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• W2164073865 abstract "Article29 October 2009free access The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway Makiko Koike-Kumagai Makiko Koike-Kumagai Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Kei-ichiro Yasunaga Kei-ichiro Yasunaga Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Rei Morikawa Rei Morikawa Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Takahiro Kanamori Takahiro Kanamori Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Kazuo Emoto Corresponding Author Kazuo Emoto Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan PRESTO, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Makiko Koike-Kumagai Makiko Koike-Kumagai Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Kei-ichiro Yasunaga Kei-ichiro Yasunaga Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Rei Morikawa Rei Morikawa Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Takahiro Kanamori Takahiro Kanamori Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Search for more papers by this author Kazuo Emoto Corresponding Author Kazuo Emoto Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan Department of Genetics, SOKENDAI, Mishima, Japan PRESTO, Japan Science and Technology Agency, Saitama, Japan Search for more papers by this author Author Information Makiko Koike-Kumagai1, Kei-ichiro Yasunaga1, Rei Morikawa1, Takahiro Kanamori1 and Kazuo Emoto 1,2,3 1Neural Morphogenesis Laboratory, National Institute of Genetics, Mishima, Japan 2Department of Genetics, SOKENDAI, Mishima, Japan 3PRESTO, Japan Science and Technology Agency, Saitama, Japan *Corresponding author. Neural Morphogenesis Laboratory, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan. Tel.: +81 55 981 5860; Fax: +81 55 981 5860; E-mail: [email protected] The EMBO Journal (2009)28:3879-3892https://doi.org/10.1038/emboj.2009.312 There is a Have you seen ...? (December 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To cover the receptive field completely and non-redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. We here report that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo. Introduction The target of rapamycin (TOR) is an evolutionarily conserved Ser/Thr protein kinase that functions in two distinct multiprotein complexes referred as TOR complex 1 (TORC1) and complex 2 (TORC2). TORC1 is composed of TOR, Raptor, and LST8 (also know as GβL), whereas TORC2 contains TOR, Rictor, LST8, and Sin1 (Sarbassov et al, 2005a; Wullschleger et al, 2006; Bhaskar and Hay, 2007). TORC1 regulates cell growth by phosphorylating ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein (4E-BP) in a rapamycin-sensitive manner. The function of TORC2 is less well-defined than that of TORC1, but some studies suggest that TORC2 is involved in actin cytoskeleton reorganization (Jacinto et al, 2004; Sarbassov et al, 2004). Both TORC1 and TORC2 are evolutionarily conserved in the functions and the compositions. Indeed, recent studies in both mammalian and Drosophila cell cultures have indicated that TORC2 can directly phosphorylate the serine residue (Ser473 and Ser505 in humans and Drosophila Akt, respectively) in the hydrophobic motif of Akt/PKB (Hresko and Mueckler, 2005; Sarbassov et al, 2005b; Jacinto et al, 2006). In addition to the growth control in proliferating cells, TOR has critical functions in non-proliferating cells. In particular, recent genetic and pharmacological studies have shown that mammalian TOR (mTOR) is involved in various processes in the nervous system, including cell size control (Kwon et al, 2003), local protein synthesis in dendrites (Takei et al, 2004; Raab-Graham et al, 2006), synaptic plasticity (Tang et al, 2002; Cammalleri et al, 2003; Hou and Klann, 2004), and dendrite arborization (da) (Jaworski et al, 2005). These mTOR functions in neurons are believed to be mediated by TORC1 because rapamycin, a potential TORC1-specific inhibitor, can mimic the neuronal defects induced by mTOR ablation. In contrast, much less is known regarding the function