Moreover, autophagy is involved in the immune defense system targeting intracellular viruses, parasites, or bacteria and impaired autophagy may be detrimental during microbial infections 8, 9, 10, 12. autophagy-related protein 16L (ATG16L), a protein necessary for autophagy induction 16. Hereditary diseases like Crohn’s disease are linked to mutations in e.g. Diseases like cancer and neuropathies are characterized by dysregulation of autophagy 14, 15. Recent studies revealed that aberrant autophagy impacts several disorders 12, 13. Thus, it acts as an innate immune defense pathway 8, 9, 10 and eventually provides peptides for display on major histocompatibility complex (MHC) molecules on antigen-presenting cells 11. In addition, autophagy targets foreign compounds such as viral proteins in the cytoplasm (Xenophagy). to the release of anti-microbial peptides 7. Furthermore, cellular proteins processed by autophagy may lead e.g. Diverse cellular signaling pathways are regulated by autophagy, including the turnover of key signaling components 6. Overall, autophagy primarily has a pro-survival, cytoprotective role 5. Specific autophagy targets are usually earmarked for destruction by ubiquitin chains. This process is either specifically (selective autophagy) targeting compounds recognized by so-called autophagy receptors like SQSTM1 (p62) or leads to bulk degradation of parts of the cytoplasm 4. Their contents along with the inner membrane are degraded at a low pH by lysosomal peptidases and hydrolases. Upon induction of autophagy, cytoplasmic cargo is engulfed by double-layered membranes, which upon elongation and closure form so-called autophagosomes, eventually fusing with lysosomes. Autophagy is active in almost all cells of the body. Basal levels of autophagy are required to maintain homeostasis of eukaryotic cells, mediating continuous turnover of organelles and proteins. Macroautophagy (hereafter called autophagy) is a catabolic cellular process 1, 2, 3. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. (II) Monitoring of autophagy upon infection with e.g. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense.
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