Developmental Cell, Vol. 9, 75–86, July, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.devcel.2005.04.003
The Dynein Light Chain Tctex-1
Has a Dynein-Independent Role
in Actin Remodeling during Neurite Outgrowth
Jen-Zen Chuang,1Ting-Yu Yeh,1Flavia Bollati,3
Cecilia Conde,3Federico Canavosio,3
Alfredo Caceres,3and Ching-Hwa Sung2,*
1Department of Ophthalmology
2Department of Cell and Development Biology
Weill Medical College of Cornell University
1300 York Avenue
New York, New York 10012
Avenue Friuli 2434
ment of enhanced actin-based motility has been further
supported by the finding that the RhoGTPase family
proteins, key players in regulating actin cytoskeleton
(Ridley et al., 1992), as well as various effectors and
guanine nucleotide exchange factors of RhoGTPase
proteins have drastic effects on neurite extension (re-
viewed in Fukata et al., 2002c). It is generally thought
that Rho is a negative regulator and Rac is a positive
regulator of neurite growth. Nevertheless, little is known
about how the local activation (or inactivation) of these
RhoGTPase family proteins is regulated. Very recent
data suggest that selective enrichment of mPar3/mPar6/
atypic protein kinase C complex and phosphatidylinosi-
tol 3-kinase activity at axonal neurites may play a cru-
cial role in axonal specification through a mechanism
upstream of RhoGTPase-mediated cytoskeletal modu-
lation (Shi et al., 2003).
Microtubules are also involved in establishing axonal
polarity. This idea has been demonstrated by the ob-
servation that nascent axons had more stable microtu-
bules (Ferreira et al., 1989) and that more microtubules
were selectively shipped into neurites fated to be axons
(Baas et al., 1993). More recently, CRMP-2 (Collapsin
response mediator protein-2) was found to selectively
accumulate in the distal region and the growth cones
of nascent axons (Inagaki et al., 2001). CRMP-2’s ability
to bind free tubulin subunits may assist in microtubule
assembly, thereby enhancing axonal neurite extension
(Fukata et al., 2002b). The microtubule plus-end bind-
ing protein APC (Adenomatous polyposis coli) has also
been shown to play roles in nerve growth factor-
induced axonal outgrowth through its ability to promote
microtubule assembly (Zhou et al., 2004).
Studies on the dynamics of growth cones in Aplysia
neurons have lent support to the idea that microtubule
redistribution into the leading lamellipodium is second-
ary to F-actin disassembly (Forscher and Smith, 1988).
Moreover, evidence suggests that the growing microtu-
bules stimulate actin polymerization via Rac1 activation
at the leading edge of migrating fibroblasts (Waterman-
Storer et al., 1999). The interaction between the micro-
tubule plus-end binding protein CLIP170 and Rac1 ef-
fector IQGAP1 may be involved in these processes of
cell protrusion (Fukata et al., 2002a). In contrast to non-
neuronal motile cells, the molecular machinery respon-
sible for the coordination between microtubules and
microfilaments during neurite outgrowth of the verte-
brate neuron is poorly understood.
Cytoplasmic dynein is the major motor protein com-
plex responsible for minus-end, microtubule-based
motile processes, and hence for an extremely wide
range of cellular events (Hirokawa, 1998). Each dynein
complex consists of two heavy chains (DHC) that have
ATPase and motor activities, plus a group of accessory
polypeptides including dynein intermediate chains (DIC),
light intermediate chains, and light chains (Vallee et al.,
2004). The intermediate chain links accessory proteins
and the dynactin complex (which participates in the
processivity and cargo binding of the dynein motor) to
the dynein heavy chain (King and Schroer, 2000; Water-
Coordinated microtubule and microfilament changes
are essential for the morphological development of
neurons; however, little is know about the underlying
molecular machinery linking these two cytoskeletal
systems. Similarly, the indispensable role of Rho-
GTPase family proteins has been demonstrated, but
it is unknown how their activities are specifically reg-
ulated in different neurites. In this paper, we show
that the cytoplasmic dynein light chain Tctex-1 plays
a key role in multiple steps of hippocampal neuron
development, including initial neurite sprouting, axon
specification, and later dendritic elaboration. The neu-
ritogenic effects elicited by Tctex-1 are independent
from its cargo adaptor role for dynein motor trans-
port. Finally, our data suggest that the selective high
level of Tctex-1 at the growth cone of growing axons
drives fast neurite extension by modulating actin dy-
namics and also Rac1 activity.
Typical vertebrate neurons have a single thin axon that
can grow very long and multiple, thick, tapering den-
drites that are comparatively short. Although the extreme
polarization of neurons has long been recognized, how
neurons develop this polarity remains largely elusive.
Specific internal cues that are crucial for polarity deter-
mination have been demonstrated in a model system
of cultured embryonic hippocampal neurons. In this
system, cellular asymmetry develops in the absence of
extracellular spatial cues (Fukata et al., 2002a).
Previous studies have suggested that enhancement
of neurite elongation directs axon specification (Esch
et al., 1999), and modulation of the dynamics of both
actin microfilaments and microtubules is likely to be in-
volved in axonal elongation (Fukata et al., 2002c). Phar-
macological evidence suggested that actin in the growth
cone of the nascent axon is much more labile than that
in the growth cones of minor neurites that later develop
into dendrites (Bradke and Dotti, 1999). The involve-
man-Storer et al., 1995). Several light chain subunits
have been identified (Vallee et al., 2004). Among these
light chains, Tctex-1 has clearly been shown to be in-
volved in cargo binding. For example, Tctex-1 binds
specifically to the cytoplasmic tail of rhodopsin and
links rhodopsin-bearing vesicles to the dynein motor
complex for transport (Tai et al., 1999; Tai et al., 2001).
However, biochemical evidence revealed that a signifi-
cant amount of Tctex-1 also exists outside the dynein
complex (Li et al., 2004; Tai et al., 1998). To date, the
cellular function of the complex-free Tctex-1 and the
regulation of Tctex-1 assembly into dynein complex
The abundance and expression pattern of Tctex-1 in
postmitotic young hippocampal neurons (Chuang et al.,
2001) prompted us to examine the role of Tctex-1 in
neuronal development. In this report, we investigated
the expression and function of Tctex-1 in hippocampal
neuronal cultures, and we show that the asymmetric
and dynamic distributions of Tctex-1 play an essential
role in multiple steps of neuronal morphogenesis. Neu-
rons failed to develop neurites when Tctex-1 was sup-
pressed; Tctex-1 overexpression prevented axonal po-
larity formation and induced multiple, abnormally long
axon-like neurites. Our data argue that dynein motor
activity is not critical for Tctex-1-mediated neurite out-
growth. Finally, our findings support the idea that Tctex-
1-mediated neurite development depends on its ability
to modulate actin dynamics and Rac1 activity, indicat-
ing that Tctex-1 may act as a regulatory link between
the microtubule and microfilament cytoskeletons in the
growth cones of growing axons.
ticular enrichment within axons or growth cones. Fur-
thermore, little or no labeling for dynein light chain rp3,
a Tctex-1 homolog, was found in young neuronal cul-
tures (data not shown).
