al., 2003). Remarkably,
transfection of CDK5RAP2 into E2 caused the formation of
long MT bundles emanating from the centrosomes; both
CDK5RAP2 and EB1 were enriched on the bundles (Figure
6C). In addition, the CDK5RAP2 expression gave rise to
intense acetylation of MTs (Figure 6C), pointing to MT sta-
bilization. In contrast, expression of the L938A/P939A mu-
tant did not show such effects (Figure 6C). Transfection of
the EB1-binding fragment 926-1208 and its L938A/P939A
mutant yielded the same results as the respective full-length
proteins (Figure 6D). Such effects of CDK5RAP2 expression
also were observed using other selected EB1-GFP clones. In
a control assay, expression of CDK5RAP2 or 926-1208 in
parental U2OS did not induce MT bundling and acetylation
(Figure 6E). Therefore, the interaction between CDK5RAP2
and EB1 generates notable impacts on MT structures in the
in EB1-GFP stable lines transfected with CDK5RAP2 might be
caused by cell apoptosis, we carried out the following assays.
As a control, apoptosis of EB1-GFP stable cells was induced
with staurosporine. First, we examined the morphology of
nuclear DNA and did not find chromatin condensation
and fragmentation in the stable cells transfected with
CDK5RAP2 (Supplemental Figure 2, A and C). Second, we
analyzed the subcellular localization of pericentrin, as it
disperses from centrosomes in apoptotic cells (Moss et al.,
2006; Sanchez-Alcazar et al., 2007). In CDK5RAP2-trans-
fected stable cells, pericentrin was readily observed at the
centrosomes (Supplemental Figure 2A). In contrast, pericen-
trin did not exhibit centrosomal localization in EB1-GFP cells
undergoing apoptosis induced with staurosporine (Supple-
mental Figure 2B). Third, we analyzed caspase activation by
using an antibody specifically recognizing active caspase-3.
Active caspase-3 was undetectable in CDK5RAP2-trans-
fected stable cells, whereas it was readily detected in apo-
ptotic EB1-GFP cells (Supplemental Figure 2, C and D).
Fourth, EB1-GFP stable cells transfected with CDK5RAP2
displayed MT patterns different from those of apoptotic
cells. MT bundles were found throughout the cytoplasm in
the coexpressing cells (Supplemental Figure 2, A and C),
whereas MTs occurred only along the plasma membranes in
apoptotic cells (Moss et al., 2006; Sanchez-Alcazar et al.,
2007). We conclude that the coexpression of CDK5RAP2 and
EB1 did not induce apoptosis.
To gain insight into the mechanism of CDK5RAP2 action,
we conducted MT assembly assays in vitro by using EB1, the
EB1-binding fragment 926-1208, and its L938A/P939A mu-
tant. These proteins were prepared by bacterial expression
and purified (Figure 7A). The assembly assays were first
conducted in the presence of MT seeds to test MT assembly
by elongation from the seeds. The addition of either EB1 or
926-1208 into the assays did not promote MT polymeriza-
tion; but the addition of both proteins dramatically en-
hanced the extent of polymerization (Figure 7B). When the
L938A/P939A mutant was used instead of the wild type, the
combination with EB1 failed to increase light scattering (Fig-
ure 7B). Clearly, the association of CDK5RAP2 with EB1
promotes MT polymerization. For morphological examina-
tion by microscopy, MTs were polymerized with a mixture
of rhodamine-labeled and unlabeled tubulin. Long MT fila-
ments and bundles were extensively formed in the sample
containing both 926-1208 and EB1, but not in the others
(Figure 7C). These results revealed that the CDK5RAP2–EB1
complex not only promotes polymerization but also bundles
We then performed MT assembly assays in the absence
of MT seeds to explore MT-nucleating activity. However,
the addition of both 926-1208 and EB1 had no significant
effect on MT assembly (Figure 7D). Moreover, the combi-
nation of 926-1208 and EB1 displayed polymerizing activ-
ities dependent on the doses of MT seeds (Figure 7D).
