© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience
ARTICLES
Calcium-dependent interaction of Lis1 with IQGAP1
and Cdc42 promotes neuronal motility
Stanislav S Kholmanskikh1, Hajira B Koeller1, Anthony Wynshaw-Boris2, Timothy Gomez3, Paul C Letourneau4
& M Elizabeth Ross1
Lis1 gene defects impair neuronal migration, causing the severe human brain malformation lissencephaly. Although much is
known about its interactions with microtubules, microtubule-binding proteins such as CLIP-170, and with the dynein motor
complex, the response of Lis1 to neuronal motility signals has not been elucidated. Lis1 deficiency is associated with deregulation
of the Rho-family GTPases Cdc42, Rac1 and RhoA, and ensuing actin cytoskeletal defects, but the link between Lis1 and Rho
GTPases remains unclear. We report here that calcium influx enhances neuronal motility through Lis1-dependent regulation of
Rho GTPases. Lis1 promotes Cdc42 activation through interaction with the calcium sensitive GTPase scaffolding protein IQGAP1,
maintaining the perimembrane localization of IQGAP1 and CLIP170 and thereby tethering microtubule ends to the cortical actin
cytoskeleton. Lis1 thus is a key component of neuronal motility signal transduction that regulates the cytoskeleton by complexing
with IQGAP1, active Cdc42 and CLIP-170 upon calcium influx.
Rho-family GTPases are thought to regulate cell motility through a general scheme in which Cdc42 promotes cell polarization, Cdc42 and Rac1
promote the extension of filopodia and lamellipodia, respectively, at the
leading edge, and RhoA favors actin-myosin contraction in the cell body
and trailing process1–3. RhoA activity is elevated and Cdc42/Rac1 activities are depressed in association with defects in F-actin at the leading
edge of migrating neurons that are deficient in the neuronal migration
protein Lis1 (ref. 4). However, the mechanisms by which Lis1 influences
small GTPases and the neuronal cytoskeleton remain to be elucidated.
Calcium (Ca2+) signaling is another important regulator of cell
motility, including neuronal movement and growth cone guidance5–8,
in a wide range of cell types. One way in which Ca2+ might signal
through Lis1 is in the coordinate regulation of actin polymerization
and microtubule growth9,10. In considering whether Lis1 is also
involved in Ca2+-mediated regulation of neuronal motility,
IQGAP1—a Ca2+/calmodulin regulated protein known to be an
important Ca2+ sensor in cells—may have a key role. IQGAP1 binds
and stabilizes GTP-bound Cdc42 in its active form11 and, like Lis1,
binds directly to CLIP-170 (ref. 12). IQGAP1, in complex with CLIP170, may link microtubule plus-ends and the cortical actin meshwork,
downstream of Cdc42 and Rac1 (ref. 13). Moreover, IQGAP1 links
Cdc42/Rac1 and actin filaments during polarization and migration of
non-neuronal Vero cells14. Thus, the mechanisms regulating neuronal
motility may involve interactions of Ca2+, Lis1, CLIP-170, IQGAP1 and
Rho GTPases.
Because a complete loss of Lis1 leads to early embryonic death,
neurons from Lis1+/– mice15 were used in the present study. We found
that Lis1 was required for the proper activation of Rho GTPases and
actin polymerization at the leading edge of locomoting cerebellar
neurons and postmigratory hippocampal neurons in response to
Ca2+ influx triggered via NMDA receptors (NMDAR). In cerebellarslice culture, granule neurons express and upregulate NMDARs during
granule cell migration16. Moreover, Ca2+ influx triggered by the
NMDAR coagonist glycine can stimulate granule cell migration in a
response that is blocked by NMDAR antagonists or Ca2+ chelation17.
Although functioning NMDARs are not expressed in cerebral cortex
until after neurons reach the cortical plate18,19, NMDARs participate in
the morphogenesis of dendritic branches and spines during synaptogenesis and long-term potentiation (LTP) by a Ca2+-dependent
mechanism that involves Rho GTPases20,21. We used D-serine as a
more specific and probable endogenous agonist of the ‘glycine site’ of
NMDARs during development22–24, to provoke Ca2+ influx and to
stimulate the migration of cultured cerebellar granule neurons and
process elaboration of postmigratory, neonatal hippocampal neurons.
The data supported a model in which Lis1 enhanced neuronal motility
and cytoskeletal regulation through a multimeric complex involving
Lis1, IQGAP1 and Cdc42. This complex responded to Ca2+ influx and
locally promoted actin polymerization, probably integrating F-actin
with microtubules, at the leading edge of the neuron.
RESULTS
Lis1 and Rho GTPases regulate calcium dependent motility
The NMDAR agonist D-serine (10 mM) increased the locomotion
of granule cells from wild-type mice on a poly-D-lysine–laminin
1Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA. 2Department of Pediatrics,
University of California San Diego, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA. 3Department of Neuroscience, University of
Wisconsin Medical School, 1300 University Avenue, Madison, Wisconsin 53706, USA. 4Department of Neuroscience, University of Minnesota, 321 Church Street,
Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to M.E.R. ([email protected]).
Received 22 September; accepted 22 November; published online 20 December 2005; doi:10.1038/nn1619
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+/–
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+/–
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, pCdc42ca
, pLis1
Control
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Above baseline (%)
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Transients (h–1)
© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience
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+/–
0''
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0''
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, D-Serine
High
Lis1+/–,
pCdc42ca
45''
90''
pCdc42dn
f
Low
+/–
Lis1 ,
pLis1
0''
45''
90''
0''
45''
90''
Figure 1 D-Serine requires Lis1 to increase neuronal migration in a Cdc42-dependent manner. Wild-type and Lis1+/– cerebellar granule neuron migration,
compared in the presence and absence of 10 mM D-serine in time-lapse images. (a) Light micrographs followed single neurons (arrows) in successive frames.