of TORC2 in neurons, although Sin1 and Rictor are enriched in the developing neurons (Makino et al, 2006; Shiota et al, 2006). Neurons in the same functional class are often organized in characteristic spatial patterns throughout the nervous system (Wassle and Boycott, 1991; Jan and Jan, 2003; Parrish et al, 2007). In many sensory circuits, a complete and non-redundant representation of sensory information is attained by a tiling arrangement of the dendrites, such that the dendritic arbors of the same cell type show little or no overlap. For example, the mammalian retina contains more than 20 distinct functional classes of retinal ganglion cells (RGCs), and the dendritic fields of some RGCs of the same subclass typically cover the retina with little overlap between neighbouring neurons, whereas RGCs of different subtypes have extensively overlapping arbors (Wassle and Boycott, 1991; Rockhill et al, 2002). Tiling of dendritic fields has also been observed in the sensory neurons of the leech Hirudu medicinalis, Manduca, Drosophila, and Caenorhabditis elegans (Gan and Macagno, 1995; Grueber et al, 2001, 2003; Gallegos and Bargmann, 2004), suggesting that tiling is a general mechanism that organizes the dendritic fields. The Drosophila peripheral nervous system contains identifiable neurons with cell-type-specific dendritic morphologies, including da neurons (Bodmer and Jan, 1987). Dendrites of class IV da neurons tile the body wall in a cell-type-specific manner (Grueber et al, 2003; Parrish et al, 2007). Time-lapse analysis has indicated that terminal dendrites of these class IV neurons often stop growing or turn when they encounter dendrites of the same type (Grueber et al, 2003; Sugimura et al, 2003; Emoto et al, 2004). In addition, laser ablation of class IV neurons causes an invasion of the vacated dendritic territories by neighbouring class IV neurons (Grueber et al, 2003; Sugimura et al, 2003). Conversely, duplication of class IV neurons results in a partitioning of the receptive field. These observations indicate that dendritic tiling in class IV neurons arises from homotypic repulsive interactions between dendrites of neighbouring cells. This tiling mechanism functions in class IV neurons to avoid crossing of homotypic branches in the same neurons (iso-neuronal tiling) as well as between neighbouring neurons (hetero-neuronal tiling). Recent studies indicate that in addition to the tiling mechanism in class IV neurons, the self-avoidance mechanism functions in all da neurons to ensure proper spacing of dendritic branches (Gao, 2007). From the results of a genetic screen for genes that regulate dendritic tiling, the NDR family kinase Tricornered (Trc) and its activator Furry (Fry) were identified as important components of the intracellular signalling cascade that regulates homotypic repulsion in class IV da neurons (Emoto et al, 2004). Dendrites of trc and fry mutants fail to avoid homologous dendritic branches, resulting in a significant overlap of dendritic fields. The Trc kinase signalling is required for the homotypic repulsion between neighbouring dendrites (hetero-neuronal tiling) and also between dendritic branches within single neurons (iso-neuronal tiling) (Emoto et al, 2004; Gao, 2007; Parrish et al, 2007). The C. elegans Trc (Sax-1) and Fry (Sax-2) homologues have also been found to serve a similar function in mechano-sensory neurons (Gallegos and Bargmann, 2004), indicating an evolutionarily conserved function for the Trc signalling in dendritic tiling. Hence, a more detailed understanding of Trc signalling may provide new insights into dendritic tiling. The NDR family of kinases including Trc is activated by the phosphorylation of a conserved serine in the kinase domain (Ser292 in Trc) and a conserved threonine within the hydrophobic motif (Thr449 in Trc). Recent genetic and biochemical studies have indicated that the Ste20 family of MST kinases can contribute to phosphorylation of this conserved threonine (Mah et al, 2001; Stegert et al, 2005; Emoto et al, 2006; Seiler et