Tctex-1 Mediates Neuritogenesis
Antisense (AS) oligonucleotide against Tctex-1 was used
to carry out loss-of-function experiments to study its
role during hippocampal neuronal development. The re-
duction of Tctex-1 protein, but not control proteins
(e.g., DHC, tubulin), was confirmed by immunoblotting
(Figure 2E) and quantitative immunofluorescence (Sup-
plemental Table S1). Almost all cells treated with no oli-
gonucleotide (data not shown) or with a scrambled con-
trol oligonucleotide (Figures 2A and 2D) were able to
reach stage 2 or stage 3 of neuritic development after
24–36 hr; in contrast, the majority of Tctex-1-AS-treated
cells failed to develop neurites (Figures 2B–2D, Supple-
mental Table S2). The Tctex-1-suppressed neurons had
segmented lamellipodia but neither typical neurites nor
growth cones. Some Tctex-1-suppressed neurons dis-
played short, thin tubulin+processes that penetrated
the lamellipodial veil (Figures 2B and 2C). Morphomet-
ric analysis revealed that although more than 80% of
36 hr control neurons reached stage 3, less than 2% of
AS-treated cells reached this stage (Figure 2D, Supple-
mental Table S2).
Similar results were also obtained by examining neu-
rons with Tctex-1 suppressed by RNA interference.
Neurons transfected with Tctex-1-siRNA oligonucleo-
tides, identified by lower levels of Tctex-1 immunolabel-
ing, were almost always associated with cells arrested
at stage 1 of neuritic development (Supplemental Fig-
ures S2A and S2B).
In order to unambiguously identify neurons targeted
with Tctex-1-siRNA, we generated a plasmid harboring
both the Tctex-1 hairpin siRNA and the GFP cDNA (i.e.,
Tctex-1-siRNA/GFP). The silencing effect of this plasmid
was confirmed in both transfected HEK cells (Supple-
mental Figure S2C) and transfected neurons (Figure 2F).
When neurons were transfected with Tctex-1-siRNA/GFP
plasmid 2 hr after plating and examined 20–22 hr later,
almost all GFP+neurons had specific reduction of
Tctex-1 immunofluorescence but not tubulin immuno-
fluorescence (Figures 2G–2J, arrows; Supplemental Ta-
ble S1). Again, the majority of the GFP+, siRNA-targeted
cells failed to develop neurites and arrested at stage 1.
These siRNA-targeted cells are neurons because they
were labeled by TuJ1 antibody, which recognizes the
neuron-specific β-III tubulin. By contrast, control neu-
rons transfected with scrambled control-siRNA/GFP
plasmid developed neurites normally (Supplemental Ta-
ble S2). These results consistently suggested that
proper Tctex-1 dosage is essential for the initial events
of neurite development, such as neurite sprouting from
the spherical cell bodies.
To examine Tctex-1’s role in neurite extension, neurons
were treated with either Tctex-1-AS or Tctex-1-siRNA/
GFP w15 hr after plating and analyzed 20 hr later. While
most of control cells reached stage 3, the majority of
Tctex-1-suppressed cells were arrested at stage 2 (Fig-
ures 2K–2N; Supplemental Table S2). These results
clearly suggested that Tctex-1 suppression could effec-
tively inhibit neurite extension and, hence, suppress
differentiation during stage 2–3 development.
Tctex-1 Is Enriched in Growing Axons of Cultured
Within the first 24 hr after plating, cultured hippocampal
neurons develop several relatively symmetric minor pro-
cesses 20–30 ?mavein length (stage 2 cells); Tctex-1
was evenly distributed throughout the cell body and mi-
nor neurites during this stage (data not shown). How-
ever, during development through stages 2–3, Tctex-1
immunoreactivity increased considerably in the longest
neurites that had large growth cones and abundant
microtubules (Figures 1A–1C), morphological charac-
teristics of nascent axons. Quantification of Tctex-1 la-
beling intensity relative to that of tubulin confirmed the
Tctex-1 signal had a gradient in nascent axons: low at
the proximal end and high at the distal region (Figure
1H). By contrast, very little Tctex-1 signal was detected
in the growth cones of minor neurites. At the axonal
growth cone, Tctex-1 colocalized with tyrosinated
(tyr)-tubulin in the central region (Figures 1A and 1B).
However, Tctex-1-labeled puncta were also arrayed on
F-actin microfilaments that extended into the periph-
eral lamellipodial veil (Figures 1D and 1E).
Upon reaching stage 3, Tctex-1’s distribution ap-
peared to be even more polarized, as it became con-
siderably enriched in the distal axonal shaft and its
growth cone (Figures 1F and 1G). In contrast to Tctex-1,
immunolabeling for DHC (see Supplemental Figure S1
available with this article online), DIC, or dynactin sub-
unit p150glued(data not shown) did not reveal any par-
Tctex-1 in Neurite Development
Figure 1. Asymmetric Distribution of Tctex-1
in Hippocampal Neurons
(A–C) A stage 2–3 neuron immunostained for
Tctex-1 (A), tyr-tubulin (B), and F-actin (C).
Arrows point to the neurite of the future
axon, and open arrows point to the axonal
(D and E) Magnified views of axonal growth
cones shown in (A) and (C). Arrowheads indi-
cate the codistributed Tctex-1 and F-actin at
in the insert.
(F and G) Confocal images of a stage 3 neu-
ron colabeled for Tctex-1 (F), tyr-tubulin (blue
in [G]), and F-actin (red in [G]). Arrow marks
the axon growth cone.
(H) Quantification of Tctex-1 expression levels
in the neurites and growth cones of stage
2–3 neurons. The Tctex-1 labeling index rep-
resents the ratio of Tctex-1 labeling intensity
relative to the tyr-tubulin labeling intensity.
Both the minor neurites and the axonal neu-
rites of these cells were subdivided into three
even parts, which were designated as immediate segment (IS), middle segment (MS), and distal segment (DS), according to their distance
from the cell body. The Tctex-1 labeling in the central regions (CR) of growth cones (GC) was also considered for quantification. However, the
Tctex-1 labeling index in the peripheral regions of growth cones was not shown, because the tyr-tubulin labeling in these regions was often
undetectable. n >100. Scale bars equal 10 ?m in (A)–(C), (F), and (G) and 5 ?m in (D) and (E).
Ectopic Tctex-1 Expression Promotes Neurite
Outgrowth and Abolishes Neuronal Polarity
Gain-of-function studies were subsequently performed
to examine the phenotypes of neurons overexpressing
Tctex-1. For controls, dissociated neurons were singly
transfected with GFP, GFP-DIC, or Flag-DIC (data not
shown) or cotransfected with Flag-DIC and GFP (Fig-
ures 3L–3O; Supplemental Table S3). The kinetics of
neurite development and the morphological parame-
ters of all control neurons were undistinguishable from
Figure 2. Tctex-1 Suppression Inhibits Neu-
(A–C) Confocal images of neurons treated
with a control AS for 24 hr (A), Tctex-1-AS for
24 hr (B), or Tctex-1-AS for 36 hr (C), fol-
lowed by immunostaining for tyr-tubulin (red)
and F-actin (green). The cell in (A) has the
morphological appearance of stage 2–3 cell.
In (C), arrow points to a segmented lamelli-
podium, and the arrowheads point to the
short tubulin+processes that penetrate the
(D) Chart showing the percentage of cells at
stage 1, 2, and 3 of neuritic development af-
ter a 24 hr or 36 hr treatment with control (C)
or Tctex-1 AS-oligonucleotides. The scoring
of stage 2 and stage 3 cells in control experi-
ments were based on their typical morpho-
logical features (Dotti et al., 1988; Goslin and
Banker, 1991). However, for the purpose of
quantification, AS-treated cells carrying mul-
tiple tubulin+processes with lengths similar
to either minor neurites or axonal neurites
were considered for classification, in spite of
the fact that most of these processes are not
typical neurites and lack growth cones.
(E and F) Immunoblotting of equal amount of
proteins extracted from neurons treated with
(+) or without (−) AS for 24 hr (E) or neurons
transfected with control-siRNA/GFP or Tctex-
1-siRNA/GFP (F) for 36 hr. The indicated anti-
bodies were used.
