Developmental Cell 11, 411–421, September, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.07.008

GTP Hydrolysis by the Rho Family GTPase TC10Promotes Exocytic Vesicle Fusion

Kazuho Kawase,1,2 Takeshi Nakamura,1,3,6,*

Akiyuki Takaya,1 Kazuhiro Aoki,1,3

Kazuhiko Namikawa,4 Hiroshi Kiyama,4

Shuichiro Inagaki,2 Hiroshi Takemoto,2

Alan R. Saltiel,5 and Michiyuki Matsuda1,3,6

1Department of Signal TransductionResearch Institute for Microbial DiseasesOsaka UniversityOsaka 565-0871Japan2Discovery Technologies 2Discovery Research LaboratoriesShionogi & Co., Ltd.Osaka 553-0002Japan3Department of Pathology and Biology of DiseasesGraduate School of MedicineKyoto UniversityKyoto 606-8501Japan4Department of Anatomy and NeurobiologyGraduate School of MedicineOsaka City UniversityOsaka 545-8585Japan5Life Sciences Institute andDepartment of Internal Medicine and PhysiologyThe University of Michigan Medical CenterAnn Arbor, Michigan 48109

Summary

TC10, a Rho family GTPase, has been shown to play animportant role in the exocytosis of GLUT4 and other

proteins, primarily by tethering the vesicles at theplasma membrane. Using a newly developed probe

based on fluorescence resonance energy transfer,we found that TC10 activity at tethered vesicles drop-

ped immediately before vesicle fusion in HeLa cellsstimulated with epidermal growth factor (EGF), sug-

gesting that GTP hydrolysis by TC10 is a critical stepin vesicle fusion. In support of this model, a GTPase-

deficient TC10 mutant potently inhibited EGF-inducedvesicular fusion in HeLa cells and depolarization-

induced neuronal secretion. Furthermore, we foundthat GTP hydrolysis by TC10 in the vicinity of the

plasma membrane was dependent on Rac and the re-dox-regulated Rho GAP, p190RhoGAP-A. We propose

that an EGF-stimulated GAP accelerates GTP hydroly-

sis of TC10, thereby promoting vesicle fusion.

Introduction

Membrane trafficking is of great importance in a widerange of physiological and pathological processes. To

*Correspondence: tnakamr@path1.med.kyoto-u.ac.jp6 Lab address: http://iisi.path1.med.kyoto-u.ac.jp/mm/index.htm

date, numerous molecules have been identified as im-portant regulators of vesicular trafficking via genetic,biochemical, and cell biological studies (Schekman,2002; Rothman, 2002). Recently, Rho GTPases, whichregulate a number of diverse cellular functions includingcytoskeletal organization, transcriptional regulation,and cell growth control (Bishop and Hall, 2000; Van Aelstand D’Souza-Schorey, 1997), have also been identifiedas being involved in various aspects of membrane traf-ficking (Symons and Rusk, 2003; Ridley, 2001).

Rab and Arf have been regarded as principal classes ofGTPases that regulate membrane trafficking (Takai et al.,2001). An archetypal function of Rab proteins is vesicletethering by interaction with specific effectors on targetmembranes (Jahn et al., 2003; Zerial and McBride,2001). Arf regulates vesicle budding mainly through theassembly of coat proteins (Chavrier and Goud, 1999;Nie et al., 2003). In contrast to Rab and Arf, a mechanisticview of how Rho GTPases control vesicular traffickingremains largely unknown. To address this issue, wetook advantage of activity imaging using a fluorescentresonance energy transfer (FRET) probe. TC10, a Rhofamily GTPase, has been shown to play a significantrole in the exocytosis of GLUT4 (Chiang et al., 2001; Salt-iel and Pessin, 2002) and other proteins (Cuadra et al.,2004; Cheng et al., 2005). Furthermore, TC10 has beenshown to be mainly localized to vesicular structures(Michaelson et al., 2001), which makes it suitable formonitoring activity changes on vesicles.

In the present study, we report the visualization of GTPhydrolysis of TC10 immediately before vesicle fusion us-ing a combination of a newly developed FRET probe andtotal internal reflection fluorescence (TIRF) microscopy.We postulate that GTP hydrolysis by TC10 triggers ves-icle fusion. In support of this model, a GTPase-deficientTC10 mutant potently inhibited epidermal growth factor(EGF)-induced vesicular fusion in HeLa cells and depo-larization-induced secretion of neuropeptide Y (NPY) inPC12 cells. Our study also indicated that GTP-TC10 isrequired for loading its binding partners onto vesiclesand the delivery of vesicles to target membranes.Thus, TC10 could play roles in three separate steps ofexocytosis: loading of the cargo, tethering to the plasmamembrane, and triggering vesicle fusion.

Results

Development of a Probe for TC10 ActivityIn order to elucidate the precise role of TC10 in exocyticprocesses, we developed a green fluorescent protein(GFP)-based FRET probe, designated Raichu-TC10.The basic structure of Raichu-TC10 was identical tothat of the Rac1 reporter Raichu-Rac1 (Itoh et al.,2002), except that the Rac binding domain (RBD) ofPOSH and TC10 were substituted for the RBD of PAKand Rac1, respectively (Figure 1A).