al, 2006), whereas the serine residue appears to be phosphorylated by NDR kinases themselves. In Drosophila, for example, the Ste20 kinase Hippo (Hpo) directly phosphorylates Trc on Thr449 in vivo and in vitro (Emoto et al, 2006), yet the regulatory mechanism for Trc activation in neurons still remains elusive. In this study, we report that TORC2 genes function cell-autonomously to regulate the dendritic tiling of Drosophila class IV da neurons. Mutations in the TORC2 genes cause significant defects in dendritic tiling of class IV da neurons, which are similar to those observed in trc and fry mutants. TORC2 mutations genetically interact with trc mutations to affect dendritic tiling, and both Trc and its human homologue NDR1 can form a complex with TORC2 in neurons and cultured cells. Furthermore, we provide genetic and biochemical evidence that TORC2 is required for Trc activation both in vitro and in vivo. These findings establish TORC2 as a critical regulator of dendritic tiling in Drosophila sensory neurons through the Trc signalling pathway. Results Sin1 and Rictor are required cell-autonomously to control dendritic tiling To isolate the genes required for dendritic tiling of class IV neurons, we carried out a genetic screen using the pickpocket-EGFP (ppk-EGFP) reporter, which specifically labels class IV da neurons (Grueber et al, 2003). From ∼300 mutant lines carrying PiggyBac transposon (PBc) insertions on the second chromosome (Thibault et al, 2004), we isolated one PBc insertion line with a robust dendritic tiling defect in class IV neurons (Figure 1A and B). This PBc is inserted into the single coding exon of sin1 (Hietakangas and Cohen, 2007), and is therefore likely to eliminate the Sin1 activity (hereafter this PBac insertion line is referred as sin1PBac). Homozygosity of sin1PBac or trans-heterozygous combinations of sin1PBac and a chromosomal deficiency (Df) that uncovers sin1 showed identical dendritic tiling defects (Figure 1F). In contrast, a heterozygosity of sin1PBac or hemizygosity of sin1 caused no such defects, indicating that the tiling defects we observed in sin1PBac result from the loss of sin1 functions. Quantification of the crossing points between dendritic branches indicated that ∼10% of dendritic branches crossed one another in both sin1PBac homozygotes (11.8±2.8%, n=25) and sin1PBac/Df heterozygotes (12.6±2.1%, n=25), compared with ∼1% of crossing in wild-type (WT) dendrites (1.2±0.2%, n=15) (Figure 1F). The excessive overlap of mutant dendrites is unlikely to result from abnormal stratification of terminal branches, as the terminal branches were sandwiched between the epidermis and muscles, which were typically ∼1 μm apart in both mutant and WT larvae. In addition to the dendritic tiling defect, the total number of dendrite branches in sin1 mutants was reduced to ∼80% of WT (146.0±11.4; sin1PBac/sin1PBac, 101.5±10.7; and sin1PBac/Df, 102.3±12.2/4 × 104 μm2) (Figure 1A, B, D, and E). Thus, in addition to the dendritic tiling, Sin1 may have a function in dendritic branching of class IV da neurons. Figure 1.sin1 and rictor function cell-autonomously in regulation of dendritic tiling in class IV neurons. (A–C) Live images of ddaC dendrites visualized by the pickpocket-EGFP (ppk-EGFP) reporter in wild-type (WT) (A), sin1PBac homozygote (B), rictorΔ2 homozygote (C). Anterior is left and dorsal is up. Arrows indicate crossing points of dendritic branches. Scale bar=50 μm. (D–F) Quantification of the total branch length (D), the branch number (E), and the crossing points (F) of WT and mutant ddaC dendrites. Error bars indicate mean±s.d. (WT, n=15; others, n=25), *P<0.01 (Student's t-test). Note that larvae heterozygous for sin1PBac over a deletion [Df(2R)BSC11] uncovering the sin1 gene show dendritic tiling defects identical to those of sin1 homozygotes. Genotypes: (A) yw; +/+; ppk-EGFP/ppk-EGFP, (B) yw; sin1PBac/sin1PBac; ppk-EGFP/ppk-EGFP, and (C) yw, rictor Δ2/yw, rictorΔ2; +/+; ppk-EGFP/ppk-EGFP. (G–I) MARCM clones of WT (G), sin1 (H), and rictor (J) are shown. Arrows