D-Serine sped the movement of wild-type but not Lis1+/– neurons. (b) D-Serine increased wild-type migration (n ¼ 78 Lis1+/+, 88 Lis1+/–, 73 Lis1+/+ + D-serine,
114 Lis1+/– + D-Ser), whereas pCdc42dn-transfected wild-type neurons reduced migration to Lis1+/– levels (n ¼ 64) and ablated the response to D-serine
(n ¼ 84 Lis1+/+ transfected, D-serine treated). Expression of pCdc42ca in Lis1+/– neurons restored migration (n ¼ 40) and D-serine further increased migration
rate 10% (n ¼ 30). In Lis1+/– neurons, pEGFPC3Lis1 in the presence of pCdc42dn prevented rescue of neuronal migration (n ¼ 21 Lis1+/–, pLis1; 22 Lis1+/–,
pLis1, pCdc42dn). (c,d) The frequency (c) and amplitude (d) of Ca2+ transients were equally high in Lis1+/– and wild-type granule cells (Lis1+/+ control/treated
n ¼ 87/62, Lis1+/– control/treated n ¼ 72/59) (* P o 0.01). Pseudocolor image shows three frames (15-s intervals) of Fluo-4–loaded neurons. (e) Wild-type
neurons transfected with dominant negative Cdc42 (pCdc42dn) moved little and were no faster with D-serine, whereas Lis1+/– neurons transfected with
constitutively active Cdc42 (pCdc42ca) moved substantially faster. (f) Lis1+/– neurons cotransfected with pCdc42dn blocked pEGFPC3-Lis1 rescue of the
migration defect. (* P o 0.001, wild-type control; d P o 0.001, Lis1+/– control.) Error bars, s.e.m.
substratum (+25%, Supplementary Fig. 1 online) and on processes of
neurons or glia (35% increase, Fig. 1a,b). By contrast, it did not
stimulate the motility of cerebellar granule neurons from Lis1+/– mice
(43% slower than for the wild-type control). Next, we assessed the
effects of D-serine on Ca2+ influx into granule cells using the Ca2+sensitive fluorescent dye Fluo-4 AM and time-lapse microscopy7,25.
Addition of D-serine similarly increased the amplitudes and frequency
of Ca2+ transients in cerebellar granule cells from wild-type and Lis1+/–
littermates (Fig. 1c,d). Therefore, the inability of D-serine to enhance
migration of Lis1-deficient neurons must involve signaling events
downstream from Ca2+ influx.
Rho GTPases are disregulated in Lis1+/– neurons, and this contributes to the migration deficit in these cells4. Here, the effect of plasmidexpressed constitutively active (pCdc42ca) or dominant negative Cdc42
(pCdc42dn) and Lis1 (pLis1) on cerebellar neuronal motility was
examined in dissociated cell culture (Fig. 1b,e,f). We recorded granule
cell migration on processes in the presence or absence of D-serine.
pCdc42dn decreased wild-type neuronal movement by 46%. Notably,
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D-serine
did not enhance the migration of pCdc42dn-transfected wildtype neurons (Fig. 1b,e). Lis1+/– neurons transfected with pCdc42ca
increased their migration rate by 81%, and the addition of D-serine
enhanced this effect only slightly (10%) (Fig. 1b,e). Moreover,
pCdc42dn transfection blocked the ability of pLis1 to rescue the
Lis1+/– migration defect (Fig. 1b,e).
We examined the effects of D-serine on the activities of GTPases of
the Rho family. D-serine increased active Cdc42, Rac1 and RhoA in
wild-type neurons 3.5-, 2.6- and 1.5-fold, respectively, and the intracellular Ca2+ chelator BAPTA-AM or the NMDAR antagonist D-2amino-5-phosphonopentanoate (AP5) blocked these increases
(Fig. 2a–c). Thus, the D-serine stimulation of small GTPases was
NMDAR and Ca2+ dependent. However, compared to the wild type,
Rac1 and Cdc42 were less active in Lis1+/– cells, and their activities did
not significantly increase upon D-serine treatment. RhoA was more
active in Lis1+/– cells, and RhoA activity actually decreased (relative to
untreated Lis1+/–) in response to D-serine (Fig. 2b), suggesting that
normal Lis1 levels were required for D-serine to activate GTPases.
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Cdc42-GTP
RhoA-GTP
b
*
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200
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BAPTA +
D-Serine
AP5 +
D-Serine
BAPTA +
D-Serine
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Cdc42-GTP
D-Serine
100
Control
F-actin and filopodia in Lis1+/– neurons, and this rescue was blocked by
dominant negative Cdc42 (N17Cdc42-GST or –Cdc42) (Fig. 3d–g).
Effects of D-serine were also blocked by –Cdc42, reducing F-actin and
filopodia content in the wild type to Lis1+/– levels, whereas Lis1+/–
neurons failed to respond (Fig. 3h–k).
N.S.
Rac1-GTP
c
Wild type
*
300
Control
Percentage wild type
© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience
actin
Cdc42-GTP
We compared the levels of F-actin in wild-type and Lis1+/– neurons
in response to D-serine–mediated Ca2+ influx after loading with mutant
Cdc42 protein in dissociated neonatal hippocampal neurons. Lis1+/–
neurons contained less F-actin and fewer filopodia than did the wildtype, and constitutively active Cdc42 (L61Cdc42-GST or +Cdc42)
restored these to wild-type levels (Fig. 3a–d). pLis1 also restored
Figure 3 D-Serine and Lis1 regulate F-actin
content in a Cdc42-dependent manner.
