Research Article
3601
Drosophila Rheb GTPase is required for cell cycle
progression and cell growth
Parthive H. Patel1,*, Nitika Thapar2,*, Lea Guo2, Monica Martinez3, John Maris3, Chia-Ling Gau2,
Judith A. Lengyel1,3,‡ and Fuyuhiko Tamanoi1,2,‡
1Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095-1489, USA
2Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California,
Los Angeles,
CA 90095-1489, USA
3Department of Molecular, Cell and Developmental Biology, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA
90095-1489, USA
*These authors contributed equally to this work
‡Authors for correspondence (e-mail: [email protected]; [email protected])
Accepted 8 May 2003
Journal of Cell Science 116, 3601-3610 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00661
Summary
Precise body and organ sizes in the adult animal are
ensured by a range of signaling pathways. In a screen
to identify genes affecting hindgut morphogenesis in
Drosophila, we identified a P-element insertion in dRheb, a
novel, highly conserved member of the Ras superfamily of
G-proteins. Overexpression of dRheb in the developing fly
(using the GAL4:UAS system) causes dramatic overgrowth
of multiple tissues: in the wing, this is due to an increase in
cell size; in cultured cells, dRheb overexpression results in
accumulation of cells in S phase and an increase in cell size.
Using a loss-of-function mutation we show that dRheb is
required in the whole organism for viability (growth) and
for the growth of individual cells. Inhibition of dRheb
activity in cultured cells results in their arrest in G1 and a
reduction in size. These data demonstrate that dRheb is
required for both cell growth (increase in mass) and cell
cycle progression; one explanation for this dual role would
be that dRheb promotes cell cycle progression by affecting
cell growth. Consistent with this interpretation, we find
that flies with reduced dRheb activity are hypersensitive to
rapamycin, an inhibitor of the growth regulator TOR. In
cultured cells, the effect of overexpressing dRheb was
blocked by the addition of rapamycin. These results imply
that dRheb is involved in TOR signaling.
Introduction
Regulation of cell size and number is required to establish a
normal, correctly proportioned adult body size (Coelho and
Leevers, 2000; Johnston and Gallant, 2002; Kozma and
Thomas, 2002). Although a great deal is known about cell
cycle regulation, little is known about the regulation of cell
size. Recently, it has become clear that, although growth
(increase in mass) usually occurs concomitantly with cell
division, it is actually a distinct process (Edgar, 1999; Coelho
and Leevers, 2000). Work in Drosophila has made it possible
to characterize the separate effects of genes on these two
processes during development of the whole organism (Edgar,
1999; Johnston and Gallant, 2002). The genes string (cdc25),
cyclin E and E2F, for example, have been shown to be
required for cell cycle progression but not for cellular growth
(Neufeld et al., 1998).
Recently, the insulin and TOR (target of rapamycin)
signaling pathways have been shown to regulate growth in
Drosophila (Kozma and Thomas, 2002; Oldham and Hafen,
2003). Mutants in these pathways affect total organ and body
size, as well as individual cell size. A downstream target of
both pathways is dS6K, mutations in which affect only cell
size, not cell number (Montagne et al., 1999). Most known
components of the insulin and TOR signaling pathways affect
both cell size and number; current understanding is that these
effects on cell number are primarily via their effects on cell
growth (Coelho and Leevers, 2000; Johnston and Gallant,
2002; Kozma and Thomas, 2002).
The Ras-like gene Rheb (Ras homolog enriched in brain)
was recently shown to be required for growth of the yeast
Schizosaccharomyces pombe. The rheb mutants (rhb1)
arrest in G0/G1 as small, rounded cells (Mach et al., 2000;
Yang et al., 2001), suggesting a role for Rheb in cell cycle
progression and cell growth. Highly conserved Rheb genes
have been described throughout the metazoa (Yamagata et al.,
1994; Reuther and Der, 2000; Urano et al., 2000; Urano
et al., 2001; Im et al., 2002; Panepinto et al., 2002). To
investigate the cellular role of Rheb, we have taken
advantage of the unique suitability of Drosophila for both
genetic and biochemical studies. Here, we show that
Drosophila dRheb has both GTP binding and GTPase
activities. Overexpression of dRheb results in tissue
overgrowth and increased cell size in the whole organism, and
transition into S phase and cell growth in culture. Conversely,
reduction of dRheb activity results in reduced tissue growth
and smaller cell size in the whole organism, as well as a G1
arrest and smaller cell size in culture. The results of treating
S2 cells and flies with rapamycin, an inhibitor of TOR,
suggest that the effects of dRheb are probably mediated by
dTOR.
Key words: Cell cycle, Growth, Drosophila, Rheb, TOR
3602
Journal of Cell Science 116 (17)
Materials and Methods
Fly stocks, dRheb overexpression, dRheb loss-of-function
clones
Flies were cultured at 25°C on standard food, in a yw or w genetic
background. Overexpression of dRheb in the embryonic hindgut, in
the third instar larval eye disc posterior to the morphogenetic furrow,
in the dorsal compartment of the wing disc, in the posterior
compartment of the wing disc or in clones of cells in the early eye
imaginal disc was effected by combining the dRhebAV4 allele or a
UASdRheb construct with bynGAL4,UASGFP (Iwaki and Lengyel,
2002), GMR-GAL4 (III) (Ellis et al., 1993; Tapon et al., 2001),
apGAL4 (Montagne et al., 1999), enGAL4 (Neufeld et al., 1998) or
eyFLP;act<y+<GAL4,UAS-GFP (Pignoni and Zipursky, 1997). The
dRheb coding sequence was amplified from the GH15143 expressed
sequence tag clone using primers 5′-GCTAAGATCTATGCCAACCAAGGAGCGCCACATA and 5′-GCGCCTCGAGTTACGATACAAGACAACC, and subcloned into XhoI/BglII-digested pUAST (Brand
and Perrimon, 1993) to generate the UASdRheb construct. Germ-line
transformation was by standard procedures (Ashburner, 1989), as
described previously (Green et al., 2002). Loss-of-function clones in
the eye disc were produced by combining eyFLP with FRT(82B),w+
M(3)95A and FRT(82B) dRhebAV4 [generated by recombination
between dRhebAV4 and FRT(82B),Sb (Xu and Rubin, 1993)]. Excision
of the P-element in dRhebAV4 was effected by introduction of
transposase using Ki [∆2-3].
