cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.10732844
title: Streamlined particle quantification (SParQ) plug-in is an automated fluorescent vesicle quantification plug-in for particle quantification in Fiji/ImageJ

description: The endolysosomal system is critical for protein homeostasis in cells. A common way of studying protein transport and degradation (e.g. via autophagy) is by labeling vesicular structures such as endosomes, autophagosomes, lysosomes, or model substrates with fluorescent tags or by fluorescent antibody staining. Detailed analyses require quantification of hundreds of structures under various conditions. Typically, the images are analyzed individually with software such as the widely available Fiji/ImageJ (https://imagej.net/Fiji/Downloads), adjusting and thresholding each image and channel independently, which is a very labor intensive and fastidious task. To streamline the process, we developed a plug-in that, integrated into Fiji, enables the automated quantification of vesicular (i.e. punctate) structures. Importantly, the process still allows the operator to evaluate and have control over all the phases of quantification process. CMA: chaperone-mediated autophagy; CSV: comma separated values; eMI: endosomal microautophagy; Fiji: Fiji is just ImageJ; MA: macroautophagy; SParQ: Streamlined Particle Quantification
DOI: 10.6084/m9.figshare.10732844.v2
title: Streamlined particle quantification (SParQ) plug-in is an automated fluorescent vesicle quantification plug-in for particle quantification in Fiji/ImageJ

description: The endolysosomal system is critical for protein homeostasis in cells. A common way of studying protein transport and degradation (e.g. via autophagy) is by labeling vesicular structures such as endosomes, autophagosomes, lysosomes, or model substrates with fluorescent tags or by fluorescent antibody staining. Detailed analyses require quantification of hundreds of structures under various conditions. Typically, the images are analyzed individually with software such as the widely available Fiji/ImageJ (https://imagej.net/Fiji/Downloads), adjusting and thresholding each image and channel independently, which is a very labor intensive and fastidious task. To streamline the process, we developed a plug-in that, integrated into Fiji, enables the automated quantification of vesicular (i.e. punctate) structures. Importantly, the process still allows the operator to evaluate and have control over all the phases of quantification process. CMA: chaperone-mediated autophagy; CSV: comma separated values; eMI: endosomal microautophagy; Fiji: Fiji is just ImageJ; MA: macroautophagy; SParQ: Streamlined Particle Quantification
DOI: 10.6084/m9.figshare.10732844.v1
title: Streamlined Particle Quantification (SParQ) plug-in is an automated fluorescent vesicle quantification plug-in for particle quantification in Fiji/ImageJ.

description: The endolysosomal system is critical for protein homeostasis in cells. A common way of studying protein transport and degradation (e.g. via autophagy) is by labeling vesicular structures such as endosomes, autophagosomes, lysosomes, or model substrates with fluorescent tags or by fluorescent antibody staining. Detailed analyses require quantification of hundreds of structures under various conditions. Typically, the images are analyzed individually with software such as the widely available Fiji/ImageJ (https://imagej.net/Fiji/Downloads), adjusting and thresholding each image and channel independently, which is a very labor intensive and fastidious task. To streamline the process, we developed a plug-in that, integrated into Fiji, enables the automated quantification of vesicular (i.e. punctate) structures. Importantly, the process still allows the operator to evaluate and have control over all the phases of quantification process.
DOI: 10.6084/m9.figshare.11684685
title: Loss of HSPA9 induces peroxisomal degradation by increasing pexophagy

description: Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes <i>in vitro</i> and <i>in vivo</i>. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in <i>Drosophila</i>, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.
DOI: 10.6084/m9.figshare.11684685.v1
title: Loss of HSPA9 induces peroxisomal degradation by increasing pexophagy

description: Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes <i>in vitro</i> and <i>in vivo</i>. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in <i>Drosophila</i>, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.
DOI: 10.6084/m9.figshare.11567379.v1
title: Age-dependent autophagy induction after injury promotes axon regeneration by limiting NOTCH