(G–J) Neurons transfected with Tctex-1-siRNA/
GFP 2 hr after plating and followed by
immunostaining for TuJ1 (red) and Tctex-1
(blue) 22 hr later. GFP was visualized directly.
(K–N) An example of a typical neuron transfected with Tctex-1-siRNA/GFP w15 hr after plating and immunolabeled 20 hr later. The cell has
a weak Tctex-1 signal (blue) and a normal level of TuJ1 (red); it was arrested at stage 2 of neuritic development. Scale bar equals 10 ?m.
Figure 3. Tctex-1 Overexpression Caused
Multiple Long Axons in Hippocampal Cul-
(A–C) Dissociated neurons were transfected
with Flag-Tctex-1 right before plating. Cells
fixed 24 hr posttransfection were immunola-
beled for Flag, Tau1, and phalloidin. The
Flag-Tctex-1-transfected neurons exhibited
abnormally long neurites and more than one
are Tau1+, whereas the nontransfected cells
had one single Tau1+axon (arrow). Arrow-
heads indicated that Tau1+long neurites
traveled long distances in and out of the fo-
cal plane. Note that cells ectopically express-
ing Tctex-1 consistently had much weaker
labeling for phalloidin, particularly in the neu-
rite tips. Open arrow points to the abundant
phalloidin labeling at the neurite tips of un-
(D–I) Neurons transfected with GFP-DIC (D–F)
or Flag-DIC (G–I) were immunolabeled as in-
dicated 18 hr after transfection. Scale bar
equals 10 ?m.
(J and K) Time-lapse images of a live neuron
cotransfected with GFP and Flag-Tctex-1
and imaged at 8 and 18 hr after plating. The
numbers indicate neurites that elongated
during the period of imaging.
(L–O) Histograms displaying the morpho-
logical analyses of cultures cotransfected
with GFP/Flag-Tctex-1 or GFP/Flag-DIC (2 ?g
each) 2 hr after plating and processed for im-
munolabeling at the indicated time points
posttransfection. A neurite that is Tau1+and
R50 ?m is considered to be an axon. Each
value represents the mean ± SEM of at least
50 axonal processes for each experimental
condition (*p < 0.05).
those of nontransfected neurons. The cells exhibited
normal development through stage 2 (Figures 3D–3I)
and stage 3, at which time they developed a single
Tau1+axon and several much shorter minor neurites
By contrast, almost all Flag-Tctex-1-transfected neu-
rons displayed multiple abnormally long Tau1+axon-
like neurites (Figures 3A–3C). These Tau1+neurites
were also positive for several other axonal markers in-
cluding APC, Cdc42, synapsin 1, and synaptotagmin
(Supplemental Figures S3 and S4; Zhou et al., 2004;
Fletcher et al., 1991; Schwamborn and Puschel, 2004).
Statistical analysis confirmed that the axon numbers
(Figure 3L) and the lengths (Figure 3N) were signifi-
cantly higher in Flag-Tctex-1-transfected cells than in
Flag-DIC-transfected control cells (also see Supple-
mental Table S3). Despite the increased numbers of ax-
ons, the total number of primary neurites extended
from Tctex-1-transfected cells was not significantly dif-
ferent from that of control cells (Figure 3M; Supplemen-
tal Table S3). Taken together, these results suggest that
the ability of Tctex-1 to enhance neurite outgrowth is
rather specific, and ectopic expression of other dynein
subunit such as DIC cannot imitate the axogenic ef-
fects mediated by Tctex-1. Furthermore, Tctex-1 over-
expression enhanced neurite extension, rather than in-
creased neurite sprouting.
Live neurons transfected with GFP (data not shown)
or GFP together with Flag-Tctex-1 (Figures 3J and 3K)
were also observed by time-lapse video microscopy and
sequential photography. In these experiments, neurite
development could be traced in the same cells over a
period of time, and our results confirmed the accelerated
rate of neurite outgrowth in Tctex-1-expressing neurons.
We then sought to determine whether Tctex-1’s abil-
ity to promote neurite outgrowth remains throughout
neuronal differentiation. For this purpose, neurons at 3
days in vitro (DIV) were transfected with Tctex-1 or GFP
and harvested 18 hr later. Flag-Tctex-1-expressing neu-
rons displayed significantly longer axons (Tau1+/MAP2−
neurites) than those of GFP-transfected cells (Figures
4A and 4B). The mean length of axons (2113 ± 219 ?m)
of Tctex-1-transfected neurons was almost 3-fold longer
than in GFP-transfected cells (720 ± 38 ?m; Table 1). In
addition, the 4 DIV Tctex-1-transfected neurons bore
more than 2.5aveaxons compared to the control neu-
rons with 1.2aveaxons (Table 1).
Tctex-1-transfected neurons also had significantly
longer and more elaborated MAP2+dendrites relative
to the control neurons (Figures 4C–4L). Quantitative
morphometric analysis showed that Tctex-1- and GFP-
transfected neurons had 18.6 ± 2.2 versus 8.4 ± 1.8
branches per dendrite, respectively.
Both the axogenic and dendritogenic effect elicited
by Tctex-1 appeared to be dosage dependent: neurons
transfected with a smaller amount of Tctex-1-express-
ing plasmid exhibited a less prominent, albeit still sta-
tistically significant, effect (Table 1).
Tctex-1 in Neurite Development
Figure 4. Tctex-1 Regulates Both Axon and
Dendrite Morphology in Older Neuronal Cul-
Cultures at 3 DIV were transfected with either
GFP (2 ?g) (A, C, D, G–I) or Flag-Tctex-1
(2 ?g) (B, E, F, J–L) for 18 hr before process-
ing. Neurons were either directly visualized
for GFP (A, D, H), or immunolabeled for Flag
(B, F, K) or MAP2 (C, E, G, J). Arrows and
arrowheads in (A) and (B) point to axons and
dendrites, respectively, based on their mor-
phological features. The numbers in (B) cor-
respond to the length of axonal processes.
Scale bars equal 40 ?m in (A)–(F) and 10 ?m
Tctex-1-Mediated Neurite Growth
Is Dynein Independent
To explore the mechanism underlying Tctex-1-medi-
ated neuritic development, we first asked whether the
inhibitory effect caused by Tctex-1 knockdown was re-
lated to impaired cargo binding for dynein transport.
Our first approach to test this possibility was asking
whether interference with general dynein activity by
overexpression of dynactin subunit p50 dynamitin (Ech-
everri et al., 1996) would produce a similar phenotype
to that caused by loss of Tctex-1. Consistent with the
notion that the centrosomal Golgi localization required
dynein motor activity (Corthesy-Theulaz et al., 1992),
neurons overexpressing myc-p50 had a dispersed
Golgi apparatus (Supplemental Figure S5). Despite that,
and unlike Tctex-1-suppressed neurons, neurons trans-
fected with p50 were capable of extending minor neu-
rites and developing an axon (Supplemental Figure S6),
even though alterations in neuritic caliber including
swellings and a slight reduction of axonal length were
commonly detected (data not shown).