Mapping and lethal phase of dRhebAV4
Genomic DNA at the 3′ end of the P{y+,5XUAS} insert in dRhebAV4
was isolated by inverse PCR (Sullivan et al., 2000). Amplified
product was cloned into the pCR4-TOPO vector (Invitrogen) and
sequenced. The insertion site was identified by FlyBLAST
(http://www.fruitfly.org/blast/). For determination of lethal phase,
eggs were collected from dRhebAV4/TM6-GFP adults on yeasted apple
juice agar plates for 2 hours. After aging for various times, larvae were
washed onto fresh plates and scored under a fluorescence-equipped
dissecting microscope for genotype, based on the presence or absence
of green fluorescent protein (GFP). For effect of rapamycin on time
of eclosion, 2-hour collections of 100 embryos each were placed in
multiple vials containing standard food with and without 1 µM
rapamycin (Calbiochem).
Histology and phenotypic analysis
In situ hybridization to whole-mount embryos was performed as
described previously (Tautz and Pfeifle, 1989). Digoxigenin RNA
probes (Roche Molecular Biochemicals) were generated from cDNA
templates of the dRheb open reading frame. BrdU incorporation into
embryos for 1 hour was carried out as described (Smith and Orr-Weaver,
1991) and detected with anti-BrdU antibody (1:40, Becton Dickinson)
using standard techniques (Ashburner, 1989). Scanning electron
microscopy and fixation, embedding and sectioning of adult eyes were
performed as described (Wolff and Ready, 1991; Sullivan et al., 2000).
Light microscopy was performed with a Zeiss Axiophot and images
were acquired with a Sony DKC-500 digital camera and processed with
Adobe Photoshop. Cell size and number in the wing were determined
as described by Montagne et al. (Montagne et al., 1999). Wing areas
were measured using Image J software on 10-12 wings for each
genotype; anterior was defined by wing vein L2 and anterior wing
margin, posterior by vein L3 and posterior wing margin. Wing hair
density was determined by counting wing hairs in a 0.0275 mm2 area
in the anterior and in a 0.0544 mm2 area in the posterior of each wing.
Sample standard error was ~5% of the mean for all measurements.
Drosophila cell culture, transfection, RNAi, cell cycle analysis
S2 cells (1×106) were seeded and maintained in Schneider’s
Drosophila medium (Gibco) supplemented with 10% fetal bovine
serum (Gibco) at 25°C. Transfection was carried out using Cellfectin
(Invitrogen) with 1 µg each of plasmids pPacFLAG-dRheb
(expressing FLAG-dRheb using the actin 5C promoter) and pRmHaGFP (transfection marker, expressing GFP using metallothionein
promoter). For RNAi, dRheb template containing T7 promoter
sequence was generated by PCR from the cDNA; double-stranded
RNA (dsRNA) corresponding to the entire coding sequence (546 bp)
was synthesized using T7 Megascript kit (Ambion). S2 cells were
treated with 10 µg dRheb dsRNA with and without pPacFLAG-dRheb
according to Clemens et al. (Clemens et al., 2000). For cell cycle
analysis by fluorescence-activated cell sorting (FACS), transfected
cells were fixed in 1% formalin/PBS and 70% ethanol, whereas RNAitreated cells were fixed in 70% ethanol alone. After fixing, cells were
stained with 25 µg ml–1 propidium iodide and FACS was performed
on a Becton Dickinson FACScan. For immunoblotting, total cell
extracts were prepared in insect cell lysis buffer (BD Pharmingen) and
electrophoresed on a 12% SDS polyacrylamide gel; after blotting,
proteins were immunodetected with anti-FLAG antibody (Sigma).
Expression and purification of dRheb and biochemical
analyses
dRheb coding sequence, amplified from a plasmid cDNA library
(provided by F. Laski, University of California, Los Angeles) by PCR,
was inserted into vector pET28a(+) (Novagen). Total cell lysates of
bacteria expressing His-tagged dRheb, induced by IPTG, were
prepared in lysis buffer (20 mM sodium phosphate, pH 7.4, 0.5 M
NaCl, 0.5 mM MgCl2, 10 µM GTP) using a French press. Protein was
purified using Probond resin (Invitrogen) and stored in 50% glycerol
at –20°C. GTP binding was determined by nitrocellulose filtration
assay (Finlin et al., 2001). His-dRheb (2 µg) was incubated with [γ35S]GTP (1250 Ci mmol–1, Amersham) in binding buffer (20 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 40 µg
ml–1 bovine serum albumin) in the presence of 1 mM MgCl2 at 37°C.