Under the assay conditions, the interaction between EB1
Alignment of CDK5RAP2(926-956) with several known basic and
Ser-rich sequences. Asterisks denote residues conserved in the mo-
tif. (B) HEK293T was double transfected with FLAG-tagged
CDK5RAP2 and EB1-GFP for anti-FLAG immunoprecipitation. The
immunoprecipitates (50%) and the cell extracts (5%) were analyzed
on immunoblots. The CDK5RAP2 constructs used were the full-
length (CDK5RAP2FL) and the 926-1208 fragment. WT, wild type;
938/939A, L938A/P939A mutant.
CDK5RAP2 from different mammalian species. GenBank acces-
sion numbers are as follows: chimpanzee, NP_001035901; bovine,
XP_584826; dog, XP_855524; rat, XP_575844; and mouse, NP_666102.
(D) HEK293T extracts coexpressing EB1 and CDK5RAP2 were sub-
jected to anti-GFP immunoprecipitation. The immunoprecipitates
are the human protein (FLAG-tagged) and the mouse counterpart
CDK5RAP2 contains the basic and Ser-rich motif. (A)
K.-W. Fong et al.
Molecular Biology of the Cell3664
and the CDK5RAP2 fragment promoted MT elongation
but did not induce nucleation.
In the present study, we have described the association of
CDK5RAP2 with growing MT tips and the regulatory role of
CDK5RAP2 in MT dynamics. At growing plus ends, ?TIPs
form various protein complexes with EB1, which acts as a
mediator of the dynamic interaction network. EB1 is present
at all growing MT ends and is required for a number of
?TIPs to associate with the plus ends (Vaughan, 2005; Lans-
bergen and Akhmanova, 2006). Here, our data have demon-
strated that CDK5RAP2 requires EB1 interaction for plus-
end association. Therefore, CDK5RAP2 uses a hitchhiking
mechanism, which is similar to several of the other ?TIPs.
CDK5RAP2 belongs to a class of ?TIPs that contain the
basic and Ser-rich motif responsible for interaction with EB1
(Galjart, 2005; Akhmanova and Steinmetz, 2008). We have
shown that CDK5RAP2 binds to the EB1 tail, a region that
contains a protein-binding domain. Several structural stud-
ies have revealed that the carboxy-terminal region of EB1
homodimerizes through a coiled-coil domain to form a four-
helix bundle (Hayashi et al., 2005; Honnappa et al., 2005; Slep
et al., 2005). At the junction of the coiled-coil and four-helix
bundle, a hydrophobic pocket comprising a cluster of con-
in a manner dependant on EB1 interaction. (A)
Stable U2OS lines were analyzed on immuno-
blots (IBs). Parental, parental U2OS; W1, a sub-
line expressing GFP-CDK5RAP2; M3, a subline
expressing the L938A/P939A mutant (GFP-
938/939A). (B) The stable lines were immuno-
stained for MTs (anti-?-tubulin). (C) Time-lapse
microscopy was performed on the stable cells.
mCherry-?-tubulin was transiently expressed at
low levels. Time series of the boxed areas are
enlarged. Bars, 10 ?m.
CDK5RAP2 targets to MT distal ends
CDK5RAP2 Associates with EB1
Vol. 20, August 15, 20093665
served residues provides the protein-binding site. In the EB1
interaction with APC, the dipeptide Ile2805-Pro2806 within
the basic and Ser-rich sequence of APC plays a critical role in
EB1 association by interacting perhaps with the conserved
residues of the hydrophobic pocket (Honnappa et al., 2005;
Slep et al., 2005). We have found that Ala substitution for
Leu938-Pro939, an equivalent to Ile2805-Pro2806 of APC,
ablates the EB1-binding activity of CDK5RAP2, confirming
that CDK5RAP2 binds to EB1 in a similar mode.
