Crumbs Complex Research Articles

PALS1 Is Essential for Retinal Pigment Epithelium Structure and Neural Retina Stratification

The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.

Crumbs regulates rhodopsin transport by interacting with and stabilizing myosin V

The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas.

Protein complexes that control renal epithelial polarity

Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions.

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

BackgroundCell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis.Methodology/Principal FindingsHere, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs).Conclusions/SignificanceThese results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor.

The Crumbs Complex Couples Cell Density Sensing to Hippo-Dependent Control of the TGF-β-SMAD Pathway

The Hippo pathway senses cell density information to control tissue growth by regulating the localization of the transcriptional regulators TAZ and YAP (TAZ/YAP). TAZ/YAP also regulate TGF-β-SMAD signaling, but whether this role is linked to cell density sensing is unknown. Here we demonstrate that TAZ/YAP dictate the localization of active SMAD complexes in response to cell density-mediated formation of polarity complexes. In high-density cell cultures, the Hippo pathway drives cytoplasmic localization of TAZ/YAP, which sequesters SMAD complexes, thereby suppressing TGF-β signaling. We show that during mouse embryogenesis, this is reflected by differences in TAZ/YAP localization, which define regions of active SMAD2/3 complexes. Interfering with TAZ/YAP phosphorylation drives nuclear accumulation of TAZ/YAP and SMAD2/3. Furthermore, we demonstrate that the Crumbs polarity complex interacts with TAZ/YAP, which relays cell density information by promoting TAZ/YAP phosphorylation, cytoplasmic retention, and suppressed TGF-β signaling. Accordingly, disruption of the Crumbs complex enhances TGF-β signaling and predisposes cells to TGF-β-mediated epithelial-to-mesenchymal transitions.

Antagonistic Functions of Two Stardust Isoforms inDrosophilaPhotoreceptor Cells

Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

BackgroundVertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.ResultsWe examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size.ConclusionsOur observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain.

APKC Phosphorylation of Bazooka Defines the Apical/Lateral Border in Drosophila Epithelial Cells

SummaryBazooka (PAR-3), PAR-6, and aPKC form a complex that plays a key role in the polarization of many cell types. In epithelial cells, however, Bazooka localizes below PAR-6 and aPKC at the apical/lateral junction. Here, we show that Baz is excluded from the apical aPKC domain in epithelia by aPKC phosphorylation, which disrupts the Baz/aPKC interaction. Removal of Baz from the complex is epithelial-specific because it also requires the Crumbs complex, which prevents the Baz/PAR-6 interaction. In the absence of Crumbs or aPKC phosphorylation of Baz, mislocalized Baz recruits adherens junction components apically, leading to a loss of the apical domain and an expansion of lateral. Thus, apical exclusion of Baz by Crumbs and aPKC defines the apical/lateral border. Although Baz acts as an aPKC targeting and specificity factor in nonepithelial cells, our results reveal that it performs a complementary function in positioning the adherens junction in epithelia.

Epithelial Polarity Proteins Regulate Drosophila Tracheal Tube Size in Parallel to the Luminal Matrix Pathway

Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). Here, we show that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. Our data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size.

Stepwise polarisation of the Drosophila follicular epithelium

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, beta-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.

The Crumbs complex: from epithelial-cell polarity to retinal degeneration

The evolutionarily conserved Crumbs protein complex is a key regulator of cell polarity and cell shape in both invertebrates and vertebrates. The important role of this complex in normal cell function is illustrated by the finding that mutations in one of its components, Crumbs, are associated with retinal degeneration in humans, mice and flies. Recent results suggest that the Crumbs complex plays a role in the development of other disease processes that are based on epithelial dysfunction, such as tumorigenesis or the formation of cystic kidneys. Localisation of the complex is restricted to a distinct region of the apical plasma membrane that abuts the zonula adherens in epithelia and photoreceptor cells of invertebrates and vertebrates, including humans. In addition to the core components, a variety of other proteins can be recruited to the complex, depending on the cell type and/or developmental stage. Together with diverse post-transcriptional and post-translational mechanisms that regulate the individual components, this provides an enormous functional diversity and flexibility of the complex. In this Commentary, we summarise findings concerning the organisation and modification of the Crumbs complex, and the conservation of its constituents from flies to mammals. In addition, we discuss recent results that suggest its participation in various human diseases, including blindness and tumour formation.

Crumbs proteins in epithelial morphogenesis

Cell polarity is an essential feature of most eukaryotic cells, especially epithelial cells in multicellular animals. Polarity protein complexes that regulate epithelial organization have been identified. In this review, it is proposed to describe how the Crumbs complex acts in the process of cell polarity and epithelial organization. During the last decade, several partners of Crumbs, an apical transmembrane protein, have been identified and their direct or indirect associations with the cytoplasmic domain of Crumbs have been dissected. In addition, mutants of several of the genes encoding proteins belonging to the Crumbs network have been obtained in animals ranging from flies to mouse, which have led to a better understanding of their functions in vivo. These functions include polarity axis formation, stabilization of epithelial apico-lateral junctions, photoreceptor organization and ciliogenesis. Since human CRUMBS1 mutations are associated with retina degeneration, it has become essential to define Crumbs network and to understand exactly how this network acts in polarized cells, with a view to developing suitable therapeutic approaches for treating this severe degenerative disease.

Crosstalk between small GTPases and polarity proteins in cell polarization

Cell polarization is crucial for the development of multicellular organisms, and aberrant cell polarization contributes to various diseases, including cancer. How cell polarity is established and how it is maintained remain fascinating questions. Conserved proteins of the partitioning defective (PAR), Scribble and Crumbs complexes guide the establishment of cell polarity in various organisms. Moreover, GTPases that regulate actin cytoskeletal dynamics have been implicated in cell polarization. Recent findings provide insights into polarization mechanisms and show intriguing crosstalk between small GTPases and members of polarity complexes in regulating cell polarization in different cellular contexts and cell types.