At various time points, samples were diluted into ice-cold wash buffer
(20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT)
and nitrocellulose-bound radioactivity was determined by scintillation
counting. Specificity of nucleotide binding was assessed with [γ35S]GTP in the presence of a 20-fold excess of unlabeled competing
nucleotide for 30 minutes, normalized to binding in the absence of
competitor. GTPase activity (Tanaka et al., 1991) was assayed by
measuring loss of 32P from His-dRheb preloaded with [γ-32P]GTP
(3000 Ci mmol–1) in binding buffer containing 0.1% Triton X-100, 4
µM GTP and 1 mM MgCl2 at 37°C for 30 minutes. Unlabeled ATP
(5 mM) was included to inhibit nonspecific phosphatase activity.
Hydrolysis was initiated by adjusting the MgCl2 concentration to 10
mM. Reactions were terminated at the indicated time points and
bound radioactivity was assessed by nitrocellulose binding. All
biochemical assays described here were carried out in triplicate.
Results
Isolation of a mutation in dRheb that affects growth
Ectopic expression screens can be used to identify and
characterize genes that, when expressed at a particular time or
region, generate a phenotype of interest (Rorth et al., 1998).
Lines containing random insertions of the Drosophila mobile
element P (engineered to contain the 5XUAS binding site for
the yeast transcription factor GAL4) are screened for
phenotype in the presence of a particular GAL4 driver. Because
P-elements usually insert near the transcription initiation site,
this allows ectopic expression and/or overexpression of a
particular gene (Rorth et al., 1998; Toba et al., 1999; Duchek
and Rorth, 2001).
dRheb in cell cycle progression and cell growth
3603
Fig. 1. dRheb allele allowing GAL4-driven ectopic
expression. dRhebAV4 is a P-element (P{y+,5XUAS})
insertion at position +85 within the 5′ UTR of
dRheb (A; exons are shown as boxes, with open
reading frames shaded, whereas introns are shown
as lines). When the hindgut-specific driver,
bynGAL4,UASGFP, is combined with dRhebAV4,
the level of dRheb mRNA in the embryonic hindgut
(detected by in situ hybridization) is increased (C)
and the hindgut (expressing GFP) of the first instar
larva is enlarged (D) relative to that of the wild type
(B); B and D are at the same magnification.
Anterior extent of hindgut is indicated by arrows
(B,D); hindgut overexpressing dRheb is indicated
by arrowhead (C).
We screened approximately 5000 P{y+,5XUAS} lines for
lethality in combination with bynGAL4, which drives
expression specifically in the developing posterior gut (Iwaki
and Lengyel, 2002), to identify genes affecting hindgut
morphogenesis. Among the roughly 7% of these lines that were
lethal with bynGAL4, we identified one (designated AV4) that
gave a dramatically enlarged (both in width and length) hindgut
in the first instar larva (Fig. 1B,D). As determined by inverse
PCR, this chromosome carries an insertion of the P-element in
the 5′ UTR of dRheb; this allele is designated dRhebAV4 (Fig.
1A). In situ hybridization confirms that, as expected from the
orientation of the inserted 5XUAS, combining dRhebAV4 with
bynGAL4 causes overexpression of dRheb mRNA in the
hindgut (Fig. 1C). When the P-element insert of dRhebAV4 is
mobilized by transposase and precisely excised (confirmed by
PCR), combination of the excision chromosome with bynGAL4
has no effect on hindgut size (data not shown), again
confirming that the effects observed in combination with the
bynGAL4 driver are due to the P{y+,5XUAS} insertion in
dRheb.
dRheb is an active GTPase
The dRheb gene is predicted to encode a 182 amino acid
protein that contains G-boxes characteristic of the Ras
superfamily of G-proteins. Fig. 2A shows the predicted amino
acid sequence of dRheb, together with those of the yeast and
human Rhebs. Yeast and fly each have a single Rheb gene; the
most similar human gene is hRheb1 (Gromov et al., 1995;
Mizuki et al., 1996), which maps to chromosome 7 (Mizuki et
al., 1996). We have identified an additional, more divergent
human Rheb gene, designated hRheb2, which maps to
chromosome 12. The overall sequence identity between
Drosophila and S. pombe Rheb is 51%, and that between
Drosophila and human Rheb1 is 63% (Fig. 2A). Consistent
with this high sequence conservation, dRheb, hRheb1 and
hRheb2 can replace the function of S. pombe Rheb (data not
shown).
Recombinant dRheb binds [γ-35S]GTP (a non-hydrolysable
GTP analog) in a manner that is both time and nucleotideconcentration dependent (Fig. 2B, data not shown). The
binding of GTP to dRheb is enhanced by addition of Mg2+
(data not shown) and is specific for guanine nucleotides, with
a preference for the binding of GTP over GDP (Fig. 2C).
dRheb also exhibits intrinsic GTPase activity, as shown by the
slow but significant loss of radioactivity from dRheb preloaded with [γ-32P]GTP (Fig. 2D). Hydrolysis of GTP to GDP
is confirmed by the appearance of 32P in GDP (revealed by thin
layer chromatography, data not shown) after incubation of
dRheb with [α-32P]GTP. Our detection of Rheb GTPase
activity shows that the presence of arginine at the position
corresponding to amino acid 12 of Ras [at which most Rasfamily members have glycine (Bourne et al., 1991)] does not
eliminate GTP-hydrolysing activity of the protein.
Overexpression of dRheb promotes tissue and cellular
growth and progression into S-phase
Overexpression of dRheb leads to enlargement of the larval
hindgut, so we tested the generality of this effect by driving
dRheb expression in other tissues. In the larval salivary glands,
dRheb overexpression also results in significant enlargement
(data not shown). When dRheb is overexpressed in the
developing eye imaginal disc, using the GMR-GAL4 driver,
dramatically enlarged eyes are seen in the adult (Fig. 3A-F).