To directly test the role of dynein motor activity in
Table 1. Quantative Analyses of Morphological Changes of Transfected Neurons
Overexpressed Protein Number AxonsMean Axonal Length Number Dendritic BranchesMean Dendritic Length
Tctex-1-WT (2 ?g)
Tctex-1-WT (1 ?g)
Tctex-1 + CA-Rho
Tctex-1 + DN-Rac
1.24 ± 0.2
2.45 ± 0.3*
1.85 ± 0.2*
2.21 ± 0.2*
1.38 ± 0.2
1.08 ± 0.2
1.20 ± 0.2
1.20 ± 0.2
1.1 ± 0.2
720 ± 38
2113 ± 219*
1228 ± 46*
1915 ± 147*
840 ± 26
240 ± 16*
298 ± 24*
585 ± 84
510 ± 68
8.4 ± 1.8
18.6 ± 2.2*
14.2 ± 2.4*
16.8 ± 1.6*
9.2 ± 1.2
4.2 ± 0.8*
2.4 ± 0.8*
6.2 ± 0.6
2.2 ± 0.2*
238 ± 24
604 ± 18*
378 ± 28*
524 ± 34*
256 ± 16
80 ± 12*
40 ± 10*
220 ± 46
68 ± 10*
All measurements were performed at 4 DIV. Cells were transfected at 3 DIV (at which time point the mean axonal length of control cells was
540 ± 62 ?m) and fixed 18–20 hr later. 2 ?g of plasmid was typically used, except 1 ?g of Tctex-1 was used in one set of experiments to
demonstrate the dosage effect. Each value represents the mean ± SEM of at least 50 cells for each experimental condition. Asterisk represents
value significantly different from that of GFP-transfected control neuron (p < 0.01) as determined by one-way ANOVA.
Figure 5. Tctex-1’s Neuritic Effect Is Dynein
(A–F) Confocal images of 1 DIV neurons co-
transfected with DHC-siRNA and GFP 2 hr
after plating. The GFP+ targeted, TuJ1+ neu-
rons were capable of reaching stage 2 (A–C)
or 3 (D–F) of neuritic development.
(G) Immunoblots of the DIC immunoprecipi-
tates obtained from mock-transfected 293T
cells or 293T transfected with either Flag-
WT, T94A, or T94E for either DIC or Flag. To-
tal inputs are also shown.
(H) The DIC immunoprecipitates obtained from
the 20 S fractions of (I) were immunoblotted
with anti-DIC and anti-Tctex-1 Abs. Tctex-1
Ab recognized both Flag-tagged and endog-
enous (Endo) Tctex-1, which migrated dif-
(I) Lysates of 293T cells transfected with the
in a 5%–20% linear sucrose gradient. Each
fraction was analyzed by SDS-PAGE and im-
munoblotted with indicated Abs.
(J and K) Immunolabeling of 4 DIV neurons
transfected with Flag-T94E and Flag-T94A
using anti-Flag Ab. Scale bars equal 10 ?m.
neuron development, we performed morphometric analy-
ses on neurons cotransfected with DHC-siRNA and
GFP plasmid. The gene silencing and function blocking
effects of this DHC-siRNA plasmid has been demon-
strated by Shu et al. (2004) and confirmed by us (Sup-
plemental Figure S7). As shown in Figures 5A–5F, 1 DIV
neurons targeted with DHC-siRNA developed rather
normally through stages 2 and 3, by which time a single
axon was extended. Taken together, these results sug-
gested that dynein motor activity was not critically re-
quired for the initial elaboration of neurites and/or axon
specification. These results further indicated that nei-
Tctex-1 in Neurite Development
ther the inhibitory effect of Tctex-1 silencing nor the
axogenic effect of Tctex-1 overexpression is likely to be
due to its dynein-related role.
Previous biochemical evidence revealed that a pool
of Tctex-1 exists that is not associated with dynein
complex (Tai et al., 1998). To date, the cellular function
of the complex-free Tctex-1 and the regulation of
Tctex-1 assembly into dynein complex are unknown. To
gain insight into whether the “complex” or the “free”
form of Tctex-1 is involved in neurite development, we
attempted to identify a Tctex-1 mutant unable to incor-
porate into the dynein complex and examine its effect
on neurite outgrowth and axonal polarity in trans-
Tctex-1 is assembled into dynein complex through its
interaction with DIC (Mok et al., 2001; Tai et al., 2001).
The DIC binding region of Tctex-1 has been mapped to
the region between amino acids 55 and 95 (Mok et al.,
2001). In this region, we detected a consensus protein
kinase C (PKC) phosphorylation site at threonine 94
(T94). Because purified Tctex-1 can be phosphorylated
by PKC in an in vitro assay (our unpublished results),
we reasoned that it was probable that the phosphoryla-
tion at T94 serves as a mechanism regulating its in-
teraction with DIC. Site-directed Tctex-1 mutants hav-
ing the T94 replaced with alanine (T94A) or glutamic
acid (T94E) were generated to mimic the unphosphory-
lated and the phosphorylated Tctex-1, respectively, and
these mutants were then tested for their ability to bind
endogenous DIC and dynein complex in transiently
transfected 293T cells. Both Flag-wild-type (Flag-WT)
Tctex-1 and Flag-T94A, but not Flag-T94E, were coim-
munoprecipitated with endogenous DIC using anti-DIC
antibody (Figure 5G). Converse experiments consis-
tently showed that anti-Flag Ab coprecipitated DIC
from cells coexpressing either Flag-WT or Flag-T94A,
but not Flag-T94E (data not shown). The inability of
T94E binding to DIC in these experiments predicted
that T94E was not able to be incorporated into dynein
The organization of the ectopically expressed Tctex-1
related to the dynein complex was also examined by
velocity density gradient sedimentation. Gradient frac-
tions of cell lysates containing transfected Flag-tagged,
wt, T94E, or T94A were immunoblotted for p150Glued,
DIC, and Flag. As shown in Figure 5I, in all cases, both
markers for dynein complexes (e.g., DIC) and dynactin
complexes (e.g., p150Glued) were sedimented at 19–20
S, suggesting that overexpression of Tctex-1 and its
mutants did not perturb the integrity of dynein com-
plexes. Moreover, both Flag-WT and T94A cosedimented
with the dynein/dynactin complexes at 19–20 S (Figure
5I); immunoprecipitation of these dynein-containing
fractions with anti-DIC antibody coprecipitated both
endogenous as well as the transfected Flag-WT or
Flag-T94A (Figure 5H). These results suggested that the
ectopically expressed Flag-WT partially replaced the
endogenous Tctex-1 and incorporated into the dynein
complex, even though, as predicted, a fraction of Flag-
WT and Flag-T94A were detected outside the dynein
complexes and distributed on the top of the gradient
(fractions 9–12; Figure 5I).
In contrast, Flag-T94E was absent from the dynein-
containing 19–20 S fractions but exclusively detected
in the light fractions of the gradient. Consistently, only
endogenous Tctex-1, but not Flag-T94E, was detected
in the DIC immunoprecipitates of the 19–20 S, dynein
complex (Figure 5H). These results collectively demon-
strated that phosphorylation at T94 may be a mecha-
nism to dissociate Tctex-1 from dynein complexes, and
therefore the T94E mutant was only found in the frac-
tions outside the dynein complex pool.
We then examined how overexpression of the T94E
and T94A mutant affects neurite extension. Like wt
Tctex-1-transfected neurons, 4 DIV neurons ectopically
expressing T94E for 18 hr displayed multiple axon-like
processes (Figure 5J). Other morphological features in-
duced by T94E were also indistinguishable from neu-
rons transfected with wt Tctex-1. Namely, the axons
and dendrites were significantly longer, and the den-
drites had more branches compared to those of GFP-
transfected control neurons (Table 1). In striking con-
trast, T94A mutant overexpression did not induce the
axogenic/dendritogenic phenotype caused by wt and
T94E mutant overexpression (Figure 5K). Altogether,
these results argued that the T94 phospho-mimic, com-
plex-free form of Tctex-1 was likely to be an active form
of Tctex-1 for the enhanced neurite outgrowth activity.
