Pub Date : 2025-01-06Epub Date: 2024-11-01DOI: 10.1083/jcb.202403116
Charles Bond, Siewert Hugelier, Jiazheng Xing, Elena M Sorokina, Melike Lakadamyali
Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins are uniformly present on each LEL, or if there are cell-type-dependent LEL subpopulations with unique protein compositions is unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging to examine the distribution of seven key LEL proteins (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, and LAMTOR4). While LAMP1, LAMP2, and Cathepsin D were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type-specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts.
{"title":"Heterogeneity of late endosome/lysosomes shown by multiplexed DNA-PAINT imaging.","authors":"Charles Bond, Siewert Hugelier, Jiazheng Xing, Elena M Sorokina, Melike Lakadamyali","doi":"10.1083/jcb.202403116","DOIUrl":"10.1083/jcb.202403116","url":null,"abstract":"<p><p>Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins are uniformly present on each LEL, or if there are cell-type-dependent LEL subpopulations with unique protein compositions is unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging to examine the distribution of seven key LEL proteins (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, and LAMTOR4). While LAMP1, LAMP2, and Cathepsin D were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type-specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"224 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11533445/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-06Epub Date: 2024-10-30DOI: 10.1083/jcb.202404052
Victoria E von Saucken, Stefanie E Windner, Giovanna Armetta, Mary K Baylies
The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (size scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show that local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.
{"title":"Postsynaptic BMP signaling regulates myonuclear properties in Drosophila larval muscles.","authors":"Victoria E von Saucken, Stefanie E Windner, Giovanna Armetta, Mary K Baylies","doi":"10.1083/jcb.202404052","DOIUrl":"10.1083/jcb.202404052","url":null,"abstract":"<p><p>The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (size scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show that local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"224 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11530350/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142545720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.
{"title":"Definition of phosphatidylinositol 4,5-bisphosphate distribution by freeze-fracture replica labeling.","authors":"Takuma Tsuji, Junya Hasegawa, Takehiko Sasaki, Toyoshi Fujimoto","doi":"10.1083/jcb.202311067","DOIUrl":"10.1083/jcb.202311067","url":null,"abstract":"<p><p>Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"224 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11535894/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-11-18DOI: 10.1083/jcb.202407193
Ravi Chidambaram, Kamal Kumar, Smriti Parashar, Gowsalya Ramachandran, Shuliang Chen, Susan Ferro-Novick
Here, we report that the RTN3L-SEC24C endoplasmic reticulum autophagy (ER-phagy) receptor complex, the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L, and the FIP200 autophagy initiating protein, target mutant proinsulin (Akita) condensates for lysosomal delivery at ER tubule junctions. When delivery was blocked, Akita condensates accumulated in the ER. In exploring the role of tubulation in these events, we unexpectedly found that loss of the Parkinson's disease protein, PINK1, reduced peripheral tubule junctions and blocked ER-phagy. Overexpression of the PINK1 kinase substrate, DRP1, increased junctions, reduced Akita condensate accumulation, and restored lysosomal delivery in PINK1-depleted cells. DRP1 is a dual-functioning protein that promotes ER tubulation and severs mitochondria at ER-mitochondria contact sites. DRP1-dependent ER tubulating activity was sufficient for suppression. Supporting these findings, we observed PINK1 associating with ER tubules. Our findings show that PINK1 shapes the ER to target misfolded proinsulin for RTN3L-SEC24C-mediated macro-ER-phagy at defined ER sites called peripheral junctions. These observations may have important implications for understanding Parkinson's disease.