Similarly, overexpression of dRheb in clones of cells in the eye
and antennal discs, using the ‘flip-out’ method (Pignoni and
Zipursky, 1997) and eyFLP, results in an enlarged eye, antenna
and head (Fig. 3G-J). When compared with control eyes (Fig.
3K), the ommatidia of these eyes are much larger than normal,
are not organized into the normal hexagonal array and are
frequently flanked by extra bristles (Fig. 3L). The larger overall
eye and ommatidial size, and the extra bristles further support
the notion that dRheb overexpression promotes growth. The
fact that bristles in the dRheb-overexpressing eyes are larger
(thicker) than normal (Fig. 3K,L), together with the fact that
each bristle is derived from a single cell (Wolff and Ready,
1993), suggests that dRheb might promote growth at the level
of the individual cell.
Overexpression of dRheb also promotes growth in the wing,
an organ consisting of two opposed, flat, single-layered
epithelial sheets. Overexpression of dRheb in the dorsal
epithelial compartment causes the wing to curve downwards
(Fig. 4C,D), presumably owing to an increase in area of the
dorsal compartment. When dRheb is overexpressed in the
posterior compartment of the wing, the posterior area is
increased by 36% (Fig. 4A,B), further supporting a role for
dRheb in growth. To distinguish between effects on cell
number versus cell size, we counted wing hairs (each of which
is produced by a single cell) in normal and dRheb-
3604
Journal of Cell Science 116 (17)
overexpressing compartments (Fig. 4E,F). Because the cell
density in the dRheb-overexpressing compartment is only 67%
of that in the wild-type compartment, we conclude that dRheboverexpressing cells are significantly larger (i.e. each cell
occupies more area) than wild-type cells. Wing hair density,
combined with compartment area measurements, shows that
total cell number in the posterior compartment is the same
whether or not dRheb is being overexpressed. Thus, in the
wing, dRheb overexpression results in an increase in cell size
but not cell number. This is similar to what has been described
for overexpression of activated Ras1 and Myc; in Drosophila,
these genes are believed to affect cell cycle progression via
their primary function as regulators of cell growth (Johnston et
al., 1999; Prober and Edgar, 2000; Prober and Edgar, 2001).
In the developing embryo, dRheb expression correlates with
DNA replication. Immediately after fertilization, as rapid,
syncytial nuclear divisions take place (Foe et al., 1993), dRheb
mRNA is present at a high, uniform level. This level decreases
as rates of nuclear division slow, begins increasing again
during stage 11 and becomes significantly higher in tissues
undergoing endocycles (i.e. midgut) and mitoses (i.e. central
A
nervous system). Later in embryogenesis (stages 13-16), as
revealed by in situ hybridization and BrdU incorporation,
the same regions that show strong dRheb expression are
also carrying out DNA synthesis (Fig. 5A, Flybase). Thus,
embryonic mRNA expression patterns and levels are consistent
with a role for dRheb in promoting S-phase.
To characterize the effect of dRheb further at the cellular level,
Drosophila S2 cells were co-transfected with vectors expressing
dRheb and GFP (a marker for transfection). Examination of
the cell cycle profile of transfected (GFP plus dRheboverexpressing) cells by FACS reveals that increased levels of
dRheb result in an increase in the proportion of cells in S-phase
(Fig. 5B). In addition, analysis of cell size by forward scatter
analysis reveals that S2 cells overexpressing dRheb are slightly
larger than control (GFP alone) cells (Fig. 5C).
The overexpression studies described above show that
dRheb can promote two processes: increase in cell size and cell
cycle progression. The correlation between dRheb expression
and DNA synthesis in the embryo, and the effect of dRheb
overexpression in cultured cells both suggest that dRheb
promotes progression into S-phase. The increased size of
G1
G2
G3
dRheb
hRheb1
hRheb2
SpRheb
MP-TKERHIAMMGYRSVGKSSLCIQFVEGQFVDSYDPTIENTFTKIERVKSQDYIVKLIDTAGQD
MPQSKSRKIAILGYRSVGKSSLTIQFVEGQFVDSYDPTIENTFTKLITVNGQEYHLQLVDTAGQD
MPLVRYRKVVILGYRCVGKTSLAHQFVEGEFSEGYDPTVENTYSKIVTLGKDEFHLHLVDTAGQD
MAPIKSRRIAVLGSRSVGKSSLTVQYVENHFVESYYPTIENTFSKNIKYKGQEFATEIIDTAGQD
*. : *::.::* *.***:** *:**..* :.* **:***::*
::: .::******
G4
64
65
65
65
dRheb
hRheb1
hRheb2
SpRheb
EYSIFPVQYSMDYHGYVLVYSITSQKSFEVVKIIYEKLLDVMGKKYVPVVLVGNKIDLHQERTVS
EYSIFPQTYSIDINGYILVYSVTSIKSFEVIKVIHGKLLDMVGKVQIPIMLVGNKKDLHMERVIS
EYSILPYSFIIGVHGYVLVYSVTSLHSFQVIESLYQKLHEGHGKTRVPVVLVGNKADLSPEREVQ
EYSILNSKHSIGIHGYVLVYSITSKSSFEMVKIVRDKILNHTGTEWVPIVVVGNKSDLHMQRAVT
****:
. :. :**:****:** **:::: : *: : *. :*:::**** ** :* :
129
130
125
130
CaaX
G5
dRheb
hRheb1
hRheb2
SpRheb
TEEGKKLAESWRAAFLETSAKQNESVGDIFHQLLILIENE-NGNP-QEKSGCLVS
YEEGKALAESWNAAFLESSAKENQTAVDVFRRIILEAEKM-DGAASQGKSSCSVM
AVEGKKLAESWGATFMESSARENQLTQGIFTKVIQEIARV--ENSYGQERRCHLM
AEEGKALANEWKCAWTEASARHNENVARAFELIISEIEKQANPSPPGDGKGCVIA
*** **:.* .:: *:**:.*: .
* ::
.