These results provided additional support that the neu-
ronal phenotype caused by Tctex-1 occurs through a
Tctex-1 Modulates the Actin Cytoskeleton
through a RhoGTPase-Dependent Pathway
During the course of the above-described experiments,
it became obvious to us that Tctex-1-transfected neu-
rons had consistently less F-actin labeling than control
nontransfected neurons, or those transfected with con-
trol proteins such as GFP-DIC (Figure 3C versus Figure
3F) or Flag-DIC (Figure 6B). The loss of the F-actin sig-
nal was particularly prominent in neurite tips. Quantifi-
cation of phalloidin-labeled F-actin confirmed these
observations (Figure 6A). In fact, the phalloidin staining
of Tctex-1-transfected cells closely resembles that of
cytochalasin D-treated neurons, which had no F-actin
enrichment at the axonal tip. Moreover, while the tu-
bulin level was normally tapered off at the very distal
end of control cells, the tubulin levels were relative con-
stant throughout the distal ends of axons in both Tctex-
1-transfected and cytochalasin D-treated cells. Intrigu-
ingly, a previous report showed that various treatments
that disassemble actin also induce multiple axon-like
processes on cultured hippocampal neurons (Bradke
and Dotti, 1999). The common phenotypes shared by
Tctex-1-transfected and cytochalasin D-treated neu-
rons prompted us to speculate that Tctex-1 may regu-
late axonal outgrowth by attenuating the stability of ac-
It is now well established that RhoGTPase family pro-
teins play pivotal roles in regulating actin cytoskeleton
organization (Ridley et al., 1992). Rho and Rac have op-
posing effects on actin organization and differentially
affect neurite outgrowth and axon formation. For exam-
ple, neurite outgrowth is promoted by Rac1 and inhib-
ited by RhoA (see Luo, 2000; Nikolic, 2002). To test
whether Tctex-1-induced neurite outgrowth and axonal
formation involve a modulation of actin assembly through
Figure 6. Tctex-1 Regulates the Actin Cy-
toskeleton by Modulating RhoGTPase
(A) Quantification of F-actin and tubulin fluo-
rescence intensity at the neuritic tip of 1 DIV
control neurons, neurons transfected with Flag-
Tctex-1, or neurons treated with cytocha-
lasin D (0.5 ?g/ml) for 6 hr. Measurements
were performed pixel by pixel (y axis) along
the distal end of axonal processes (x axis).
taken at 5 ?m intervals are shown in the
graph. Each value represents the mean ±
SEM of at least 50 axonal processes for each
(B) Representative images of distal axonal
processes of neurons transfected with Flag-
DIC and Flag-Tctex-1 and labeled for F-actin
and Flag. All images were taken using iden-
tical confocal settings to quantify paladin la-
beling. Arrows mark the axonal tips for the
zero points used in the quantification de-
scribed in (A).
(C) Equal amounts of protein extracted from
3T3 fibroblasts infected with adenovirus en-
coding Tctex-1/GFP or GFP (left) or neurons
transfected with Tctex-1-si/GFP or control-
si/GFP (right) were subjected to the Rac1
activity assay. A representative immunoblot
shows the total and GTP bound Rac1.
(D–K) 1 DIV neurons singly transfected with
either HA-CA-RhoA (D, E) or HA-DN-Rac1 (F,
G) or double-transfected with Tctex-1/HA-
CA-RhoA (H, I) or Tctex-1/HA-DN-Rac1. (J)
and (K) were immunolabeled for either HA (D, F, I, K) together with MAP2 (E, G) or together with Flag (H, J).
(L–N) Confocal images of cultured neurons transfected with 1 ?g (L), 2 ?g (M), or 4 ?g (N) of myc-CA-Rac1 and labeled with anti-myc Ab.
(O–Q) Confocal images showing the morphology of a neuron cotransfected with myc-CA-Rac1 and Tctex-1-si/GFP (2 ?g each; red, myc;
green, GFP; blue, Tctex-1). Note that the transfected neuron, which displays very faint Tctex-1 immunofluorescence, extended a single axon
(arrows) and several “veiled” shorter neurites. All cells shown in this figure were transfected 2 hr after plating and fixed 24 hr later. Scale bars
equal 10 ?m.
a RhoGTPase-dependent pathway, we asked if the con-
stitutively active (CA) form of RhoA (G14V) or the domi-
nant-negative (DN) form of Rac1 (T17N) could reverse
Tctex-1-mediated phenotypes in cultured neurons.
As expected (Fukata et al., 2002c), neurons singly
transfected with either HA-tagged CA-RhoA (Figures
6D and 6E) or DN-Rac1 (Figures 6F and 6G) mutants
displayed a dramatic inhibition of axon extension and
formation of MAP2+dendrites (also see Table 1). How-
ever, neurons coexpressing CA-RhoA together with
Tctex-1 had a single axon with an average length inter-
mediate between those of neurons singly transfected
with CA-RhoA or Tctex-1 (Figures 6H and 6I; Table 1).
Similarly, Tctex-1 overexpression partially reversed the
inhibition of axon elongation induced by coexpression
of DN-Rac1, even though these neurons still failed to
elaborate MAP2+dendrite processes (Figures 6J and
6K). Collectively, these results suggested that Tctex-1-
induced phenotypes in neurons mimic Rac1 activation
or RhoA inactivation or both.
Tctex-1 overexpression in fibroblasts yielded morpho-
logical changes—enhanced membrane ruffle and loss of
stress fibers (data not shown)—reminiscent of those
induced by the active form of Rac1. Pull-down assay
revealed that Tctex-1 overexpression in 3T3 cells in-
duced a w2-fold increase in Rac1 activity, but not RhoA
activity (data not shown), without affecting total Rac1
levels (Figure 6C). Furthermore, converse experiments
showed that neurons transfected with Tctex-1-siRNA/
GFP had significantly lower Rac1 activity compared to
the control-siRNA/GFP-transfected cells (Figure 6C).
These biochemical results consistently suggested that
Tctex-1 upregulated Rac1 activity.
To further test the possibility that Tctex-1 could regu-
late the actin cytoskeleton through Rac1 activation, we
asked whether CA-Rac1 overexpression could com-
pensate for the Tctex-1-siRNA-mediated inhibitory ef-
fect on neurite development. To this end, we first
showed that neurons transfected with myc-CA-Rac1
alone induced dosage-dependent phenotypic changes.
Cells transfected with a lower amount (1 ?g) of CA-
Rac1 extended a single long axon and several short
minor processes, with growth cones that display promi-
nent lamellipodial veils (Figure 6L). Higher doses of CA-
Rac1, mimicking Tctex-1 overexpression, also induced
multiple axon-like neurites (2 ?g in Figure 6M, 4 ?g in
Figure 6N). Many of these axon-like neurites exhibited
excess lamellipodial “waves,” similar to those de-
scribed in neurons overexpressing Rac-GEF (guanine
nucleotide exchange factor)-Tiam1 (T cell lymphoma
invasion and metastasis protein) (Kunda et al., 2001).
These structures were not detected in Tctex-1-overex-
As shown in Figures 6O–6Q, neurons cotransfected
with equal amounts of Tctex-1-siRNA/GFP and myc-
Rac1-CA plasmids (2 ?g each) displayed a single axon
Tctex-1 in Neurite Development
with several minor neurites. These results suggested
that Rac1 is able to overcome the neuritic inhibitory
effected exerted by Tctex-1-siRNA. The minor neurites
in these double-transfected cells displayed “veiled”
growth cones, closely resembling neurons with lower-
level Rac1 overexpression (Figure 6L). These results,
together with the preceding results, collectively sug-
gested that the neurite and axon development induced
by Tctex-1 was likely to be mediated through Rac1-
duced in Tctex-1-overexpressing neurons. The loss of
F-actin signal was especially profound at the neuronal
tips, consistent with the notion that the actin filaments
located in these regions of the neuron are generally
more labile (Bradke and Dotti, 1999). In fact, the phalloi-
din staining of Tctex-1-transfected cells closely resem-
bles that of cytochalasin D-treated hippocampal neu-
rons. Third, the phenotype of multiple long axons
induced by Tctex-1 overexpression can be, to a large
extent, reversed by the coexpression with mutants of
RhoGTPase proteins, prime regulators of actin reorga-
nization. Finally, Tctex-1 levels modulate Rac1 activity.