CDK5RAP2 is related to several proteins in lower organ-
isms, including Drosophila centrosomin (Cnn) and Schizosac-
charomyces pombe Mto1p and Pcp1p, in terms of ?-tubulin
complex binding (Sawin et al., 2004; Fong et al., 2008). Cnn
and Mto1p have been found to exist in satellite particles that
move back-and-forth in MT-dependent manners (Megraw et
al., 2002; Sawin et al., 2004). The back-and-forth moving
behaviors of Cnn and Mto1p are different from CDK5RAP2
tracking of MT tips, which is a unidirectional movement. In
addition, we did not find the basic and Ser-rich motif in the
sequences of Cnn, Mto1p, and Pcp1p, implying that the
tip-tracking function is not conserved in these proteins.
The EB1-binding motif found in human CDK5RAP2 is
well conserved in the homologues from chimpanzee, bo-
vine, and dog but not in those from rat and mouse. As a
result, we have found that mouse CDK5RAP2 does not have
EB1-binding and plus-end tracking properties. Therefore,
the MT tip-associated function of CDK5RAP2 is a gain-of-
function during the evolution of mammals. In line with this
idea, CDK5RAP2 is a rapidly evolved protein with the mo-
lecular evolutionary rate significantly higher in primates
than in rodents (Evans et al., 2006). CDK5RAP2 has been
implicated to play a role in brain development, as its muta-
tions cause primary microcephaly (Bond et al., 2005). Rodent
brains have substantial differences in size and structure from
the brains of primates, bovine, and dog. For example, the
cerebral cortexes of primates, bovine, and dog have many
grooves (i.e., sulci and fissures) to significantly enlarge the
surface area compared with the smooth brains of rodents. It
is plausible that CDK5RAP2 acts in brain development in
part by associating with MT distal tips. Similarly, APC2 and
EB1, ?TIPs at the adherens junctions of Drosophila epithelial
with an eb1-targeting siRNA or the control. The protein levels of EB1, GFP-CDK5RAP2, and ?-tubulin were detected. (B) Live cells of W1 were
imaged for GFP-CDK5RAP2. Below are time series of the boxed areas. Bar, 10 ?m. (C) Histogram shows the fluorescence intensity ratios of
growing MT tips to cytoplasm. Eight control and twelve EB1-depleted cells were analyzed; 10 MTs were chosen from each cell for analysis.
Statistical analysis was performed using Student’s unpaired two tails t test (p ? 0.001).
Depletion of EB1 inhibits CDK5RAP2 tracking MT plus ends. (A) The stable line of GFP-CDK5RAP2 (clone W1) was transfected
Table 1. MT dynamic properties
Time in growth (%)
Time in shrinkage (%)
Time in pause (%)
12.0 ? 3.3 12.1 ? 4.2 12.3 ? 5.8
18.4 ? 12.6 17.9 ? 10.816.8 ? 11.2
0.335 ? 0.146 0.301 ? 0.147 0.360 ? 0.169
0.077 ? 0.039 0.067 ? 0.023 0.073 ? 0.032
U2OS cells stably expressing YFP-?-tubulin were transfected with
the control or cdk5rap2-targeting siRNA (CDK5RAP2si). The
L938A/P939A mutant construct (938/939A) is resistant to the
siRNA duplex used and expresses the mCherry-tagged protein.
Live cells were imaged 72 h after transfection for 90 s at a 2-s
interval. MT tips near the cell periphery were chosen to plot the MT
life histories. Statistical analysis was performed using Student’s
unpaired two-tailed t test for comparison between two groups.
Presented are mean values ? SD for growth and shrinkage rates and
rescue and catastrophe frequencies. Asterisks indicate p ? 0.05
compared with the control data.
K.-W. Fong et al.
Molecular Biology of the Cell3666
cells and male germline stem cells, regulate the orientation
of mitotic spindles during the asymmetric division (Lu et al.,
2001; Yamashita et al., 2003).