(a–i) Neonatal hippocampal neurons were stained
for F-actin. (a) Wild-type neurons had multiple
filopodia (a subset of these are indicated by
arrowheads). (b) Lis1+/– neurons had much fewer
and shorter filopodia. (c) Constitutively active
Cdc42 (+Cdc42) added to Lis1+/– neurons
restored filopodia and F-actin staining. (d) Lis1+/–
neurites contained less F-actin than did the wild
type (Lis1+/–/wild-type control, n ¼ 27/24).
+Cdc42 enhanced F-actin signal in Lis1+/–
neurites (n ¼ 38). pLis1 restored F-actin to
Lis1+/– neurites (n ¼ 15) and –Cdc42 inhibited
this effect (n ¼ 12). (e,f) When added to Lis1+/–
neurons, pLis1 plasmid (pEGFPC3-Lis1) restored
filopodia and F-actin staining. (g) Dominant
negative Cdc42 (–Cdc42) added to pLis1transfected Lis1+/– neurons prevented pLis1 from
promoting actin polymerization or filopodial
numbers. (h) D-Serine enhanced filopodial
numbers in wild-type neurons, but was blocked by
–Cdc42 (i). (j) D-Serine did not increase filopodial
numbers in Lis1+/– neurons. (k) D-Serine (10 mM)
enhanced F-actin fluorescence in wild-type
(n ¼ 24) but not Lis1+/– neurons (n ¼ 49). Added
–Cdc42 blocked the effects of D-serine on F-actin
in wild-type but not Lis1+/– neurons (n ¼ 24 each)
(* P o 0.01 compared to wild-type untreated
control; z P o 0.01 compared to Lis1+/– control).
Error bars, s.e.m.
52
a
Wild type
b
Lis1 impacts cellular distribution of CLIP-170 and IQGAP1
Both Cdc42 and Rac1 interact with IQGAP1 at the leading edge of
motile fibroblasts13, and IQGAP1 can bind to stabilize GTP-Cdc42 and
GTP-Rac1 in the active state26. IQGAP1 is of particular interest because
it is involved in Ca2+/calmodulin-dependent signaling to the cytoskeleton27,28. We compared IQGAP1 distribution in wild-type and Lis1+/–
neurons from neonatal hippocampus (Fig. 4a–i). Fixation conditions
favored the retention of cytoskeletal-associated proteins. In wild-type
cells, antibody to IQGAP1 (anti-IQGAP1) heavily labeled the soma and
the neurite and overlapped with F-actin to the very edge of the cell
margin (Fig. 4a,c). In Lis1+/– neurons, IQGAP1 immunostaining did
not extend into the rim of the plasma membrane (Fig. 4b,d). Although
IQGAP1 immunolabeling heavily decorated the leading process in wildtype neurons, it was virtually absent from neurites of Lis1+/– neurons
(Fig. 4a,b,e,f) as quantified by fluorescence-area measurements
Lis1+/–
c
Lis1+/–
+ Cdc42
d
Threshold area per
–1
neurite length (pixels µm )
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Rac1-GTP
‡
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Wild type
e
Lis1+/–
f
+/–
Lis1
pLis1
g
+/–
Lis1
Lis1+/–
Lis1
pLis1
–Cdc42
+/–
, pLis1
Lis1+/–, pLis1
–Cdc42
Lis1+/–
+Cdc42
k
h
Wild type
D-Serine
i
Wild type
–Cdc42
D-Serine
j
+/–
Lis1
D-Serine
Threshold area per
–1
neurite length (pixels µm )
a
Figure 2 D-Serine activation of Rho GTPases is impaired in Lis1+/– neurons.
(a) D-Serine (10 mM) increased GTP-bound Rac1, Cdc42 and RhoA in wildtype neurons. Disregulated basal Rho GTPase activities in Lis1+/– neurons
either slightly increased or decreased in response to D-serine. (b) As a
percentage of wild-type unstimulated control, Rac1 and Cdc42 activities
were reduced in Lis1+/– cells and changed little upon D-serine treatment,
whereas RhoA activity was increased in Lis1+/– cells and decreased with
D-serine. Significant change is indicated (n ¼ 3 experiments). (c) D-Serine
activation of Rac1 and Cdc42 in wild-type cultures (and RhoA, not shown)
was blocked by either 20 mM BAPTA (intracellular Ca2+ chelator) or 200 mM
AP5 (NMDA-R antagonist). Error bars, s.e.m.
Lis1+/–
Wild type
15
10
*
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Wild type
Figure 4 Lis1 levels modulate IQGAP1 and CLIP-170 distribution. Neonatal
post-migratory neurons from hippocampus or motile cerebellar granule
cells were labeled with anti-IQGAP1 (green, or yellow in the dual labeled,
deconvolved image) and phalloidin (red, F-actin). (a) Wild-type neurites were
speckled with IQGAP1 (arrows). (b) Lis1+/– neurites were simplified and
devoid of IQGAP1. (c) Area boxed in a. Somal IQGAP1 overlapped F-actin
to the edge of the plasma membrane (cortical actin). Dashed lines indicate
cortical actin rim. (d) Boxed area in b. IQGAP1 of a Lis1+/– neuron does not
overlap F-actin at the plasma membrane (dashed lines). (e,f) Enlarged detail
(arrows in a and b, respectively). IQGAP1 in the wild-type neurite (e) was
nearly absent in Lis1+/– process (f). (g) Comparison of IQGAP1 and F-actin
levels in wild-type (n ¼ 42) and Lis1+/– (n ¼ 45) neurons. (h,i) Rescue of
the IQGAP1 defect in Lis1+/– cerebellar neurons by exogenous Lis1. IQGAP1
(green), F-actin (red) and Lis1-GFP (blue). Image deconvolution colocalized
F-actin and IQGAP1 (overlap yellow in h) or Lis1-GFP and F-actin (overlap
white in i). Next to untransfected neurons, a Lis1+/– neuron with pEGFPC3Lis1 accumulated IQGAP1 (green) in the leading neurite (arrows) and at the
inner plasma membrane (upper enlargement) (h). Lis1-GFP fusion protein
in transfected neurons, identified by GFP (blue) immunostaining (i). Arrows
in h and i indicate IQGAP1 and Lis1-GFP colocalization. (* P o 0.01).
