Publications by authors named "Lukas A Huber"

111 Publications

Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations.

J Clin Med 2021 Apr 28;10(9). Epub 2021 Apr 28.

Institute of Cell Biology, Medical University of Innsbruck, A-6020 Innsbruck, Austria.

Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.
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http://dx.doi.org/10.3390/jcm10091901DOI Listing
April 2021

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia.

J Allergy Clin Immunol 2021 Apr 20. Epub 2021 Apr 20.

Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

Background: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality.

Objective: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects.

Methods: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements.

Results: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES.

Conclusions: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.
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http://dx.doi.org/10.1016/j.jaci.2021.03.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056780PMC
April 2021

Congenital Diarrhea and Cholestatic Liver Disease: Phenotypic Spectrum Associated with MYO5B Mutations.

J Clin Med 2021 01 28;10(3). Epub 2021 Jan 28.

Emma Children's Hospital/AMC, 1105 Amsterdam, The Netherlands.

Myosin Vb (MYO5B) is a motor protein that facilitates protein trafficking and recycling in polarized cells by RAB11- and RAB8-dependent mechanisms. Biallelic MYO5B mutations are identified in the majority of patients with microvillus inclusion disease (MVID). MVID is an intractable diarrhea of infantile onset with characteristic histopathologic findings that requires life-long parenteral nutrition or intestinal transplantation. A large number of such patients eventually develop cholestatic liver disease. Bi-allelic MYO5B mutations are also identified in a subset of patients with predominant early-onset cholestatic liver disease. We present here the compilation of 114 patients with disease-causing MYO5B genotypes, including 44 novel patients as well as 35 novel MYO5B mutations, and an analysis of MYO5B mutations with regard to functional consequences. Our data support the concept that (1) a complete lack of MYO5B protein or early MYO5B truncation causes predominant intestinal disease (MYO5B-MVID), (2) the expression of full-length mutant MYO5B proteins with residual function causes predominant cholestatic liver disease (MYO5B-PFIC), and (3) the expression of mutant MYO5B proteins without residual function causes both intestinal and hepatic disease (MYO5B-MIXED). Genotype-phenotype data are deposited in the existing open MYO5B database in order to improve disease diagnosis, prognosis, and genetic counseling.
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http://dx.doi.org/10.3390/jcm10030481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865828PMC
January 2021

Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA.

Sci Rep 2021 Jan 27;11(1):2331. Epub 2021 Jan 27.

Institute of Cell Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, 6020, Innsbruck, Austria.

The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1-306) and p50 (aa 1-367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (K) of 2.228 × 10 M and 0.794 × 10 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.
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http://dx.doi.org/10.1038/s41598-021-81743-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840993PMC
January 2021

Overcoming limitations in the availability of swabs systems used for SARS-CoV-2 laboratory diagnostics.

Sci Rep 2021 01 26;11(1):2261. Epub 2021 Jan 26.

Department of Internal Medicine II, Infectious Diseases, Immunology, Rheumatology, Pneumology, Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria.

The diagnosis of COVID-19 relies on the direct detection of SARS-CoV-2 RNA in respiratory specimens by RT-PCR. The pandemic spread of the disease caused an imbalance between demand and supply of materials and reagents needed for diagnostic purposes including swab sets. In a comparative effectiveness study, we conducted serial follow-up swabs in hospitalized laboratory-confirmed COVID-19 patients. We assessed the diagnostic performance of an in-house system developed according to recommendations by the US CDC. In a total of 96 serial swabs, we found significant differences in the accuracy of the different swab systems to generate a positive result in SARS-CoV-2 RT-PCR, ranging from around 50 to 80%. Of note, an in-house swab system was superior to most commercially available sets as reflected by significantly lower Ct values of viral genes. Thus, a simple combination of broadly available materials may enable diagnostic laboratories to bypass global limitations in the supply of swab sets.
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http://dx.doi.org/10.1038/s41598-021-81782-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838421PMC
January 2021

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Cell 2021 Feb 25;184(3):655-674.e27. Epub 2021 Jan 25.

Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck 6020, Austria.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
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http://dx.doi.org/10.1016/j.cell.2020.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868890PMC
February 2021

CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.

Front Immunol 2020 14;11:2010. Epub 2020 Aug 14.

Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.
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http://dx.doi.org/10.3389/fimmu.2020.02010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457048PMC
August 2020

A Dedicated Evolutionarily Conserved Molecular Network Licenses Differentiated Cells to Return to the Cell Cycle.

Dev Cell 2020 10 7;55(2):178-194.e7. Epub 2020 Aug 7.

Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Differentiated cells can re-enter the cell cycle to repair tissue damage via a series of discrete morphological and molecular stages coordinated by the cellular energetics regulator mTORC1. We previously proposed the term "paligenosis" to describe this conserved cellular regeneration program. Here, we detail a molecular network regulating mTORC1 during paligenosis in both mouse pancreatic acinar and gastric chief cells. DDIT4 initially suppresses mTORC1 to induce autodegradation of differentiated cell components and damaged organelles. Later in paligenosis, IFRD1 suppresses p53 accumulation. Ifrd1 cells do not complete paligenosis because persistent p53 prevents mTORC1 reactivation and cell proliferation. Ddit4 cells never suppress mTORC1 and bypass the IFRD1 checkpoint on proliferation. Previous reports and our current data implicate DDIT4/IFRD1 in governing paligenosis in multiple organs and species. Thus, we propose that an evolutionarily conserved, dedicated molecular network has evolved to allow differentiated cells to re-enter the cell cycle (i.e., undergo paligenosis) after tissue injury. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.devcel.2020.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606764PMC
October 2020

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

Elife 2020 08 3;9. Epub 2020 Aug 3.

Institute for Cell Biology, Medical University of Innsbruck, Innsbruck, Austria.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.
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http://dx.doi.org/10.7554/eLife.58246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449699PMC
August 2020

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

Nature 2020 09 1;585(7826):597-602. Epub 2020 Jul 1.

Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.
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http://dx.doi.org/10.1038/s41586-020-2444-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610377PMC
September 2020

Novel PCNT variants in MOPDII with attenuated growth restriction and pachygyria.

Clin Genet 2020 09 7;98(3):282-287. Epub 2020 Jul 7.

Department of Pediatrics and Adolescent Medicine, Division of Pediatric Pulmonology, Allergology and Endocrinology, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria.

Biallelic loss-of-function mutations in the centrosomal pericentrin gene (PCNT) cause microcephalic osteodysplastic primordial dwarfism type II (MOPDII), which is characterized by extreme growth retardation, microcephaly, skeletal dysplasia, and dental anomalies. Life expectancy is reduced due to a high risk of cerebral vascular anomalies. Here, we report two siblings with MOPDII and attenuated growth restriction, and pachygyria. Compound heterozygosity for two novel truncated PCNT variants was identified. Both truncated PCNT proteins were expressed in patient's fibroblasts, with a reduced total protein amount compared to control. Patient's fibroblasts showed impaired cell cycle progression. As a novel finding, 20% of patient's fibroblasts were shown to express PCNT comparable to control. This was associated with normal mitotic morphology and normal co-localization of mutated PCNT with centrosome-associated proteins γ-tubulin and centrin 3, suggesting some residual function of truncated PCNT proteins. These data expand the clinical and molecular spectrum of MOPDII and indicate that residual PCNT function might be associated with attenuated growth restriction in MOPDII.
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http://dx.doi.org/10.1111/cge.13797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497047PMC
September 2020

AP1S1 missense mutations cause a congenital enteropathy via an epithelial barrier defect.

Hum Genet 2020 Oct 18;139(10):1247-1259. Epub 2020 Apr 18.

Institute of Cell Biology, Biocenter, Medical University of Innsbruck, 6020, Innsbruck, Austria.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.
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http://dx.doi.org/10.1007/s00439-020-02168-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497319PMC
October 2020

Lysosomal size matters.