?TIPs are well positioned to regulate MT dynamics. The
RNAi experiments from the current study revealed that
CDK5RAP2 is a factor promoting MT growth and prevent-
ing MT pause, which is reminiscent of the EB1 effects on
MTs (Rogers et al., 2002; Tirnauer et al., 2002; Busch and
Brunner, 2004; Kita et al., 2006). The CDK5RAP2 function
associated with MT dynamics is exerted largely by binding
with EB1 and plus-end tracking, because the EB1-binding–
deficient mutant rescued the dynamics defects only to a
small extent. Moreover, the CDK5RAP2–EB1 complex pro-
motes MT growth and induces MT bundling in vitro, and it
stabilizes MTs in transfected cells via the formation of MT
bundles. These observations suggest that CDK5RAP2 exists
in one of the various complexes formed by EB1 to promote
MT growth and dynamics at the plus ends.
EB1 has an intrinsic activity of promoting MT assembly,
which can be stimulated upon binding APC or p150gluedto
EB1-GFP (clone E2) and parental U2OS cells (Pa-
rental) were analyzed on immunoblots (IBs). (B)
The stable line E2 was imaged for EB1-GFP and
MTs (anti-?-tubulin). (C and D) The E2 line was
transiently transfected with the wild type or the
L938A/P939A mutant (938/939A) of FLAG-
CDK5RAP2. The constructs transfected were the
(anti-acetylated ?-tubulin). (E) CDK5RAP2 and
926-1208 were transiently expressed in parental
CDK5RAP2 Associates with EB1
Vol. 20, August 15, 20093667
the EB1 tail (Nakamura et al., 2001; Ligon et al., 2003; Ha-
yashi et al., 2005). Our MT assembly experiments have
shown that the CDK5RAP2–EB1 complex but not EB1 alone
or in combination with the EB1-binding–deficient mutant of
CDK5RAP2 promotes the elongation of MTs. Because
CDK5RAP2 binds to the EB1 tail, the interaction presumably
relieves EB1 autoinhibition, which is similar to the binding
effects of APC and p150glued. It has been reported that EB1
and its fission yeast homologue Mal3, when applied alone,
promote the spontaneous assembly of MTs (des Georges et
al., 2008; Vitre et al., 2008). In our assays, EB1 alone did not
exhibit any effect. We noted that the concentrations of EB1
and tubulin in our assembly assays are different from those
used in these reports. Our assays were performed with 18
?M tubulin, which is below the critical concentration of
tubulin, and 0.5 ?M EB1, which is close to the physiological
concentration (?0.27 ?M) measured from Xenopus eggs (Tir-
nauer et al., 2002). In the above-mentioned reports, 0.9 ?M
EB1 and 15 ?M tubulin were used or a minimum of 2 ?M
Mal3 was applied to 4 ?M in heterodimer concentration of S.
pombe tubulin to stimulate MT assembly (des Georges et al.,
2008; Vitre et al., 2008). Therefore, the discrepancy of the
observed EB1 effects is probably due to the lower concen-
tration of EB1 and the lower ratio of EB1 to tubulin in our
assays. Indeed, EB1 displays concentration-dependent ef-
fects on MTs both in vitro and in transfected cells (Mimori-
Kiyosue et al., 2000a; Bu and Su, 2001; Ligon et al., 2003; Vitre
et al., 2008). Seemingly, EB1 can be auto-stimulatory if its
concentration is high enough.
As a highly dynamic structure, MT cytoskeleton is capable
of rapid remodeling in response to changes in the cellular
environment. CDK5RAP2 is enriched at growing MT tips in
MT polymerization. (A) The purified recombinant
proteins were examined on a SDS-PAGE gel stained
with Coomassie Blue. 938/939A, the L938A/P939A
mutant of CDK5RAP2(926-1208). (B) MT assembly
was performed in the presence of 0.1 ?g/?l MT
seeds. Recombinant proteins used included 926-1208
or 938/939A, 1.5 ?M; and His6-EB1, 0.5 ?M. (C) MTs
were polymerized as specified in B from a mixture of
rhodamine-labeled and unlabeled tubulin for exam-
ination by fluorescence microscopy. (D) MT assem-
bly assays contained various amounts of MT seeds.