Error bars, s.e.m.
Lis1+/–
pLis1
i
F-actin
Lis1+/–
pLis1
normalized to neurite length (Fig. 4g). Next, Lis1+/– granule cells
were transfected with pEGFPC3-Lis1. Untransfected neurites were
devoid of IQGAP1, whereas exogenous Lis1 restored IQGAP1 to the
neurite and colocalized with Lis1-GFP (Fig. 4h,i). Moreover, IQGAP1
labeling extended to the edge of the cell margin of pEGFPC3-Lis1–
transfected Lis1+/– cells but not to that of the untransfected
mutant neurons (enlargements in Fig. 4h). Thus, Lis1 facilitated
IQGAP1 recruitment to the cortical actin meshwork and leading
process of neurons.
IQGAP1 is part of a complex found at the leading edge of
fibroblasts where it directly interacts with Cdc42 and CLIP-170 to
capture microtubule plus-ends into the cortical actin meshwork13.
Microtubule ends exhibiting dynamic instability are associated with a
protein complex called +TIPs (plus-end tracking proteins)29,30. Among
+TIPs, CLIP-170 is a nonmotor protein that transiently links membranes to microtubule ends, thereby regulating molecular motors29,31.
Because IQGAP1 was reduced in Lis1+/– neurons in the leading edge at
the inner surface of the plasma membrane and because Lis1 directly
interacts with CLIP-170, we next examined the distribution of IQGAP1
and CLIP-170 in the insoluble fraction of lysates from cells with or
without 10 mM D-serine. D-Serine substantially shifted both CLIP-170
and IQGAP1 to the insoluble membrane fraction from the soluble
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fraction of wild-type neuronal lysates (Fig. 5a–d). Moreover, when
neurons were protein loaded with wild-type Cdc42 (wtCdc42) or
dominant negative Cdc42 (dnCdc42), the D-serine–induced displacement of CLIP-170 and IQGAP1 to the insoluble fraction was blocked
by dnCdc42 (Fig. 5e,f). Thus the D-serine–triggered shift in the
subcellular distribution of CLIP-170 and IQGAP1 required both Lis1
and the activation of Cdc42.
Lis1 augments IQGAP1, CLIP-170 and GTP-Cdc42 interaction
If Lis1 were indeed required for the D-serine–mediated activation of
Cdc42 and the recruitment of IQGAP1 to the plasma membrane, then
Lis1 might associate with these proteins. Because both Lis1 and
IQGAP1 bind to CLIP-170 (ref. 13,32) and IQGAP1 binds activated
Cdc42 (ref. 26), Lis1 might participate in a multimeric complex. We
sought to coprecipitate proteins with active Cdc42 using beads coated
with the GTP-Cdc42 binding domain of WASP (WASP-CRIB-domain
or WCD), which is specific for GTP-bound Cdc42 and not for Rac1 or
RhoA33. WCD beads precipitated Cdc42, Lis1 and IQGAP1 (Fig. 6a,b)
and CLIP-170 (Supplementary Fig. 2 online). However, other soluble
proteins in the lysates that should not bind to WCD or Cdc42,
including FAK, RhoA and cyclin D2, were not pulled down, indicating
that this binding was specific. The relationship was further supported
by the colocalization of endogenous IQGAP1 and Lis1 at the leading
edge of wild-type fibroblasts and neurons in culture (Fig. 6c–e).
Moreover, binding of Lis1 to IQGAP1 and of CLIP-170 to both Lis1
and IQGAP1 was detected by coimmunoprecipitation with either
antibody to Lis1 (anti-Lis1) or anti-IQGAP1 (Fig. 6f). These independent pull-down methods—WCD beads and immunoprecipitation—
provided strong biochemical evidence for the participation of Lis1 in a
multimeric protein complex with active Cdc42, IQGAP1 and CLIP170, and for its direct binding to both IQGAP1 and CLIP-170. The
interaction of IQGAP1, Lis1 and active Cdc42 was enhanced in
cultured cerebellar granule neurons by D-serine treatment, and the
effect was blocked by the NMDAR antagonist AP5 and by BAPTA-AM
(Fig. 6b). Therefore, Lis1 participation in this complex was dependent
on Ca2+.
The specificity of the WCD complex was further tested by targeted
downregulation of CLIP-170 and IQGAP1 with shRNAi plasmids.
NIH3T3 cells were transfected with pS-shCLIP170 or pRNAT-shIQGAP1 (Fig. 7 and Supplementary Figs. 2 and 3 online). Levels of
IQGAP1 or CLIP-170 were markedly reduced in 3T3 cells expressing
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dnCdc42
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dnCdc42
the appropriate shRNAi (Fig. 7a). Loss of CLIP-170 sharply decreased
the amount of Lis1 and reduced IQGAP1 and Cdc42 in the WCD pulldown (Fig. 7b). Similarly, decreased IQGAP1 reduced Lis1 and Cdc42
in the pull-down (Fig. 7b). Expression of pR-shIQGAP1 in granule cells
reduced the migration speed of wild-type neurons to 55% of that of the
control and blocked the ability of D-serine to enhance migration
(Fig. 7c). Moreover, knockdown of either CLIP-170 or IQGAP1 with
the shRNA constructs slowed wild-type granule neuron migration to
35% and 45%, respectively, that in wild-type controls, and haploinsufficient Lis1+/– neurons were 30% slower than the wild-type neurons
DISCUSSION
Here, Ca2+ influx enhanced neuronal motility through Lis1-dependent
regulation of Rho GTPases and cytoskeletal modulation. The small
GTPases regulate polymerization of actin (F-actin), driving extension
of the leading edge of motile cells1,34. Disregulation of Rho GTPases
disrupts actin organization and dynamics and is part of the migration
deficit in Lis1+/– neurons4. Rho GTPases are also implicated in the
modulation of microtubule polymerization and dynamics10,35. Indeed,
Rac1 and Cdc42 activities are important for the capture of microtubule
SL
a
WCD
250
150
100
IQGAP1
54
250
150
100
FAK
50
Lis1
c
37
25
50
cyclin
37
D2
25
25
Cdc42
20
15
e
25
RhoA
D-Serine
BAPTA/D-Serine
Control
BAPTA/D-Serine
D-Serine
b
d
20
15
Control
Figure 6 Lis1 and IQGAP1 colocalize in cells and coprecipitate with WASPCD agarose (WCD) along with Cdc42-GTP, and by direct immunoprecipitation. (a) Cdc42-GTP bound to WCD also pulled down Lis1 and IQGAP1.
