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Scientific Reports Jun 2024Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral...
Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.
Topics: Endoplasmic Reticulum; Autophagy; Saccharomyces cerevisiae; Membrane Microdomains; Cellular Senescence; Saccharomyces cerevisiae Proteins; Vacuoles; Membrane Proteins
PubMed: 38871812
DOI: 10.1038/s41598-024-64493-8 -
Frontiers in Molecular Biosciences 2024Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy,... (Review)
Review
Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker L, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to L. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.
PubMed: 38859931
DOI: 10.3389/fmolb.2024.1378656 -
International Journal of Biological... 2024Autophagy plays a critical role in maintaining cellular homeostasis and responding to various stress conditions by the degradation of intracellular components. In this... (Review)
Review
Autophagy plays a critical role in maintaining cellular homeostasis and responding to various stress conditions by the degradation of intracellular components. In this narrative review, we provide a comprehensive overview of autophagy's cellular and molecular basis, biological significance, pharmacological modulation, and its relevance in lifestyle medicine. We delve into the intricate molecular mechanisms that govern autophagy, including macroautophagy, microautophagy and chaperone-mediated autophagy. Moreover, we highlight the biological significance of autophagy in aging, immunity, metabolism, apoptosis, tissue differentiation and systemic diseases, such as neurodegenerative or cardiovascular diseases and cancer. We also discuss the latest advancements in pharmacological modulation of autophagy and their potential implications in clinical settings. Finally, we explore the intimate connection between lifestyle factors and autophagy, emphasizing how nutrition, exercise, sleep patterns and environmental factors can significantly impact the autophagic process. The integration of lifestyle medicine into autophagy research opens new avenues for promoting health and longevity through personalized interventions.
Topics: Autophagy; Humans; Life Style; Animals; Aging; Neurodegenerative Diseases
PubMed: 38725847
DOI: 10.7150/ijbs.95122 -
Developmental Cell Jun 2024Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the...
Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.
Topics: Lysosomes; Endoplasmic Reticulum; Humans; Endosomal Sorting Complexes Required for Transport; Calcium-Binding Proteins; COP-Coated Vesicles; Microautophagy; Vesicular Transport Proteins; Cell Cycle Proteins; Protein Transport; HeLa Cells; Apoptosis Regulatory Proteins; Autophagy; TOR Serine-Threonine Kinases; Calcium
PubMed: 38593803
DOI: 10.1016/j.devcel.2024.03.027 -
Journal of Fungi (Basel, Switzerland) Feb 2024Temperature and moisture belong to the most important environmental factors affecting the growth and development of fungi. However, the effect of temperature on the...
Temperature and moisture belong to the most important environmental factors affecting the growth and development of fungi. However, the effect of temperature on the mycelia of the edible mushrooms has not been determined. Here, a comprehensive analysis was performed to determine the influence of culture temperature on 13 strains of mycelia of three species (, , and ) at 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C. The mycelial branching and growth rate data showed that 15-20 °C was a suitable temperature range for the mycelial growth of the 13 strains. RNA sequences revealed that a total of 2843, 2404, 1973, 1572, and 1866 differentially expressed genes (DEGs) were identified at 5 °C, 10 °C, 15 °C, 25 °C, and 30 °C compared with 20 °C. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis further indicated that the purine nucleotide and tyrosine metabolism pathways were crucial for mycelium development. Moreover, the enrichment of autophagy of mitochondria, regulation of cell morphogenesis, and piecemeal microautophagy of the nuclei at 25 °C (vs. 20 °C) indicated the damage caused by heat stress in mycelia. Notably, a total of four unique module eigengenes (MEs) were identified through a weighted gene coexpression network analysis (WGCNA). Among them, 2293 genes in the turquoise module were significantly positively correlated with temperature (r = 0.946, < 0.001), whereas 739 genes in the blue module were significantly negatively correlated with temperature (r = -0.896, < 0.001), suggesting that the effect of high temperatures on mycelial genes was significantly greater than that of low temperatures. Moreover, the coexpression network indicated that high culture temperatures accelerated the oxidative stress response and energy metabolism in mycelia, while upregulation of purine nucleotide catabolism and ribosomal protein-related genes were improved by low-temperature tolerance. In addition, the upregulated expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and heat shock protein (HSP) genes in mycelia was associated with reactive oxygen species (ROS)-mediated damage at high temperatures. Overall, this study provides an important theoretical basis and application value for optimizing cultivation techniques.