Traffic 2020 01 6;21(1):60-75. Epub 2019 Dec 6.

Institute of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.
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http://dx.doi.org/10.1111/tra.12714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972631PMC
January 2020

LAMTOR/Ragulator regulates lipid metabolism in macrophages and foam cell differentiation.

FEBS Lett 2020 01 26;594(1):31-42. Epub 2019 Aug 26.

Division of Cell Biology, Biocenter, Medical University of Innsbruck, Austria.

Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism.
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http://dx.doi.org/10.1002/1873-3468.13579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003824PMC
January 2020

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Traffic 2019 09;20(9):674-696

Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.
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http://dx.doi.org/10.1111/tra.12679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771566PMC
September 2019

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking.

Front Immunol 2019 18;10:497. Epub 2019 Mar 18.

Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using -Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using -Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice.
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http://dx.doi.org/10.3389/fimmu.2019.00497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431647PMC
September 2020

β-1,3-glucan-lacking mediates an efficient antifungal immune response by activating complement and dendritic cells.

Virulence 2019 12 29;10(1):957-969. Epub 2018 Oct 29.

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.

Complement system and dendritic cells (DCs) form - beside neutrophils and macrophages - the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with lacking ß-1,3-glucans ( under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking β-glucan synthase, making the mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the mediated induction of an inflammatory cytokine profile.Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms.: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal-regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.
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http://dx.doi.org/10.1080/21505594.2018.1528843DOI Listing
December 2019

Loss of MYO5B Leads to Reductions in Na Absorption With Maintenance of CFTR-Dependent Cl Secretion in Enterocytes.

Gastroenterology 2018 12 23;155(6):1883-1897.e10. Epub 2018 Aug 23.

Departments of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee; Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee; Nashville Veterans Affairs Medical Center, Nashville, Tennessee. Electronic address:

Background & Aims: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters.

Methods: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCre;Myo5b mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry.

Results: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCre;Myo5b mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCre;Myo5b mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCre;Myo5b mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity.

Conclusions: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
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http://dx.doi.org/10.1053/j.gastro.2018.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279525PMC
December 2018

Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.

Traffic 2018 08 21;19(8):639-649. Epub 2018 May 21.

Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.
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http://dx.doi.org/10.1111/tra.12575DOI Listing
August 2018

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

J Cell Biol 2017 12 9;216(12):4199-4215. Epub 2017 Oct 9.

Division of Cell Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.
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http://dx.doi.org/10.1083/jcb.201703061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716276PMC
December 2017

Crystal structure of the human lysosomal mTORC1 scaffold complex and its impact on signaling.

Science 2017 10 21;358(6361):377-381. Epub 2017 Sep 21.

Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.
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http://dx.doi.org/10.1126/science.aao1583DOI Listing
October 2017

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Division of Cell Biology, Medical University of Innsbruck, Innsbruck, Austria.

Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied. Morphology of duodenal sections was analyzed by electron and fluorescence microscopy. Small intestinal enterocytes and organoid-derived monolayers displayed the subcellular characteristics of MVID. For the analyses of Munc18-2-dependent SNARE-protein interactions, a Munc18-2 CaCo2-KO model cell line was generated by applying CRISPR/Cas9 technology. Munc18-2 is required for Slp4a/Stx3 interaction in fusion of cargo vesicles with the apical plasma membrane. Cargo trafficking was investigated in patient biopsies, patient-derived organoids, and the genome-edited model cell line. Loss of Munc18-2 selectively disrupts trafficking of certain apical brush-border proteins (NHE3 and GLUT5), while transport of DPPIV remained unaffected. Here, we describe the molecular mechanism how the loss of function of Munc18-2 leads to cargo-selective mislocalization of brush-border components and a subapical accumulation of cargo vesicles, as it is known from the loss of polarity phenotype in MVID.
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http://dx.doi.org/10.1172/jci.insight.94564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518552PMC
July 2017

A novel but frequent variant in LPA KIV-2 is associated with a pronounced Lp(a) and cardiovascular risk reduction.

