Full text of DOI:10.1093/oxfordjournals.hmg.a018911

© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 15 2205–2213
ARTICLE
Interaction between LIS1 and doublecortin,
two lissencephaly gene products
Michal Caspi, Roee Atlas, Ayelet Kantor, Tamar Sapir and Orly Reiner+
Department of Molecular Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel
Received 7 June 2000; Revised and Accepted 24 July 2000
Mutations in either LIS1 or DCX are the most common cause for type I lissencephaly. Here we report that LIS1
and DCX interact physically both in vitro and in vivo. Epitope-tagged DCX transiently expressed in COS cells
can be co-immunoprecipitated with endogenous LIS1. Furthermore, endogenous DCX could be co-immuno-
precipitated with endogenous LIS1 in embryonic brain extracts, demonstrating an in vivo association. The two
protein products also co-localize in transfected cells and in primary neuronal cells. In addition, we demonstrate
homodimerization of DCX in vitro. Using fragments of both LIS1 and DCX, the domains of interaction were
mapped. LIS1 and DCX interact with tubulin and microtubules. Our results suggest that addition of DCX and
LIS1 to tubulin enhances polymerization in an additive fashion. In in vitro competition assays, when LIS1 is
added first, DCX competes with LIS1 in its binding to microtubules, but when DCX is added prior to the addition
of LIS1 it enhances the binding of LIS1 to microtubules. We conclude that LIS1 and DCX cross-talk is important
to microtubule function in the developing cerebral cortex.
INTRODUCTION
a large group of proteins known to be involved in multiple
protein interactions (9). Although LIS1 was isolated as a
subunit of platelet-activating factor acetylhydrolase Ib (10),
the protein also interacts with microtubules and affects micro-
tubule dynamics (11). The product of the doublecortin gene
associates with and stabilizes microtubules (12–14). We have
identified the tubulin-binding domain of DCX and found it to
be an evolutionarily conserved repeat (15). Interestingly, most
lissencephaly-causing amino acid substitutions in DCX cluster
within the repeated domain (15). Our combined results emphasize
the importance of LIS1–microtubule and DCX–microtubule
interactions during normal and abnormal brain development.
As LIS1 and DCX have similar functions, we raised the
hypothesis that they may interact directly. Our results suggest
that LIS1 and DCX are co-expressed, interact and can function
in the same protein complex in the developing brain.
Development of the human brain is a complex process, which
initiates in the prenatal stage and is completed in the first years
of childhood. Corticogenesis involves processes such as
proliferation, migration and differentiation that leads to the
formation of the mature convoluted cortex. Defects in neuronal
migration may result in lissencephaly that is a severe brain
malformation characterized by the lack of most gyri and sulci.
There are two types of lissencephaly: type I or ‘classical lissence-
phaly’, where four layers of abnormally positioned neurons are
observed in the neocortex, and type II or ‘cobblestone’
lissencephaly, where the cortex is unlayered (1). Mutations in
two different genes may result in type I lissencephaly: LIS1, an
autosomal gene located on chromosome 17p13.3 (2), and
doublecortin, an X-linked gene (3,4). The phenotypic presen-
tation of the patients is very similar, with delayed development
and magnetic resonance imaging (MRI) analysis revealing
both agyria and pachygyria. However, whereas the frontal
brain is more affected in patients with mutations in
doublecortin, the parietal and occipital cortex are more
affected in the case of mutations in LIS1 (5). Mutations in the
X-linked DCX gene result in lissencephaly in males or subcortical
laminar heterotopia, i.e. ‘double cortex’, in females (3,4). The
milder phenotype seen in females consists of a brain with bilateral
plates or bands of gray matter located beneath the cortex and
ventricle but separated from both, hence the descriptive term