PubMed: 38535187
DOI: 10.3390/jof10030178 -
Cell and Tissue Research Apr 2024Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles....
Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.
Topics: Male; Animals; Mice; Humans; Caenorhabditis elegans; Tissue Distribution; CRISPR-Cas Systems; Membrane Proteins; Mammals; HSP40 Heat-Shock Proteins
PubMed: 38403745
DOI: 10.1007/s00441-024-03875-w -
Developmental Cell Mar 2024Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via...
Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.
Topics: Saccharomyces cerevisiae; Lipid Droplets; Saccharomyces cerevisiae Proteins; Vacuoles; Lipid Metabolism; Autophagy
PubMed: 38354739
DOI: 10.1016/j.devcel.2024.01.014 -
Autophagy May 2024The endoplasmic reticulum (ER) extends to the outer (ONM) and the inner (INM) nuclear membrane forming the nuclear envelope (NE) that delimits the nucleoplasm containing...
The endoplasmic reticulum (ER) extends to the outer (ONM) and the inner (INM) nuclear membrane forming the nuclear envelope (NE) that delimits the nucleoplasm containing the cell genome. Unfolded protein responses (UPRs) and reticulophagy responses increase or reduce ER size and activities, respectively. If dynamic changes of the ER are transmitted to the contiguous NE was not known. In our recent publication, we report on the transmission of stress-induced ER expansion to the NE, which requires disassembly of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes deputed to ensure a physical connection between the cytoplasmic cytoskeleton and the nuclear lamina and to maintain the width between INM and ONM within 50 nm. LINC complexes disassembly relies on reduction of the disulfide bond that covalently links SUN proteins in the INM and KASH proteins (SYNE/NESPRIN proteins in mammals) in the ONM by the ONM-resident reductase TMX4. Upon stress resolution, the physiological shape of the NE is reestablished by SEC62-driven ONM-phagy, where ONM-derived vesicles are directly captured by RAB7- and LAMP1-positive endolysosomes in processes that proceed via asymmetric microautophagy of the NE.
Topics: Nuclear Envelope; Autophagy; Endoplasmic Reticulum Stress; Humans; Animals; Cytoskeleton; Endoplasmic Reticulum
PubMed: 38153175
DOI: 10.1080/15548627.2023.2299123 -
Trends in Cell Biology Dec 2023Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e.,... (Review)
Review
Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e., macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. In macroautophagy, a portion of the cytoplasm is wrapped by the autophagosome, which then fuses with lysosomes and delivers the engulfed cytoplasm for degradation. In CMA, the translocation of cytosolic substrates to the lysosomal lumen is directly across the limiting membrane of lysosomes. In microautophagy, lytic organelles, including endosomes or lysosomes, take up a portion of the cytoplasm directly. Although macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become evident that microautophagy plays a variety of cellular roles from yeast to mammals. Here we review the very recent updates of microautophagy. In particular, we focus on the feature of the degradative substrates and the molecular machinery that mediates microautophagy.
PubMed: 38104013
DOI: 10.1016/j.tcb.2023.11.005 -
Cell Reports Dec 2023Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes,...
Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.
Topics: Chaperone-Mediated Autophagy; Microautophagy; Autophagy; Endosomes; Lysosomes; Molecular Chaperones
PubMed: 38060380
DOI: 10.1016/j.celrep.2023.113529