Eur Heart J 2017 Jun;38(23):1823-1831

Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Schoepfstrasse 41, 6020 Innsbruck, Austria.

Aims: Lp(a) concentrations represent a major cardiovascular risk factor and are almost entirely controlled by one single locus (LPA). However, many genetic factors in LPA governing the enormous variance of Lp(a) levels are still unknown. Since up to 70% of the LPA coding sequence are located in a difficult to access hypervariable copy number variation named KIV-2, we hypothesized that it may contain novel functional variants with pronounced effects on Lp(a) concentrations. We performed a large scale mutation analysis in the KIV-2 using an extreme phenotype approach.

Methods And Results: We compiled an discovery set of 123 samples showing discordance between LPA isoform phenotype and Lp(a) concentrations and controls. Using ultra-deep sequencing, we identified a splice site variant (G4925A) in preferential association with the smaller LPA isoforms. Follow-up in a European general population (n = 2892) revealed an exceptionally high carrier frequency of 22.1% in the general population. The variant explains 20.6% of the Lp(a) variance in carriers of low molecular weight (LMW) apo(a) isoforms (P = 5.75e-38) and reduces Lp(a) concentrations by 31.3 mg/dL. Accordingly the odds ratio for cardiovascular disease was reduced from 1.39 [95% confidence interval (CI): 1.17-1.66, P = 1.89e-04] for wildtype LMW individuals to 1.19 [95%CI: 0.92; 1.56, P = 0.19] in LMW individuals who were additionally positive for G4925A. Functional studies point towards a reduction of splicing efficiency by this novel variant.

Conclusion: A highly frequent but until now undetected variant in the LPA KIV-2 region is strongly associated with reduced Lp(a) concentrations and reduced cardiovascular risk in LMW individuals.
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http://dx.doi.org/10.1093/eurheartj/ehx174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837733PMC
June 2017

Abnormal Rab11-Rab8-vesicles cluster in enterocytes of patients with microvillus inclusion disease.

Traffic 2017 07 17;18(7):453-464. Epub 2017 May 17.

Division of Histology and Embryology, Medical University of Innsbruck, Innsbruck, Austria.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.
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http://dx.doi.org/10.1111/tra.12486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693299PMC
July 2017

Deficiency of the sphingosine-1-phosphate lyase SGPL1 is associated with congenital nephrotic syndrome and congenital adrenal calcifications.

Hum Mutat 2017 04 6;38(4):365-372. Epub 2017 Mar 6.

Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria.

We identified two unrelated consanguineous families with three children affected by the rare association of congenital nephrotic syndrome (CNS) diagnosed in the first days of life, of hypogonadism, and of prenatally detected adrenal calcifications, associated with congenital adrenal insufficiency in one case. Using exome sequencing and targeted Sanger sequencing, two homozygous truncating mutations, c.1513C>T (p.Arg505*) and c.934delC (p.Leu312Phefs*30), were identified in SGPL1-encoding sphingosine-1-phosphate (S1P) lyase 1. SGPL1 catalyzes the irreversible degradation of endogenous and dietary S1P, the final step of sphingolipid catabolism, and of other phosphorylated long-chain bases. S1P is an intracellular and extracellular signaling molecule involved in angiogenesis, vascular maturation, and immunity. The levels of SGPL1 substrates, S1P, and sphingosine were markedly increased in the patients' blood and fibroblasts, as determined by liquid chromatography-tandem mass spectrometry. Vascular alterations were present in a patient's renal biopsy, in line with changes seen in Sgpl1 knockout mice that are compatible with a developmental defect in vascular maturation. In conclusion, loss of SGPL1 function is associated with CNS, adrenal calcifications, and hypogonadism.
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http://dx.doi.org/10.1002/humu.23192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384969PMC
April 2017

New Mouse Models for Microvillus Inclusion Disease (MVID): Where Do the Inclusions Come From and Are They Cause or Consequence?