The absence of proteins not expected to interact (RhoA, FAK, and cyclin D2)
confirmed the specificity of the pull-down assay. CLIP-170, which binds Lis1
and IQGAP1, was also in the complex (Supplementary Fig. 2). Note that
less Lis1, Cdc42-GTP and IQGAP1 were pulled down from the Lis1+/– cell
lysates. Straight-lysate (SL) levels of IQGAP1, FAK, total Cdc42, total RhoA
and cyclin D2 were similar between genotypes. (b) In wild-type cerebellar
neurons, D-serine increased IQGAP1, Lis1 and pulled-down Cdc42, and this
was blocked by AP5. Levels in SL were unchanged by treatments. Calcium
chelation with BAPTA-AM blocked D-serine from increasing IQGAP1, Lis1 and
Cdc42 in the WCD pull-down. (c) Lis1 (green) and IQGAP1 (red) overlapped
at the leading fibroblast edge (arrows), but not in the cytoplasm. (d) IQGAP1
(red) and Lis1 (green) colocalized in axon (arrows) and growth cone (inset) of
a cultured cerebellar neuron. (e) Enlargement of boxed area in c shows yellow
overlap of IQGAP1 and Lis1 in this deconvolved image. (f) Lis1, IQGAP1 and
CLIP-170 were directly immunoprecipitated (IP). Anti-Lis1 coprecipitated
IQGAP1, CLIP-170 and actin, whereas IQGAP1 antibody reciprocally
coprecipitated Lis1, CLIP-170 and actin, demonstrated by immunoblotting
(IB) with anti-IQGAP1, anti-Lis1, anti-actin and antibody to CLIP-170. pGFP
transfected cultures were precipitated with anti-GFP as negative control.
Scale bars, 15 mm.
f
IQGAP1
191
IQGAP1
IP
Straight
Soluble
Lis1
Insoluble
Cdc42-GST
IQGAP1
Soluble
GFP
wtCdc42
dnCdc42
Control
wtCdc42
dnCdc42
Cdc42-GST
Soluble
IQGAP1
D-Serine
f
and were further slowed only a few percent by shIQGAP1 or shCLIP170
RNA (Fig. 7d). Thus, loss of Lis1 reduced the amount of active Cdc42,
IQGAP1 or CLIP-170 pulled down in the WCD complex, and knockdown of either IQGAP1 or CLIP-170 decreased the amounts of Lis1
and GTP-Cdc42 in the complex. Furthermore, neuronal movement
was slowed to a similar degree whether Lis1, IQGAP1 or CLIP-170 was
removed from the system. Finally, the effect on migration when both
Lis1 and IQGAP1 or CLIP-170 were lost was not simply additive,
consistent with their concerted function as a complex.
BAPTA/D-Serine
*
Control
N.S
100
Insoluble
D-Serine
Control
D-Serine
*
0
CLIP-170
e
Insoluble
Soluble
IQGAP1
D-Serine
Lis1+/–
700
BAPTA/D-Serine
Wild type
Lysate
*
Control
Soluble
N.S.
100
Lis1+/–
D-Serine
Control
D-Serine
Control
Insoluble
N.S.
200
Insoluble
d
Lis1+/–
Lis1+/–
+D-Serine
Lis1+/+
Lis1
Wild type
300
D-Serine
c
400
0
+/–
IQGAP1
Wild type
Wild type
+D-Serine
Lis1+/–
Control
Wild type
Control
© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience
Lysate
*
Lis1+/–
Soluble
Percentage control, OD
Insoluble
500
Figure 5 Ca2+-dependent redistribution of CLIP-170 and IQGAP1 to
the insoluble fraction is impaired in Lis1+/– neurons. (a–d) Treatment of
cerebellar cultures with 10 mM D-serine shifted CLIP-170 (a,b) and IQGAP1
(c,d) from the soluble fraction of cell lysates to the insoluble fraction in
wild-type but not in Lis1+/– neurons. CLIP-170 or IQGAP1 bands in straight
lysate of control or D-serine treated cultures demonstrated protein loading for
fractionation. (b,d) Three separate experiments were compared using optical
density of the western blot bands expressed as percentage of controls (*P o
0.01 compared to wild-type untreated control). (e,f) Wild-type cerebellar
granule neurons, protein loaded with either GST-tagged wild-type Cdc42
(wtCdc42) or mutant dominant negative Cdc42 (dnCdc42), were treated with
vehicle or D-serine. Whereas neurons containing wtCdc42 displaced CLIP170 and IQGAP1 to the insoluble fraction upon D-serine treatment, neurons
containing dnCdc42 did not. Anti-GST antibody staining showed that cells
contained equivalent amounts of tagged protein; whole cell (straight) lysate
provided a loading control. Error bars, s.e.m.
