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1.
J Cell Biol ; 223(6)2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38506714

ABSTRACT

The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.


Subject(s)
Electron Microscope Tomography , Extracellular Matrix , Biological Transport , Cell Movement , Cytosol , Electron Microscope Tomography/methods , Extracellular Matrix/ultrastructure
2.
J Struct Biol ; 212(3): 107633, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32987119

ABSTRACT

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.


Subject(s)
Cell Culture Techniques/methods , Cryoelectron Microscopy/methods , Specimen Handling/methods , Printing, Three-Dimensional , Reproducibility of Results , Workflow
3.
Nat Commun ; 8(1): 295, 2017 08 18.
Article in English | MEDLINE | ID: mdl-28821724

ABSTRACT

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing.The protease Atg4 mediates Atg8 lipidation, required for autophagosome biogenesis, but also triggers Atg8 release from the membranes, however is unclear how these steps are coordinated. Here the authors show that phosphorylation by Atg1 inhibits Atg4 at autophagosome formation sites.


Subject(s)
Autophagy-Related Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Autophagosomes/metabolism , Autophagosomes/ultrastructure , Autophagy , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Blotting, Western , Microscopy, Electron , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Phosphatidylethanolamines/metabolism , Phosphorylation , Protein Kinases/genetics , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
4.
EMBO Rep ; 18(5): 765-780, 2017 05.
Article in English | MEDLINE | ID: mdl-28330855

ABSTRACT

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8.


Subject(s)
Autophagosomes/metabolism , Autophagy-Related Protein 8 Family/chemistry , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/metabolism , Autophagy , Microtubule-Associated Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Proteins/genetics , Membranes/chemistry , Membranes/metabolism , Microtubule-Associated Proteins/genetics , Phagosomes/metabolism , Phosphatidylethanolamines/metabolism , Protein Binding , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics
5.
Elife ; 52016 11 23.
Article in English | MEDLINE | ID: mdl-27879200

ABSTRACT

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.


Subject(s)
Autophagy-Related Protein 5/chemistry , Autophagy-Related Protein 8 Family/chemistry , Autophagy-Related Proteins/chemistry , Autophagy/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Autophagy-Related Protein 12/genetics , Autophagy-Related Protein 12/metabolism , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Binding Sites , Biological Transport , Cell Cycle Proteins , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Membrane Transport Proteins , Molecular Docking Simulation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Signal Transduction , Transcription Factor TFIIIA/genetics , Transcription Factor TFIIIA/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism
6.
Methods ; 75: 37-43, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25461810

ABSTRACT

Macroautophagy is a major bulk degradation pathway for cytoplasmic material in eukaryotic cells. During macroautophagy, double membrane-bound organelles called autophagosomes are formed in a de novo manner. In the course of their formation autophagosomes capture cytoplasmic material, which is subsequently degraded upon fusion with the lysosomal system in complex eukaryotes or the vacuole in yeast. Several proteins are required for autophagosome formation. Among these are the components of two ubiquitin-like conjugation reactions that collectively mediate the conjugation of the ubiquitin-like Atg12 to the Atg5 protein and of the ubiquitin-like protein Atg8 to the headgroup of the membrane lipid phosphatidylethanolamine. The lipidated form of Atg8 is membrane-bound and marks the growing autophagosomal membrane as well as the completed autophagosome. Here we describe assays for the in vitro reconstitution of the Atg8 lipidation reaction using recombinantly expressed and purified proteins derived from Saccharomycescerevisiae in combination with small and giant unilamellar vesicles. The assays enable the study of the biochemical mechanisms of action of the Atg8 lipidation machinery and to analyze the impact of mutations and post-translational modifications of the conjugation machinery on Atg8 lipidation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy/genetics , Microfilament Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Molecular Biology/methods , Saccharomyces cerevisiae Proteins/metabolism , Autophagy-Related Protein 8 Family , Cell Membrane/metabolism , In Vitro Techniques , Lipid Metabolism , Microtubule-Associated Proteins/genetics , Mutation , Phagosomes/genetics , Phagosomes/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin
7.
Nat Cell Biol ; 16(5): 425-433, 2014 May.
Article in English | MEDLINE | ID: mdl-24705553

ABSTRACT

Autophagy protects cells from harmful substances such as protein aggregates, damaged mitochondria and intracellular pathogens, and has been implicated in a variety of diseases. Selectivity of autophagic processes is mediated by cargo receptors that link cargo to Atg8 family proteins on the developing autophagosomal membrane. To avoid collateral degradation during constitutive autophagic pathways, the autophagic machinery must not only select cargo but also exclude non-cargo material. Here we show that cargo directly activates the cargo receptor Atg19 by exposing multiple Atg8 binding sites. Furthermore, Atg19 mediates tight apposition of the cargo and Atg8-coated membranes in a fully reconstituted system. These properties are essential for the function of Atg19 during selective autophagy in vivo. Our results suggest that cargo receptors contribute to tight membrane bending of the isolation membrane around the cargo.


Subject(s)
Autophagy , Cell Membrane/metabolism , Microtubule-Associated Proteins/metabolism , Receptors, Cell Surface/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism , Autophagy-Related Protein 8 Family , Autophagy-Related Proteins , Binding Sites , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microtubule-Associated Proteins/genetics , Mutation , Phosphatidylethanolamines/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Unilamellar Liposomes/metabolism , Vesicular Transport Proteins/genetics
8.
Cell Stem Cell ; 14(6): 824-37, 2014 Jun 05.
Article in English | MEDLINE | ID: mdl-24704494

ABSTRACT

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.


Subject(s)
Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Stem Cell Niche , Aged , Animals , Humans , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID
9.
Int J Oncol ; 40(5): 1331-8, 2012 May.
Article in English | MEDLINE | ID: mdl-22366868

ABSTRACT

Epidemiological evidence on the chemopreventive activity of mesalazine against colitis-associated cancer has accumulated in recent years. Together with the variety of mesalazine molecular antitumor effects this has prompted the development of novel mesalazine derivatives. The objective of this study was to test five novel derivatives (compounds 2-14, 2-17, 2-28, 2-34L, 2-39) for their effect on cell proliferation, their capability to scavenge superoxide anions, to induce a cell cycle arrest and to improve replication fidelity in cultured colorectal cells. Compound 2-14 was identified as the strongest inhibitor of cell proliferation and functioned as a potent superoxide scavenger, as did 2-17 and 2-34L. 2-14 induced a G2/M-arrest in HCT116 and a G0/G1-arrest in HT29 cells. 2-17 caused a G0/G1-arrest and 2-34L a G2/M-arrest in HT29 cells. 2-17 and 2-34L reduced mutation rates at a (CA)13 repeat in a dose-dependent fashion. These data suggest that certain mesalazine derivatives share important antitumor effects. From this experimental profile compounds 2-17 and 2-34L both improve replication fidelity, which is biologically relevant not only for colitis-associated cancer but also potentially for individuals with hereditary non-polyposis colorectal cancer.


Subject(s)
Anticarcinogenic Agents/pharmacology , Colorectal Neoplasms/prevention & control , DNA Replication/drug effects , Mesalamine/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Checkpoint Kinase 1 , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Dose-Response Relationship, Drug , Free Radical Scavengers/pharmacology , HCT116 Cells , HT29 Cells , Humans , MutL Protein Homolog 1 , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidative Stress/drug effects , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/drug effects , Superoxides/metabolism , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
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