.
* :
B
C
D
Nucleotide Specificity
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
Time (min)
50
60
70
GTP hydrolysis
140
100
120
90
% of control
% of control
pmol [35S]GTPγS bound
GTP binding
0.6
182
184
183
185
100
80
60
40
20
80
70
60
50
40
0
(-)
ATP
CTP
GTP
UTP
GDP
Competing cold nucleotide (2µM)
0
20
40
60
80
Time (min)
Fig. 2. dRheb is a highly conserved GTPase. Sequence alignment of S. pombe, D. melanogaster and two human Rheb proteins by Clustal (A)
indicates that dRheb is a highly conserved Ras-like GTPase. The G boxes (G1-G5) and the CaaX box are indicated by lines above the
sequences; asterisks and dots indicate identical and similar residues, respectively (A). GTP binding of dRheb was examined by incubating Histagged dRheb with [γ-35S]GTP in binding buffer in the presence of 1 mM MgCl2; radioactivity bound to dRheb was assessed as described in
Materials and Methods (B). To examine nucleotide specificity, radioactivity in [γ-35S]GTP bound to His-tagged dRheb in the presence of 20fold excess unlabeled ATP, CTP, GTP, UTP or GDP was compared with [γ-35S]GTP bound in the absence of nucleotide (set to 100%), as
described in Materials and Methods (C). GTPase activity of dRheb was examined as described in Materials and Methods: His-tagged dRheb
was preloaded with [γ-32P]GTP in 1 mM MgCl2 and hydrolysis initiated by increasing MgCl2 concentration to 10 mM; bound radioactivity was
determined by nitrocellulose filter assay (D).
dRheb in cell cycle progression and cell growth
3605
Fig. 3. dRheb overexpression causes eye and head overgrowth.
Combination of GMR-GAL4 (which drives expression in the eye
imaginal disc) with dRhebAV4 results in marked overgrowth of the eye
(E,F) compared with the wild type (A,B) or driver alone (C,D). Eye
and head overgrowth is even more dramatic (G-J) when ey-FLPinduced GAL4-expressing clones overexpress dRheb in dRhebAV4/+
eye and antennal discs. (A,C,E,G,I) Lateral views of eye. (B,D,F,H,J)
Frontal views of head. Scanning electron microscopy shows that,
compared with the wild-type (K), dRheb-overexpressing eyes have
enlarged ommatidia and bristles, as well as irregular and occasionally
fused ommatidia and extra bristles (L). Solid arrows, bristles; dashed
arrows, extra bristles; black arrowhead, fused ommatidia.
cultured cells, eye bristles and wing cells overexpressing
dRheb suggests that dRheb promotes increase in mass of
individual cells (cell growth). These two distinct effects of
dRheb raise the question of whether dRheb affects the cell
cycle and cell growth independently or via a pathway that
coordinates both of these processes.
Loss of dRheb function results in reduced cell and tissue
size
Although the results of overexpression studies described above
suggest a role for dRheb in regulation of cell growth and cell
cycle progression, removal of gene activity is necessary
unequivocally to establish required function. Of great utility is
the fact that the dRhebAV4 allele, in addition to allowing ectopic
expression when combined with a GAL4 driver, is also
homozygous lethal. Precise excision of the P-element in the
dRhebAV4 chromosome results in a reversion to viability,
confirming that the dRhebAV4 lethal phenotype is due to
disruption of dRheb activity resulting from the P-element
insertion. To determine the lethal phase of dRhebAV4
homozygotes, we collected eggs for 2 hours from heterozygous
parents and determined the number of surviving homozygous
mutant larvae relative to heterozygous sibs as a function of time
(Fig. 6A). Embryos homozygous for dRhebAV4 hatch into first
instar larvae that grow very slowly, move lethargically, and die
by 72 hours without molting into second instar larvae (Fig.
6B,C). dRheb is thus required for growth of the whole organism.
To assess the requirement for dRheb in individual cells, we
made clones of cells lacking dRheb in the eye. For this purpose,
we recombined FRT(82B) onto the dRhebAV4 chromosome;
combination of this chromosome with eyFLP should, in
principle, allow generation of w;dRheb–/– clones in the
background of a w+;dRheb+/– eye. In this background,
however, dRheb–/– clones were not detected, similar to what
has been reported for clones lacking Ras1 function (Prober and
Fig. 4. dRheb overexpression causes
increased cell size in the wing.
Overexpression of dRheb in the dorsal
compartment of the wing disc (using the
UASdRheb construct and apGAL4)
results in enlargement of the dorsal
compartment, as shown by downward
curvature of the wing (C,D). When
dRheb is overexpressed in the posterior
of the wing using UASdRheb and
enGAL4, the area of the posterior
compartment (B) is larger than that of the
control enGAL4 wing (A) (1.0 mm2 and
0.73 mm2, respectively). Cell density is
lower in the posterior compartment of the
dRheb-overexpressing (F) than in the
control (E) wing (4300 hairs mm–2 and 6400 hairs mm–2, respectively), indicating that the dRheb-overexpressing cells are larger. From cell
density and area measurements, we find that the number of cells per compartment is not significantly different for dRheb-overexpressing and
control wings (6100 and 6100, respectively, anterior; 8900 and 9300, respectively, posterior). Our measurements also reveal apparent
compensation for dRheb-driven posterior overgrowth; anterior compartment area is somewhat reduced in UASdRheb,enGAL4 wings relative to
control (0.42 mm2 and 0.49 mm2) whereas cell density is increased (7300 hairs mm–2 and 6300 hairs mm–2).
