TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth

Abstract

Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila melanogaster, we have identified the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis. We present genetic evidence that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, we find that disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. Our data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling.

Figure 1.

Mutations in endocytic regulators modify TOR overexpression phenotypes. (A–F) Disruption of Hsc70-4 or shibire dominantly enhances the small rough eye phenotype caused by ey TOR. Scanning electron microscopy images of D. melanogaster compound eyes: wild-type (A), eyTOR/+ (B), eyTOR/+; Tsc1²⁹/+ (C), eyTOR/+; Hsc70-4^e19/+ (D), UAS-Shi^K44A/+; eyGAL4/UAS-Shi^K44A (E), and UAS-Shi^K44A/+; eyTOR eyGAL4/UAS-Shi^K44A (F). (G) Mutations in the e3/e19 complementation group disrupt the ATPase domain of the Hsc70-4 clathrin-uncoating ATPase. Shown is the domain structure of Hsc70-4, with the mutated residues indicated. (H and I) Mutation of Hsc70-4 does not lead to increased levels of TOR protein. (H) Loss of GFP expression marks a clone of cells homozygous for Hsc70-4^e3 (arrow) from an eye imaginal disc expressing eyeless-driven FLAG-tagged TOR. Genotype: hsflp¹²²/+; eyTOR/+; FRT82B Ubi-GFP/FRT82B Hsc70-4^e3. (I) Extracts from wild-type and Hsc70-4^e3/e19 third instar larvae were blotted with anti-TOR antibodies to detect endogenous TOR levels. (J–L) Expression of Shi^K44A causes relocalization of TOR to endocytic vesicles. FLAG-tagged TOR expressed clonally in fat body cells (J) shows altered distribution upon coexpression of UAS-Shi^K44A (K). TOR-containing vesicles near the cell surface can incorporate the endocytic tracer TR-avidin (L, arrow). Genotype: hsflp¹²²/+; Act>CD2>Gal4, UAS-TOR/TM3-UAS-Shi^K44A. Bars: (A–F) 100 μm; bar in (L) is 12.5 μm in H–K, and 6.25 μm in L.

Figure 2.

TOR signaling affects endocytosis. (A–C) Starvation and TOR overexpression affect GFP-Rab5 localization. Flp-dependent, spontaneously induced clones of GFP-Rab5–expressing cells in mid–third instar fat body tissue are shown. (A) Under fed conditions, GFP-Rab5 is uniformly distributed throughout both the cytoplasm and surface of these cells. (B) In larvae starved for 4.5 h, higher levels of GFP-Rab5 are observed near the cell surface. (C) TOR overexpression leads to the formation of large GFP-Rab5 aggregates at the cell surface. Images in A–C show the nuclear focal plane. Inset in C shows image taken at a focal plane just below the plasma membrane. Genotypes: hsflp¹²²/+; Act>CD2>Gal4/UAS-GFP-Rab5 (A and B) and hsflp¹²²/+; UAS-TOR, Act>CD2>Gal4/UAS-GFP-Rab5 (C). (D) TOR overexpression increases levels of GFP-Hsc70-4. The image shows a clone of cells expressing FLAG-tagged TOR (marked by FLAG staining in inset), resulting in increased levels of endogenously expressed GFP-Hsc70-4. Genotype: hsflp¹²²/+; UAS-TOR, Act>CD2>Gal4/Hsc70-4^Wee-P1. (E–J) Endocytic uptake in D. melanogaster fat body cells. Fat body tissue containing clones of overexpressing or mutant cells was incubated with fluorescently labeled avidin to monitor endocytic uptake. Images show surface (E and F) or cytoplasmic (E′, F′, and G–J) focal planes. Clones are demarcated by GFP expression (insets) and are indicated by yellow arrows. (E–F) Endocytosis is inhibited in response to disruption of shibire or Hsc70-4 function. A clone of Shi^K44A-expressing cells (E) is marked by GFP expression, and a clone homozygous mutant for a null allele of Hsc70-4 (F) is marked by the absence of GFP. (G–J) TOR signaling positively affects bulk endocytic uptake. Shown are clones of cells homozygous for null mutations in Tor (G), Tsc1, (H), S6k (I), and a clone overexpressing 4E-BP (J). Bar, 25 μm. Genotypes: hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/TM3-UAS-Shi^K44A (E), hsflp¹²²/+; Cg-Gal4/+; FRT82B UAS-GFP/FRT82B Hsc70-4 ^Δ16 (F), hsflp¹²²/+; UAS-2XeGFP FRT40A fb-GAL4/Tor ^ΔP FRT40A (G), hsflp¹²²/+; Cg-Gal4/+; FRT82B UAS-GFPnls/FRT82B Tsc1²⁹ (H), hsflp¹²²/+; Cg-Gal4/+; UAS-2XeGFP FRT80B/S6K^l1 FRT80B (I), and hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-4E-BP (J). Bar, 25 μm.