Characterization of Raichu-TC10 was conducted inthe same way as for other Raichu probes that we devel-oped previously (Mochizuki et al., 2001; Itoh et al., 2002;Yoshizaki et al., 2003; Takaya et al., 2004). Raichu

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probes for wild-type, Q75L, and T31N mutants of TC10were expressed in 293F cells and their emission profileswere examined with an excitation wavelength of 433 nm.In Raichu-type probes, FRET was typically observed asan increase in the emission peak of 527 nm (sensitizedFRET channel) and a concurrent decrease in the emis-sion peak of 475 nm (CFP channel) (Mochizuki et al.,2001). Therefore, the emission ratio of 527 nm to 475 nmis used to represent FRET efficiency. Compared withthe wild-type TC10 probe, Raichu-TC10-Q75L, whichlacks GTPase activity, had an increased FRET ef-ficiency, while Raichu-TC10-T31N, which shows a re-duced affinity to guanine nucleotides, had a decreasedFRET efficiency (Figure 1B). The GTP loading of theTC10 probes correlated well with that of the authenticTC10 proteins (Figure 1C). Next, we tested the specific-ity of Raichu-TC10 on a panel of Rho family GTPaseGAPs and GEFs (Figure 1D). p50RhoGAP, previously

Figure 1. Basic Properties of Raichu-TC10

(A) Schematic representation of Raichu-TC10 bound to GDP or GTP.

YFP and CFP denote a yellow- and cyan-emitting mutant of GFP, re-

spectively. RBD, GEF, and GAP indicate the Rac binding domain of

POSH, the guanine nucleotide exchange factor for TC10, and the

TC10 GTPase-activating protein, respectively.

(B) Emission spectra of Raichu-TC10 (excited at 433 nm) expressed

in 293F cells. WT, Q75L, and T31N denote wild-type, constitutively

active mutant, and dominant-negative mutant, respectively.

(C) 293T cells expressing Raichu-TC10 or EGFP-TC10 were labeled

with 32Pi. The guanine nucleotides bound to the GTPases were ana-

lyzed by TLC, and the average of two samples is shown with SEM.

(D) FRET efficiency of Raichu-TC10 coexpressed with various GAPs

and GEFs for Rho family GTPases in 293F cells was obtained by flow

cytometry and is shown as the mean 6 SEM.

(E) Negative linear correlation between the FRET efficiency of Rai-

chu-TC10 and the amount of coexpressing p50RhoGAP. The FRET

efficiency was obtained from spectrograms of 293T cells cotrans-

fected with pRaichu-TC10 and varying amounts of pIMR21-p50Rho-

GAP. The bars are SEM (n = 2).

shown to have GAP activity toward TC10 (Neudaueret al., 1998), and p190RhoGAP potently stimulated theGTPase activity of Raichu-TC10. We also observed anegative linear correlation between the level of p50Rho-GAP expression and the FRET efficiency of Raichu-TC10 (Figure 1E). KIAA0053 and TCGAP moderatelydecreased the FRET efficiency. CdGAP, a GAP specificfor Cdc42 (Itoh et al., 2002), had no clear effect on FRET.In contrast, Raichu-TC10 in the presence of DOCK180and Dock4 showed a reproducible increase in FRET ef-ficiency (Figure 1D). Dock2 and Tiam1 did not increasethe level of FRET. The effect of C3G was modest inthis assay. These results indicate that Raichu-TC10could monitor the balance between GEF and GAP activ-ities acting on TC10.

Localization-Specific Differences in TC10 ActivityAs TC10 protein is expressed in a wide range of celllines, including both secretory and nonsecretory cells(see Figure S1A in the Supplemental Data availablewith this article online), we mainly used HeLa cells inthe present study. Raichu-TC10 was localized to vesi-cles, perinuclear compartments (PNC), and the plasmamembrane (Figure 2A, DIC and sensitized FRET im-ages). This distribution of Raichu-TC10 was indistin-guishable from that of authentic TC10 (Figures S1Band S1C; see also Movie S1) and consistent with a previ-ous report (Michaelson et al., 2001). Colocalization stud-ies showed very little overlap between EGFP-TC10-ex-pressing vesicles and the vesicular structures positivefor mRFP-Rab5a or mRFP-Rab7, which were used asmarkers for early and late endosomes, respectively (Fig-ures S1D and S1E). In contrast, TC10-expressing vesi-cles partially overlapped with vesicular structures posi-tive for Rab11 and transferrin, markers for recyclingendosomes (Figures S1D–S1G) as described previously(Michaelson et al., 2001). Moreover, a substantial part ofTC10 colocalized with VSV-G and NPY, used as markersfor exocytosing vesicles (Figures S1H–S1K). To summa-rize, TC10 was localized to exocytosing vesicles and re-cycling endosomes but was absent from early and lateendosomes, a pattern that is consistent with TC10having a role in exocytosis.

Using the Raichu-TC10/TC10-CT probe, we foundthat TC10 activity at the vesicles was markedly higherthan that at the plasma membrane, and moderatelyhigher than that in the PNC (Figures 2A–2C). The lowTC10 activity at the plasma membrane was confirmedin confluent MDCK cells (Figure S2). In cells expressinga negative control probe, Raichu-TC10-T31N/TC10-CT,such regional differences in the FRET signal were notobserved (data not shown). To exclude the possibilitythat the high FRET efficiency was caused by probe ac-cumulation at the vesicles, we generated correlationplots of the sensitized FRET intensities versus the CFPintensities (Figure 2D), in which the slopes reflectedthe level of FRET. The results confirmed that the levelof FRET was significantly higher at the vesicles than inthe PNC or at the plasma membrane. As the vesiculardistribution of TC10 is restricted to the exocytosis path-way, one possibility is that TC10 is converted to theGTP-bound state on loading onto vesicles from PNC,and then to the GDP-bound form on unloading fromvesicles to the plasma membrane.

Vesicle Fusion Promoted by GTP Hydrolysis of TC10413

Figure 2. Distribution of TC10 Activity in

HeLa Cells

(A) DIC (left), sensitized FRET (middle), and

FRET (right) images of a Raichu-TC10-ex-

pressing HeLa cell. The FRET image is shown

in the pseudocolor mode. The upper and

lower limits of the FRET efficiency are shown

on the bottom. The scale bars represent

25 mm.

(B) The left and right panels show a high-

magnification view of the boxed region in

the middle and right panels in (A), respec-

tively. The scale bars represent 5 mm.