3606
Journal of Cell Science 116 (17)
Fig. 5. dRheb expression correlates with S-phase
during embryogenesis and promotes S-phase in
S2 cells. In stage 13 embryos, the same domains
express high levels of dRheb and incorporate
BrdU into DNA: (A, left) in situ hybridization;
(A, right) staining with anti-BrdU. At this stage,
mitoses are taking place in the ventral nerve
cord and brain (arrows), and endoreplication is
occurring in the gut, including a central domain
of the midgut (black arrowheads). To test the
effects of overexpressing dRheb at the cellular
level, S2 cells were transfected with pRmHaGFP either alone or in conjunction with
pPacFLAG-dRheb, and GFP expression was
induced 16 hours later by addition of 500 µM
CuSO4. Cells were harvested 48 hours after
induction and FACS was performed after gating
for GFP-positive cells as described in Materials
and Methods. Cells overexpressing dRheb
exhibit an increased proportion of S phase (B); a
small panel on the right summarizes the cell
cycle profile of these cells. Forward scatter
analysis of the same cells reveals that
overexpression of dRheb results in an increase in
cell size (C); quantitation of the forward scatter
results is shown on the right. For FACS analysis
of untransfected cells, 10,000 cells were
collected and single cells were gated using an
FL2-width versus FL2-area density plot for cell
cycle progression. A gate using SSC-H versus
FSC-H was used for cell size analysis. For FACS analysis of transfected cells, 5000-7000 GFP-positive cells were collected and cells were
gated using FL1-H versus FL2-area density plot. Cell size was analysed using the SSC-H versus FSC-H gate. Mean FSC-H of the GFP-positive
cell population was calculated from three independent experiments; error bars represent standard deviation from the mean.
Edgar, 2000). In both cases, the absence of detectable w clones
is probably due to cell competition, a process in which fastergrowing cells out-compete slowly growing cells, which are
then eliminated by apoptosis (Morata and Ripoll, 1975). To
give dRheb–/– clones a growth advantage, we generated them
in a Minute (M+/–) background (Minute genes regulate protein
synthesis; thus M+/+ cells grow more rapidly than surrounding
M+/– cells) (Andersson et al., 1994). Heads and eyes containing
multiple dRheb–/– M+/+ clones are dramatically smaller than
those containing dRheb+/+clones (Fig. 7H,I). Sections of these
eyes show that the dRheb–/– ommatidia are smaller overall
because they are composed of dramatically smaller cells (Fig.
7J cf. C).
Two important inferences can be drawn from analysis of
these dRheb loss-of-function clones. First, because eyes
bearing multiple dRheb–/– clones are smaller than wild-type,
Fig. 6. dRheb is required in the whole organism for
viability and growth. The lethal phase of dRhebAV4
homozygous mutants was determined by counting
dRheb–/– (identified by lack of GFP expression) and
dRheb+/– (identified by GFP expression) sibs at
various times after 2 hour egg collections [A; after
egg lay (AEL)]. The dRheb–/– larvae grow more
slowly, as shown by comparison with wild-type at 72
hours AEL (B,C; same magnification). Comparison
of mouth hooks shows that wild-type larvae (B) have
progressed to second instar, whereas dRheb–/– larvae
(C) remain in first instar.
dRheb must play a required role in tissue growth. Second, the
smaller size of individual dRheb–/– M+/+ cells (Fig. 7J)
indicates that dRheb is required in a cell autonomous manner
for cell growth (increase in mass).
To investigate whether these inferred roles of dRheb can be
demonstrated at the cellular level, we used dsRNA (Clemens
et al., 2000) to inhibit Rheb function in Drosophila S2 cells.
Addition of dsRNA corresponding to the entire coding
sequence of dRheb to the culture medium almost completely
inhibits expression of FLAG-tagged dRheb (Fig. 8A).
Characterization of cell cycle profiles by FACS showed a
dramatic increase in the proportion of cells in G1-phase by day
four to five in dRheb dsRNA-treated cells; this effect persisted
to at least day 8 (Fig. 8B; notice the increased proportion of
cells in G2-phase in controls). In addition to having an effect
on the cell cycle, inhibition of dRheb also has a significant
dRheb in cell cycle progression and cell growth
3607
Fig. 7. dRheb is required for cell and tissue
growth. As controls, wild-type eyes and heads
(A,B) and sections of wild-type eyes viewed by
light microscopy (C) are shown. dRheb loss-offunction clones (dRheb–/–) were detected only
when generated in a Minute (M+/–) background.
In this M+/– background, eyes and heads
composed primarily of dRheb+/+,M+/+ clones
(white tissue), generated by FLP-FRT mediated
recombination in a M+/– background, are
approximately the same size as those of wildtype (A,B,D,E). Eyes and heads containing
dRheb–/–,M+/+ clones are dramatically reduced
in size (D-I; white tissue is dRheb–/–, red tissue
is dRheb+/–). Sections of these eyes show
disorganized, smaller ommatidia composed of
smaller cells (C,J; arrow indicates small,
Rheb–/– photoreceptor cell, arrowhead indicates
normal size, Rheb+/– photoreceptor cell).
A,D,H, lateral view of eye; B,E,I, frontal view
of head; F,G, dorsal view of head.
effect on cell growth. Forward scatter analysis reveals a
dramatic reduction in cell size after the addition of dRheb
dsRNA (Fig. 8C). Both the diminution in cell size and the
accumulation of cells in G1-phase after dRheb inhibition
follow roughly the same time course (i.e. both are maximal by
day five) (Fig. 8B,C).