Figure 3.

Endocytic regulators and TOR signaling control Lsp2 internalization. Images show surface (A and B) or cytoplasmic (A′, B′, and C–F) focal planes of third instar larval fat body stained with anti-Lsp2. (A and B) Endocytosis of Lsp2 from the larval hemolymph is inhibited in response to disruption of shibire (A) or Hsc70-4 (B). Note the accumulation of Lsp2 at the surface of Shi^K44A-expressing cells, but not in Hsc70-4–null cells. (C–F) TOR signaling is required for Lsp2 uptake. Shown are clones of cells homozygous for null mutations in Tor (C), Tsc1 (D), S6k (E), and a clone overexpressing 4E-BP (F). Arrows indicate mutant or overexpressing cells. Bar, 25 μm. Genotypes are as in Fig. 2 E–J.

Figure 4.

TOR signaling inhibits endocytic down-regulation of the amino acid transporter Slimfast. Fat body tissue from mid–third instar larvae stained with Slimfast antibody, which is shown in red. Nuclei are shown in blue. Insets show cellular genotypes; transgene-expressing clones (A, D–H, and J) are marked by GFP expression (green) and loss-of-function clones (B, C, and I) are marked by the absence of GFP. (A–C) High levels of Slimfast accumulate near the surface of cells expressing Shi^K44A (A), but not in cells homozygous for two different alleles of Hsc70-4 (B and C). Mutant cells are indicated by arrows. (D and E) Slimfast levels are regulated by TOR signaling. (D) Overexpression of the positive TOR regulator Rheb leads to increased cell surface levels of Slimfast. (E) Overexpression of the negative TOR regulators Tsc1 and Tsc2 leads to decreased Slimfast levels. (F–H) Slimfast down-regulation resulting from TOR inactivation requires endocytosis. Coexpression of Shi^K44A inhibits TSC1–TSC2-induced Slimfast down-regulation (F; compare to E). Expression of dominant-negative Rab^S43N does not affect Slimfast levels in fed animals (G), but prevents Slimfast down-regulation in response to 24-h starvation (H). (I and J) Slimfast turnover requires endocytic targeting/sorting factors. Loss of Hrs function (I) or expression of dominant-negative Nedd4 (J) leads to increased Slimfast at the plasma membrane. Slimfast is imaged just below the plasma membrane focal plane in these images; GFP expression in insets is imaged at the nuclear focal plane. Arrows mark mutant cells. Genotypes: hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Shi^K44A (A), hsflp¹²²/+; Cg- Gal4/+; FRT82B UAS-GFP/FRT82B Hsc70-4^e3 (B), hsflp¹²²/+; Cg-Gal4/+; FRT82B UAS-GFP/FRT82B Hsc70-4^Δ16 (C), hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Rheb^EP50.084 (C and D), hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Tsc1, UAS-Tsc2, (F) hsflp¹²²/+; Tubulin>CD2>Gal4, UAS-GFP/+; TM3-UAS-Shi^K44A/UAS-Tsc1, UAS-Tsc2 (E), hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Rab5^S43N (G and H), hsflp122/+; Hrs^D28 FRT40A/UAS-2XeGFP FRT40A fb-Gal4 (I), and hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Nedd4^C97411S (J). Bar, 25 μm.

Figure 5.