indicate the crossing points of the dendrites. Scale bar=50 μm. (J–L) Quantification of the branch length (J), the branch points (K), and the crossing points (L) of MARCM clones. (WT, n=5; sin1, n=11; rictor, n=9) Clone genotypes: (G) hsFLP, elav-Gal4, UAS-mCD8-GFP/+; FRT42D, (H) hsFLP, elav-Gal4, UAS-mCD8-GFP/+; FRT42D, sin1PBac, AND (I) FRT19A, rictorΔ2; UAS-Gal4[109(2)80], UAS-mCD8GFP/hsFLP. Error bars indicate mean±s.d., *P<0.01 (Student's t-test). Download figure Download PowerPoint Sin1 is implicated in various signalling processes through its formation of a complex with different partners, including stress-activating protein kinase (Wilkinson et al, 1999; Schroder et al, 2005), Ras small GTPase (Lee et al, 1999), and the components of TORC2 Rictor and TOR (Jacinto et al, 2006; Yang et al, 2008). To determine whether Sin1 functions together with any of these known interactors to control dendritic tiling, we examined dendrite phenotypes in mutants for the potential Sin-binding partners and found a prominent tiling defect of class IV dendrites in mutants for rictor, which encodes a component unique to TORC2 (Figure 1C). The phenotypes observed in rictor dendrites were quantitatively similar to those observed in sin1 mutants: the number of dendritic crossings was significantly higher (8.1±1.7%, n=25) than that in WT, whereas the terminal branch number was decreased to ∼80% (114.2±13.4/4 × 104 μm2) of that in WT (Figure 1D–F). Consistent with the earlier finding that Sin1 and Rictor function together to regulate tiling, trans-heterozygous combinations of sin1 and rictor alleles caused significant dendritic defects that were qualitatively similar to sin1 and rictor null mutants, whereas heterozygosity of sin1 or rictor had no obvious dendritic phenotype on its own (Figure 1D–F). Finally, sin1 rictor double mutants showed dendritic tiling defects that were indistinguishable from the single mutants (Figure 1D–F). Hence, Sin1 and Rictor most probably function in the same signalling pathway to regulate dendritic tiling. As sin1 appears to be expressed ubiquitously (Supplementary Figure S1), the dendritic phenotypes in sin1 and rictor mutants may reflect a cell-autonomous requirement of these genes in neurons or could be a consequence of non-autonomous functions of these genes in surrounding tissues such as the epidermis and muscles. To distinguish between these two possibilities, we carried out MARCM (mosaic analysis with a repressible cell marker) analysis (Lee and Luo, 1999) to generate single-cell clones that are homozygous for null mutations in sin1 or rictor in a heterozygous background and analysed the effects on dendritic tiling. Similar to the sin1 and rictor homozygous mutants, dorsal class IV neuron MARCM clones of sin1 or rictor mutants showed defects in dendritic tiling, indicating that TORC2 genes are cell-autonomously required for dendritic tiling (Figure 1G–I). In contrast, dendrites of class I MARCM clones were not significantly affected in sin1 or rictor mutants (Supplementary Fugure S2). Class IV neuron-specific expression of sin1 and rictor largely rescued the dendritic phenotypes of sin1 and rictor MARCM clones, respectively (Figure 1J–L), further confirming the cell-autonomous functions of Sin1 and Rictor in class IV da neurons. In general, the tiling defects in sin1 or rictor clones were less severe than those observed in the sin1 or rictor homologous mutant larvae. This could be an effect of protein perdurance in MARCM clones (Lee and Luo, 1999). Alternatively, there may be cell-nonautonomous functions of the TORC2 genes in dendrite development. Sin1 and Rictor are required for dendritic tiling between neighbouring class IV neurons Given the essential roles of Sin1 and Rictor in tiling of terminal branches from the same neuron (iso-neural tiling), we next tested for the requirement in tiling of dendrites from different class IV neurons (hetero-neural tiling). The dendrites of the three class IV neurons found in each hemisegment normally cover the whole epidermis with very little