double cortex (6). Mutations in LIS1 are hemizygous and the
disease results from reduced gene dosage (2). The primary
amino acid sequence reveals that LIS1 contains seven WD
(tryptophan–aspartic acid) repeats (2,7,8), positioning LIS1 in
RESULTS
Co-expression and co-localization of LIS1 and DCX
To follow expression of LIS1 and DCX during brain development
we examined RNA expression in the dorsal telencephalon of
mouse embryos beginning at embryonic day E10.5–E15.5,
when the process of neurogenesis is active. Although LIS1 is
clearly expressed at all time points examined, the expression of
DCX initiates at E11.5 and increases later on (Fig. 1). This
result reflects the notion that both genes are expressed in the
developing brain as has been shown (13,16). Next, we examined
whether the two protein products are co-expressed in the same
+To whom correspondence should be addressed. Tel: +972 8 9342319; Fax: +972 8 9344108; Email: orly.reiner@weizmann.ac.il

2206 Human Molecular Genetics, 2000, Vol. 9, No. 15
neurons. As can be seen (Fig. 2), the staining in neurons suggested
co-localization of both proteins. However, the co-localization in
the neurons is partial: quite strikingly, LIS1 antibodies react
with thin neurites that DCX antibodies do not recognize. Both
antibodies stain the soma and the thick neurites. Nevertheless,
DCX immunostaining is lacking from at least one of the neurites
that may be the future axon, suggesting polarity in DCX neuronal
localization. In order to further investigate the co-localization, we
transiently transfected COS-7 cells that endogenously express
LIS1 with FLAG-tagged DCX and immunostained them. The
transfected cells depict a significant overlap in the staining
pattern (Fig. 3). The DCX-transfected cells exhibit a marked
change in their morphology in comparison with untransfected
cells, caused by the bundled microtubules. This co-localization
prompted us to investigate further potential interactions
between LIS1 and DCX.
Figure 1. Expression of Lis1 and DCX RNA in the dorsal telencephalon. Total
RNA (7.5 µg) from the dorsal telencephalon of several mouse embryos from
different embryonic days (E10.5–E15.5) was loaded onto an agarose gel and
northern blotted. The blot was probed with Lis1, DCX and GAPDH probes as
indicated. The four Lis1 mRNA species are indicated by arrowheads. DCX
expression is visible at E11.5. Loading was assessed by hybridization with
GAPDH.
Figure 2. Cellular localization of DCX and LIS1 proteins in neurons. Rat hippocampal primary neurons were co-stained with rabbit anti-LIS1 (affinity purified)
and mouse anti-DCX antibodies. (A, D and G) Primary mouse anti-DCX antibodies and secondary anti-mouse–rhodamine. (B, E and H) Primary rabbit anti-LIS1
and secondary anti-rabbit–FITC. (J) Primary mouse anti-DCX and secondary anti-mouse–FITC. (K) Primary rabbit anti-LIS1 and secondary anti-rabbit–rhodamine.
(C, F, I and L) Computer-derived overlay. (A–F) Olympus microscope; 100× magnification; (G–L) confocal microscope; 40× magnification.

Human Molecular Genetics, 2000, Vol. 9, No. 15 2207
Figure 4. LIS1–DCX interaction using surface plasmon resonance. LIS1 was
immobilized to the sensor chip at two densities [7700 and 6200 resonance
units (RU)]. The time is in minutes. A blank channel was prepared by a flow
buffer instead of protein solution during the immobilization phase. GST–DCX
(5 mM) was introduced to the immobilized LIS1 at a constant flow rate of
10 ml/min. As can be seen in the two channels GST–DCX is bound by the
LIS1 protein in a dose-specific manner.
Figure 3. Cellular localization of DCX and LIS1 in COS-7 cells. COS-7 cells
were transfected with FLAG–DCX and fixed after 48 h. Co-staining was
performed with either anti-LIS1 and anti-FLAG antibodies or anti-LIS1 and anti-
DCX. (A and D) Primary rabbit anti-LIS1 and secondary anti-rabbit–FITC.
(B and E) Primary mouse anti-FLAG and secondary anti-mouse–rhodamine.
(G) Primary rabbit anti-LIS1 and secondary anti-rabbit–rhodamine. (H) Primary
mouse anti-DCX and secondary anti-mouse–FITC. (C, F and I) Overlap of co-
staining.