All together, these results indicate that Tctex-1 is not
only physically associated, but also functionally in-
volved, with both the microtubule and the microfila-
ment cytoskeletal systems.
Our data also provide multiple lines of evidence ar-
guing that dynein motor activity is not critical for Tctex-
1-mediated neurite initiation and extension. First, the
selective distribution of Tctex-1 in growing axons and
the axonal tips was not shared by the DHC and DIC,
implying that part of the “enriched” Tctex-1 signal may
be free from the dynein complex. Second, p50 dynami-
tin overexpression and DHC gene silencing, which dis-
rupts general dynein function, did not phenocopy the
Tctex-1 knockdown effect. Third, Tctex-1 overexpres-
sion did not disrupt the integrity of the dynein complex,
yet produced a dramatic phenotype. Fourth, the Tctex-
1-mediated neuritic effect can be precisely reproduced
by a phospho-mimic variant, T94E, which fails to incor-
porate into dynein. These results coherently suggested
that the neuritic effect exerted by Tctex-1 is largely in-
dependent from its cargo adaptor role that is involved
in dynein transport.
Nevertheless, the lack of a dramatic effect on neurite
sprouting and axon formation in neurons with DHC ex-
pression/function suppressed does not necessarily in-
dicate a lack of participation in these events, but may
reflect the existence of some compensatory mecha-
nism. Multiple dynein species or minus-end kinesins
have been described, and some of them might be re-
quired for microtubule-based transport during neurite
formation (Hirokawa, 2000) and axon retraction (Ahmad
et al., 2000). In our experiments, impairing dynein activ-
ity by p50 overexpression in cultured hippocampal neu-
rons causes swollen axons, consistent with the histo-
logical changes seen in the axons of motor neurons
in p50-overexpressing transgenic mice (LaMonte et al.,
2002). Finally, p50-overexpressing hippocampal neu-
rons had slightly shorter axons (data not shown), con-
sistent with those described in granule neurons overex-
pressing p50 (Solecki et al., 2004).
This report reveals that Tctex-1, a cytoplasmic dynein
light chain subunit, plays pivotal roles in multiple steps
of hippocampal neuronal development, including neu-
rite initiation, neurite growth, axonal polarity establish-
ment, and dendritic elaboration. We therefore predict
that similar or overlapping mechanisms based on
Tctex-1 are involved in these cellular events during neu-
ronal morphogenesis. Tight control of the asymmetric
and dynamic localization of Tctex-1 during neuronal de-
velopment appears to be a key element providing
forces that shape the neuron. For example, high-level
Tctex-1 accumulation in the distal region and growth
cones of a given neuron at stage 2–3 empowers that
neurite to elicit the fastest growth rate and hence be-
come “committed” to the axonal fate. Conversely, the
near absence of Tctex-1 in minor neurites could prevent
their growth. However, what happens to the axon, hap-
pens later in the minor neurites. Tctex-1 reappears in
the minor neurites at stage 4, thereby allowing dendritic
extension and branching. The ability of Tctex-1 to affect
dendrite development was consistent with Tctex-1 im-
munoreactivity being readily detected in dendritic shafts
in 3 DIV cultures (data not shown), even though Tctex-1
is low in minor neurites of young cultured neurons. The
importance of Tctex-1 in neurite development is well
correlated with its abundance in fetal brains (Kai et al.,
1997) and postmitotic young neurons in adult brain
(Chuang et al., 2001).
Molecular Mechanism Mediating Tctex-1’s
Tctex-1 is known as a light chain of cytoplasmic dynein,
a microtubule-based motor complex. Tctex-1’s associ-
ation with microtubules has been demonstrated both
immunocytochemically and biochemically. Moreover,
the ability of Tctex-1 in docking cargoes onto cytoplas-
mic dynein and, hence, microtubules has been con-
firmed (Tai et al., 1999; Tai et al., 2001).
However, several pieces of evidence described in this
paper led us to propose that Tctex-1 is also involved in
actin organization in undifferentiated neuroblasts and
developing neurons. First, the Tctex-1 punctate signals
at the filopodia edges of neurites were nicely colocalized
with phalloidin-labeled microfilaments. The abundance
of Tctex-1 at axonal tips correlates well with previous
ultrastructural analysis showing a strong Tctex-1 signal
in mossy fibers projecting to CA3 pyramidal neurons
(Chuang et al., 2001). Second, Tctex-1 expression levels
were inversely correlated with the abundance of F-actin:
the level of overall F-actin labeling was significantly re-
Molecular Basis Underlying Tctex-1 Regulation
of Actin Dynamics
Our data suggest that Tctex-1 may participate in more
than one step of actin/microtubule remodeling at the
neurite growth cone. First, Tctex-1 could induce local
actin instability, imitating the effect of cytochalasin D.
Second, Tctex-1 could enhance F-actin and microtu-
bule polymerization by locally activating Rac1. The lat-
ter possibility is highly conceivable because of the en-
richment of Tctex-1 at the axonal growth cones and
its ability to regulate Rac activity. Nevertheless, how
Tctex-1 coordinates these steps of actin organization
in still an open question.
A study of migrating fibroblasts suggests that micro-
tubule growth activates Rac1 and hence actin polymer-
ization (Waterman-Storer et al., 1999). The proteins
preferentially associated with the plus-end of growing
microtubules thus are good candidates to regulate
Rac1 activity. Tctex-1 is also highly concentrated at
microtubule plus ends (Tai et al., 2001). It is thus con-
ceivable that Tctex-1 dissociates from the dynein com-
plex near the microtubule growing end, perhaps via
phosphorylation at Thr94, and locally activates Rac1.
Our data show that the ectopically expressed unphos-
phorylated mimic T94A mutant is distributed in both the
dynein complex and complex-free pools, but it has
no axogenic effect. It is therefore likely that both the
phosphorylation at Thr94 and its dissociation from the
dynein complex are required for the “activation” of
Tctex-1 for its neuritic role.
Tctex-1-enhancing Rac1 activity would be consistent
with the fact that overexpressed Rac1 (this paper) or
Tiam1 (Kunda et al., 2001) also enhanced neurite out-
growth and had an axogenic effect on hippocampal
neuron cultures. Nevertheless, consistent with a previ-
ous report (Threadgill et al., 1997), Rac1 also induces
lamellipodial wave structures located along axons and
has a strong impact on primary dendrites that Tctex-1
does not have, indicating that Tctex-1 only participates
in a subset of the Rac1 signaling pathways. These re-
sults also dovetail with our observations that Tctex-1
overexpression can only reverse the DN-Rac1-medi-
ated axogenic, but not dendritic, effect. In addition,
Tctex-1 suppression hardly inhibited the wave struc-
tures induced by CA-Rac1. The molecular link between
the regulator(s) of Rac1 and Tctex-1 is presently un-
known. Tctex-1 itself is not likely to be a Rac1-GEF be-
cause it lacks a Dbl homology domain, which is required
for GEF activity (Hart et al., 1991). Indeed, our unpub-
lished observations showed that the Rac activation
ability possessed by Tctex-1 was less potent than that
possessed by Rac1GEFs, such as Tiam1. Tctex-1 most
likely regulates Rac1 activity through an indirect path-
way, such as recruiting RacGEF(s) and/or Rac effec-
tor(s). Alternatively, Tctex-1 may sequester RacGTPase
activating protein (GAP) and enhance Rac1 activation.
