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First published online July 31, 2003
doi: 10.1242/10.1242/jcs.00661

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Drosophila Rheb GTPase is required for cell cycle progression and cell growth

Parthive H. Patel^1,*, Nitika Thapar^2,*, Lea Guo², Monica Martinez³, John Maris³, Chia-Ling Gau², Judith A. Lengyel^1,3, and Fuyuhiko Tamanoi^1,2,

¹ Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095-1489, USA
² Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095-1489, USA
³ Department of Molecular, Cell and Developmental Biology, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095-1489, USA

View larger version (24K): [in a new window]   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).

View larger version (38K): [in a new window]   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 His-tagged dRheb with [-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 [-35S]GTP bound to His-tagged dRheb in the presence of 20-fold excess unlabeled ATP, CTP, GTP, UTP or GDP was compared with [-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 [-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).

View larger version (86K): [in a new window]   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-FLP-induced 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.

View larger version (52K): [in a new window]   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).

View larger version (38K): [in a new window]   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 pRmHa-GFP 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.

View larger version (19K): [in a new window]   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.

View larger version (74K): [in a new window]   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-of-function 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 wild-type (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.

View larger version (36K): [in a new window]   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 anti-FLAG 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).

View larger version (26K): [in a new window]   Fig. 9. dTOR is required for dRheb-mediated growth. Time of eclosion (hatching from pupal case) in days after egg lay (AEL) is compared for siblings carrying either one (dRheb/+) or two (+/TM3) wild-type copies of dRheb in the presence or absence of 1 µM rapamycin; the proportions of the total eclosed for each genotype and growth condition are indicated (A). Mean eclosion time of 314 +/TM3 and 202 dRheb/+ sibs in medium without rapamycin was 9.6±0.8 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 13.6±1.7 days, respectively. Treatment of S2 cells with rapamycin results in a decrease in cell size, as determined by forward scatter analysis (B). Although overexpression of dRheb leads to an increase in cell size, treatment of cells overexpressing dRheb with rapamycin inhibits dRheb-mediated cell growth (C).

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