overlap (Grueber et al, 2003; Emoto et al, 2004; Parrish et al, 2007). For example, the adjacent v'ada and vdaB neurons appeared to restrict themselves to their respective dendritic territories and rarely branched into dendritic fields of their neighbours (Figure 2A, B and H). However, in sin1 and rictor null mutants, the v'ada and vdaB dendrites often invaded the neighbouring fields (Figure 2C–F). Furthermore, the hetero-neuronal tiling defects in sin1 and rictor mutants were largely rescued by the neuronal expression of sin1 or rictor, respectively (Figure 2G). These observations suggest that Sin1 and Rictor regulate both iso-neuronal and hetero-neuronal tiling, presumably through the same mechanisms. Figure 2.Hetero-neuronal dendritic tiling defect in sin1 and rictor mutants. (A–F) Live images and their traces of adjacent v'ada and vdaB dendrites. In wild-type (WT) larvae (A), the dendrites of the adjacent class IV neurons, v'ada and vdaB, do not overlap; however, class IV dendrites overlap extensively in sin1 (C) and rictor (E) mutants, as evident from the tracing of dendrites derived from v'ada (red) and vdaB (blue) neurons in WT (B), sin1 (D), and rictor (F) larvae. Arrows indicate the crossing points of dendritic branches between the neighbouring neurons. Scale bar=50 μm. Genotypes: (A) yw; +/+; ppk-EGFP/ppk-EGFP, (C) yw; sin1PBac/sin1PBac; ppk-EGFP/ppk-EGFP, and (E) yw, rictorΔ2/yw, rictorΔ2; +/+; ppk-EGFP/ppk-EGFP. (G) Quantification of the crossing points in v'ada and vdaB dendrites of the WT and the mutant third instar larvae. We normalized the crossing number to the total branch length of the ventral area of v'ada dendrites and the dorsal area of vdaB dendrites. Error bars indicate mean±s.d. (WT, n=15, sin1, n=11; rictor, n=9), *P<0.01 (Student's t-test). Rescue genotypes: sin1PBac, UAS-sin1-Flag/sin1PBac, ppkGal4; ppk-EGFP/ppk-EGFP and rictorΔ2/rictorΔ2; +/ppkGal4; UAS-rictor, ppk-EGFP/ppk-EGFP. (H) Schematic representation of an abdominal hemisegment of the Drosophila larval peripheral nervous system (PNS). Dendritic arborization (da) neurons are indicated by diamonds; triangles, other multidendritic neurons; circles, extra sensory neurons; and cylinders, chordotonal organs. Download figure Download PowerPoint TOR controls dendritic arborization and tiling through distinct complexes Sin1 and Rictor form a complex together with the TOR kinase referred as the TORC2 (Sarbassov et al, 2005a; Wullschleger et al, 2006; Bhaskar and Hay, 2007). We thus next examined Tor mutant MARCM clones for defects in dendritic tiling and found that unlike sin1 and rictor mutant MARCM clones, Tor MARCM clones showed a severe and highly penetrant simplification of dendritic arbors, with significant reductions in the number and length of dendritic branches, and hence in the overall size of the receptive field (Figure 3B, G and H). In addition to TORC2, TOR is also found in the functionally distinct TORC1, which is composed of TOR, Raptor, and LST8 (Sarbassov et al, 2005a; Wullschleger et al, 2006) and has recently been reported to regulate the elaboration of dendritic arbors by phosphorylating ribosomal S6K and 4E-BP in cultured hippocampal neurons (Jaworski et al, 2005). We thus next examined S6K null mutant MARCM clones and observed dendritic defects similar to Tor MARCM clones (Figure 3C, G, and H). Furthermore, Tor and S6K trans-heterozygotes showed simplified dendrites qualitatively similar to Tor and S6K mutant MARCM clones (Figure 3D, I, and J). Thus, as observed in cultured neurons, the TORC1-S6K signalling seems to have a critical function in dendrite growth and branching in post-mitotic class IV neurons. In contrast to the Tor/S6K trans-heterozygotes, a significant tiling defect was observed in larvae trans-heterozygous for mutations in Tor and either sin1 or rictor (Figure 3E, F, I and J), supporting the model in which TORC2 composed of TOR, Sin1, and Rictor together regulates the dendritic tiling of class IV neurons. Collectively, our data indicate that TORC1 and TORC2 have distinct functions