Many Tctex-1-interacting proteins have so far been iso-
lated (reviewed in Vallee et al., 2004), and whether any
of these proteins play roles in Tctex-1-mediated Rac
activation remains an open question. Finally, because
Rac and Rho activities feed back to each other, we can-
not rule out the possibility that Tctex-1 may also attenu-
ate Rho activity.
Although our knowledge about neurite extension has
improved immensely in the past several years, relatively
little is known about the mechanisms of the first step
of neural development (reviewed in da Silva and Dotti,
2002; Dehmelt and Halpain, 2004). The inhibition of
neurite initiation by Tctex-1 knockdown shown in this
paper is dramatic; almost all neurons that had Tctex-1
suppressed were arrested at stage 1 in 24 hr cultures.
More specifically, the Tctex-1-suppressed neurons re-
sembled those at the substage called stage 1.2 (Deh-
melt and Halpain, 2004). At stage 1.2, the lamellipodia
are segmented and the microtubules have already in-
vaded into the lamellipodia. These processes are in
contrast to stage 1.1 cells, where lamellipodia are uni-
formly distributed around the circumference of the cell.
The arrangement is also unlike stage 1.3 cells, which
have microtubules aligned into parallel arrays (or bun-
dles) at the selective segment. These results are in
agreement with the hypothesis that microtubule poly-
merization takes place subsequent to actin disassem-
bly, further supporting the role of Tctex-1 in regulating
actin dynamics. Nevertheless, because the two cy-
toskeleton systems often feed back to each other (Witt-
mann and Waterman-Storer, 2001), future studies will
be needed to examine the possibility that Tctex-1 could
modulate microtubule stability and hence, microfila-
Last but not least, it is our prediction that the dual
function of Tctex-1 as a motor cargo adaptor in the
microtubule transport system and a microfilament
modulator is also very likely to be an important part of
other cellular events (e.g., cell division, cell migration)
that require a coordinated collaboration between the
two cytoskeleton systems (Rodriguez et al., 2003).
Antisense RNA, siRNA Oligonucleotide, Antibody,
Plasmid Construct, and Virus Production
All antibodies, siRNAs, plasmids, and the details of their construc-
tion are listed in Supplemental Data. Adenoviruses encoding Tctex-1
and GFP were produced using the AdEasy system (Stratagene, La
Jolla, CA). The shuttle vector was prepared by placing Tctex-1 into
pTrack Shuttle vector (gift of Dr. B. Vogelstein, Johns Hopkins Uni-
versity). Adenovirus was amplified and purified as described (Tai et
al., 2001). Adenovirus encoding GFP was a gift of Dr. F. Packe-
Peterson (Weill Medical College, Cornell).
Culture, Transfection, and Immunochemical Analyses
of Hippocampal Neurons
Embryonic hippocampal neuron cultures were prepared as de-
scribed (Goslin and Banker, 1991). AS oligos (5 ?M) were added
twice into culture medium, at 2 hr and 12 hr after plating. For trans-
fection, either plasmid (1–4 ?g) or siRNA oligonucleotide (133 nM)
were mixed with Lipofectamine 2000 and added into either freshly
trypsin-dissociated hippocampal neurons, neurons 2 hr after plat-
ing, or neurons cultured 3 DIV. Neurons were fixed at the indicated
time and processed for immunolabeling as described (Paglini et al.,
1998). FITC- or TRITC-phalloidin were added during the secondary
antibody incubation. All immunostained cells were analyzed by
Leica TCS SP2 spectral confocal system (Nussloch, Germany) or
Zeiss confocal microscope. At least three independent experi-
ments were conducted for each manipulation, with 15–40 cover-
slips and 50–100 cells examined in each experiment. Quantification
of labeling intensities and morphometric analyses were carried out
by using Metamorph software (Universal Imaging Co., Downing-
town, PA) as described (Kunda et al., 2001). In some experiments,
transfected neurons were cultured and prepared according to the
procedures described by Paglini et al. (1998) for time-lapse im-
Rac1 Activity Assays
Protein extracts of siRNA-transfected neurons or adenovirus-
infected NIH 3T3 cells were subjected to Rac1 activity assays fol-
lowing the manufacturer’s instructions (Cytoskeleton, Denver, CO).
Briefly, the GTP bound form of Rac1 was affinity purified by GST
agarose containing the Rac binding domain of Pak1. GTP bound
Rac1 or total cell lysates were immunoblotted with anti-Rac1 anti-
body (BD Transduction Lab, San Diego, CA) using ECL method.
Tctex-1 in Neurite Development
Site-directed mutagenesis of Flag-Tctex-1 was carried out using
Quickchange (Stratagene, La Jolla, CA). Velocity density gradient
sedimentation, immunoprecipitation, and immunoblotting assays
were carried out essentially as described (Tai et al., 1998).
the organization of actin filaments and microtubules in a neuronal
growth cone. J. Cell Biol. 107, 1505–1516.
Fukata, M., Watanabe, T., Noritake, J., Nakagawa, M., Yamaga, M.,
Kuroda, S., Matsuura, Y., Iwamatsu, A., Perez, F., and Kaibuchi, K.
(2002a). Rac1 and Cdc42 capture microtubules through IQGAP1
and CLIP-170. Cell 109, 873–885.
Fukata, Y., Itoh, T.J., Kimura, T., Menager, C., Nishimura, T., Shiro-
mizu, T., Watanabe, H., Inagaki, N., Iwamatsu, A., Hotani, H., and
Kaibuchi, K. (2002b). CRMP-2 binds to tubulin heterodimers to pro-
mote microtubule assembly. Nat. Cell Biol. 4, 583–591.
Fukata, Y., Kimura, T., and Kaibuchi, K. (2002c). Axon specification
in hippocampal neurons. Neurosci. Res. 43, 305–315.
Goslin, K., and Banker, G. (1991). Rat Hippocampal Neurons in
Low-Density Culture (Cambridge, MA: MIT).
Hart, M.J., Eva, A., Evans, T., Aaronson, S.A., and Cerione, R.A.
(1991). Catalysis of guanine nucleotide exchange on the CDC42Hs
protein by the dbl oncogene product. Nature 354, 311–314.
Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and
the mechanism of organelle transport. Science 279, 519–526.
Hirokawa, N. (2000). Stirring up development with the heterotri-
meric kinesin KIF3. Traffic 1, 29–34.
Inagaki, N., Chihara, K., Arimura, N., Menager, C., Kawano, Y.,
Matsuo, N., Nishimura, T., Amano, M., and Kaibuchi, K. (2001).
CRMP-2 induces axons in cultured hippocampal neurons. Nat.
Neurosci. 4, 781–782.
Kai, N., Mishina, M., and Yagi, T. (1997). Molecular cloning of Fyn-
associated molecules in the mouse central nervous system. J. Neu-
rosci. Res. 48, 407–424.
King, S.J., and Schroer, T.A. (2000). Dynactin increases the proces-
sivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24.
Kunda, P., Paglini, G., Quiroga, S., Kosik, K., and Caceres, A. (2001).
Evidence for the involvement of Tiam1 in axon formation. J. Neu-
rosci. 21, 2361–2372.
LaMonte, B.H., Wallace, K.E., Holloway, B.A., Shelly, S.S., Ascano,
J., Tokito, M., Van Winkle, T., Howland, D.S., and Holzbaur, E.L.
(2002). Disruption of dynein/dynactin inhibits axonal transport in
motor neurons causing late-onset progressive degeneration. Neu-
ron 34, 715–727.
Li, M.G., Serr, M., Newman, E.A., and Hays, T.S. (2004). The Dro-
sophila tctex-1 light chain is dispensable for essential cytoplasmic
dynein functions but is required during spermatid differentiation.
Mol. Biol. Cell 15, 3005–3014.