Divergent polarization mechanisms during vertebrate epithelial development mediated by the Crumbs complex protein Nagie oko

The zebrafish MAGUK protein Nagie oko is a member of the evolutionarily conserved Crumbs protein complex and functions as a scaffolding protein involved in the stabilization of multi-protein assemblies at the tight junction. During zebrafish embryogenesis, mutations in nagie oko cause defects in both epithelial polarity and cardiac morphogenesis. We used deletion constructs of Nagie oko in functional rescue experiments to define domains essential for cell polarity, maintenance of epithelial integrity and cardiac morphogenesis. Inability of Nagie oko to interact with Crumbs proteins upon deletion of the PDZ domain recreates all aspects of the nagie oko mutant phenotype. Consistent with this observation, apical localization of Nagie oko within the myocardium and neural tube is dependent on Oko meduzy/Crumbs2a. Disruption of direct interactions with Patj or Lin-7, two other members of the Crumbs protein complex, via the bipartite L27 domains produces only partial nagie oko mutant phenotypes and does not impair correct junctional localization of the truncated Nagie oko deletion protein within myocardial cells. Similarly, loss of the evolutionarily conserved region 1 domain, which mediates binding to Par6, causes only a subset of the nagie oko mutant epithelial phenotypes. Finally, deletion of the C-terminus, including the entire guanylate kinase and the SH3 domains, renders the truncated Nagie oko protein inactive and recreates all features of the nagie oko mutant phenotype when tested in functional complementation assays. Our observations reveal a previously unknown diversity of alternative multi-protein assembly compositions of the Crumbs-Nagie-oko and Par6-aPKC protein complexes that are highly dependent on the developmental context.

Polarized Subcellular Localization of JAK/STAT Components Is Required for Efficient Signaling

Three protein complexes control polarization of epithelial cells: the apicolateral Crumbs and Par-3 complexes and the basolateral Lethal giant larvae complex. Polarization results in the specific localization of proteins and lipids to different membrane domains. The receptors of the Notch, Hedgehog, and WNT pathways are among the proteins that are polarized, with subcellular receptor localization representing an important aspect of signaling regulation. For example, in the WNT pathway, differential DFz2 receptor localization results in activation of either the canonical or the planar polarity pathway. Despite the large body of research on the vertebrate JAK/STAT pathway, there are no reports indicating polarized signaling. By using the conserved Drosophila JAK/STAT pathway as a system, we find that the receptor and its associated kinase are located in the apical membrane of epithelial cells. Unexpectedly, the transcription factor STAT is enriched in the apicolateral membrane domain of ectoderm epithelial cells in a Par-3-dependent manner. Our results indicate that preassembly of STAT and the Receptor/JAK complex to specific membrane domains is a key aspect for signaling efficiency. Our results also suggest that receptor polarization in the ectoderm cell membrane restricts the cell's response to ligands provided by neighboring cells.

The transcription factor snail represses Crumbs3 expression and disrupts apico-basal polarity complexes

In epithelial cells, the tight junction divides the plasma membrane into distinct apical and basolateral domains. Polarization is essential for epithelial cell function, and apico-basal cell polarity is lost during the epithelial to mesenchymal transition (EMT), a program of events characterized not only by loss of cell polarity, but also by enhanced cell motility and increased cell invasion. Among several apically localized protein complexes, the Crumbs and Par protein complexes have pivotal roles in control of epithelial polarity and apical membrane formation. Here, we demonstrate that the Snail transcriptional repressor antagonizes expression of the Crumbs polarity complex. We show that Snail abolishes localization of the Crumbs and Par complexes to the tight junction, decreases Crumbs complex protein levels and suppresses Crumbs3 transcription. Evidence that Snail acts directly to antagonize Crumbs3 promoter activity is presented. Strikingly, we note that reexpression of exogenous Crumbs3 in Snail-expressing Madin-Darby Canine Kidney cells partially restores cell-cell junctions. Moreover, we find that the EMT inducer transforming growth factor-beta elicits transcriptional repression of Crumbs3 and results in a measurable loss of Crumbs3 protein. Our findings provide new insights into the links between the transcriptional repression function of Snail and its role in antagonizing key apico-basal polarity factors during EMT.

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin -7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin -7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.

FERM protein EPB41L5 is a novel member of the mammalian CRB–MPP5 polarity complex

Cell polarity is induced and maintained by separation of the apical and basolateral domains through specialized cell–cell junctions. The Crumbs protein and its binding partners are involved in formation and stabilization of adherens junctions. In this study, we describe a novel component of the mammalian Crumbs complex, the FERM domain protein EPB41L5, which associates with the intracellular domains of all three Crumbs homologs through its FERM domain. Surprisingly, the same FERM domain is involved in binding to the HOOK domain of MPP5/PALS1, a previously identified interactor of Crumbs. Co-expression and co-localization studies suggested that in several epithelial derived tissues Epb4.1l5 interacts with at least one Crumbs homolog, and with Mpp5. Although at early embryonic stages Epb4.1l5 is found at the basolateral membrane compartment, in adult tissues it co-localizes at the apical domain with Crumbs proteins and Mpp5. Overexpression of Epb4.1l5 in polarized MDCK cells affects tightness of cell junctions and results in disorganization of the tight junction markers ZO-1 and PATJ. Our results emphasize the importance of a conserved Crumbs–MPP5–EPB41L5 polarity complex in mammals.

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex

In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. On the other hand, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. Here we report, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2.