(C) The bars represent the mean 6 SD (n = 13)

of the relative FRET efficiency (sensitized

FRET/CFP ratio) of Raichu-TC10 at the

plasma membrane (PM), vesicles, and PNC.

(D) Correlation plots of the sensitized FRET

intensities versus the CFP intensities of Rai-

chu-TC10 at the vesicles, PNC, and PM. The

slopes and correlation coefficients calculated

by linear regression are 1.23 and 0.95 (vesi-

cles), 0.96 and 0.90 (PNC), and 0.81 and

0.98 (PM), respectively.

GTP Hydrolysis by TC10 Immediately

before Vesicular FusionThe difference in TC10 activity between vesicles and theplasma membrane prompted us to visualize thechanges in TC10 activity during vesicular fusion, usingTIRF microscopy (Ma et al., 2004; Tsuboi et al., 2004;Fix et al., 2004) (Figure S3A; see also Movie S2). EGFstimulation strongly accelerated the exocytosis of vesi-cles containing the probe in HeLa cells (see Figure 5A,control, below). The procedure for obtaining ratio im-ages of vesicles (Figure S4A) is described in Experimen-tal Procedures. Figure 3A and Figures S4B and S5Ashow sequential images of sensitized FRET and the ratio(sensitized FRET/CFP) during the EGF-induced fusionprocess. Time point zero was set to the first frame show-ing the highest fluorescence intensity of the vesicles.Before time point zero in Figure 3A, the vesicles hadstopped lateral movement; that is, they had been teth-ered to the plasma membrane (Lizunov et al., 2005) buthad not yet fused to the plasma membrane. After timepoint zero, the fluorescence diffused into the plasmamembrane, indicating that vesicular fusion began be-tween 0 and 0.2 s. The FRET level of the vesicles drop-ped immediately before fusion. In this experiment, weused Raichu-TC10-NC as a negative control probe, be-cause a dominant-negative form, Raichu-TC10-T31N,mostly accumulated in the perinuclear region. When

Raichu-TC10-NC was used in this assay, the FRET leveldid not change during the fusion process (Figure 3B;Figures S4C and S5B). We further analyzed the FRETlevel on the vesicles in a more quantitative manner (Fig-ures 3C–3E). The method of image analysis is describedin Experimental Procedures. The FRET efficiency wasnormalized to the level observed at time point zero tocompare the time-dependent changes of FRET effi-ciency between Raichu-TC10 and Raichu-TC10-NC(Figure 3E). The FRET level of vesicles containing Rai-chu-TC10, but not Raichu-TC10-NC, was significantlydecreased between 20.2 and 0 s (p < 0.01). Therefore,vesicular fusion occurred within 0.4 s of GTP hydrolysisby TC10.

EGF-Induced TC10 GTP Hydrolysis at the Plasma

MembraneTo investigate the mechanism of GTP hydrolysis byTC10 prior to fusion, we examined the changes inTC10 activity in EGF-stimulated cells. In HeLa cells,EGF treatment rapidly decreased the level of FRET, sug-gesting a decrease in GTP-TC10 (Figures 4A and 4G).This inactivation of TC10 was confirmed by a pull-downexperiment using an immobilized GST-PAK-CRIB do-main fusion protein (Figure 4J, 2DPI). Because thedecrease in the FRET level was most prominent atthe plasma membrane, including nascent lamellipodia

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Figure 3. GTP Hydrolysis by TC10 Immediately before Fusion

(A and B) The sensitized FRET intensity and ratio images of Raichu-TC10 (A) and Raichu-TC10-NC (B) in fusing vesicles obtained by TIRF mi-

croscopy. The first and second rows show plots of the sensitized FRET intensity scanned across the center of the fusing vesicles and pseudo-

colored 3D plots of the sensitized FRET intensity, respectively (see details in Figures S3B–S3E).

(C and D) Time course of normalized sensitized FRET and CFP intensities of vesicles containing Raichu-TC10 (C) or Raichu-TC10-NC (D).

(E) Time-dependent changes in the normalized FRET efficiency of Raichu-TC10- or Raichu-TC10-NC-expressing vesicles. The bars in (C)–(E) are

SEM (Raichu-TC10, n = 8; Raichu-TC10-NC, n = 11).

(Figure 4G; Figure S6), we used Raichu-TC10/K-RasCT,the localization of which was largely restricted to theplasma membrane, in the following quantitative analysis(Figure 4H, CFP). In cells expressing Raichu-TC10/K-RasCT, the level of FRET dropped to its nadir within5 min of EGF addition (Figures 4B and 4H). BecauseEGF stimulation modulates the activities of several Rasand Rho GTPases (Mochizuki et al., 2001; Kurokawaet al., 2004), we examined the effects of a panel of dom-inant-negative mutants of Ras and Rho GTPases onTC10 inactivation following EGF stimulation. We foundthat a dominant-negative mutant of Rac1, Rac1-N17, in-hibited EGF-induced TC10 downregulation, particularlyin the early phase (Figure 4C). Rac1 activates p190Rho-GAP via reactive oxygen species (ROS) (Nimnual et al.,2003). Because p190RhoGAP stimulated the GTPaseactivity of TC10 (Figure 1D), it is possible that the Rac/ROS/p190RhoGAP pathway is involved in EGF-inducedTC10 inactivation at the plasma membrane. In supportof this idea, diphenyleneiodonium chloride (DPI), an in-hibitor of ROS generation, almost completely blocked

the EGF-induced TC10 inactivation (Figure 4D). Theblockage of TC10 inactivation by DPI treatment wasconfirmed by a pull-down experiment (Figure 4J). Ithas been shown that EGF-induced p190RhoGAP activa-tion depends on p120RasGAP (Ellis et al., 1990). Inagreement with this, overexpression of a dominant-neg-ative mutant of p120RasGAP consisting mostly of SH2and SH3 domains (p120RasGAP-232) also inhibitedEGF-induced TC10 inactivation (Figure 4E). Further-more, knockdown of p190RhoGAP-A, one of the twop190RhoGAP isoforms, by RNAi (Figure S7A) was foundto abrogate TC10 inactivation (Figure 4F; and timecourse in Figure 4I). These data strongly suggest thatGTP hydrolysis by TC10 was caused primarily by EGF-induced activation of p190RhoGAP-A at the plasmamembrane.

