Over the last two decades we and others could show that the LAMTOR complex (late endosomal/lysosomal adaptor, MAPK and MTOR activator) is strictly recruited to the membrane of late endocytic compartments, from where it actively influences MAPK, mTORC signaling and endosomal trafficking. The complex is involved in several biological processes including immunity, early embryogenesis, tissue homeostasis, cellular proliferation and migration. We use complementary molecular and cellular biology methods, stretching from proteomics, to live and confocal microscopy, electron microscopy, and structural biology, in order to functionally characterize the LAMTOR complex at the molecular and cellular levels. In addition, we use model organisms such as yeast and knockout mice to determine the biological roles of the complex in vivo, at the organism level.
As a result of an interaction proteomics screen using TAP-MS (Tandem Affinity Purification, coupled to Mass Spectrometry) we have identified several proteins interacting with the LAMTOR complex. The core interactome includes all members of the LAMTOR complex, the RAG GTPases (that mediate the translocation of mTORC1 to endosomes/lysosomes ), and SLC38A9 (a previously uncharacterized member of the solute carrier family 38, that we recently identified as an integral component of the amino acid-sensing machinery that controls the activation of mTORC1 (Nature, in press). We are currently analysing this comprehensive interactome data functionally with a special focus on the interplay between signaling and endosomal biogenesis.
Fig.1: Core interactome of the LAMTOR complex
The use of animal models led to great progresses in understanding the role of the endosomal adaptor Lamtor2 in endosomal/lysosomal trafficking. In a first approach LAMTOR2 knockout mice were generated, but severe defects during embryogenesis resulted in no viable offspring (Teis et al., 2006). During this time, 4 patients, all siblings, suffering from a primary immunodeficiency syndrome, due to a hypomorph LAMTOR2 allele, were identified and demanded for further investigations on the function of LAMTOR2 for the immune system (Bohn et al., 2007). Therefore, we have established conditional knockout mouse models to investigate the role of LAMTOR2 for the immune system. A special interest was put on antigen presenting cells including macrophages and dendritic cells (DCs). The correct uptake and processing of pathogens and antigen presentation in the context of the immune response is strictly regulated by the endosomal/lysosomal system. Using a mouse model where Lamtor2 was specifically depleted in macrophages, key players in the immune system, we have been able to demonstrate that LAMTOR2 is a host defense factor against pathogens (Fig.2) (Taub et al, J Cell Sci. 2012) and our findings correlate with the previously described immunodeficiency syndrome. We are currently following this line of results combining the mouse genetic approach with a proteomic approach aimed at deepening our understanding of the Lamtor2 interaction partners and downstream targets involved in phagolysosomal maturation underlying an efficient antimicrobial response.
DCs are initiators of adaptive immunity and unlike macrophages also able to prime naïve T cells. Investigation of a DC specific knockout mouse revealed a crucial role of LAMTOR2 for DC homeostasis. While soon after birth the epidermal Langerhans cell (LC) network is dispersed due to increased apoptosis and a proliferation defect (Sparber et al., 2014), the aging animals suffer from a massive expansion of conventional (cDCs) and plasmacytoid DCs (pDCs) cumulating in a myeloid proliferation syndrome (MPD) (Fig. 3). As cellular mechanism causing those phenotypes a deregulation of late endosomal LAMTOR complex dependent MAPKinase and mTORC1 signaling were identified. Loss of LCs was related to a decreased signaling, while cDC and pDC expansion was caused by boosted mTORC1 activation (Scheffler et al., 2014).
Fig. 2: Primary LAMTOR2+/+ and LAMTOR 2-/- macrophages infected with Salmonella (green). After 24 h an increased bacteria load in LAMTOR2-/- macrophages was observed. Actin (red) and Hoechst (blue). Scale bars: 10 mm (Taub et. al., 2012).
Fig. 3: Dendritic cell infiltrate in a spleen section of a DC specific LAMTOR2 knockout mouse model at the age of three months. red: CD11c, blue: hematoxylin.
Bohn, G., A. Allroth, G. Brandes, J. Thiel, E. Glocker, A.A. Schaffer, C. Rathinam, N. Taub, D. Teis, C. Zeidler, R.A. Dewey, R. Geffers, J. Buer, L.A. Huber, K. Welte, B. Grimbacher, and C. Klein. 2007. A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14. Nat Med. 13:38-45.
Scheffler, J.M., F. Sparber, C.H. Tripp, C. Herrmann, A. Humenberger, J. Blitz, N. Romani, P. Stoitzner, and L.A. Huber. 2014. LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling. Nat Commun. 5:5138.
Sparber, F., J.M. Scheffler, N. Amberg, C.H. Tripp, V. Heib, M. Hermann, S.P. Zahner, B.E. Clausen, B. Reizis, L.A. Huber, P. Stoitzner, and N. Romani. 2014. The late endosomal adaptor molecule p14 (LAMTOR2) represents a novel regulator of Langerhans cell homeostasis. Blood. 123:217-27.
Taub, N., M. Nairz, D. Hilber, M.W. Hess, G. Weiss, and L.A. Huber. 2012. The late endosomal adaptor p14 is a macrophage host-defense factor against Salmonella infection. J Cell Sci. 125:2698-708.
Teis, D., N. Taub, R. Kurzbauer, D. Hilber, M.E. de Araujo, M. Erlacher, M. Offterdinger, A. Villunger, S. Geley, G. Bohn, C. Klein, M.W. Hess, and L.A. Huber. 2006. p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation during tissue homeostasis. J Cell Biol. 175:861-8.
Microvillus inclusion disease is an autosomal recessive enteropathy characterized by intractable diarrhea setting on within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid-Schiff (PAS)-positive vesicles in enterocytes.
Together with our collaborators from the Department of Pediatrics I, MUI and the Division of Histology and Embryology, MUI, we were the first to identify mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients (Mueller et al., Nature Genetics 2008). In a follow-up study, we described further 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients (Ruemmele et al., Human Mutation 2010).
Further investigations have focused on the role of Myosin Vb and its interplay with Rab Small GTPases in the establishment of correct epithelial polarity by making use of a CaCo2 RNAi cell model (Thoeni et al., Traffic 2014).
Recently, we have identified novel mutations in the STX3 gene, causing a variant form of MVID in patients negative for mutations in MYO5B (Wiegerinck et al., Gastroenterology 2014). Syntaxin 3 is an apical t-SNARE protein pivotal for polarized apical exocytosis and secretion in epithelial cells.
By using a CaCo2 cell model for epithelial/enterocyte polarity and state of the art genome-editing technologies we focus on the intracellular cascade and the proteins involved, which ensure correct polarized intracellular traffic in order to maintain proper epithelial polarity.
Fig. 4: Polarized epithelial CaCo2 cyst showing basolateral proteins (green), apical proteins (white and red) and nuclei (blue).