Luo, L. (2000). Rho GTPases in neuronal morphogenesis. Nat. Rev.
Neurosci. 1, 173–180.
Mok, Y.K., Lo, K.W., and Zhang, M. (2001). Structure of Tctex-1 and
its interaction with cytoplasmic dynein intermediate chain. J. Biol.
Chem. 276, 14067–14074.
Nikolic, M. (2002). The role of Rho GTPases and associated kinases
in regulating neurite outgrowth. Int. J. Biochem. Cell Biol. 34, 731–
Paglini, G., Kunda, P., Quiroga, S., Kosik, K., and Caceres, A. (1998).
Suppression of radixin and moesin alters growth cone morphology,
motility, and process formation in primary cultured neurons. J. Cell
Biol. 143, 443–455.
Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., and Hall,
A. (1992). The small GTP-binding protein rac regulates growth fac-
tor-induced membrane ruffling. Cell 70, 401–410.
Rodriguez, O.C., Schaefer, A.W., Mandato, C.A., Forscher, P., Be-
ment, W.M., and Waterman-Storer, C.M. (2003). Conserved micro-
tubule-actin interactions in cell movement and morphogenesis.
Nat. Cell Biol. 5, 599–609.
Schwamborn, J.C., and Puschel, A.W. (2004). The sequential activ-
ity of the GTPases Rap1B and Cdc42 determines neuronal polarity.
Nat. Neurosci. 7, 923–929.
Shi, S.H., Jan, L.Y., and Jan, Y.N. (2003). Hippocampal neuronal
polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase
activity. Cell 112, 63–75.
Shu, T., Ayala, R., Nguyen, M.D., Xie, Z., Gleeson, J.G., and Tsai,
Supplemental Data include seven figures, two tables, and Supple-
mental Experimental Procedures and can be found with this article
online at http://www.developmentalcell.com/cgi/content/full/9/1/
We are indebted to the kind gifts given by our colleagues as men-
tioned in the text. This work was supported by Research To Prevent
Blindness, The Irma T. Hirsch Trust, The Ruth and Milton Steinbach
Fund, and NIH EY11307 to C.-H.S. and FONCyT, John Simon Gug-
genheim Foundation Fellowship, and the Howard Hughes Medical
Institute (HHMI 75197-553201 International Research Scholars Pro-
gram) to A.C.
Received: July 22, 2004
Revised: January 19, 2005
Accepted: April 13, 2005
Published: July 5, 2005
Ahmad, F.J., Hughey, J., Wittmann, T., Hyman, A., Greaser, M., and
Baas, P.W. (2000). Motor proteins regulate force interactions be-
tween microtubules and microfilaments in the axon. Nat. Cell Biol.
Baas, P.W., Ahmad, F.J., Pienkowski, T.P., Brown, A., and Black,
M.M. (1993). Sites of microtubule stabilization for the axon. J. Neu-
rosci. 13, 2177–2185.
Bradke, F., and Dotti, C.G. (1999). The role of local actin instability
in axon formation. Science 283, 1931–1934.
Chuang, J.Z., Milner, T.A., and Sung, C.H. (2001). Subunit hetero-
geneity of cytoplasmic dynein: differential expression of 14 kDa dy-
nein light chains in rat hippocampus. J. Neurosci. 21, 5501–5512.
Corthesy-Theulaz, I., Pauloin, A., and Pfeffer, S. (1992). Cytoplas-
mic dynein participates in the centrosomal localization of the Golgi
complex. J. Cell Biol. 118, 1333–1345.
da Silva, J.S., and Dotti, C.G. (2002). Breaking the neuronal sphere:
regulation of the actin cytoskeleton in neuritogenesis. Nat. Rev.
Neurosci. 3, 694–704.
Dehmelt, L., and Halpain, S. (2004). Actin and microtubules in neu-
rite initiation: are MAPs the missing link? J. Neurobiol. 58, 18–33.
Dotti, C.G., Sullivan, C.A., and Banker, G.A. (1988). The establish-
ment of polarity by hippocampal neurons in culture. J. Neurosci. 8,
Echeverri, C.J., Paschal, B.M., Vaughan, K.T., and Vallee, R.B.
(1996). Molecular characterization of the 50-kD subunit of dynactin
reveals function for the complex in chromosome alignment and
spindle organization during mitosis. J. Cell Biol. 132, 617–633.
Esch, T., Lemmon, V., and Banker, G. (1999). Local presentation of
substrate molecules directs axon specification by cultured hippo-
campal neurons. J. Neurosci. 19, 6417–6426.
Ferreira, A., Busciglio, J., and Caceres, A. (1989). Microtubule for-
mation and neurite growth in cerebellar macroneurons which de-
velop in vitro: evidence for the involvement of the microtubule-
associated proteins, MAP-1a, HMW-MAP2 and Tau. Brain Res. Dev.
Brain Res. 49, 215–228.
Fletcher, T.L., Cameron, P., De Camilli, P., and Banker, G. (1991).
The distribution of synapsin I and synaptophysin in hippocampal
neurons developing in culture. J. Neurosci. 11, 1617–1626.
Forscher, P., and Smith, S.J. (1988). Actions of cytochalasins on
Developmental Cell Download full-text
L.H. (2004). Ndel1 operates in a common pathway with LIS1 and
cytoplasmic dynein to regulate cortical neuronal positioning. Neu-
ron 44, 263–277.
Solecki, D.J., Model, L., Gaetz, J., Kapoor, T.M., and Hatten, M.E.
(2004). Par6alpha signaling controls glial-guided neuronal migra-
tion. Nat. Neurosci. 7, 1195–1203.
Tai, A.W., Chuang, J.-Z., and Sung, C.-H. (1998). Localization of
Tctex-1, a cytoplasmic dynein light chain, to the Golgi apparatus
and evidence for dynein complex heterogeneity. J. Biol. Chem. 273,
Tai, A.W., Chuang, J.-Z., Bode, C., Wolfrum, U., and Sung, C.-H.
(1999). Rhodopsin’s carboxy-terminal cytoplasmic tail acts as a
membrane receptor for cytoplasmic dynein by binding to the dy-
nein light chain Tctex-1. Cell 97, 877–887.
Tai, A.W., Chuang, J.-Z., and Sung, C.-H. (2001). Cytoplasmic dy-
nein regulation by subunit heterogeneity and its role in apical trans-
port. J. Cell Biol. 153, 1499–1509.
Threadgill, R., Bobb, K., and Ghosh, A. (1997). Regulation of den-
dritic growth and remodeling by Rho, Rac, and Cdc42. Neuron 19,
Vallee, R.B., Williams, J.C., Varma, D., and Barnhart, L.E. (2004).
Dynein: an ancient motor protein involved in multiple modes of
transport. J. Neurobiol. 58, 189–200.
Waterman-Storer, C.M., Karki, S., and Holzbaur, E.L. (1995). The
p150Glued component of the dynactin complex binds to both
microtubules and the actin-related protein centractin (Arp-1). Proc.
Natl. Acad. Sci. USA 92, 1634–1638.
Waterman-Storer, C.M., Worthylake, R.A., Liu, B.P., Burridge, K.,
and Salmon, E.D. (1999). Microtubule growth activates Rac1 to pro-
mote lamellipodial protrusion in fibroblasts. Nat. Cell Biol. 1, 45–50.
Wittmann, T., and Waterman-Storer, C.M. (2001). Cell motility: can
Rho GTPases and microtubules point the way? J. Cell Sci. 114,
Zhou, F.Q., Zhou, J., Dedhar, S., Wu, Y.H., and Snider, W.D. (2004).
NGF-induced axon growth is mediated by localized inactivation of
GSK-3beta and functions of the microtubule plus end binding pro-
tein APC. Neuron 42, 897–912.