A Critical Role for TC10 GTP Hydrolysis in Vesicle

FusionThe following observations suggest to us that GTPhydrolysis by TC10 is an essential step during vesicle

Vesicle Fusion Promoted by GTP Hydrolysis of TC10415

Figure 4. EGF-Induced GTP Hydrolysis by TC10 at the Plasma Membrane

(A–F) HeLa cells were transfected with pRaichu-TC10/TC10-CT (A) or pRaichu-TC10/K-RasCT (B–F), cotransfected with pERedNLS-Rac1N17

(C), pERedNLS-p120RasGAP-232 (E), pSUPER-p190RhoGAP-A (F), or empty pSUPER vector (F), stimulated with EGF, and then imaged every

1 min. In (D), the cells were treated with 5 mM DPI for 30 min before addition of EGF. The relative FRET efficiency was expressed by measuring the

decrease against the basal activity, which was averaged over 10 min before the EGF addition. The bars are SEM (n > 5).

(G and H) FRET images of Raichu-TC10/TC10-CT- or Raichu-TC10/K-RasCT-expressing cells before and after EGF addition are shown in the

pseudocolor mode. The CFP image at time point zero is also shown. The scale bars represent 25 mm.

(I) FRET images of HeLa cells expressing Raichu-TC10/K-RasCT and pSUPER-p190RhoGAP-A before and after EGF addition are as shown in (G)

and (H). The scale bars represent 25 mm.

(J) HeLa cells expressing HA-TC10 were treated with EGF for the indicated periods in the absence or presence of DPI and then examined by

Bos’s pull-down method. The bars represent the mean 6 SD (n = 3) of the relative TC10 activity as the number of times it decreased compared

with the values of serum-starved cells at 0 min.

fusion (Figures 5A–5C). First, expression of a GTPaseactivity-deficient mutant of TC10, TC10-Q75L, markedlyinhibited EGF-induced vesicular fusion in HeLa cells.Second, inhibition of GTP hydrolysis by TC10 using ex-pression of a dominant-negative mutant of Rac1 (Rac1-N17), DPI treatment, and RNAi-mediated knockdown ofp190RhoGAP-A prevented vesicle fusion. Our observa-tions agree with a previous report that constitutivetransport of VSV-G protein to the plasma membrane isseverely impaired by TC10-Q75L expression in 3T3L1cells (Kanzaki et al., 2002). Intriguingly, a dominant-neg-ative mutant of TC10, TC10-T31N, and RNAi-mediatedknockdown of TC10 (Figure S7B) also inhibited vesicularfusion (Figures 5A and 5C). This is consistent with thefinding that TC10 is required to recruit the exocyst

complex to the plasma membrane through its bindingto Exo70 (Inoue et al., 2003), which is a component ofthe exocyst complex (TerBush et al., 1996).

To further explore the possibility that GTP hydrolysisby TC10 is a critical step in vesicle fusion, we measuredthe lifetime of vesicles that were tethered on the plasmamembrane after EGF stimulation. To process the TIRFimages, we developed an algorithm to count the numberof vesicles that appeared in the time-lapse images afterEGF stimulation and remained at the same location formore than 0.5 or 2.5 min (see Figure S8 for the criteriaof vesicles). Expression of TC10-Q75L not only in-creased the number of vesicles that were tethered tothe plasma membrane after EGF stimulation, but alsoextended their lifetime (Figure 5D). Knockdown of

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p190RhoGAP-A also increased the number of newlytethered vesicles and increased their lifetime, albeit toa lesser extent (Figure 5E). In contrast, depletion ofTC10 reduced the number of newly tethered vesicles(Figure 5F).

The inhibitory effect of TC10-Q75L and TC10-T31N onexocytosis was also demonstrated in depolarization-mediated secretion of NPY. Expression of TC10-Q75Lor TC10-T31N perturbed NPY secretion by PC12 cells(Figure 6A). Under similar conditions, a decrease inGTP-TC10 was observed using the Raichu-TC10/K-RasCT probe (Figures 6B and 6C).

Effect of GTP Loading on the SubcellularLocalization of TC10

Our proposal that TC10 GTP hydrolysis is a critical stepin the initiation of vesicle fusion does not exclude thepossibility that TC10 functions in other aspects of vesic-

Figure 5. Requirement of GTP Hydrolysis by TC10 for Vesicle Fusion

(A) Raichu-TC10 cDNA was cotransfected into HeLa cells with an

empty vector, pERedNLS-Flag-TC10-Q75L, pERedNLS-Flag-

TC10-T31N, or pERedNLS-Rac1-N17. In one sample, the Raichu-

TC10-expressing cells were pretreated with 5 mM DPI. Fusion events

during a 6 min observation period were counted blind before or after

EGF stimulation (control, n = 13; TC10-WT, n = 11; TC10-Q75L, n =

13; TC10-T31N, n = 9; Rac1-N17, n = 12; DPI, n = 8).

(B) Raichu-TC10 cDNA was cotransfected with pSUPER-p190Rho-

GAP-A or pSUPER. After puromycin selection, the numbers of fusion

events were counted as described in (A) (vector, n = 9; p190Rho-

GAP-A knockdown, n = 10).

(C) HeLa cells were transfected with 40 nM TC10 or scramble siRNA.

After 24 hr, the cells were transfected with cDNA encoding a Raichu-

TC10 siRNA-resistant form that contained a silent mutation. The

numbers of fusion events were counted as described in (A) (scram-

ble siRNA, n = 8; TC10 siRNA, n = 10).