Lis1+/+
Lis1+/–
CLIP-170
600
D-Serine
Wild type
Percentage control, OD
D-Serine
b
Control
Control
D-Serine
CLIP-170
a
Lis1
97
Actin
51
IB
Lis1
39
CLIP-170
28
Cdc42
19
SL
WCD
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relatively normal18,19,37, perhaps facilitated
by other sources of Ca2+ influx37. However,
in postmigratory neurons, NMDAR activity
pS-shCLIP170
upregulates the Rho GTPases associated
with dendritic branches and spines during
pR-eGFP
pR-shIQGAP1
synaptogenesis and in LTP in adults21,38.
Here, we presented evidence that Ca2+ influx
c
b
acts through Lis1 and the activation of
140
D-Serine
Control
*
Rho GTPases to regulate the cytoskeleton at
100
the leading edge of moving neurons and
* *
growth cones.
IQGAP1
60
Lis1 and Rho GTPase connections were
Lis1
20
supported by the observations that dominant
0
Cdc42
negative Cdc42 reduced F-actin in wild-type
pR-eGFP, D-Serine
pR-eGFP
Rac1
pR-shIQGAP1,
hippocampal neurons and slowed cerebellar
pR-shIQGAP1
D-Serine
0"
45"
90"
0"
45"
90"
granule neuron migration, emulating Lis1WCD
Lysate
deficient cells. Moreover, either constitutively
pR-eGFP
pR-shCLIP170
PR-shIQGAP1
active Cdc42 or exogenous Lis1 normalized
d
100
F-actin distribution and the motility of Lis1+/–
*
* *
Wild
*
*
60
neurons. Both restoration by the addition of
type
Lis1 and D-serine effects in wild-type neurons
20
were blocked by dominant negative Cdc42,
0
+/–,
indicating that Lis1 and D-serine acted
+/–
Lis1
Wild
type
Lis1
pR-shIQGAP1
pR-eGFP
upstream of Cdc42. The D-serine–mediated
Wild type,
Lis1+/–,
rise in Cdc42/Rac1 activities and the recruit0"
45" 90"
0"
45" 90"
0"
45" 90"
PR-shCLIP170
pR-eGFP
ment of IQGAP1 to the leading edge were
+/–
Wild type
Lis1 ,
pR-shIQGAP1
consistent with previous observations in nonpR-shCLIP170
neuronal cells in which Rac1 and Cdc42 were
Figure 7 RNAi-targeted decreases in CLIP-170 or IQGAP1 reduce Lis1 and Cdc42 in WCD-complexes.
found to be important for the capture of
D-Serine–enhanced neuronal migration is blocked by IQGAP1-RNAi. (a,b) shRNAi targeted
dynamic microtubule ends by the cortical
downregulation of IQGAP1 or CLIP-170 in transfected NIH3T3 cells. (a) NIH3T3 transfected cultures
actin meshwork13 to support migration14.
showed decreased immunolocalization (arrows) of CLIP-170 in pS-shCLIP170–expressing cells and of
Lis1
involvement in this recruitment was
IQGAP1 labeling in pR-shIQGAP1–expressing cells. (b) Reduced IQGAP1 or CLIP-170 levels diminished
suggested here by the failure of D-serine treatboth Lis1 and GTP-Cdc42 in the protein complex pulled down with WCD. Reduced CLIP-170 also
decreased IQGAP1 in the complex. Absence of Rac1 in the WCD lanes demonstrated specificity of the
ment of Lis1+/– neurons to redistribute CLIPpull-down. (c) IQGAP1 RNA interference by pR-shIQGAP1 slowed migration of wild-type cerebellar
170 and IQGAP1 to the insoluble neuronal
neurons, and transfected cells did not respond to D-serine (n ¼ 18 pR-eGFP control; 22 pR-shIQGAP1;
fraction. Moreover, D-serine treatment of
20 pR-shIQGAP1, D-ser). (d) shRNAi-mediated knockout of IQGAP1 (by pR-shIQGAP1) or CLIP-170
wild-type neurons increased the amounts of
+/–
(by pR-shCLIP170) only slightly worsened the migration defect in Lis1 neurons (n ¼ 91 wild-type
Lis1, IQGAP1 and Cdc42 pulled down with
pR-eGFP control; 118 Lis1+/– pR-eGFP; 82 wild-type pR-shIQGAP1; 91 Lis1+/– pR-shIQGAP1; 44 wildWCD, which was decreased in Lis1+/– neurons
type pR-shCLIP170; 59 Lis1+/– pR-shCLIP170). *P o 0.001 compared to control. Error bars, s.e.m.
and was blocked in wild-type neurons by AP5,
BAPTA and shRNAi to CLIP-170 or IQGAP1.
ends by CLIP-170 complexes13, and RhoA activation facilitates micro- Although Lis1 was shown here to interact with CLIP-170 and IQGAP1/
tubule polarization and stabilization at the leading edge of migrating Cdc42 in a Ca2+-dependent manner to affect F-actin in neurons,
cells36. Together with our previous observations4, the present data demonstration of Lis1 involvement in microtubule capture with
supported a role for Lis1 in the regulation of actin polymerization and IQGAP1 and CLIP-170 awaits further investigation of microtubule
the probable coordination with microtubules through its impact on dynamics in these cells.
Our results here indicate that Lis1 has an integral role in the
RhoA, Rac1 and Cdc42.