Authors:
Lukas A Huber

Cell Mol Gastroenterol Hepatol 2016 Mar 18;2(2):112-113. Epub 2016 Jan 18.

Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.

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http://dx.doi.org/10.1016/j.jcmgh.2015.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980747PMC
March 2016

TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation.

BMC Biol 2016 10 25;14(1):95. Epub 2016 Oct 25.

Division of Cell Biology, Biocenter, Medical University Innsbruck, Innrain 80-82, A-6020, Innsbruck, Austria.

Background: TPA Induced Sequence 7 acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. We and others have shown that TIS7 regulates adult myogenesis through MyoD, one of the essential myogenic regulatory factors.

Results: Here, we present data identifying ICln as the specific, novel protein downstream of TIS7 controlling myogenesis. We show that TIS7/ICln epigenetically regulate myoD expression controlling protein methyl transferase activity. In particular, ICln regulates MyoD expression via its interaction with PRMT5 by an epigenetic modification that utilizes symmetrical di-methylation of histone H3 on arginine 8. We provide multiple evidences that TIS7 directly binds DNA, which is a functional feature necessary for its role in transcriptional regulation.

Conclusion: We present here a molecular insight into TIS7-specific control of MyoD gene expression and thereby skeletal muscle differentiation.
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http://dx.doi.org/10.1186/s12915-016-0318-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080701PMC
October 2016

Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response.

Proc Natl Acad Sci U S A 2016 Apr 5;113(16):4326-31. Epub 2016 Apr 5.

Division of Cell Biology, Biocenter, Medical University of Innsbruck, A-6020 Innsbruck, Austria

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.
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http://dx.doi.org/10.1073/pnas.1522372113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843428PMC
April 2016

Towards understanding microvillus inclusion disease.

Mol Cell Pediatr 2016 Dec 29;3(1). Epub 2016 Jan 29.

Department of Paediatrics I, Medical University of Innsbruck, Anichstrasse 35, 6020, Innsbruck, Austria.

Microvillus inclusion disease (MVID) is characterised by onset of intractable life-threatening watery diarrhoea during infancy. Transmission electron microscopy demonstrates shortening or absence of apical microvilli, pathognomonic microvillus inclusions in mature enterocytes and subapical accumulation of periodic acid-Schiff-positive granules or vesicles confirming diagnosis. Mutations in MYO5B have been found to cause MVID. In two patients with MVID, whole-exome sequencing of DNA revealed homozygous truncating mutations in STX3. Mutations in these genes disrupt trafficking between apical cargo vesicles and the apical plasma membrane. Thus, disturbed delivery of certain brush border membrane proteins is a common defect in MVID.
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http://dx.doi.org/10.1186/s40348-016-0031-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733813PMC
December 2016

Lysosomal signaling in control of degradation pathways.

Curr Opin Cell Biol 2016 Apr 28;39:8-14. Epub 2016 Jan 28.

Division of Cell Biology, Biocenter, Medical University of Innsbruck, Austria. Electronic address:

Autophagy mediates the (non-)selective bulk degradation of cytoplasm, protein aggregates, damaged organelles and certain pathogens. The endosomal membrane system uses multivesicular bodies (MVBs) to selectively deliver ubiquitinated membrane proteins together with extracellular components into lysosomes. Microautophagy (MA) and chaperone-mediated autophagy (CMA) additionally contribute to the selective delivery of cargo into lysosomes. The coordinated function of these lysosomal degradation pathways is essential to maintain cellular homeostasis. Their activity is controlled by mTOR (mammalian target of rapamycin) signaling and thus coupled to metabolic processes during cell growth. Here, we will discuss how TORC1 on lysosomes and TORC2 at the plasma membrane coordinate the different membrane biogenesis pathways with cargo selection, vesicle transport and fusion with lysosomes in response to intracellular and extracellular cues.
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http://dx.doi.org/10.1016/j.ceb.2016.01.006DOI Listing
April 2016