(D–F) The numbers of vesicles that appeared after EGF stimulation

and remained more than 0.5 or 2.5 min were counted as described

in the text. The numbers of video images used for the analysis are

as follows: control, n = 8; TC10-WT, n = 11; TC10-Q75L, n = 11;

TC10-T31N, n = 6; empty shRNA vector (pSuper), n = 6; pSuper-

p190RhoGAP-A, n = 8; scramble siRNA, n = 6; TC10 siRNA, n = 6.

Averaged data are shown with SD.

ular trafficking. Indeed, in adipocytes, TC10 has beenshown to promote insulin-induced GLUT4 translocationat the step of the vesicle tethering to the plasma mem-brane (Saltiel and Pessin, 2002; Inoue et al., 2003). Togain insight into the role of TC10 in vesicular trafficking,we examined the subcellular localization of constitu-tively active and dominant-negative mutants of TC10in HeLa cells and MDCK cells (Figure S9). We foundthat TC10-T31N was enriched in perinuclear vesicles.Conversely, TC10-Q75L localized mostly at the plasmamembrane, and its vesicular distribution was markedlydiminished. The accumulation of GTP-TC10 at theplasma membrane probably reflects the increased num-ber and longer lifetime of tethered vesicles (Figure 5D).Furthermore, the fusion of vesicles carrying GTPase-de-ficient TC10 may be mediated by endogenous TC10, orother molecules possessing a similar function, presenton the same vesicles. The possibility of redundancy inthe triggering machinery of vesicle fusion is supportedby the observation of incomplete inhibition of theexocytosis process by the dominant-negative TC10mutant (Figure 5A).

Figure 6. Involvement of TC10 in Depolarization-Induced Secretion

of Neuropeptide Y

(A) NPY-Venus cDNA was cotransfected into PC12 cells with vector

alone, pERedNLS-Flag-TC10-Q75L, or pERedNLS-Flag-TC10-

T31N. The bars represent the mean 6 SD (n = 3) of the normalized

NPY-Venus secretion induced by depolarization stimulation for

20 min.

(B and C) PC12 cells expressing Raichu-TC10/K-RasCT were incu-

bated in low-potassium buffer for 20 min, and then depolarized by

substituting high-potassium buffer. Images were obtained every

1 min. In (B), time-dependent changes in relative FRET efficiency

are expressed by measuring the decrease against the basal activity,

which was averaged over 10 min before depolarization. The bars are

SEM (n = 11). In (C), representative FRET (top) and DIC (bottom)

images before and 5 min after depolarization are shown. The scale

bar represents 25 mm.

Vesicle Fusion Promoted by GTP Hydrolysis of TC10417

Figure 7. Effect of TC10 Depletion on Lamel-

lipodial Extension and Cell Migration

(A and B) HeLa cells were transfected with

40 nM TC10 siRNA or scrambled siRNA. After

serum starvation, the cells were stimulated

with EGF, and DIC images were obtained ev-

ery 1 min for 30 min (scramble, n = 58; TC10

siRNA, n = 85). (A) Representative images of

the control (left) and TC10-depleted cells

(right) 30 min after EGF addition. White and

black dotted lines indicate cell contours be-

fore and after EGF stimulation, respectively.

The scale bars represent 25 mm. In (B), the ra-

tio of cell area after stimulation to cell area be-

fore stimulation was divided into eight seg-

ments as indicated under the horizontal

axes. Histograms plotting the percentage of

cell numbers in each segment are presented

for the control (white bars) and TC10-de-

pleted cells (black bars).

(C and D) HeLa cells were transfected with 40

nM TC10 siRNA or scrambled siRNA and re-

plated on collagen-coated glass-bottomed

dishes. After 30 min incubation, DIC images

were obtained every 3 min for 4 hr (scramble,

n = 22; TC10 RNAi, n = 30). (C) shows repre-

sentative examples of migration tracks of

the control and knockdown cells cultured

on collagen and tracked for 4 hr. Randomly selected individual migration tracks were copied and combined into a single figure to avoid empty

spaces. The scale bars represent 50 mm. In (D), histograms of the migration distance of 25 mm windows are shown for the control (white bars) and

TC10-depleted cells (black bars).

Inhibition of Lamellipodial Extension and CellMigration by Depletion of TC10

Inhibition of SNARE-mediated trafficking during pro-cesses such as exocytosis and secretion has beenshown to impair cell protrusion and migration (Bretscherand Aguado-Velasco, 1998; Tayeb et al., 2005). Thus, weexamined the effect of TC10 depletion on EGF-inducedlamellipodial extension and cell migration. Upon EGFstimulation, control cells extended membrane protru-sions, but TC10-depleted cells did not show suchmorphological changes (Figure 7A). The inhibition of la-mellipodial protrusion in TC10-depleted cells was quan-titatively analyzed by calculating the ratio of cell area af-ter stimulation to cell area before stimulation (Figure 7B).We also found that cell migration was markedly impairedin TC10-depleted cells (Figures 7C and 7D). We postu-late that the loss of EGF-induced lamellipodial exten-sion and cell migration by TC10 depletion was largelydue to the decreased rate of vesicular trafficking in theabsence of TC10.