From the loss-of-function studies in the whole organism and
in cultured cells described above, we conclude that dRheb
affects both cell growth (mass increase) and cell cycle
progression promoting transition of cells from G1 to S phase.
dTOR is required for dRheb-mediated cell growth
Control of cell growth and cell cycle progression can, in
principle, be regulated by parallel independent pathways or
through a signal that coordinates both (Coelho and Leevers,
2000). In both yeast and Drosophila, mutations in cell-cyclespecific genes (such as cyclin E) result in cell cycle arrest with
an associated increase in cell size owing to continued cell
growth (Johnston et al., 1977; Neufeld et al., 1998), although
overexpression of these genes results in smaller cells. Because
loss of dRheb function in both cultured cells and in the whole
organism results in reduced cell size, it is likely that dRheb
coordinates cell cycle and cell growth. We therefore considered
the possibility that dRheb might impinge on the insulin and
TOR signaling pathways, which are major contributors to the
regulation of cell growth in both Drosophila and mammalian
cells (Kozma and Thomas, 2002; Oldham and Hafen, 2003).
Because dRheb larvae exhibit a growth arrest similar to dTOR
mutants and larvae starved for amino acids, we used rapamycin
treatment to investigate whether dRheb interacts,
either in the whole organism or in cultured cells,
with dTOR. Under normal growth conditions, flies
with either one or two copies of dRheb eclose
(emerge from the pupal case) at the same time; in
the presence of rapamycin, however, larvae with
only one wild-type copy of dRheb grow more slowly,
eclosing two days later than their wild-type sibs (Fig.
9A). Reduced dRheb thus sensitizes the organism to
the growth-inhibiting effect of rapamycin. We also
Fig. 8. dRheb is required in cultured cells for G1/S
progression and cell growth. The effect of inhibiting
dRheb expression by dsRNA was tested in S2 cells
transfected with pPAC-FLAG or pPAC-FLAG-dRheb.
After 72 hours, dsRNA-treated or untreated cells were
lysed. Western-blot analysis of total protein with antiFLAG antibody revealed inhibition of FLAG-dRheb
expression in the presence of dRheb dsRNA (A). When
cells treated with dRheb dsRNA were harvested one to
eight days after treatment and subjected to flow
cytometry, they were found to gradually arrest at G0/G1
(B). Forward scatter analysis of cells collected two, three,
four and five days after the addition of dRheb dsRNA
shows that it causes a reduction in cell size (C).
3608
Journal of Cell Science 116 (17)
examined possible involvement of dTOR in dRheb
function in S2 cells, in which, as expected, treatment
with rapamycin causes cells to decrease in size (Fig.
9B). Significantly, the cell-growth-promoting effect
of overexpressing dRheb is blocked by rapamycin
(Fig. 9C). This latter result indicates that the effect
of dRheb overexpression depends on the functional
activity of dTOR; in other words, dTOR is epistatic
to (downstream of) dRheb.
A
Cell Counts
Discussion
We show here that, both in vivo and in culture, dRheb
is required for cell growth. A role for dRheb in cell
30
cycle progression in S2 cells in culture is indicated
C
B
by observations that dRheb overexpression drives
GFP+dRheb
+rapamycin
GFP + rapamycin
cells into S-phase, whereas inhibition of dRheb
expression leads to G1 arrest. Consistent with this,
GFP+dRheb
dRheb expression in the embryo correlates with
domains carrying out DNA replication. A role for
GFP
dRheb in promoting cell growth is shown in vivo by
the enlarged cell size (but not cell number) in wings
1000 0
1000
0
overexpressing dRheb, and the smaller cell size in
FSC-H
dRheb–/– clones in the eye. In culture, dRheb
overexpression leads to a small increase in cell size,
Fig. 9. dTOR is required for dRheb-mediated growth. Time of eclosion
whereas inhibition of dRheb expression causes a
(hatching from pupal case) in days after egg lay (AEL) is compared for siblings
dramatic reduction in cell size. The effect of dRheb
carrying either one (dRheb/+) or two (+/TM3) wild-type copies of dRheb in the
on growth is probably mediated by dTOR. Mutants
presence or absence of 1 µM rapamycin; the proportions of the total eclosed for
in dTOR exhibit phenotypes similar to those of
each genotype and growth condition are indicated (A). Mean eclosion time of
dRheb loss-of-function mutants, namely small cell
314 +/TM3 and 202 dRheb/+ sibs in medium without rapamycin was 9.6±0.8
size and growth arrest similar to what is seen during
days and 9.2±0.6 days, respectively, whereas the mean eclosion time of 190
+/TM3 and 126 dRheb/+ sibs in medium with rapamycin was 12.6±1.5 and
amino acid starvation (Britton and Edgar, 1998;
13.6±1.7 days, respectively. Treatment of S2 cells with rapamycin results in a
Oldham et al., 2000; Zhang et al., 2000). Our
decrease in cell size, as determined by forward scatter analysis (B). Although
experiments using rapamycin show that flies with
overexpression of dRheb leads to an increase in cell size, treatment of cells
reduced levels of dRheb are hypersensitive to the
overexpressing dRheb with rapamycin inhibits dRheb-mediated cell growth (C).
drug and that the effect of overexpressing dRheb in
S2 cells is blocked by rapamycin. These results,
activity. Our results are in agreement with the reported intrinsic
taken together, suggest that Rheb might function upstream of
GTPase activity of mammalian Rheb by Im et al. (Im et al.,
TOR.