LIS1 and DCX interact in vitro
Several assays performed with recombinant proteins demon-
strated a possible interaction between LIS1 and DCX. We used
BIAcore, a system that measures changes in surface plasmon
resonance that are proportional to the changes in the mass of
molecular species bound to the surface (17–20). The mass
differences measured reflect the kinetic events including asso-
ciation and dissociation of complexes that are composed of
immobilized molecules and other molecules brought into
contact with them in the flow cell. Binding of DCX to immobilized
LIS1 is specific and dose dependent as it is proportional to the
amount of bound LIS1 protein (ligand levels) (Fig. 4). This
was the first indication that LIS1 and DCX can interact directly
without additional mediators.
Figure 5. Interaction of DCX and LIS1 in vitro using pull-down assays.
(A) Schematic presentation of the different recombinant GST–DCX
fragments used in the experiment. (B) 6×His–LIS1 translated in a rabbit
reticulocyte system labeled with [35S]methionine and pulled-down by GST–DCX
and GST control. (C) 6×His–LIS1 expressed and purified from insect cells
was pulled down by GST fusion full-length and shorter fragments of DCX.
(D) LIS1 was also pulled down from embryonic brain extract. (E) Reciprocally,
DCX from embryonic brain extract was pulled down by 6×His–LIS1. Localization
of the interaction site was done by using GST–pep(1), –pep(2) and –pep(1+2)
of DCX (C and D). From both panels it is clear that the minimal fragment
required for this in vitro interaction is pep(1) (amino acids 51–135) of DCX.
Mapping of interaction domains
LIS1–DCX heterodimerization and DCX homodimerization.
To map the region within DCX that interacts with LIS1, we
used several glutathione S-transferase (GST)–DCX fragments
(schematic presentation shown in Fig. 5A): full-length DCX
(amino acids 1–360), amino acids 110–360, amino acids 1–213
(12), pep1 (amino acids 51–135), pep2 (amino acids 178–259),
and pep(1+2) (amino acids 51–259) (15). Full-length GST–DCX
pulled down recombinant LIS1 (Fig. 5B), as well as in vitro
translated LIS1 (Fig. 5C) and endogenous LIS1 from embryonic
brain extract (Fig. 5D). The smallest portion of DCX that pulls

2208 Human Molecular Genetics, 2000, Vol. 9, No. 15
Figure 6. In vitro DCX homodimerization. (A) 6×His–DCX expressed and
purified from bacterial cells was pulled down by GST fusion full-length and
shorter fragments of DCX (pep1, pep2 and pep1+2). GST was used as a
negative control. (B) FLAG-tagged DCX was transfected to 293T cells and
pulled down by GST fusion full-length and shorter fragments of DCX (pep1,
pep2 and pep1+2). GST was used as a negative control. From both panels it is
clear that the minimal fragment required for this in vitro interaction is pep(1)
(amino acids 51–135) of DCX.
down LIS1 is pep1. Our previous results (15) identified this
region as an evolutionarily conserved domain. This peptide
was also shown to pull down tubulin from brain extract, but did
not pull down purified tubulin (15). The peptide probably
recruited additional proteins from the extract that mediated its
interaction with tubulin. It may be that LIS1 mediates pep1–
tubulin interaction.
In addition, we detected in vitro homodimerization of
DCX. This was demonstrated by pull-down of recombinant DCX
tagged with 6×His or GST (Fig. 6A), and in COS cell extracts
where FLAG–DCX was pulled down by GST–DCX (Fig. 6B).
The homodimerization domain was mapped to the pep1
domain of DCX as described previously (Fig. 6). Our finding
of a common interaction region responsible for DCX
homodimerization and LIS1–DCX interaction predicted that
LIS1 and DCX compete.
Figure 7. Localization of DCX interaction site within the LIS1 protein. The
different WDs were translated in a rabbit reticulocyte system labeled with
[35S]methionine and pulled down by GST–DCX, GST and beads control.
(A) Schematic presentation of the different LIS1 peptides used in the experiment.
(B) The percentage of each LIS1 fragment pulled down by DCX was calculated
using densitometer analysis. (C) An autoradiograph of the N-terminal and
WD7 peptides (WD4 was omitted due to high background binding).