在这里,我们报告了RTN3L-SEC24C内质网自噬(ER-phagy)受体复合物、泛素化RTN3L的CUL3KLHL12 E3连接酶以及FIP200自噬启动蛋白在ER小管连接处靶向突变型胰蛋白酶(秋田)凝集物进行溶酶体递送。当输送受阻时,秋田凝集物就会在 ER 中积累。在探索小管在这些事件中的作用时,我们意外地发现,帕金森病蛋白 PINK1 的缺失会减少外周小管连接并阻止 ER 吞噬。在缺失 PINK1 的细胞中,过量表达 PINK1 激酶底物 DRP1 增加了连接,减少了秋田凝集物的积累,并恢复了溶酶体输送。DRP1 是一种具有双重功能的蛋白质,它能促进ER管化,并在ER-线粒体接触点处切断线粒体。依赖 DRP1 的 ER 管化活性足以抑制线粒体。为支持这些发现,我们观察到 PINK1 与 ER 小管有关联。我们的研究结果表明,PINK1能在被称为外周连接点的确定的ER位点上塑造ER,将错误折叠的前胰岛素作为RTN3L-SEC24C介导的大ER吞噬的目标。这些观察结果可能对理解帕金森病具有重要意义。
{"title":"PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.","authors":"Ravi Chidambaram, Kamal Kumar, Smriti Parashar, Gowsalya Ramachandran, Shuliang Chen, Susan Ferro-Novick","doi":"10.1083/jcb.202407193","DOIUrl":"https://doi.org/10.1083/jcb.202407193","url":null,"abstract":"<p><p>Here, we report that the RTN3L-SEC24C endoplasmic reticulum autophagy (ER-phagy) receptor complex, the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L, and the FIP200 autophagy initiating protein, target mutant proinsulin (Akita) condensates for lysosomal delivery at ER tubule junctions. When delivery was blocked, Akita condensates accumulated in the ER. In exploring the role of tubulation in these events, we unexpectedly found that loss of the Parkinson's disease protein, PINK1, reduced peripheral tubule junctions and blocked ER-phagy. Overexpression of the PINK1 kinase substrate, DRP1, increased junctions, reduced Akita condensate accumulation, and restored lysosomal delivery in PINK1-depleted cells. DRP1 is a dual-functioning protein that promotes ER tubulation and severs mitochondria at ER-mitochondria contact sites. DRP1-dependent ER tubulating activity was sufficient for suppression. Supporting these findings, we observed PINK1 associating with ER tubules. Our findings show that PINK1 shapes the ER to target misfolded proinsulin for RTN3L-SEC24C-mediated macro-ER-phagy at defined ER sites called peripheral junctions. These observations may have important implications for understanding Parkinson's disease.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-11-12DOI: 10.1083/jcb.202411005
Roger Pons Lanau, Felix Campelo
VPS13 proteins mediate lipid transfer across membranes. Among them, VPS13B is associated with Golgi membranes, and VPS13B mutations cause Cohen syndrome. In this issue, Ugur et al. (https://doi.org/10.1083/jcb.202311189) and Du et al. (https://doi.org/10.1083/jcb.202402083) reveal new VPS13B interactors and their functions in Golgi organization and trafficking.
{"title":"Stairway to the Golgi: Two paths VPS13B can go by.","authors":"Roger Pons Lanau, Felix Campelo","doi":"10.1083/jcb.202411005","DOIUrl":"10.1083/jcb.202411005","url":null,"abstract":"<p><p>VPS13 proteins mediate lipid transfer across membranes. Among them, VPS13B is associated with Golgi membranes, and VPS13B mutations cause Cohen syndrome. In this issue, Ugur et al. (https://doi.org/10.1083/jcb.202311189) and Du et al. (https://doi.org/10.1083/jcb.202402083) reveal new VPS13B interactors and their functions in Golgi organization and trafficking.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11557495/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-11-18DOI: 10.1083/jcb.202408061
Yongjuan Sang, Boran Li, Tinglin Su, Hanyu Zhan, Yue Xiong, Zhiming Huang, Changjing Wang, Xiaoxia Cong, Mengjie Du, Yang Wu, Hang Yu, Xi Yang, Kezhi Ding, Xuhua Wang, Xiaolong Miao, Weihua Gong, Liang Wang, Jingwei Zhao, Yiting Zhou, Wei Liu, Xinyang Hu, Qiming Sun
ER-phagy is an evolutionarily conserved mechanism crucial for maintaining cellular homeostasis. However, significant gaps persist in our understanding of how ER-phagy and the ER network vary across cell subtypes, tissues, and organs. Furthermore, the pathophysiological relevance of ER-phagy remains poorly elucidated. Addressing these questions requires developing quantifiable methods to visualize ER-phagy and ER architecture in vivo. We generated two transgenic mouse lines expressing an ER lumen-targeting tandem RFP-GFP (ER-TRG) tag, either constitutively or conditionally. This approach enables precise spatiotemporal measurements of ER-phagy and ER structure at single-cell resolution in vivo. Systemic analysis across diverse organs, tissues, and primary cultures derived from these ER-phagy reporter mice unveiled significant variations in basal ER-phagy, both in vivo and ex vivo. Furthermore, our investigation uncovered substantial remodeling of ER-phagy and the ER network in different tissues under stressed conditions such as starvation, oncogenic transformation, and tissue injury. In summary, both reporter models represent valuable resources with broad applications in fundamental research and translational studies.