in the dendrite developments of class IV neurons: TORC1 for dendritic growth and branching, and TORC2 for dendritic tiling. Figure 3.TORC1 and TORC2 regulate dendritic growth/branching and tiling, respectively. (A–C) Tor and S6K MARCM clones are defective in both dendritic arborization and branching. MARCM clones of (A) wild-type (WT), (B) TorΔP, and (C) S6Kl−1 are shown. Bar represents 50 μm. Clone genotypes: (A) hsFLP, elavGal4, UAS-mCD8-GFP/+; FRT40A, (B) hsFLP, elavGal4, UAS-mCD8-GFP/+; FRT40A, TorΔP; (C) hsFLP, elavGal4, UAS-mCD8-GFP/+; +/+; FRT82B, S6Kl−1. (D–F) Live images of ddaC dendrites visualized by the ppk-EGFP reporter in third instar larvae trans-heterozygous for TorΔP and S6Kl−1 (D), for TorΔP and sin1PBac (E), and for TorΔP and rictorΔ2 (F). Arrows in (E) and (F) indicate the crossing points of the dendritic branches. Genotypes: (D) TorΔP/+; S6Kl−1, ppk-EGFP/ppk-EGFP, (E) TorΔP/sin1PBac; ppk-EGFP/ppk-EGFP, and (F) rictorΔ2/+; TorΔP/+; ppk-EGFP/ppk-EGFP. (G, H) Quantification of the total branch points (G) and the branch length (H) of MARCM clones. Error bars indicate the mean±s.d. (WT, n=6; Tor, n=11; S6K, n=5), *P<0.01 relative to WT controls (Student's t-test). (I, J) Quantification of the branch points (I) and the crossing points (J) per μm2 (4 × 104) of the dendritic branches at the third instar larval stage in ddaC neurons. Error bars indicate the mean±s.d. (n=15), *P<0.01 (Student's t-test). Download figure Download PowerPoint TORC2 interacts with the Trc kinase signalling pathway to control dendritic tiling Previous studies have shown that the NDR family kinase Trc/Sax-1 and its activator Fry/Sax-2 control both the iso-neuronal and hetero-neuronal dendritic tiling of sensory neurons in Drosophila and C. elegans (Emoto et al, 2004, 2006; Gallegos and Bargmann, 2004). To examine whether TORC2 genes and trc might function in the same genetic pathway to regulate dendritic tiling, we examined genetic interactions between trc and the TORC2 genes. As mentioned above, heterozygosity for null mutations in the TORC2 genes sin1, rictor, or Tor caused no significant defects in dendritic arborization including tiling (Figure 1D–F). Similarly, heterozygosity for null alleles of trc caused no discernable defects in dendrite development (Figure 4B; Emoto et al, 2004). However, trans-heterozygous combinations of mutations in trc together with sin1 caused a significant tiling defect that was comparable to trans-heterozygous combinations of TORC2 mutants (Figure 4C and F). Similarly, trans-heterozygous combinations of trc together with rictor or Tor caused similar tiling defects (Figure 4D–F). Thus, Trc and the TORC2 genes genetically interact to regulate dendritic tiling. Figure 4.TORC2 genetically and physically interacts with Trc. (A–F) Live images of third instar class IV neuron visualized using the pickpocket-EGFP reporter in (A) wild-type (WT), (B) trc/+, (C) trc/sin1 trans-heterozygous, (D) trc/rictor trans-heterozygous, (E) and trc/Tor trans-heterozygous larvae. Anterior is left and dorsal is up. Bar=50 μm. Genotypes: (A) yw; +/+; ppk-EGFP/ppk-EGFP, (B) yw; +/+; trc1, ppk-EGFP/ppk-EGFP, (C) yw; sin1PBac/+; trc1, ppk-EGFP/ppk-EGFP, (D) yw, rictorΔ2/+; +/+; trc1, ppk-EGFP/ppk-EGFP, and (E) yw; TorΔP/+; trc1, ppk-EGFP/ppk-EGFP. (F) Quantification of the crossing points in ddaC of WT and trans-heterozygotes. Error bars indicate the mean±s.d. (n=15) (G) Trc can form a complex with Sin1 in Drosophila neurons. Trc and Wts were co-immunoprecipitated with neuronally expressed Sin1-Flag from transgenic fly embryos as indicated by western blot analysis using anti-Trc and anti-Wts antibodies, respectively. (H) Association of endogenous TORC2 and NDR1 in human HeLa cells. The cells were lysed in buffer containing either 0.3% CHAPS or 1% Triton X-100 as indicated. −, immunoprecipitation control (no primary antibody was used). Co-immunoprecipitation of the TORC components was detected by specific antibodies as described. (I) Morphologies of phalloidin-labeled S2 cells on concanavalin (Con) A-coated