2002), who also described that the ratio of GTP to GDP bound
An important question is whether Rheb affects downstream
to Rheb in mammalian cells is higher than that bound to Ras.
effectors of TOR, most particularly S6K, which modulates
The possibility that dRheb might also have high GTP:GDP
growth by controlling protein synthesis. Recent studies have
ratio could explain why overexpression of wild-type dRheb has
demonstrated a link between dRheb and the dTOR/dS6K
significant growth promoting effects, as shown here. In our
pathway (Saucedo et al., 2003; Stocker et al., 2003). Transient
experiments, we did not use dRhebQ63L, a mutant analogous
expression of dRheb in Drosophila S2 cells was found to lead
to the constitutively active Ras, because it has previously been
to the activation of p70S6K, and inhibition of dRheb expression
shown that expression of S. pombe RhebQ64L or RhebS20N
by small interfering RNA blocked amino-acid-induced
(analogous to a dominant negative Ras) in S. pombe cells has
activation of p70S6K (Saucedo et al., 2003). In addition, in
no detectable effect (Mach et al., 2000). Similar observations
Drosophila dRheb mutants, p70S6K activity was reported to
were made with mammalian Rheb (Clark et al., 1997).
be decreased (Stocker et al., 2003). We have recently observed
Our finding of GTPase activity for dRheb raises the
that transient expression of human Rheb in mammalian
possibility that there is a dRheb GTPase-activating protein
cells (HEK293) induces activation (phosphorylation) of S6K
(GAP). A candidate for such an activity is the TSC1/TSC2
(C.L.G. and F.T., unpublished). Although these results might
complex; TSC1 and TSC2 are tumor suppressors that act to
be taken to suggest that Rheb acts solely through TOR and
regulate TOR negatively (Gao and Pan, 2001; Potter et al.,
S6K, it is important to realize that dS6K–/– flies develop
2001; Tapon et al., 2001; Radimerski et al., 2002).
into adults with reduced body size (Montagne et al., 1999),
Interestingly, the C-terminal portion of TSC2 contains a GAP
whereas, as shown here, dRheb–/– flies do not survive. Thus,
domain that is reported to function as a GAP for Rap1 and
dS6K might not be the only target of dRheb.
Rab5 (Wienecke et al., 1995; Xiao et al., 1997). Our results
As shown here, dRheb exhibits intrinsic GTPase activity
linking dRheb to TOR raise the possibility, currently under
despite having arginine at the position corresponding to
investigation, that TSC2 is a GAP for Rheb.
glycine-12 of Ras, a residue known to be crucial for GTPase
dRheb in cell cycle progression and cell growth
We have previously reported that, in S. pombe, Rheb
inhibition leads to cell cycle arrest at G0/G1 phase (Yang et
al., 2001). Because this growth arrest can be rescued by
expression of dRheb, there is conservation of function between
Rhebs in two evolutionarily very distant organisms. It is
therefore significant that TOR and TSC homologues exist in S.
pombe (Kawai et al., 2001; Weisman and Choder, 2001;
Matsumoto et al., 2002). Notably, disruption of tor2+ in S.
pombe leads to growth arrest, whereas disruption of tsc2+ leads
to amino acid uptake defect (Weisman and Choder, 2001;
Matsumoto et al., 2002); both of these phenotypes are
reminiscent of those seen for yeast rheb mutants (Urano et al.,
2000; Yang et al., 2001). Further experiments are needed to
examine possible genetic interactions between the tor, tsc and
rhb1 genes in S. pombe. As for mammalian Rheb, it has been
reported that Rheb inhibits activation of Raf kinase and that
overexpression of Rheb in NIH3T3 cells antagonizes Ras
transformation (Clark et al., 1997; Im et al., 2002). It will be
important to examine whether mammalian Rheb also functions
in the insulin/mTOR signaling pathway.
Our observations suggesting that Rheb is involved in the
TOR pathway could have implications for cancer therapy,
because TOR activity appears to be upregulated in several
human cancers (Hidalgo and Rowinsky, 2000). Because of its
interaction with TOR, Rheb might be an important player
in tumorigenesis. As previously reported, farnesylation is
required for Rheb activity as well as its cellular localization
(Clark et al., 1997; Urano et al., 2000; Yang et al., 2000);
farnesyltransferase inhibitors (FTIs), anticancer drugs
currently being evaluated in clinical trials, might block Rheb
function. It is interesting to note that FTI has been shown to
inhibit p70S6K activation in mammalian cells (Law et al.,
2000). TOR function is blocked by rapamycin, initially used
as an immunosuppressant but currently under investigation as
an anticancer drug (Crespo and Hall, 2002). Therefore, both
Rheb and TOR can be targets for anticancer drug therapy.
Further investigations are expected to provide insights that will
be relevant for the design of future drug therapies.
We thank J. Clemens and L. Zipursky for advice on small
interfering RNA experiments with S2 cell lines, J. Merriam for
assistance with the ectopic expression screen, J. Canon and C. Evans
for advice on histology, G. Jackson, P. Gupta, U. Banerjee, J. Merriam
and the Bloomington Stock Center for providing fly lines, and F. Laski
and M. Smith for providing the Drosophila plasmid cDNA library and
the pPacFLAG, pRmHa-GFP vectors. This work was supported by
NIH grants CA41996, CA32737 and HD09948 to FT and JAL,
respectively, as well as by a grant from the UCLA Jonsson
Comprehensive Cancer Center to JAL. Flow cytometry performed in
the UCLA Flow Cytometry Core Facility was supported by NIH
grants CA16042 and AI28697.
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