In a similar fashion, we mapped the reciprocal regions in
LIS1 that are important for DCX binding. Previous studies
with LIS1 have indicated that simple truncations result in a
protein that is not folded properly (21). However, our recent
work suggests that LIS1 is a β-propeller similar to the β-subunit of
G proteins (21,22) and therefore it is likely that each WD will
retain its structure as an anti-parallel β sheet. Based on this
assumption, we cloned individual WD domains of LIS1 and
the N-terminal portion (schematic presentation shown in
Fig. 7A), translated them in vitro and used GST–DCX in a
pull-down experiment. The results were plotted as the percentage
of input protein that was pulled down by GST–DCX. According
to this type of analysis the main WD repeat that participates in
the LIS1–DCX interaction is WD7.
Figure 8. Interaction of DCX and LIS1 in vivo. (A) 293T cells were transfected
with normal and mutated FLAG–DCX constructs. Immunoprecipitation assay
was performed after 48 h using anti-FLAG agarose beads. The reactions were
immunoblotted and detection was done using monoclonal anti-LIS1 antibodies
(clone 210). All the different mutant, as well as the normal, DCX proteins
interact with endogenous LIS1. (B) The level of expression of the different
FLAG–DCX constructs by western blot of the extract using anti-FLAG antibodies.
(C) Immunoprecipitation of DCX from embryonic brain extract was performed
with anti-LIS1 monoclonal antibodies (clone 338). A western blot analysis
detected the presence of DCX in the immunoprecipitated material using rabbit
anti-DCX antibodies.
LIS1 and DCX interact in vivo. To examine LIS1–DCX inter-
action in vivo, 293T cells were transiently transfected with
PECE–FLAG–DCX constructs. The constructs harbor normal
DCX or mutations that were identified in patients (T203R, R192W,
Y125H, D62N, R59L and S47R) (3,4,15). Immunoprecipitation
using anti-FLAG antibodies successfully precipitated endo-
genous LIS1 from transfected cells, but not from mock-
transfected cells (Fig. 8A). DCX mutations were expressed, as

Human Molecular Genetics, 2000, Vol. 9, No. 15 2209
Figure 9. DCX and LIS1 affect tubulin assembly rate in vitro. The assembly rate of tubulin was measured using a light scattering assay. The concentration of tubulin
used was 15 mM. The optical absorbance was recorded every minute for 40 min. The recombinant proteins used were GST, GST–DCX and GST–LIS1, each at a
final concentration of 7.5 mM. The recombinant proteins were added prior to the measurement. Addition of the two proteins together (each at a concentration of
7.5 mM) shows that they have an additive effect on tubulin polymerization.
demonstrated by western blot using anti-FLAG antibodies
(Fig. 8B).
common sites, the additive affect may be explained by altered
binding affinities.
Next we wanted to test whether LIS1–DCX interactions
occurred in the developing mouse brain. LIS1 from embryonic
brain extracts was immunoprecipitated using mouse mono-
clonal anti-LIS1 antibodies, protein A/G beads or anti-GAD
antibodies (data not shown). Co-immunoprecipitation with
DCX was checked by immunoblotting with anti-DCX anti-
bodies (Fig. 8C). A DCX-immunoreactive band was detected
specifically in the anti-LIS1 precipitate but not in the control
precipitates. These studies suggest that LIS1 and DCX form a
complex in the developing brain.
To differentiate between these two possibilities we designed
a competition assay where both proteins were added to pre-
assembled microtubules. In the first experiment, increasing
amounts of DCX were added to constant amounts of LIS1
(Fig. 10A); whereas in the second experiment, increasing
amounts of LIS1 were added to constant amounts of DCX
(Fig. 10B). The distribution of LIS1 between the insoluble
(pellet) microtubule fraction and the soluble (supernatant)
fraction was examined by western blot analysis. Interestingly,
the results changed depending on the order in which the
components were added. When increasing amounts of DCX
were added to constant amounts of LIS1, DCX competes LIS1
out and more LIS1 was found in the supernatant fraction
(Fig. 10A). When increasing amounts of LIS1 were added to
constant amounts of DCX, the affinity of LIS1 to microtubules
increased and more LIS1 was found in the pellet fraction
(Fig. 10B). Addition of LIS1 did not change the affinity of
DCX to the microtubule cytoskeleton (data not shown), and
DCX was found mainly in the pellet fraction. In these experi-
ments the net amount of polymerized microtubules did not
change (Fig. 10C).