CDK5RAP2(926-1208), 1.5 ?M; His6-EB1, 0.5 ?M.
The CDK5RAP2–EB1 complex promotes
K.-W. Fong et al.
Molecular Biology of the Cell3668
cell protrusions. This and other results presented in the
current study raise the possibility that CDK5RAP2 may
participate in MT reorganization through the selective sta-
bilization of MTs, thus playing a role in MT-dependent
processes such as cell polarization and directional move-
ment. It is envisaged that the plus-end accumulation of
CDK5RAP2 is under tight regulation spatially and tempo-
rally. Such regulation of ?TIPs would afford the guidance of
MT reorganization and the sensitivity of the MT network to
We thank Drs. Yuko Mimori-Kiyosue, Roger Y. Tsien, Richard A. Kahn
(Emory University School of Medicine, Atlanta, GA), Carsten Janke, and
Donald C. Chang (The Hong Kong University of Science and Technology,
Hong Kong, China) for plasmid constructs. We also are indebted to Dr.
Weichuan Yu and Tianwei Jiang (The Hong Kong University of Science and
Technology) for assistance on protein sequence alignment. This work was
supported by the Research Grants Council (General Research Fund and
Collaborative Research Fund) and the University Grants Committee (Area of
Excellence Scheme) of Hong Kong.
Akhmanova, A., and Steinmetz, M. O. (2008). Tracking the ends: a dynamic
protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell Biol.
Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A., and
Mann, M. (2003). Proteomic characterization of the human centrosome by
protein correlation profiling. Nature 426, 570–574.
Askham, J. M., Moncur, P., Markham, A. F., and Morrison, E. E. (2000).
Regulation and function of the interaction between the APC tumour suppres-
sor protein and EB1. Oncogene 19, 1950–1958.
Askham, J. M., Vaughan, K. T., Goodson, H. V., and Morrison, E. E. (2002).
Evidence that an interaction between EB1 and p150(Glued) is required for the
formation and maintenance of a radial microtubule array anchored at the
centrosome. Mol. Biol. Cell 13, 3627–3645.
Bieling, P., Kandels-Lewis, S., Telley, I. A., van, D. J., Janke, C., and Surrey, T.
(2008). CLIP-170 tracks growing microtubule ends by dynamically recogniz-
ing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233.
Bieling, P., Laan, L., Schek, H., Munteanu, E. L., Sandblad, L., Dogterom, M.,
Brunner, D., and Surrey, T. (2007). Reconstitution of a microtubule plus-end
tracking system in vitro. Nature 450, 1100–1105.
Bond, J., et al. (2005). A centrosomal mechanism involving CDK5RAP2 and
CENPJ controls brain size. Nat. Genet. 37, 353–355.
Bu, W., and Su, L. K. (2003). Characterization of functional domains of human
EB1 family proteins. J. Biol. Chem. 278, 49721–49731.
Bu, W., and Su, L. K. (2001). Regulation of microtubule assembly by human
EB1 family proteins. Oncogene 20, 3185–3192.
Busch, K. E., and Brunner, D. (2004). The microtubule plus end-tracking
proteins mal3p and tip1p cooperate for cell-end targeting of interphase mi-
crotubules. Curr. Biol. 14, 548–559.
Carvalho, P., Tirnauer, J. S., and Pellman, D. (2003). Surfing on microtubule
ends. Trends Cell Biol. 13, 229–237.
Ching, Y. P., Qi, Z., and Wang, J. H. (2000). Cloning of three novel neuronal
Cdk5 activator binding proteins. Gene 242, 285–294.
des Georges, A., Katsuki, M., Drummond, D. R., Osei, M., Cross, R. A., and
Amos, L. A. (2008). Mal3, the Schizosaccharomyces pombe homolog of EB1,
changes the microtubule lattice. Nat. Struct. Mol. Biol. 15, 1102–1108.