This connection of Lis1 to Ca2+-dependent signaling has not been transduction of motility signals triggered by Ca2+ influx to regulate
made previously. Increased intracellular Ca2+ transients induced by the cytoskeleton through upstream regulation of small GTPases. The
the activation of glutamatergic NMDARs, voltage-dependent Ca2+ scaffolding protein IQGAP1 is a strong candidate for a partner through
channels or both provide a potent stimulus for granule neuron which Lis1 may link Ca2+ influx, increase active Cdc42 levels and target
migration in the cerebellum16,17. The 25–35% neuronal motility dynamic microtubule ends to cortical actin. IQGAP1 binding to
increases in the present experiments compare well with those calmodulin is Ca2+ regulated, but the relationship between Ca2+
previously observed17 and was surprisingly robust in view of the and IQGAP1-calmodulin binding is not completely understood. In
experimental design that neither depleted the extracellular Ca2+ nor chromaffin tissue, calmodulin more effectively blocks IQGAP1 bindsuppressed spontaneous Ca2+ fluxes from other sources. Bergmann ing to F-actin in the absence of Ca2+ (ref. 39). In studies using a
glial cells have recently been identified as an in vivo source of D-serine conjugated calmodulin reagent, elevated intracellular Ca2+ enhances
for NMDAR activation on migrating cerebellar granule neurons24. the binding of IQGAP1 to Ca2+/calmodulin, suggesting that Ca2+
NMDAR stimulation is an unlikely motogen in cerebral cortex, as should inhibit IQGAP1 interactions with cortical actin and Cdc42
functioning receptors do not appear until neurons reach the cortical (refs. 40–42). More recent studies that avoid using conjugated
plate and cortical migration in NR1-knockout mouse embryos is calmodulin show that IQGAP1 preferentially binds apo-calmodulin
AntiIQGAP1
eGFP
pR-eGFP
Average speed
(% pR-eGFP )
AntiCLIP170
Average speed
(% wild type, pR-eGFP)
pR-shIQGAP1
Control
pR-shIQGAP1
pS-shCLIP170b
pS-shCLIP170b
eGFP Anti-IQGAP1 Merge
Control
pR-shIQGAP1
© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience
a
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compared to Ca2+/calmodulin, and elevated cytosolic Ca2+ concentrations dissociate IQGAP1 from calmodulin (ref. 28), which is the most
commonly encountered action of Ca2+ on calmodulin-binding to IQdomain proteins43. That Ca2+ influx enhanced the association of
IQGAP1 with the insoluble cell fraction was most consistent with
previous observations28 that calmodulin avidly binds IQGAP1 in the
absence of Ca2+ and that the influx of free Ca2+ triggers the dissociation
of IQGAP1 from calmodulin.
A model emerges in which Ca2+ influx releases IQGAP1 from
calmodulin, freeing it to bind and stabilize active Cdc42 and Rac1
(Supplementary Fig. 4 online). Lis1, perhaps activated by Ca2+dependent phosphorylation (Supplementary Fig. 5 online), forms a
complex with IQGAP1 and +TIPs proteins (CLIP-170, dynactin
subunit p150glued, cytoplasmic dynein) that link the cortical actin
meshwork and growing microtubule ends. Insufficient Lis1, IQGAP1
or CLIP-170 destabilizes this complex, preventing the sustained presence of active Cdc42/Rac1 to drive actin polymerization and protrusion of the leading edge. Our data from neurons containing half the
normal Lis1 complement and the recent demonstration that more
drastic Lis1 knockout by RNAi disrupts neural cell division and axon
growth44 underscore the importance of Lis1 to cytoskeletal dynamics.
The present study establishes Lis1 as a key signal transduction molecule
connecting extracellular neuronal motility signals with intracellular
regulators of the cytoskeleton.
METHODS
Cell culture and videomicroscopy. All protocols involving mice were reviewed
and approved by the Institutional Animal Care and Use Committee. P5–P7
cerebellar or P0–P3 hippocampal neurons from Lis1+/– mice or their wild-type
siblings15 were cultured using established procedures45. Dissected tissue
was dissociated in papain (Worthington) and plated onto poly-D-lysine(20 mg ml–1) and laminin- (25 mg ml–1) coated surfaces. Neuronal cultures
were maintained at +37 1C in 5% CO2 in Basal Medium Eagle (BME,
Invitrogen) containing 10% horse serum, 10% fetal bovine serum (FBS),
200 mM glutamine and 6 mM glucose (GCM-R). Granule cell migration was
followed by phase-contrast video microscopy of live cultures, as described
before4. The effects of NMDAR stimulation on cell motility were assessed as
described previously17,25, except that the cells were incubated with vehicle or
10 mM D-serine (Sigma) for the duration of the video recording.
Calcium imaging. Cerebellar neuron cultures were loaded with the Ca2+sensitive fluorescent dye Fluo-4, and transient changes in intracellular Ca2+
concentration were examined using time-lapse confocal microscopy as previously described7. Briefly, a 5-mM solution of Fluo-4, AM (Molecular Probes
F-14201) in dimethylsulphoxide (DMSO) was diluted 1:1 with 20% pluronic
acid in DMSO (Molecular Probes F-3000). Of this 1,000 stock, 2 ml was
mixed with 100 ml of media, added to the cells (in 2 ml media) and incubated
for 15 min at +37 1C. The GCM-R media was then exchanged for prewarmed
recording media4. An Olympus Fluoview 500 confocal microscope was used
with 40 or 60 water objectives (NA 0.8 or 0.9, respectively). Fluo-4 was
excited with a 488-nm argon laser.
Immunocytochemistry and quantification of fluorescence intensity. After
24 h in culture, cells plated on two-chambered cover glass were fixed in 0.2%
glutaraldehyde and briefly permeabilized with 0.1% Triton X-100. Cytoskeletal
components including their bound proteins were thus retained while the
soluble protein fraction was removed. Cells were treated with rabbit antibody
to green fluorescent protein (anti-GFP; Molecular Probes), mouse antiIQGAP1 (BD), mouse anti-Lis1 (Sigma-Aldrich) followed by phalloidin–
AlexaFluor 647 and secondary antibodies conjugated to AlexaFluor 488, 568
or 647 dyes (Molecular Probes). pEGFPC3-Lis1 transfected neurons were
identified in fixed samples by anti-GFP immunostaining. Fluorescence collected
on an inverted microscope (model 200M; Zeiss) connected to a laser confocal
scanning head (UltraView, Perkin-Elmer) was quantified, and the
three-dimensional image was reconstructed using MetaMorph software
56
(Universal Imaging). The high-resolution colocalization data images in
Figures 4 and 6 were additionally deconvolved using AutoDeblur software
(AutoQuant Imaging).