coverslips were classified into three groups (stellate, serrate, and smooth). Cells were treated with dsRNA against indicated genes for 7 days and then plated on Con A and then stained with rhodamine-phalloidin to visualize filamentous actin. (J) Quantification of cell shape on knockdown of the indicated genes. RNAi knockdown of Trc- or TORC2-specific components (Sin1and Rictor) causes a significant increase in the number of stellate cells. Note that the cell morphology was not significantly affected by genetic ablation of Raptor, a TORC1-specific component. Download figure Download PowerPoint Given the genetic interactions between Trc and TORC2 components in dendritic tiling control, we next tested whether Trc could physically associate with TORC2 proteins. We expressed an epitope-tagged version of Sin1 (Sin1-Flag) in larval neurons using a nervous-system-specific Gal4 driver and found that Trc could be co-immunoprecipitated with Sin1-Flag (Figure 4G). This co-immunoprecipitation appeared to be specific, because Warts, another NDR kinase present in neurons, did not co-immunoprecipitate with Sin1-Flag (Figure 4G). These results suggest that Trc might associate with TORC2 in the Drosophila nervous system. To further examine the physical interaction between TORC2 and Trc, we evaluated whether endogenous TOR complexes can be immunoprecipitated with Trc (Figure 4H). As no reliable antibodies are available for Drosophila TORC2 components, we carried out co-immunoprecipitations using HeLa cell extracts and antibodies specific for human TORC2 components and a human Trc homologue NDR1 (Hergovich et al, 2006). We found that the mTOR protein isolated with a specific antibody interacted with NDR1 as well as with hSin1, Rictor, and a TORC1-specific component Raptor (Figure 4H). In contrast, the protein complexes isolated with hSin1 or Rictor antibodies contained mTOR and NDR1 but not Raptor, while those isolated with the Raptor antibody contained mTOR but not NDR1, hSin1, nor Rictor (Figure 4H). These results indicate that NDR1 interacts, at least in part, with TORC2 but not with TORC1. As reported previously, both TORC1 and TORC2 were stable in 0.3% CHAPS buffer but were disrupted by 1% Triton X-100 (Figure 4H). Interestingly, although the interaction between mTOR and NDR1 was disrupted by Triton X-100, the interactions between NDR1 and hSin1 or Rictor were stable under these conditions (Figure 4H), suggesting that NDR1 associates with hSin1 and/or Rictor, rather than mTOR. Previous studies suggest that the avoidance behaviour of class IV dendrites requires dynamic remodelling of the cytoskeletons (Grueber et al, 2003; Sugimura et al, 2003; Emoto et al, 2004; Parrish et al, 2007). To examine the possible function of TORC2 and Trc in the cytoskeletal organization, we used an established assay for monitoring actin network reorganization in cultured Drosophila S2 cells (Rogers et al, 2003). When placed on glass coverslips coated with the lectin concanavalin A, S2 cells reorganize their actin network to build a lamellipodium (Figure 4I, smooth). RNA interference (RNAi) knockdown of Sin1 or Rictor resulted in a significant increase in cells with aberrant organizations of their actin filaments (Figure 4I and J). These cells can be classified into three categories: cells with normal lamellae, cells that spread but showed an abnormal serrated edge, and cells that spread but showing a stellate morphology. Although the stellate morphology was observed in <5% of the control cells (3.0%, n=123) or Rap" @default.
• W2164073865 created "2016-06-24" @default.
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• W2164073865 creator A5041563757 @default.
• W2164073865 creator A5058331067 @default.
• W2164073865 creator A5073520625 @default.
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• W2164073865 date "2009-10-29" @default.
• W2164073865 modified "2023-10-16" @default.
• W2164073865 title "The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway" @default.
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