LIS1, DCX and tubulin. To explore whether LIS1 and DCX
affect microtubule formation in an additive or competitive
way, we measured the assembly rate of tubulin using a light
scattering assay (Fig. 9). This assay is based on the increase in
optical scattering as microtubules polymerize, measured as an
effective optical density. Addition of DCX to tubulin increased
the assembly rate of tubulin as shown previously (12). Addition of
LIS1 to tubulin without DCX also increased the polymerization
rate of microtubules (Fig. 9). When LIS1 was added to tubulin
in the presence of DCX the assembly rate of tubulin increased
in an additive fashion (Fig. 9). If LIS1 and DCX share the same
tubulin binding sites we would expect the net result not to be
additive since the proteins compete for the same interaction
region. The results of this experiment suggest that LIS1 and
DCX bind to different sites on tubulin or that they share
common sites. If the latter possibility is correct and they share
DISCUSSION
To investigate molecular mechanisms involved in lissencephaly,
we tested for an interaction between LIS1 and DCX using
several independent methods. The notion that two genes,

2210 Human Molecular Genetics, 2000, Vol. 9, No. 15
The actual interaction of the two gene products was demon-
strated as the recombinant proteins interacted in the BIAcore
and pull-down assays. Mapping of the interaction domains was
feasible using pull-down assays with short peptides of either
LIS1 or DCX. The minimal interacting regions are the last WD
repeat in LIS1 (WD7) and pep1 in DCX. In addition to DCX
heterodimerization, we also showed that DCX is capable of
homodimerization in vitro. The interaction domains of DCX
with both DCX or LIS1 were mapped to an evolutionarily
conserved region (pep1, amino acids 52–133) (15), which
includes the tubulin binding site as well. The existence of such
a complex in vitro suggests that it may also function in vivo.
Indeed, the LIS1–DCX interaction in vivo was demonstrated
by co-immunoprecipitation of transiently expressed DCX in
transfected cells with endogenous LIS1. Moreover, the two
proteins were co-immunoprecipitated in embryonic brain
extracts that express both LIS1 and DCX. In transfected cells,
we tested the interaction of six naturally occurring pathogenic
mutations of DCX with LIS1. Some of the mutations were not
expected to alter interactions because they are located outside
the interaction domain mapped using the pull-down experiments.
However, point mutations may affect protein folding; there-
fore, we thought that it may be useful to test all the mutated
proteins we had. The mutations that we tested did not interfere
with the association between LIS1 and DCX.
We have previously demonstrated that both LIS1 and DCX
interact with tubulin therefore, we cannot exclude the possibility
that in vivo interactions between DCX and LIS1 are mediated
by tubulin. Thus, we tested the effect of both proteins on
microtubule polymerization. Using a light scattering assay it
was observed that the addition of both proteins result in an
additive effect on microtubule polymerization. This suggested
two possibilities: (i) LIS1 and DCX have different binding
sites thus their binding is additive; or (ii) these proteins share
regions but perhaps the interaction of one with tubulin changes
the binding affinity of the other. In order to distinguish
between these possibilities we designed a competition assay
where increasing concentrations of DCX were added to constant
concentrations of LIS1 with pre-assembled microtubules or vice
versa. Interestingly, the results were dependent on the order of
the added components (a schematic model is shown in Fig. 11).