description: Macroautophagy/autophagy is essential for maintaining cellular homeostasis through the degradation of organelles and proteins. It also has a prominent role in modulating aging. However, the role of autophagy in the neuronal response to axon injury and axon regeneration, particularly in the context of aging, remains largely unknown. Our candidate genetic screen for axon regeneration regulators has identified genes in the autophagy pathway. Using a reporter that monitors autophagosomes and autolysosomes, we were able to monitor the dynamics of autophagy during axon regeneration. In response to axon injury, there was a significant increase in the number of autophagic vesicles. Injury-triggered autophagy activation and axon regeneration capacity undergo an age-dependent decline, and autophagy-activating agents partially rescued these declines. We found that DLK-1 was both required and sufficient for injury-induced autophagy activation. Autophagic vesicles co-localized with the NOTCH4 ortholog, LIN-12 receptor, a previously identified inhibitor of axon regeneration. Epistasis analyses indicate that LIN-12 might be a target of autophagy in axon regeneration. Together, our data suggest that DLK-mediated injury signaling can activate autophagy, which might limit the level of LIN-12 and NOTCH proteins to promote axon regeneration. Our findings reveal that autophagy activation can promote axon regeneration in neurons that lack maximal regrowth capacity, providing a promising therapeutic strategy for axon injury. <b>Abbreviations</b>: 3-MA: 3-methyladenine; ALs: autolysosomes; APs: autophagosomes; ARF-6: ADP-Ribosylation Factor related 6; ATG-9: AuTophaGy (yeast Atg homolog) 9; ATG9A: autophagy related 9A; BA1: bafilomycin A<sub>1</sub>; BEC-1: BEClin (human autophagy) homolog; BECN1: beclin 1; <i>C. elegans: Caenorhabditis elegans</i>; CEBP-1: C/EBP (CCAAT/enhancer-binding protein) homolog; CNS: central nervous system; DLK-1: Dual-Leucine zipper Kinase; DMSO: dimethyl sulfoxide; DRG: dorsal root ganglion; FOS: Fos proto-oncogene, AP-1 transcription factor subunit; GABA: gamma-aminobutyric acid; GFP: green fluorescent protein; HDA-3: Histone DeAcetylase; IP3: inositol trisphosphate; ITR-1: Inositol Triphosphate Receptor; KLF-2: Kruppel-Like Factor (zinc finger protein) 2; LGG-1: LC3, GABARAP and GATE-16 family; MAK-2: MAP kinase Activated protein Kinase; MAP kinase: mitogen-activated protein kinase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKK-4: mitogen activated protein kinase kinase 4; MTOR: mechanistic target of rapamycin kinase; NGM: nematode growth medium; NICD: Notch intracellular domain; NOTCH: notch receptor; PLM: posterior lateral microtubule; PMK-3: P38 Map kinase family; PNS: peripheral nervous system; SCG10: superior cervical ganglion protein 10; SCI: spinal cord injury; UNC-51: UNCoordinated 51; ULK1: unc-51 like autophagy activating kinase 1; wnd: wallenda
DOI: 10.6084/m9.figshare.11567379
title: Age-dependent autophagy induction after injury promotes axon regeneration by limiting NOTCH

