doi: 10.1242/10.1242/jcs.00208
Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth
Tomoyuki Abe1,
Masayoshi Kato1,2,
Hiroaki Miki3,5,
Tadaomi Takenawa2,4 and
Takeshi Endo1,4,*
1 Department of Biology, Faculty of Science, and Graduate School of Science and
Technology, Chiba University, Yayoicho, Inageku, Chiba, Chiba 263-8522,
Japan
2 Division of Biochemistry, Institute of Medical Science, University of Tokyo,
Shirokanedai, Minatoku, Tokyo 108-8639, Japan
3 Division of Cancer Genomics, Institute of Medical Science, University of
Tokyo, Shirokanedai, Minatoku, Tokyo 108-8639, Japan
4 CREST, Japan Science and Technology Corporation (JST), Japan
5 PRESTO, Japan Science and Technology Corporation (JST), Japan
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Fig. 1. Structural similarity and phylogenetic relationship of Tc10 and RhoT. (A)
Comparison of the amino-acid sequences of mouse Tc10 and RhoT with those of
representative Rho family members. The origins of these Rho family proteins
are mouse Cdc42 (accession number L78075), human RhoG (X61587), mouse
Rac1(X57277), mouse RhoA (AF014371), human RhoE (X95282), human Rnd1 (Y07923),
mouse RhoD (D89821) and human RhoH (Z35227). The amino-acid sequences were
aligned by the method of Lipman and Pearson
(Lipman and Pearson, 1985 ) and
by eye. Amino acids at positions of >50% identity are shown in red. G1-G4,
core motifs for GTPase activity and GTP/GDP-binding. E, effector domain.
Switch I, switch II, Rho insert regions and CaaX motif are also indicated. (B)
Phylogenetic relationship of Rho family proteins. The phylogenetic tree was
drawn by using TreeView (Page,
1996). Rho family proteins are subdivided into six subfamilies.
RhoT and Tc10 belong to the Cdc42 subfamily. (C) Comparison of the cDNA
sequences of mouse RhoT (AB060651) and TCL (AJ276568) with
the corresponding mouse genomic sequence of chromosome 12, which was searched
using the Ensembl Genome Browser. Only a portion containing the initiation
codon and corresponding amino-acid sequences of RhoT and TCL are shown.
Matched nucleotides are marked with asterisks. Gaps are indicated with
dashes.
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Fig. 2. Expression of Cdc42, Tc10 and RhoT in several tissues and cells during
differentiation. (A) Northern blots showing the expression of Cdc42 (a), Tc10
(b) and RhoT (c). (d) Ethidium bromide staining pattern of the agarose gel
electrophoresis of the total or cytoplasmic RNAs showing the 28S and 18S
rRNAs. The RNAs were derived from newborn mouse brain and adult mouse tissues;
C2 cells during differentiation and 10T1/2 cells; PC12 cells during
differentiation; and N1E-115 cells during differentiation. C2 myoblasts (Mb)
were left in the differentiation medium for the time (hours) indicated in the
figure and differentiated to form myotubes (Mt) by 96 hours. Differentiation
of PC12 cells was induced with 0.5 mM dbcAMP or with 50 ng/ml NGF.
Differentiation of N1E-115 cells was induced by serum starvation [serum (-)]
or with 2% DMSO. The time (hours) after the treatment with each reagent is
noted. The size (kb) of each mRNA band is designated at the left of the panel.
The positions of 28S and 18S rRNAs are indicated at the right. Two faint bands
with slower mobility than the 2.5 kb RhoT band in the (c) leftmost panel are
unerased Tc10 bands after rehybridization. (B) Quantitative RT-PCR analyses
for the expression of RhoT (a) and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) as a control (b) in brain and PC12 and N1E-115 cells. PC12 and N1E-115
cells were treated with NGF (N) or dbcAMP (A) and by serum starvation (S) or
with DMSO (D) for 96 hours to induce differentiation.
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Fig. 3. Distinctive forms of cellular processes generated by Cdc42, Tc10 and RhoT
in fibroblasts. Balb/3T3 cells (A) and 10T1/2 cells (B) were transfected with
the cDNAs of Cdc42(G12V) (a,b), Tc10(G18V) (c,d) and RhoT(G30V) (e,f) fused to
the pEF-BOS/Myc vector. 48 hours after the transfection, the cells were doubly
stained with the anti-Myc mAb Myc1-9E10 to detect the Myc-tagged protein
(a,c,e) and with rhodamine-phalloidin to reveal the actin filaments (b,d, f).