In this set of experiments LIS1 was usually found associated
with the microtubule pellet fraction (Fig. 11A); however, when
DCX was added it competed with LIS1 and more was found in
the soluble fraction (Fig. 11B). DCX, on the other hand was
completely associated with microtubules regardless of LIS1
concentration (Fig. 11C and D). When LIS1 was added to the
microtubule–DCX complex a larger amount of LIS1 was then
associated with the microtubule pellet fraction (Fig. 11D). This
alteration in the binding affinity of LIS1 may be due to a
conformational change in the microtubles themselves
(Fig. 11C and D). Can this experiment reflect in vivo behaviour
of these molecules? Can the presence of one molecule affect
the function of the other? Although this indeed is far fetched,
there may be some evidence to support this hypothesis. In COS
cells transfected with DCX, we noticed a shift of endogenous
LIS1 (usually partially associated with microtubules) to be
more associated with microtubules. This question will be
addressed more directly in the future using DCX transgenic mice.
Is LIS1–DCX interaction relevant to the lissencephaly
phenotype? We believe that it is. A recent study (30) of
Figure 10. LIS1, DCX and tubulin competition assays. The relative distribution of
LIS1 in the soluble fraction (s) and microtubule-associated pellet (p) was
determined by western blot analysis using anti-LIS1 monoclonal antibodies.
(A) Constant concentration of LIS1 (2.5 µM) was added to pre-assembled
microtubules, then increasing concentrations of DCX was added to the mixture.
Note that with the addition of 1.5 µM DCX, LIS1 shifts to the soluble fraction.
(B) Constant concentration of DCX (2.5 µM) was added to pre-assembled micro-
tubules, then increasing concentrations of LIS1 were added to the mixture.
Pre-incubating the pre-assembled microtubules with DCX result in the presence of
more LIS1 in the pellet fraction [for example, compare 2 µM DCX and
2.5 µM LIS1 in (A) with 2 µM LIS1 and 2.5 µM DCX in (B)]. (C) A
representative gel showing microtubule polymerization. The gels were stained
with Coomassie brilliant blue and the relative amounts of tubulin in the pellet
and supernatant are shown.
which when mutated give rise to a similar phenotype, can
physically interact has been demonstrated in the past. Two
gene products involved in autosomal dominant polycystic
disease, PKD1 and PKD2, interact via similar coiled-coil
domains in the C-terminal region (23,24). Naturally occurring
pathogenic mutations in PKD1 and PKD2 disrupt their associa-
tions (23). An additional example are two genes involved in
tuberous sclerosis (TSC) an autosomal dominant disorder
characterized by a broad phenotypic spectrum that includes
seizures, mental retardation, renal dysfunction and dermato-
logical abnormalities. Mutations in the TSC1 and TSC2 genes
were found to be responsible for the disease. The two gene
products can interact with each other (25,26). Careful investi-
gations suggested the presence of additional proteins within
this complex (27). It has been proposed that one of these
proteins modifies the self-aggregation of the other.
We have shown that LIS1 and DCX are co-expressed in the
developing brain. Furthermore, the two proteins co-localize in
transfected COS cells and partially co-localize in primary
neurons. In neuronal cells it was observed that DCX localization
is polar. We plan to investigate this issue more carefully in the
future, as polarized morphology of neurons, typified by a
single long axon and several short dendrites is a fundamental
morphological feature (28). LIS1 immunostaining, on the other
hand, was obvious in very fine dendrites. This may be related
to the recent observation that heterotopic pyramidal neurons in
Lis1+/– mice were stunted and possessed fewer dendritic
branches (29).

Human Molecular Genetics, 2000, Vol. 9, No. 15 2211
Figure 11. A schematic model for the LIS1–DCX–microtubule interaction. (A) When LIS1 was added to pre-assembled microtubules most of the LIS1 molecules
(dimers or monomers) interacted with microtubules. (B) DCX competes with LIS1 and less LIS1 is found in the microtubule-associated fraction. (C) When DCX
was added to pre-assembled microtubules most of the DCX molecules interacted with microtubules. (D) On the other hand, increasing concentrations of LIS1 added
to the DCX–microtubule complexes caused LIS1 to bind microtubules with a higher affinity (plausibly due to a conformational change that may be in the microtubule
moiety). As a result, more of LIS1 is bound to microtubule.