Evans, P. D., Vallender, E. J., and Lahn, B. T. (2006). Molecular evolution of the
brain size regulator genes CDK5RAP2 and CENPJ. Gene 375, 75–79.
Fong, K. W., Choi, Y. K., Rattner, J. B., and Qi, R. Z. (2008). CDK5RAP2 is a
pericentriolar protein that functions in centrosomal attachment of the gamma-
tubulin ring complex. Mol. Biol. Cell 19, 115–125.
Fu, X., Choi, Y. K., Qu, D., Yu, Y., Cheung, N. S., and Qi, R. Z. (2006).
Identification of nuclear import mechanisms for the neuronal CDK5 activator.
J. Biol. Chem. 281, 39014–39021.
Galjart, N. (2005). CLIPs and CLASPs and cellular dynamics. Nat. Rev. Mol.
Cell Biol. 6, 487–498.
Graser, S., Stierhof, Y. D., and Nigg, E. A. (2007). Cep68 and Cep215
(Cdk5rap2) are required for centrosome cohesion. J. Cell Sci. 120, 4321–4331.
Hayashi, I., and Ikura, M. (2003). Crystal structure of the amino-terminal
microtubule-binding domain of end-binding protein 1 (EB1). J. Biol. Chem.
Hayashi, I., Wilde, A., Mal, T. K., and Ikura, M. (2005). Structural basis for the
activation of microtubule assembly by the EB1 and p150Glued complex. Mol.
Cell. 19, 449–460.
He, L., Hou, Z., and Qi, R. Z. (2008). Calmodulin Binding and Cdk5 Phos-
phorylation of p35 Regulate Its Effect on Microtubules. J. Biol. Chem. 283,
Honnappa, S., John, C. M., Kostrewa, D., Winkler, F. K., and Steinmetz, M. O.
(2005). Structural insights into the EB1-APC interaction. EMBO J. 24, 261–269.
Hou, Z., Li, Q., He, L., Lim, H. Y., Fu, X., Cheung, N. S., Qi, D. X., and Qi, R. Z.
(2007). Microtubule association of the neuronal p35 activator of Cdk5. J. Biol.
Chem. 282, 18666–18670.
Jimbo, T., Kawasaki, Y., Koyama, R., Sato, R., Takada, S., Haraguchi, K., and
Akiyama, T. (2002). Identification of a link between the tumour suppressor
APC and the kinesin superfamily. Nat. Cell Biol. 4, 323–327.
Kita, K., Wittmann, T., Nathke, I. S., and Waterman-Storer, C. M. (2006).
Adenomatous polyposis coli on microtubule plus ends in cell extensions can
promote microtubule net growth with or without EB1. Mol. Biol. Cell 17,
Lansbergen, G., and Akhmanova, A. (2006). Microtubule plus end: a hub of
cellular activities. Traffic 7, 499–507.
Ligon, L. A., Shelly, S. S., Tokito, M., and Holzbaur, E. L. (2003). The micro-
tubule plus-end proteins EB1 and dynactin have differential effects on micro-
tubule polymerization. Mol. Biol. Cell 14, 1405–1417.
Lim, A. C., Tiu, S. Y., Li, Q., and Qi, R. Z. (2004). Direct regulation of
microtubule dynamics by protein kinase CK2. J. Biol. Chem. 279, 4433–4439.
Louie, R. K., Bahmanyar, S., Siemers, K. A., Votin, V., Chang, P., Stearns, T.,
Nelson, W. J., and Barth, A. I. (2004). Adenomatous polyposis coli and EB1
localize in close proximity of the mother centriole and EB1 is a functional
component of centrosomes. J. Cell Sci. 117, 1117–1128.
Lu, B., Roegiers, F., Jan, L. Y., and Jan, Y. N. (2001). Adherens junctions inhibit
asymmetric division in the Drosophila epithelium. Nature 409, 522–525.