{"title":"Visualizing ER-phagy and ER architecture in vivo.","authors":"Yongjuan Sang, Boran Li, Tinglin Su, Hanyu Zhan, Yue Xiong, Zhiming Huang, Changjing Wang, Xiaoxia Cong, Mengjie Du, Yang Wu, Hang Yu, Xi Yang, Kezhi Ding, Xuhua Wang, Xiaolong Miao, Weihua Gong, Liang Wang, Jingwei Zhao, Yiting Zhou, Wei Liu, Xinyang Hu, Qiming Sun","doi":"10.1083/jcb.202408061","DOIUrl":"https://doi.org/10.1083/jcb.202408061","url":null,"abstract":"<p><p>ER-phagy is an evolutionarily conserved mechanism crucial for maintaining cellular homeostasis. However, significant gaps persist in our understanding of how ER-phagy and the ER network vary across cell subtypes, tissues, and organs. Furthermore, the pathophysiological relevance of ER-phagy remains poorly elucidated. Addressing these questions requires developing quantifiable methods to visualize ER-phagy and ER architecture in vivo. We generated two transgenic mouse lines expressing an ER lumen-targeting tandem RFP-GFP (ER-TRG) tag, either constitutively or conditionally. This approach enables precise spatiotemporal measurements of ER-phagy and ER structure at single-cell resolution in vivo. Systemic analysis across diverse organs, tissues, and primary cultures derived from these ER-phagy reporter mice unveiled significant variations in basal ER-phagy, both in vivo and ex vivo. Furthermore, our investigation uncovered substantial remodeling of ER-phagy and the ER network in different tissues under stressed conditions such as starvation, oncogenic transformation, and tissue injury. In summary, both reporter models represent valuable resources with broad applications in fundamental research and translational studies.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142648286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-11-13DOI: 10.1083/jcb.202401084
Yipeng Du, Lei Wang, Lizbeth Perez-Castro, Maralice Conacci-Sorrell, Matthew Sieber
Mitochondrial reactive oxygen species (ROS) function intrinsically within cells to induce cell damage, regulate transcription, and cause genome instability. However, we know little about how mitochondrial ROS production non-cell autonomously impacts cell-cell signaling. Here, we show that mitochondrial dysfunction inhibits the plasma membrane localization of cell surface receptors that drive cell-cell communication during oogenesis. Within minutes, we found that mitochondrial ROS impairs exocyst membrane binding and leads to defective endosomal recycling. This endosomal defect impairs the trafficking of receptors, such as the Notch ligand Delta, during oogenesis. Remarkably, we found that overexpressing RAB11 restores ligand trafficking and rescues the developmental defects caused by ROS production. ROS production from adjacent cells acutely initiates a transcriptional response associated with growth and migration by suppressing Notch signaling and inducing extra cellualr matrix (ECM) remodeling. Our work reveals a conserved rapid response to ROS production that links mitochondrial dysfunction to the non-cell autonomous regulation of cell-cell signaling.