Inhibition of endocytosis leads to tissue-specific effects on cell growth. (A–F) Disruption of Hsc70-4 or shibire function in imaginal disc cells causes increased cell size and G2 population. Graphs show flow cytometric analysis of dissociated wing imaginal discs containing clones of Hsc70-4 mutant cells (B and C) or cells expressing dominant-negative Hsc70-4 or shibire (E and F). Histograms show DNA content (left) and forward light scatter (right; indicative of cell size). In (A-C) red traces represent GFP-negative Hsc70-4 mutant or control cell populations, and green traces represent GFP-positive wild-type cell populations. In D–F, green traces represent GFP-positive transgene-expressing cells and red traces represent GFP-negative wild-type cells. Genotypes: hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B (A), hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B Hsc70-4^e19 (B), hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B Hsc70-4 ^Δ16 (C), hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/+ (D), hsflp¹²²/+; UAS-Hsc70-4^K71S/+; Act>CD2>Gal4, UAS-GFP/+ (E), hsflp¹²²/UAS-Shi^K44A; UAS-Shi^K44A/+; Act>CD2>Gal4, UAS-GFP/+ (F). (G–I) Hsc70-4 mutation and Shi^K44A expression cause distinct effects on fat body cell size. Clones of Hsc70-4 mutant cells (G and H; GFP-negative cells marked by arrows) or Shi^K44A expressing cells (I; GFP-positive cells) are shown. Cell outlines are marked by phalloidin staining (red). Genotypes: hsflp¹²²/+; Cg-Gal4/+; FRT82B UAS-GFP/FRT82B Hsc70-4^e3 (G), hsflp¹²²/+; Cg-Gal4/+; FRT82B UAS-GFP/FRT82B Hsc70-4 ^Δ16 (H), hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/TM3-UAS-Shi^K44A (I). Bar, 25 μm. (J) Quantitation of fat body cell size data. Shown are area measurements of cells homozygous for Hsc70-4^e3 or Hsc70-4 ^Δ16, or expressing Shi^K44A, relative to surrounding wild-type cells. Error bars indicate the SEM. Number of cells scored are as follows: Hsc70-4^e3, n = 116 (27 mutant and 89 wild type); Hsc70-4 ^Δ16, n = 109 (22 mutant and 87 wild type); Shi^K44A, n = 175 (24 transgene-expressing and 151 wild type).

Figure 6.

Endocytosis regulates growth signaling. (A and B) Inhibition of shibire increases PI3K signaling and inhibits autophagy. (A) Antibody staining shows increased cytoplasmic localization of the transcription factor FOXO in Shi^K44A-expressing clones. (B) Induction of autophagy by 5 h starvation is blocked in Shi^K44A- expressing clones. Autophagic cells are indicated by the punctate staining of LysoTracker red. Genotype: hsflp¹²²/+; Act>CD2>Gal4, UAS-GFP/UAS-Shi^K44A. (C) Immunoblot showing levels and phosphorylation status of S6K and AKT from control (lane 1), Hsc70-4^e3/e19 (lane 2), and Tsc1²⁹ (lane 3) larval extracts. (D) Hsc70-4 mutation increases the severity of Tsc1 cell size and cell cycle phenotypes. Flow cytometric histograms of Hsc70-4^e3 and Tsc1²⁹ single- and double-mutant mitotic clones are shown. Genotypes: (Row 1) hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B Hsc70-4^e3, (Row 2) hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B Tsc1²⁹, (Row 3) hsflp¹²²/+; FRT82B Ubi-GFP/FRT82B Tsc1²⁹ Hsc70-4^e3. (E) Hsc70-4 and Tsc1 mutants show synergistic effects on tissue growth. Shown are eyes containing eye-specific clones homozygous mutant for Hsc70-4 (top), Tsc1 (middle), or Hsc70-4 and Tsc1 (bottom). Mutant clones are marked by the lack of the white ⁺ marker, and thus appear white. Note the loss of anterior tissue in Hsc70-4 mutant eyes (top, arrow) and the overgrowth of anterior tissue in Hsc70-4 Tsc1 double mutants (bottom, arrow). Genotypes: ey-FLP/+; FRT82B Pw ⁺ l(3)cl^R3/FRT82B Hsc70-4^e3 (top), ey-FLP/+; FRT82B Pw ⁺ l(3)cl^R3/FRT82B Tsc1²⁹ (middle), ey-FLP/+; FRT82B Pw ⁺ l(3)cl^R3/FRT82B Hsc70-4^e3 Tsc1²⁹ (bottom). (F) Heterozygous mutation of Hsc70-4 dominantly confers rapamycin resistance. Wild-type and Hsc70-4^e3/+ animals develop at a similar rate to adulthood (eclosion) when grown on normal media. Addition of rapamycin to the media results in a more severe delay in wild type than in Hsc70-4^e3/+ animals. Bar in B is 25 μm in A, 50 μm in B, and 200 μm in E.

Figure 7.

Inverse regulation of bulk endocytosis and targeted endocytic degradation. (A) Under conditions favorable for growth, TOR promotes bulk endocytic uptake and inhibits the endocytic turnover of specific nutrient importers such as Slimfast. (B) In growth-inhibitory conditions, inactivation of TOR leads to a decrease in bulk endocytosis and an increase in the targeted endocytic degradation of excess nutrient importers. (C) Together, the opposing actions of TOR on bulk and targeted endocytosis serve to facilitate nutrient import, providing energy and building blocks necessary for biosynthetic growth and leading to a further stimulation of TOR signaling.