Bar, 20 µm.
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Fig. 4. Generation of neurites by Tc10 or RhoT but not by Cdc42. PC12 cells (A,C)
and N1E-115 cells (B,D) were transfected with the cDNAs of wt or
constitutively active forms of Cdc42, Tc10 and RhoT fused to pEF-BOS/Myc
vector. (A,B) Fluorescent micrographs of the cells expressing the Myc-tagged
proteins. Forty-eight hours after transfection, the cells were stained with
Myc1-9E10 to detect wt (a,c,e) and constitutively active (b,d,f) forms of
Cdc42 (a,b), Tc10 (c,d) and RhoT (e,f). Bar, 20 µm. (C,D) Ratio of the
neurite-extending cells. The degree of neurite extension is expressed as a
multiple of the cell body diameter as indicated in the legends. More than 100
cells were assessed in each experiment. The values are the means±s.d.
of triplicate experiments.
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Fig. 5. Binding of Cdc42, Tc10 and RhoT to the CRIB motif of N-WASP leading to the
Arp2/3-complex-mediated actin polymerization. (A) Yeast two-hybrid interaction
assay. The yeast strain Y190 was transformed with wt, constitutively active or
dominant-negative mutants of the small GTPases in bait pGBT9 vector and with
the N-terminal portion (amino acids 1-275) of N-WASP containing the CRIB motif
in prey pACT2 vector. The interaction was analyzed by ß-galactosidase
colony-lift filter assay. (B) Pull-down assay. GST-tagged small GTPases loaded
with GTP S or GDP were immobilized to glutathione-Sepharose, and then
the lysate from Balb/3T3 cells transfected with HA-tagged N-WASP was applied
to the resin. Proteins bound to the small GTPases were eluted, and N-WASP was
detected by immunoblotting (a). Used GST-small GTPases were analyzed by
SDS-PAGE (b). (C) Fluorometric actin polymerization assay. Protein mixtures
contain 60 nM Arp2/3 complex, 100 nM His-N-WASP, 200 nM GST-VCA or 400 nM
small GTPases in X buffer. The reaction was started by adding the mixture of 2
µM unlabeled actin and 0.2 µM pyrene-actin to the preincubated protein
mixtures, and fluorescence change was measured at 407 nm.
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Fig. 8. Suppression of dbcAMP-induced neurite outgrowth in PC12 cells and
serum-starvation-induced neurite outgrowth in N1E-115 cells by
dominant-negative mutants of Cdc42, Tc10 and RhoT. PC12 and N1E-115 cells were
transfected with the cDNA of Cdc42(T17N), Tc10(T23K) or RhoT(T35N) in
pEF-BOS/Myc vector or with empty pEGFP-C1 vector (mock). Ten hours after the
transfection, PC12 cells were treated with 0.5 mM dbcAMP and the N1E-115 cells
were shifted to 0.5% FBS. They were stained with Myc1-9E10 48 hours after the
treatment. (A,B) PC12 (A) and N1E-115 (B) cells mock-transfected (a,b) or
transfected with Cdc42(T17N) (c,d), Tc10(T23K) (e,f) or RhoT(T35N) (g,h).
Shown are phase-contrast (a,c,e,g) and Myc1-9E10-stained fluorescent (b,d,f,h)
micrographs. Note that the cells with no expression of these mutants extend
neurites. Bar, 20 µm. (C,D) Ratio of the neurite-extending PC12 (C) and
N1E-115 (D) cells. The degree of neurite extension is expressed by multiples
of the cell body diameter as indicated in the legend. More than 100 cells were
assessed in each experiment. The values are the means±s.d. of
triplicate experiments.
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© The Company of Biologists Ltd 2003
cof (B) in pcDL-SR
vector.
Forty-eight hours after the transfection, the cells were doubly stained with
Myc1-9E10 to detect the expression of Cdc42(G12V) (a), Tc10(G18V) (c) and
RhoT(G30V) (e) and with anti-N-WASP pAb (b,d, f). Bar, 20 µm.