Discussion

A large number of observations suggest that RhoGTPases behave as simple on-off switches. Signalsare transmitted to downstream effectors when they arebound to GTP and are shut off when bound to GDP.However, this simple view is challenged by many in-stances in which overexpression of not only dominant-negative mutants but also constitutively active mutantsof Rho GTPases inhibits the same biological events (Sy-mons and Settleman, 2000), examples of which includechemotaxis of macrophages (Allen et al., 1998), neuriteoutgrowth (Luo et al., 1994), and the function of tightjunctions in MDCK cells (Jou et al., 1998). Here we

have added TC10-dependent exocytosis and secretionto this list (Figures 5 and 6A). These findings indicatethat GTP hydrolysis by Rho GTPases may play a positiverole in signal transduction (Symons and Settleman,2000). This idea is backed up by the example of otherGTPases. GTP hydrolysis by EF-Tu and the subsequentaccelerated accommodation of aminoacyl-tRNA intothe peptidyl transferase center guarantee the accuracyof protein synthesis (Krab and Parmeggiani, 1998).GTP hydrolysis by Arf1 is thought to trigger disassemblyof the COPI coat following vesicle budding from the do-nor membrane (Goldberg, 1999). However, until now,a positive role for GTP hydrolysis by Rho GTPases hasonly been speculated on, based on circumstantial evi-dence. In this study, we have tried a new approach toaddress this issue: visualization of the TC10 GTP hydro-lysis at the moment of vesicular fusion to the plasmamembrane.

Membrane attachment and fusion are the final steps inthe delivery of vesicles to the target membrane (Watersand Hughson, 2000; Jahn et al., 2003). Numerous factorsare known to be implicated in these two processes.Tethering complexes such as the exocyst and its regula-tors mediate membrane attachment (Whyte and Munro,2002), whereas the SNARE proteins execute membranefusion (Chen and Scheller, 2001). However, factors thattrigger SNARE assembly in a manner coupled to mem-brane tethering remains elusive, though SM proteins(Sec1/Munc18-like proteins) are thought to play a rolein linking the tethering machinery to SNARE complex as-sembly (Jahn et al., 2003). Furthermore, the existence ofa molecular switch is hypothesized to ensure that the re-action proceeds in one direction, but such a moleculehas not been identified (Whyte and Munro, 2002; Jahnet al., 2003). Here, we propose that GTP hydrolysis by

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Figure 8. Hypothetical Mechanism of Ligand-Stimulated Exocytosis of TC10-Containing Vesicles

During relocation from PNC to vesicles, TC10 is converted to a GTP-bound form and associates with effectors such as Exo70 and PIST. Vesicles

containing GTP-TC10 translocate to the vicinity of the plasma membrane. At their destination, vesicle tethering occurs through induced assem-

bly of the exocyst complex containing Exo70 prebound to GTP-TC10. On stimulation, p190RhoGAP-A accelerates GTP hydrolysis of TC10 on the

vesicles, inducing release of Exo70 and exocyst disassembly. These events trigger membrane fusion.

TC10 is the signal that turns on the membrane fusionmachinery.

We propose the following model of EGF-induced GTPhydrolysis by TC10 and the subsequent release of its ef-fectors in promoting vesicle fusion (Figure 8). (1) On EGFstimulation, vesicles loaded with GTP-TC10 and its ef-fector(s) are tethered to the plasma membrane (Figure 5).(2) Activated EGF receptor phosphorylates and stimu-lates p190RhoGAP-A in a manner dependent on Rac1.(3) The activated p190RhoGAP-A then promotes TC10GTP hydrolysis at the plasma membrane (Figures 3 and4). (4) The TC10 GTP hydrolysis releases Exo70 and in-duces a conformational change in the exocyst complex,which has been shown using another FRET probe forExo70 (unpublished data). (5) Release of Exo70 fromTC10 induces disassembly of the exocyst complex.The abortion of an interaction between GTPases andpairing exocyst components has been shown to disas-semble the exocyst complex: for example, the yeast exo-cyst component Sec15 is associated with the GTP formof Sec4, a yeast Rab protein, and assembly of exocystdepends on this interaction (Guo et al., 1999). In mam-mals, GTP-Ral binds to the exocyst components Sec5and Exo84 (Moskalenko et al., 2002, 2003; Sugiharaet al., 2002), and depletion of Ral reduces the assemblyof the complete exocyst complex (Moskalenko et al.,2002). (6) Assembly of the SNARE complex is triggeredby the disassembly of the tethering complex. Somegroups postulate that disassembly of the tethering com-

plex is a prerequisite for the formation of trans-SNAREcomplexes (Gao et al., 2003), though the mechanism bywhich tethering factors promote fusion of vesiclesremains controversial (Novick and Guo, 2002; Whyteand Munro, 2002).

This study has demonstrated that Rac1 regulatesEGF-induced vesicle fusion by promoting TC10 GTP hy-drolysis. A positive role of Rac1 in stimulated exocytosishas been reported in mast cells (Norman et al., 1996;Hong-Geller and Cerione, 2000) and chromaffin cells(Li et al., 2003), and the Rac1 signaling pathway control-ling outward membrane flow has been shown to act inparallel with cytoskeletal rearrangement (Norman et al.,1996). Therefore, TC10 may mediate this F-actin-independent function of Rac1 in a variety of cells.

The role of TC10 in trafficking has so far been inten-sively studied in insulin-induced GLUT4 translocationin adipocytes (Saltiel and Pessin, 2002; Saltiel and Pes-sin, 2003). In this model, insulin-activated GTP-TC10 re-cruits Exo70 and its associated exocyst complex to theplasma membrane, and thereby tethers GLUT4-contain-ing vesicles to the plasma membrane (Inoue et al., 2003;Kanzaki and Pessin, 2003). Although this scenario hasbeen proposed in insulin-stimulated adipocytes, ourstudy using dominant-negative and constitutively activemutants of TC10 in HeLa and PC12 cells argues that therole of GTP-TC10 in tethering may be generalized inother TC10-expressing cells. In support of this model,we found that depletion of TC10 in HeLa cells impaired

Vesicle Fusion Promoted by GTP Hydrolysis of TC10419

cell extension and migration, possibly through the pre-vention of exocytosis.