Megraw, T. L., Kilaru, S., Turner, F. R., and Kaufman, T. C. (2002). The
centrosome is a dynamic structure that ejects PCM flares. J. Cell Sci. 115,
Mimori-Kiyosue, Y., et al. (2005). CLASP1 and CLASP2 bind to EB1 and
regulate microtubule plus-end dynamics at the cell cortex. J. Cell Biol. 168,
Mimori-Kiyosue, Y., Shiina, N., and Tsukita, S. (2000b). Adenomatous polyp-
osis coli (APC) protein moves along microtubules and concentrates at their
growing ends in epithelial cells. J. Cell Biol. 148, 505–518.
Mimori-Kiyosue, Y., Shiina, N., and Tsukita, S. (2000a). The dynamic behavior
of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol.
Moss, D. K., Betin, V. M., Malesinski, S. D., and Lane, J. D. (2006). A novel role
for microtubules in apoptotic chromatin dynamics and cellular fragmenta-
tion. J. Cell Sci. 119, 2362–2374.
Nakamura, M., Zhou, X. Z., and Lu, K. P. (2001). Critical role for the EB1 and
APC interaction in the regulation of microtubule polymerization. Curr. Biol.
Rogers, S. L., Rogers, G. C., Sharp, D. J., and Vale, R. D. (2002). Drosophila EB1
is important for proper assembly, dynamics, and positioning of the mitotic
spindle. J. Cell Biol. 158, 873–884.
Rusan, N. M., Fagerstrom, C. J., Yvon, A. M., and Wadsworth, P. (2001). Cell
cycle-dependent changes in microtubule dynamics in living cells expressing
green fluorescent protein-alpha tubulin. Mol. Biol. Cell 12, 971–980.
Sanchez-Alcazar, J. A., Rodriguez-Hernandez, A., Cordero, M. D., Fernandez-
Ayala, D. J., Brea-Calvo, G., Garcia, K., and Navas, P. (2007). The apoptotic
microtubule network preserves plasma membrane integrity during the exe-
cution phase of apoptosis. Apoptosis 12, 1195–1208.
Sawin, K. E., Lourenco, P. C., and Snaith, H. A. (2004). Microtubule nucleation
at non-spindle pole body microtubule-organizing centers requires fission
yeast centrosomin-related protein mod20p. Curr. Biol. 14, 763–775.
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer,
A. E., and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow
fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat.
Biotechnol. 22, 1567–1572.
CDK5RAP2 Associates with EB1
Vol. 20, August 15, 20093669
Slep, K. C., Rogers, S. L., Elliott, S. L., Ohkura, H., Kolodziej, P. A., and Vale,
R. D. (2005). Structural determinants for EB1-mediated recruitment of APC
and spectraplakins to the microtubule plus end. J. Cell Biol. 168, 587–598.
Tirnauer, J. S., Grego, S., Salmon, E. D., and Mitchison, T. J. (2002). EB1-micro-
tubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabiliza-
tion and mechanisms of targeting to microtubules. Mol. Biol. Cell 13, 3614–3626.
Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P., and Cassimeris, L.
(1997). Nanomolar concentrations of nocodazole alter microtubule dynamic
instability in vivo and in vitro. Mol. Biol. Cell 8, 973–985.
Vaughan, K. T. (2005). TIP maker and TIP marker; EB1 as a master controller
of microtubule plus ends. J. Cell Biol. 171, 197–200.
Vitre, B., Coquelle, F. M., Heichette, C., Garnier, C., Chretien, D., and Arnal,
I. (2008). EB1 regulates microtubule dynamics and tubulin sheet closure in
vitro. Nat. Cell Biol. 10, 415–421.
Yamashita, Y. M., Jones, D. L., and Fuller, M. T. (2003). Orientation of
asymmetric stem cell division by the APC tumor suppressor and centrosome.
Science 301, 1547–1550.
K.-W. Fong et al.
Molecular Biology of the Cell3670