GTPase assay, cell fractionation, western blotting and immunoprecipitation.
Lysates were prepared from P5–P7 whole cerebella or dissociated neurons,
cultured for 24 h on 60-mm Petri dishes. Protein distribution between soluble
and insoluble fractions was assessed as previously described46. Active GTPbound forms of Rac1, Cdc42 and RhoA were measured with a pull-down assay
according to the manufacturer’s instructions (Cytoskeleton). Before cell lysis,
cultures were treated for 10 min with 10 mM D-serine, with or without either
20 mM BAPTA-AM buffer or 200 mM D-2-amino-5-phosphonopentanoate
(AP5). PAK1-PBD, WASP-CD or Rhotekin-PBD-GST agarose–bound proteins
(Cytoskeleton) were separated by SDS–polyacrylamide gel electrophoresis
(SDS-PAGE) and analyzed on western blots using mouse antibody to RhoA
(anti-RhoA) (Cytoskeleton), mouse antibody to Cdc42 (anti-Cdc42), mouse
anti-IQGAP1 and mouse antibody to FAK (anti-FAK; BD); mouse antibody to
actin (anti-actin) or rabbit antibody to cyclin D2 (anti–cyclin D2; Santa-Cruz);
mouse anti-Lis1 (Sigma-Aldrich). For Rac1 detection, one of two antibodies
was used: mouse anti-Rac1 (Upstate Cell Signaling Solutions) yields a doublet,
whereas mouse anti-Rac1 (BD) yields a single band at 21 kDa and produced the
same relative results. Similarly, one of two antibodies was used for CLIP-170
detection. Mouse anti–CLIP-170 (gift from H. Goodson, Notre Dame University, Notre Dame, Indiana) yields a doublet, whereas rabbit anti–CLIP-170
(Santa Cruz) yields a single band at 170 kDa that produced the same relative
results. Immunoprecipitations were performed from cell lysates with the
antibodies indicated using the Catch and Release system (Upstate Cell Signaling
Solutions) according to the manufacturer’s protocol. Films were scanned on an
optical densitometer and relative protein concentrations were determined using
Quantity One software (Bio-Rad). Optical densities were normalized to the
amount of actin, determined from western blot analysis of electrophoretic gels
loaded with equal amounts of lysate protein.
Constructs. The pEGFPC3-Lis1 plasmid expressing the Lis1-eGFP fusion
protein was a gift from G. Clark47 (Baylor College of Medicine, Houston).
Mutant cDNA (Guthrie cDNA Resource Center) encoding hemagglutinintagged constitutively active (GV12) or dominant negative (T17N) Cdc42 were
used to make expression constructs. Plasmids pHA.cdc42G12V and
pHA.cdc42.T17N were digested with BamHI and subcloned into the multiple
cloning site of pCXI.IEGFP to express the mutant Cdc42 and EGFP that
was translated separately from an internal ribosome entry site (IRES). All
clones were verified by sequencing and western blot analysis. Plasmids
expressing interfering short hairpin RNA (shRNAi) directed against
CLIP-170 were a gift from A. Akhmanova of Erasmus Medical Center,
Rotterdam, The Netherlands (pS-shCLIP170a, pS-shCLIP170b)48. shRNAi
constructs against IQGAP1 were designed after published short interfering
RNAs14. Plasmids expressing IQGAP1 shRNAi were prepared by GenScript in
pRNAT-U6.q/Neo vector (GenScript). All expression vectors were checked for
integrity by restriction digestion and sequencing. The function of each plasmid
was confirmed in transfected 3T3/NIH cells: pCdc42ca and pCdc42dn by
phalloidin staining to examine stress fibers (Supplementary Fig. 6 online);
pEGFPC3-Lis1 by GFP fluorescence and western blot of GFP and Lis1;
pS-shCLIP170 and pRNAT-shIQGAP1 by western blotting to confirm the
ability of these constructs to downregulate CLIP-170 or IQGAP1 (Fig. 7,
Supplementary Figs. 2 and 3).
Cell transfection. Dissociated cerebellar or hippocampal cells were transfected
using electroporation (Nucleofector II, Amaxa Biosystems) according to the
manufacturer’s protocol. Cells receiving the designated construct were imaged
24 h after transfection. Confocal imaging was performed on a Zeiss 200M
inverted microscope fitted with a spinning disk confocal laser (UltraView,
Perkin-Elmer).
Cdc42 protein loading. Constitutively active or dominant negative forms of
Cdc42 protein (L61Cdc42-GST and N17Cdc42-GST, respectively; Cytoskeleton) were loaded in the neuronal dissociated cultures using Pro-Ject protein
transfection reagent according to the manufacturer’s protocol (Pierce).
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Statistical analyses. All image data were analyzed with the operator blinded
to genotype. At least three independent trials were performed for each
videomicroscopy experiment, and the numbers of observations are given.
Statistical significance of the difference between mean values was determined
using a two-tailed Student t-test. The optical density values of western blots of
cell culture lysates were obtained from Lis1+/+ and Lis1+/– littermates of at least
three different litters. The significance of the mean variation was determined
using the non-parametric Mann-Whitney test. Error bars indicate s.e.m.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
Supported by the US National Institute of Neurological Disorders and Stroke
(RO1NS35515 to M.E.R., PO1NS39404 to M.E.R. and A.W.B.) and National
Institute of Child Health and Development (RO1HD19950 to P.C.L.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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