The level of FRET in Raichu-TC10-expressing cellswas significantly higher on the vesicles than in thePNC or at the plasma membrane (Figure 2); this observa-tion implies another role of TC10 in export from the PNCto vesicles. PIST, one of the effectors of TC10, has beenshown to bind to various proteins that are transported tothe plasma membrane, such as cystic fibrosis trans-membrane conductance regulator (Cheng et al., 2002),AMPA receptor (Cuadra et al., 2004), and frizzled (Yaoet al., 2001). These proteins are thought to be loadedonto transport vesicles through their association withPIST. Thus, these PIST binding proteins may becomevesicular cargo via GTP-TC10. In this model, TC10GTP hydrolysis at the plasma membrane could inducethe unloading of cargo proteins from the vesicles.

In conclusion, our results implicate TC10 in three dif-ferent steps of exocytosis: loading of the cargo, tether-ing to the plasma membrane, and triggering of vesiclefusion (Figure 8). Of note, TC10 GTP loading and GTP hy-drolysis both play important roles and occur simulta-neously in different subcellular compartments. As wehave shown in this study, functional imaging of GTPasesusing the FRET-based probe is very powerful for theanalysis of events that proceed in parallel within thesame cell.

Experimental Procedures

Plasmids

pRaichu-TC10, derived from the pCAGGS expression vector, en-

coded a probe, designated Raichu-TC10, that comprised Venus

(the brightest version of YFP) (Nagai et al., 2002), the RBD of

POSH (amino acids 292–396; obtained from the Kazusa DNA Re-

search Institute), TC10, CFP, and the carboxy-terminal region of

TC10 (TC10-CT; amino acids 193–213). In pRaichu-TC10-NC, the or-

der of the RBD of POSH and TC10 was reversed compared with

pRaichu-TC10. However, this probe showed no difference in FRET

efficiency when the Q75L or T31N mutant was replaced with wild-

type TC10, and could be used as a negative control. In pRaichu-

TC10/K-RasCT, TC10-CT was replaced with the carboxy-terminal

region of K-Ras (K-RasCT). The cDNAs for wild-type TC10, TC10-

Q75L, and TC10-T31N were subcloned into pCXN2-mRFP, pER-

edNLS, and pCAGGS-EGFP (Ohba et al., 2003). pERedNLS-Rac1-

N17 has been described previously (Aoki et al., 2004). p50RhoGAP

and p190RhoGAP were provided by A. Hall and H. Sabe, respec-

tively, and subcloned into pERed-NLS (Aoki et al., 2005) or

pIRM21-FLAG (Itoh et al., 2002). pVenus-N1-NPY (Nagai et al.,

2002) was provided by A. Miyawaki. The cDNAs for Dock180 (Hase-

gawa et al., 1996), Dock2 (Nishihara et al., 1999), Dock4 (Yajnik et al.,

2003), C3G (Tanaka et al., 1994), and Tiam1(C1199) (Habets et al.,

1994) were subcloned into a pCAGGS-derived mammalian expres-

sion vector. The cDNA for a dominant-negative mutant of p120Ras-

GAP, p120RasGAP-232, which encodes the first Src homology (SH)

2, SH3, and second SH2 domains of p120RasGAP (a gift from F. Mc-

Cormick), was prepared by PCR and subcloned into pERedNLS.

pSUPER-p190RhoGAP-A, an shRNA expression vector, was gener-

ated as described previously (Aoki et al., 2005). The 19 nucleotide

sequence used to target human p190RhoGAP-A mRNA was 50-

GATGGGTGTTATTCAGGAT-30.

Cell Culture and RNA Interference

HeLa, 293T, and Cos7/E3 (a gift from Y. Fukui, University of Tokyo)

cells were maintained in Dulbecco’s modified Eagle’s medium sup-

plemented with 10% fetal bovine serum. 293F cells (Invitrogen) were

maintained in FreeStyle 293 expression medium (Invitrogen). PC12

cells were cultured in RPMI1640 medium containing 10% horse se-

rum and 5% fetal bovine serum. TC10 siRNA was obtained from

iGENE. The 27 nucleotide sequence (sense) to target human TC10

mRNA was 50-AGUGGGUACCGGAACUUAAGGAAUAAG-30. HeLa

cells were transfected with 40 nM siRNA using Oligofectamine (Invi-

trogen) according to the manufacturer’s instructions and used after

48 hr. For imaging, 10 nM Alexa488-conjugated scramble siRNA

(Qiagen) was introduced along with TC10 siRNA, and the cells ema-

nating green fluorescence derived from Alexa488 were used for

time-lapse recordings. In some experiments, HeLa cells were trans-

fected with pSUPER-p190RhoGAP-A and pRaichu-TC10 using

Polyfect (Qiagen). Knockdown cells were selected with 1 mg/ml

puromycin for 2 days before analysis.

In Vitro Spectrofluorometry

Spectrograms of lysed cells were obtained as described previously

(Mochizuki et al., 2001; Takaya et al., 2004). Plasmids were trans-

fected into 293T cells. Thirty-six hours later, cells were harvested

in lysis buffer and clarified by centrifugation. Fluorescence spectra

were obtained with an FP-750 spectrometer (JASCO) using an

excitation wavelength of 433 nm.

Analysis of Guanine Nucleotides Bound to TC10

Guanine nucleotides bound to Raichu probes and EGFP-TC10 were

analyzed as described previously (Mochizuki et al., 2001; Takaya

et al., 2004). Briefly, 293T cells were transfected with pRaichu-

TC10 or pCAGGS-EGFP-TC10. Thirty-six hours after transfection,

cells were labeled with 32Pi in phosphate-free modified Eagle’s me-

dium (Invitrogen) for 4 hr. Raichu-TC10 and EGFP-TC10 were immu-

noprecipitated with anti-GFP antibody. The immunoprecipitates

were boiled and analyzed by thin layer chromatography (TLC). The

amount of GTP and GDP bound to TC10 was quantified with

a BAS-1000 image analyzer (Fuji Film).