description: Macroautophagy/autophagy is essential for maintaining cellular homeostasis through the degradation of organelles and proteins. It also has a prominent role in modulating aging. However, the role of autophagy in the neuronal response to axon injury and axon regeneration, particularly in the context of aging, remains largely unknown. Our candidate genetic screen for axon regeneration regulators has identified genes in the autophagy pathway. Using a reporter that monitors autophagosomes and autolysosomes, we were able to monitor the dynamics of autophagy during axon regeneration. In response to axon injury, there was a significant increase in the number of autophagic vesicles. Injury-triggered autophagy activation and axon regeneration capacity undergo an age-dependent decline, and autophagy-activating agents partially rescued these declines. We found that DLK-1 was both required and sufficient for injury-induced autophagy activation. Autophagic vesicles co-localized with the NOTCH4 ortholog, LIN-12 receptor, a previously identified inhibitor of axon regeneration. Epistasis analyses indicate that LIN-12 might be a target of autophagy in axon regeneration. Together, our data suggest that DLK-mediated injury signaling can activate autophagy, which might limit the level of LIN-12 and NOTCH proteins to promote axon regeneration. Our findings reveal that autophagy activation can promote axon regeneration in neurons that lack maximal regrowth capacity, providing a promising therapeutic strategy for axon injury. <b>Abbreviations</b>: 3-MA: 3-methyladenine; ALs: autolysosomes; APs: autophagosomes; ARF-6: ADP-Ribosylation Factor related 6; ATG-9: AuTophaGy (yeast Atg homolog) 9; ATG9A: autophagy related 9A; BA1: bafilomycin A<sub>1</sub>; BEC-1: BEClin (human autophagy) homolog; BECN1: beclin 1; <i>C. elegans: Caenorhabditis elegans</i>; CEBP-1: C/EBP (CCAAT/enhancer-binding protein) homolog; CNS: central nervous system; DLK-1: Dual-Leucine zipper Kinase; DMSO: dimethyl sulfoxide; DRG: dorsal root ganglion; FOS: Fos proto-oncogene, AP-1 transcription factor subunit; GABA: gamma-aminobutyric acid; GFP: green fluorescent protein; HDA-3: Histone DeAcetylase; IP3: inositol trisphosphate; ITR-1: Inositol Triphosphate Receptor; KLF-2: Kruppel-Like Factor (zinc finger protein) 2; LGG-1: LC3, GABARAP and GATE-16 family; MAK-2: MAP kinase Activated protein Kinase; MAP kinase: mitogen-activated protein kinase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKK-4: mitogen activated protein kinase kinase 4; MTOR: mechanistic target of rapamycin kinase; NGM: nematode growth medium; NICD: Notch intracellular domain; NOTCH: notch receptor; PLM: posterior lateral microtubule; PMK-3: P38 Map kinase family; PNS: peripheral nervous system; SCG10: superior cervical ganglion protein 10; SCI: spinal cord injury; UNC-51: UNCoordinated 51; ULK1: unc-51 like autophagy activating kinase 1; wnd: wallenda
DOI: 10.6084/m9.figshare.11687058
title: Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas

description: The Pacific oyster, <i>Crassostrea gigas</i>, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in <i>C. gigas</i>, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the <i>C. gigas</i> autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of <i>C. gigas</i>. This study allows the characterization of the complete <i>C. gigas</i> autophagy pathway for the first time. <b>Abbreviations:</b> ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.
DOI: 10.6084/m9.figshare.11687058.v1
title: Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas

description: The Pacific oyster, <i>Crassostrea gigas</i>, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in <i>C. gigas</i>, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the <i>C. gigas</i> autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of <i>C. gigas</i>. This study allows the characterization of the complete <i>C. gigas</i> autophagy pathway for the first time. <b>Abbreviations:</b> ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.
DOI: 10.6084/m9.figshare.11416755
title: Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia

description: The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional <i>atg7</i> knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. <b>Abbreviation:</b><i>atg7</i>: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1
DOI: 10.6084/m9.figshare.11416755.v1
title: Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia

description: The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional <i>atg7</i> knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. <b>Abbreviation:</b><i>atg7</i>: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1
DOI: 10.6084/m9.figshare.11515512
title: Crimean-Congo hemorrhagic fever virus replication imposes hyper-lipidation of MAP1LC3 in epithelial cells

description: Crimean-Congo hemorrhagic fever virus (CCHFV) is a virus that causes severe liver dysfunctions and hemorrhagic fever, with high mortality rate. Here, we show that CCHFV infection caused a massive lipidation of LC3 in hepatocytes. This lipidation was not dependent on <i>ATG5, ATG7</i> or <i>BECN1</i>, and no signs for recruitment of the alternative ATG12–ATG3 pathway for lipidation was found. Both virus replication and protein synthesis were required for the lipidation of LC3. Despite an augmented transcription of <i>SQSTM1</i>, the amount of proteins did not show a massive and sustained increase in infected cells, indicating that degradation of SQSTM1 by macroautophagy/autophagy was still occurring. The genetic alteration of autophagy did not influence the production of CCHFV particles demonstrating that autophagy was not required for CCHFV replication. Thus, the results indicate that CCHFV multiplication imposes an overtly elevated level of LC3 mobilization that involves a possibly novel type of non-canonical lipidation. <b>Abbreviations:</b> BECN1: Beclin 1; CCHF: Crimean-Congo hemorrhagic fever; CCHFV: Crimean-Congo hemorrhagic fever virus; CHX: cycloheximide; ER: endoplasmic reticulum; GFP: green fluorescent protein; GP: glycoproteins; MAP1LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; n.i.: non-infected; NP: nucleoprotein; p.i.: post-infection; SQSTM1: sequestosome 1