5′-GAAGATCTTCAATCCCATAGTTTAATGGTCATA TC-3′;
immunohistochemical expression of DCX demonstrated
abnormal localization in Miller–Dieker syndrome (involving
LIS1 deletion) and Fukuyama congenital muscular dystrophy
but not in other migration disorders. This result taken together
with our findings provides further evidence for a possible
endogenous function for LIS1–DCX–tubulin complex.
WD3, 5′-ATGCATCCATGGTTTCAGGGCTTTGAATGCATCAGA-3′ and
5′-GAAGATCTTCATTCCCACATT TTTATAGTTTTATC-3′;
WD4, 5′-ATGCATCCATGGGTGCAAACTGGCTACTGTGTGAAG-3′ and
5′-GAAGATCTT CAGACCCATACACGCACAGTCTGGTC-3′;
WD5, 5′-ATGCATCCATGGGTAGCAACAAAGGAATGCAAGGCT-3′ and
5′-GAAGATCTTCAATCCCACATCTTAATAGTCTTGTC-3′;
WD6, 5′-ATGCATCCATGGGTCAGTACTGGCATGTGCCTTATG-3′ and
5′-GAAGATCTTCAATCCCATACGCGTAGGGTCTTGTC-3′;
WD7, 5′-ATGCATCCATGGTACAAGAACAAGCGATGCATGAAG-3′ and
5′-GAAGATCTTCAACGGCACTCCCACACTTTTACTGT-3′.
MATERIALS AND METHODS
All fragments were then ligated into the expression vector
pRSET (Invitrogen, Leek, The Netherlands). FLAG-tagged
and GST-tagged DCX constructs were described previously
(12,15).
Northern blot
Total mRNA for northern analysis was isolated using Tri Reagent
(Sigma, Rehovot, Israel) according to the manufacturer’s
protocol. Northern blots were performed as described by
Ausubel et al. (31).
Antibodies
Mouse polyclonal anti-doublecortin antibodies were produced
against GST–DCX (12) using conventional protocols (32).
Rabbit polyclonal anti-LIS1 antibodies (R19) were affinity
purified (11). Anti-FLAG M2 monoclonal antibodies were
purchased from Sigma. All secondary antibodies were from
Jackson Immunoresearch (West Grove, PA); peroxidase-
conjugated affinipure goat anti-mouse IgG (H+L), lissamine
rhodamine-conjugated affinipure goat anti-mouse or anti-rabbit
IgG (H+L), FITC-conjugated affinipure goat anti-mouse or
anti-rabbit IgG (H+L).
Cell culture
COS-7 and 293T cells were grown at 37°C with 5% CO2 and
95% air in DMEM nutrient medium supplemented with 10%
fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and
0.1 mg/ml streptomycin. Cultures were split using standard
trypsinization procedures. Cells were transfected using the
BES transfection procedure (31). Transfected cells were
harvested or stained after 48 h. Efficiency of transfections was
estimated using an EGFP expression vector (Clontech, Palo
Alto, CA).
Immunostaining
Briefly, transfected COS-7 cells were plated on glass coverslides.
After 48 h they were washed twice with phosphate-buffered
saline (PBS), then fixed and permeabilized simultaneously as
described (11). Primary neurons derived from embryonic rat
hippocampus were obtained from Dr Tony Futerman. The
neurons were fixed for 20 min with 4% PFA, 4% sucrose at
Constructs
The different WDs were amplified by PCR using the following
primers:
WD1, 5′-ATGCATCCATGGATTCCCCGTCCGCCAGAAAAATAT-3′ and
5′-GAAGATCTTCAATCCCACACCTTAATTGTAGCATC-3′;
WD2, 5′-ATGCATCCATGGTATGAGACTGGAGATTTTGAACGA-3′ and

2212 Human Molecular Genetics, 2000, Vol. 9, No. 15
37°C and permeabilized for 5 min with 0.25% Triton X-100.