{"title":"Non-cell autonomous regulation of cell-cell signaling and differentiation by mitochondrial ROS.","authors":"Yipeng Du, Lei Wang, Lizbeth Perez-Castro, Maralice Conacci-Sorrell, Matthew Sieber","doi":"10.1083/jcb.202401084","DOIUrl":"10.1083/jcb.202401084","url":null,"abstract":"<p><p>Mitochondrial reactive oxygen species (ROS) function intrinsically within cells to induce cell damage, regulate transcription, and cause genome instability. However, we know little about how mitochondrial ROS production non-cell autonomously impacts cell-cell signaling. Here, we show that mitochondrial dysfunction inhibits the plasma membrane localization of cell surface receptors that drive cell-cell communication during oogenesis. Within minutes, we found that mitochondrial ROS impairs exocyst membrane binding and leads to defective endosomal recycling. This endosomal defect impairs the trafficking of receptors, such as the Notch ligand Delta, during oogenesis. Remarkably, we found that overexpressing RAB11 restores ligand trafficking and rescues the developmental defects caused by ROS production. ROS production from adjacent cells acutely initiates a transcriptional response associated with growth and migration by suppressing Notch signaling and inducing extra cellualr matrix (ECM) remodeling. Our work reveals a conserved rapid response to ROS production that links mitochondrial dysfunction to the non-cell autonomous regulation of cell-cell signaling.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11561560/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142621213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-10-01DOI: 10.1083/jcb.202402133
Luca Masin, Steven Bergmans, Annelies Van Dyck, Karl Farrow, Lies De Groef, Lieve Moons
Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration.
{"title":"Local glycolysis supports injury-induced axonal regeneration.","authors":"Luca Masin, Steven Bergmans, Annelies Van Dyck, Karl Farrow, Lies De Groef, Lieve Moons","doi":"10.1083/jcb.202402133","DOIUrl":"10.1083/jcb.202402133","url":null,"abstract":"<p><p>Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11451009/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-10-07DOI: 10.1083/jcb.202406119
Hitoshi Matakatsu, Richard G Fehon
Two protocadherins, Dachsous and Fat, regulate organ growth in Drosophila via the Hippo pathway. Dachsous and Fat bind heterotypically to regulate the abundance and subcellular localization of a "core complex" consisting of Dachs, Dlish, and Approximated. This complex localizes to the junctional cortex where it represses Warts. Dachsous is believed to promote growth by recruiting and stabilizing this complex, while Fat represses growth by promoting its degradation. Here, we examine the functional relationships between the intracellular domains of Dachsous and Fat and the core complex. While Dachsous promotes the accumulation of core complex proteins in puncta, it is not required for their assembly. Indeed, the core complex accumulates maximally in the absence of both Dachsous and Fat. Furthermore, Dachsous represses growth in the absence of Fat by removing the core complex from the junctional cortex. Fat similarly recruits core complex components but promotes their degradation. Our findings reveal that Dachsous and Fat coordinately constrain tissue growth by repressing the core complex.
{"title":"Dachsous and Fat coordinately repress the Dachs-Dlish-Approximated complex to control growth.","authors":"Hitoshi Matakatsu, Richard G Fehon","doi":"10.1083/jcb.202406119","DOIUrl":"10.1083/jcb.202406119","url":null,"abstract":"<p><p>Two protocadherins, Dachsous and Fat, regulate organ growth in Drosophila via the Hippo pathway. Dachsous and Fat bind heterotypically to regulate the abundance and subcellular localization of a \"core complex\" consisting of Dachs, Dlish, and Approximated. This complex localizes to the junctional cortex where it represses Warts. Dachsous is believed to promote growth by recruiting and stabilizing this complex, while Fat represses growth by promoting its degradation. Here, we examine the functional relationships between the intracellular domains of Dachsous and Fat and the core complex. While Dachsous promotes the accumulation of core complex proteins in puncta, it is not required for their assembly. Indeed, the core complex accumulates maximally in the absence of both Dachsous and Fat. Furthermore, Dachsous represses growth in the absence of Fat by removing the core complex from the junctional cortex. Fat similarly recruits core complex components but promotes their degradation. Our findings reveal that Dachsous and Fat coordinately constrain tissue growth by repressing the core complex.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11461286/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-02Epub Date: 2024-11-07DOI: 10.1083/jcb.202404094
Marina Barba-Aliaga, Vanessa Bernal, Cynthia Rong, Madeleine E Volfbeyn, Keguang Zhang, Brian M Zid, Paula Alepuz
Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process.
{"title":"eIF5A controls mitoprotein import by relieving ribosome stalling at TIM50 translocase mRNA.","authors":"Marina Barba-Aliaga, Vanessa Bernal, Cynthia Rong, Madeleine E Volfbeyn, Keguang Zhang, Brian M Zid, Paula Alepuz","doi":"10.1083/jcb.202404094","DOIUrl":"10.1083/jcb.202404094","url":null,"abstract":"<p><p>Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"223 12","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11551009/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142604896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}