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Cell May 2020Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad...
Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
Topics: Animals; Autophagy; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitophagy
PubMed: 32437660
DOI: 10.1016/j.cell.2020.04.025 -
Traffic (Copenhagen, Denmark) May 2022Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively... (Review)
Review
Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.
Topics: Lysosomes; Metabolic Networks and Pathways; Signal Transduction
PubMed: 35343629
DOI: 10.1111/tra.12839 -
Nature Cell Biology Apr 2022Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of...
Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.
Topics: Autophagosomes; Autophagy; Dyneins; Lysosomes; Protein Transport
PubMed: 35332264
DOI: 10.1038/s41556-022-00861-8 -
The Journal of Cell Biology Jun 2022The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs)....
The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.
Topics: Autophagosomes; Autophagy; Extracellular Vesicles; Humans; Lysosomes; Proteostasis; SARS-CoV-2; Sequestosome-1 Protein; Viroporin Proteins; rab27 GTP-Binding Proteins
PubMed: 35446347
DOI: 10.1083/jcb.202110151 -
Molecular Genetics and Metabolism Feb 2021
Topics: Humans; Lysosomes; Metabolic Diseases
PubMed: 33541581
DOI: 10.1016/j.ymgme.2020.12.293 -
Angewandte Chemie (International Ed. in... May 2021The ability to regulate membrane protein abundance offers great opportunities for developing therapeutic sites for various diseases. Herein, we describe a platform for...
The ability to regulate membrane protein abundance offers great opportunities for developing therapeutic sites for various diseases. Herein, we describe a platform for the targeted degradation of membrane-associated proteins using bispecific aptamer chimeras that bind both the cell-surface lysosome-shuttling receptor (IGFIIR) and the targeted membrane-bound proteins of interest. We demonstrate that the aptamer chimeras can efficiently and quickly shuttle the therapeutically relevant membrane proteins of Met and PTK-7 to lysosomes and degrade them through the lysosomal protein degradation machinery. We anticipate that our method will provide a universal platform for the use of readily synthesized aptamer materials for biochemical research and potential therapeutics.
Topics: Aptamers, Nucleotide; Cell Membrane; HeLa Cells; Humans; Lysosomes; Membrane Proteins
PubMed: 33634555
DOI: 10.1002/anie.202102170 -
Nature Communications May 2023Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. High levels of free fatty acids in the liver impair hepatic lysosomal...
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. High levels of free fatty acids in the liver impair hepatic lysosomal acidification and reduce autophagic flux. We investigate whether restoration of lysosomal function in NAFLD recovers autophagic flux, mitochondrial function, and insulin sensitivity. Here, we report the synthesis of novel biodegradable acid-activated acidifying nanoparticles (acNPs) as a lysosome targeting treatment to restore lysosomal acidity and autophagy. The acNPs, composed of fluorinated polyesters, remain inactive at plasma pH, and only become activated in lysosomes after endocytosis. Specifically, they degrade at pH of ~6 characteristic of dysfunctional lysosomes, to further acidify and enhance the function of lysosomes. In established in vivo high fat diet mouse models of NAFLD, re-acidification of lysosomes via acNP treatment restores autophagy and mitochondria function to lean, healthy levels. This restoration, concurrent with reversal of fasting hyperglycemia and hepatic steatosis, indicates the potential use of acNPs as a first-in-kind therapeutic for NAFLD.
Topics: Mice; Animals; Non-alcoholic Fatty Liver Disease; Autophagy; Liver; Lysosomes; Hydrogen-Ion Concentration
PubMed: 37142604
DOI: 10.1038/s41467-023-38165-6 -
The Journal of Cell Biology Dec 2023Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by...
Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.
Topics: Autophagy; Lysosomes; Macroautophagy; Microtubule-Associated Proteins; Salmonella; Autophagy-Related Protein 8 Family
PubMed: 37796195
DOI: 10.1083/jcb.202303078 -
Autophagy Oct 2019Multiple sources contribute membrane and protein machineries to construct functional macroautophagic/autophagic structures. However, the underlying molecular mechanisms...
Multiple sources contribute membrane and protein machineries to construct functional macroautophagic/autophagic structures. However, the underlying molecular mechanisms remain elusive. Here, we show that RAB2 connects the Golgi network to autophagy pathway by delivering membrane and by sequentially engaging distinct autophagy machineries. In unstressed cells, RAB2 resides primarily in the Golgi apparatus, as evidenced by its interaction and colocalization with GOLGA2/GM130. Importantly, autophagy stimuli dissociate RAB2 from GOLGA2 to interact with ULK1 complex, which facilitates the recruitment of ULK1 complex to form phagophores. Intriguingly, RAB2 appears to modulate ULK1 kinase activity to propagate signals for autophagosome formation. Subsequently, RAB2 switches to interact with autophagosomal RUBCNL/PACER and STX17 to further specify the recruitment of HOPS complex for autolysosome formation. Together, our study reveals a multivalent pathway in bulk autophagy regulation, and provides mechanistic insights into how the Golgi apparatus contributes to the formation of different autophagic structures. ACTB: actin beta; ATG9: autophagy related 9A; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BCAP31: B cell receptor associated protein 31; BECN1: beclin 1; Ctrl: control; CQ: chloroquine; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EEA1: early endosome antigen 1; GDI: guanine nucleotide dissociation inhibitor; GFP: green fluorescent protein; GOLGA2: golgin A2; HOPS: homotypic fusion and protein sorting complex; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; OE: overexpression; PtdIns3K: class III phosphatidylinositol 3-kinase; SQSTM1/p62: sequestosome 1; RAB2: RAB2A, member RAS oncogene family; RAB7: RAB7A, member RAS oncogene family; RAB11: RAB11A, member RAS oncogene family; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TBC1D14: TBC1 domain family member 14; TFRC: transferrin receptor; TGOLN2: trans-golgi network protein 2; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VPS41: VPS41, HOPS complex subunit; WB: western blot; WT: wild type; YPT1: GTP-binding protein YPT1.
Topics: Animals; Autophagosomes; Autophagy; Cells, Cultured; Eukaryotic Cells; HEK293 Cells; HeLa Cells; Humans; Lysosomes; Male; Mammals; Mice; Mice, Inbred C57BL; Mice, Knockout; rab2 GTP-Binding Protein
PubMed: 30957628
DOI: 10.1080/15548627.2019.1596478 -
Trends in Neurosciences Dec 2023Lysosomes play crucial roles in various cellular processes - including endocytosis, phagocytosis, and autophagy - which are vital for maintaining retinal health.... (Review)
Review
Lysosomes play crucial roles in various cellular processes - including endocytosis, phagocytosis, and autophagy - which are vital for maintaining retinal health. Moreover, these organelles serve as environmental sensors and act as central hubs for multiple signaling pathways. Through communication with other cellular components, such as mitochondria, lysosomes orchestrate the cytoprotective response essential for preserving cellular homeostasis. This coordination is particularly critical in the retina, given its high metabolic rate and susceptibility to photo-oxidative stress. Consequently, impaired lysosomal function and dysregulated communication between lysosomes and other organelles contribute significantly to the pathobiology of major retinal degenerative diseases. This review explores the pivotal role of lysosomes in retinal cells and their involvement in retinal degenerative diseases.
Topics: Humans; Retina; Lysosomes; Autophagy; Mitochondria; Endocytosis
PubMed: 37848361
DOI: 10.1016/j.tins.2023.09.006