After fixation the cells were incubated with 30 ml of the first
antibody for 60 min at room temperature, then washed three
times in PBS and incubated for 60 min with 30 ml of fluorescent-
conjugated secondary antibodies. The coverslips were washed
three times in PBS, drained and mounted. The immunostaining
was visualized using an Olympus microscope (IX50 model;
Hamburg, Germany) using the appropriate filters. Photography
was with Kodak 160T film. Neurons were observed under a
Bio-Rad (Hercules, CA) confocal microscope.
at 4°C and then rotated with 20 ml A/G protein beads for 2 h at
4°C. Beads were washed four times with T-T or IP buffer,
respectively. Sample buffer (×2) was added and the beads were
boiled and subjected to an SDS–PAGE gel. A western blot was
performed with anti-LIS1 (monoclonal clone 210) or anti-DCX
antibodies.
Rate measurements
Tubulin was purified as described (11). The rate of tubulin
assembly into polymers was monitored using a light scattering
assay (12,15). Purified tubulin was diluted in PEM buffer
(100 mM PIPES pH 6.9, 1 mM MgSO4, 1 mM EGTA) supple-
mented with 1 mM GTP, to a final concentration of 15 µM.
GST–DCX, GST–LIS1 and GST (7.5 µg) were added to the
purified tubulin subunits prior to measurement. Absorbance
was measured at 350 nm at 1 min intervals in a Uvicon spectro-
photometer (Kontron Instruments, Vineland, NJ) equipped
with temperature controlled cells. Switching the temperature to
37°C induced assembly.
GST pull-down assay
GST fusion proteins were used for pull-down experiments with
either recombinant LIS1 protein or brain extract. Recombinant
6×His–LIS1was expressed in insect cells by Baculovirus
[described by Sapir et al. (11)] and purified on Ni-NTA beads
(Qiagen, Hilden, Germany). LIS1 and the different WD
peptides were produced in the Rabbit Reticulocyte TnT
System (Promega, Madison, WI) with [35S]methionine. In each
pull-down experiment, 10 ml of the labeled reaction was used.
E17 brain extract was prepared in T-T buffer (20 mM Tris–
HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.1% Triton-X),
supplemented with protease inhibitors (Sigma).
Recombinant proteins or brain extract were incubated with
10 mg of GST–DCX, GST–pep1, GST–pep2, GST–pep(1+2)
and GST as control at 4°C for 15 h. Glutathione beads (20 ml)
in 300 ml of T-T buffer were added to the protein mixture and
rotated for 30 min at room temperature. After four washes with
T-T buffer, 2× sample buffer was added and the beads were
boiled and subjected to SDS–PAGE gel analysis. A western
blot was performed with anti-LIS1 antibodies (monoclonal
clone 210).
Competition assay
Sedimentation experiments were performed essentially as
described (13). After microtubules (5 µM) were pre-assembled
for 15 min at 37°C, GST–LIS1 and GST–DCX proteins were
added. In each experiment one protein was at a constant
concentration (2.5 µM) and the other at increasing concentra-
tions (0–5 µM). All reactions were divided into three: 60% for
Coomassie staining and 10% for western blot analysis with
anti-DCX and anti-LIS1 antibodies, respectively.
ACKNOWLEDGEMENTS
The authors thank Dr Tony Futerman for the neurons. This
work was supported in part by the Minerva Foundation, BSF
grant no. 97-00014 and HFSP grant no. RG283199. O.R. is an
Incumbent of Aser Rothstein Career Development Chair in
Genetic Diseases.
6×His pull-down assay
Fifteen milligrams of 6×His–LIS1 were incubated with embryonic
brain extract (prepared as described) at 4°C for 15 h. Ni–NTA
beads (20 mg; Qiagen) in 400 ml of sonication buffer (300 mM
NaCl, 50 mM sodium phosphate buffer pH 8.0) with 10 mM
imidazole were added to the protein mixture and rotated at 4°C
for 3 h. The beads were washed four times with sonication
buffer with 30 mM imidazole and eluted with 20 µl of sonication
buffer and 200 mM imidazole. After elution 5× sample buffer
was added and the beads were run on an SDS–PAGE gel. A
western blot was performed with mouse polyclonal anti-DCX
antibodies.
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