Flow Cytometry for FRET

293F cells were transfected with pRaichu-TC10 and an expression

plasmid for either a RhoGEF or a RhoGAP using 293fectin (Invitro-

gen). FRET efficiency was measured by FACSAria (BD) as described

previously (Kawai et al., 2004). Expression of the RhoGAP or Rho-

GEF was identified by the red fluorescence derived from RFP, which

was translated from the internal ribosomal entry site on the mRNA of

the RhoGAP or RhoGEF.

Imaging of TC10 Activity in Living Cells

TC10 activity in living cells was imaged using Raichu-TC10 probes

as described previously (Mochizuki et al., 2001). Expression plas-

mids were transfected into HeLa cells plated on glass-bottomed

dishes using Polyfect (Qiagen). After 24 hr, the cells were starved

for 3 hr and then stimulated with 50 ng/ml EGF. The FRET efficiency

(sensitized FRET/CFP ratio) in the subcellular compartments was

determined as follows. For vesicles and PNC, regions showing

higher CFP intensity than an appropriate threshold level were se-

lected, and their sensitized FRET and CFP intensities were obtained.

For the plasma membrane, three appropriate regions were selected

manually and the averages of their sensitized FRET and CFP inten-

sities were obtained.

TIRF studies were conducted using an Olympus IX70 inverted mi-

croscope equipped with a 442 nm HeCd laser (Omnichrome), a TIRF

illuminator, and a 1003 numerical aperture 1.45 objective lens. The

CFP and sensitized images were obtained simultaneously using an

image splitter (Dual-View, Optical Insights) and an EMCCD camera

(iXon DV887, Andor). Stacked images containing 500 planes were

continuously acquired with a 200 ms exposure.

Image Analysis of Tethering and Fusion of Exocytosing Vesicles

Images during vesicular fusion were analyzed using MetaMorph

software (Universal Imaging) according to Tsuboi et al. (2004), with

some modifications. Single exocytotic events were selected manu-

ally, and vesicular fusion was distinguished from vesicle retreat as

described previously (Fix et al., 2004) (Figures S3B–S3E). The fusing

vesicle was centered in the image (Figure S4Aa). The local back-

ground was determined as the average fluorescence of the region

between two concentric rings with diameters of 2 mm (inner) and

5 mm (outer) (Figure S4Ab). This background was subtracted from

a raw image (Figure S4Ac). Next, in the sensitized FRET image, the

threshold value appropriate for extracting vesicles was determined

Developmental Cell420

manually (Figure S4Ad). This threshold mask was applied to both the

sensitized FRET and the CFP images (Figure S4Ae). Finally, a ratio

image was obtained by dividing the sensitized FRET image by the

CFP image (Figure S4Af).

In Figures 3C and 3D, the sensitized FRET and CFP intensities of

the vesicles were obtained as the average over a 1 mm diameter cir-

cle centered on the fusing vesicles (Figure S4Db). Then, the local

background determined as in Figure S4Ab was subtracted. The in-

tensity was normalized to the level observed at time point zero. In

Figure 3E, the sensitized FRET and CFP intensities of the vesicles af-

ter background subtraction were used to calculate the FRET effi-

ciency. Normalization was performed by taking the ratio at time

point zero as 1.

Vesicles tethered after EGF stimulation were identified and

counted by the following algorithm. First, four mean projection im-

ages were prepared from the time-lapse images, from 22.0 to

20.5 min (pre-EGF stimulation period), from 0 to 1.5 min, from 2 to

3.5 min, and from 4 to 5.5 min (see Figure S8 for an example). Sec-

ond, vesicles above the threshold intensities were identified in

each mean projection image. Third, vesicles that appeared after

EGF stimulation and remained in at least two sequential mean pro-

jection images were defined as newly tethered vesicles and counted

in the >0.5 min score. Finally, among the newly tethered vesicles,

those detected in all three mean projection images were also

counted in the >2.5 min score.

In Vitro Analysis of TC10 Activity

TC10 activity in EGF-treated HeLa cells was measured by Bos’s pull-

down method using GST-PAK-CRIB domain fusion protein as de-

scribed previously (Aoki et al., 2005). Proteins bound to GST-PAK-

CRIB and total lysates were analyzed by immunoblotting with an

anti-HA antibody.

NPY Secretion Assay

A secretion assay using NPY-Venus was performed as described

previously (Nagai et al., 2002). pVenus-N1-NPY was cotransfected

into PC12 cells with the indicated TC10 constructs using Lipofect-

amine 2000 (Invitrogen). After 36 hr, the cells were washed with

HEPES-buffered saline, and incubated with low-potassium buffer

(145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM

glucose, 15 mM HEPES [pH 7.4]) for 20 min. Then, the buffer was

changed to high-potassium buffer (95 mM NaCl, 56 mM KCl,

2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 15 mM HEPES [pH

7.4]) to initiate stimulation of depolarization. After a 20 min incuba-

tion, the intensities of NPY-Venus fluorescence recovered from the

cell-free supernatants (secreted fraction) and the cell lysates (non-

secreted fraction) were measured with a FluoroSkan II fluorescence

microplate reader (Global Medical Instrumentation).

Supplemental Data

Supplemental Data include Supplemental Experimental Procedures,

Supplemental References, nine figures, and two movies, and are

available at http://www.developmentalcell.com/cgi/content/full/

11/3/411/DC1/.

Acknowledgments

We thank Y. Sako for advice on the image analysis. We are also

grateful to N. Yoshida, N. Fujimoto, and K. Fukuhara for technical as-

sistance, and to members of the Matsuda laboratory for technical

advice and helpful input. This work was supported by grants from

the Ministry of Education, Culture, Sports, Science, and Technology

of Japan, the New Energy and Industrial Technology Development

Organization, and the Japan Health Science Foundation.

Received: August 15, 2005

Revised: April 21, 2006

Accepted: July 19, 2006

Published: September 1, 2006

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