Wyatt D Miller,Andrew Manion,Abhinava K Mishra,Connor J Sheedy,Annalise Bond,Brooke M Gardner,Denise J Montell,Meghan A Morrissey
CD47 on viable cells protects against phagocytosis. CD47 is recognized by SIRPα, an inhibitory receptor expressed by macrophages and other myeloid cells. Activated SIRPα recruits SHP-1 and SHP-2 phosphatases, but the inhibitory signaling cascade downstream of these phosphatases is unclear. Here, we used time-lapse imaging to measure how CD47 impacts the kinetics of phagocytosis. Targets with IgG antibodies were primarily phagocytosed through a Rac-based reaching mechanism. Targets also containing CD47 were only phagocytosed through a less frequent Rho-based sinking mechanism. Hyperactivating Rac2 eliminated the suppressive effect of CD47, suggesting that CD47 prevents activation of Rac and reaching phagocytosis. During IgG-mediated phagocytosis, the tyrosine kinase Syk phosphorylates the GEF Vav, which activates Rac to drive F-actin rearrangement and target internalization. CD47 inhibited Vav phosphorylation without impacting Vav recruitment to the phagocytic synapse or Syk phosphorylation. Macrophages expressing a hyperactive Vav were no longer sensitive to CD47. These data suggest that Vav is a key target of the CD47 signaling pathway.
{"title":"CD47 inhibits phagocytosis through Vav dephosphorylation.","authors":"Wyatt D Miller,Andrew Manion,Abhinava K Mishra,Connor J Sheedy,Annalise Bond,Brooke M Gardner,Denise J Montell,Meghan A Morrissey","doi":"10.1083/jcb.202502206","DOIUrl":"https://doi.org/10.1083/jcb.202502206","url":null,"abstract":"CD47 on viable cells protects against phagocytosis. CD47 is recognized by SIRPα, an inhibitory receptor expressed by macrophages and other myeloid cells. Activated SIRPα recruits SHP-1 and SHP-2 phosphatases, but the inhibitory signaling cascade downstream of these phosphatases is unclear. Here, we used time-lapse imaging to measure how CD47 impacts the kinetics of phagocytosis. Targets with IgG antibodies were primarily phagocytosed through a Rac-based reaching mechanism. Targets also containing CD47 were only phagocytosed through a less frequent Rho-based sinking mechanism. Hyperactivating Rac2 eliminated the suppressive effect of CD47, suggesting that CD47 prevents activation of Rac and reaching phagocytosis. During IgG-mediated phagocytosis, the tyrosine kinase Syk phosphorylates the GEF Vav, which activates Rac to drive F-actin rearrangement and target internalization. CD47 inhibited Vav phosphorylation without impacting Vav recruitment to the phagocytic synapse or Syk phosphorylation. Macrophages expressing a hyperactive Vav were no longer sensitive to CD47. These data suggest that Vav is a key target of the CD47 signaling pathway.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"1 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145305757","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}
Aging cells functionally decline and accumulate damage through poorly understood mechanisms. In this issue, Antentor et al. (https://doi.org/10.1083/jcb.202412064) find that increased vacuolar pH in older yeast cells slows clathrin-mediated endocytosis. These findings have broad implications in aging-related plasma membrane protein quality control.
{"title":"Slowing down to take it in: Endocytosis during cellular aging.","authors":"Derek C Prosser","doi":"10.1083/jcb.202510010","DOIUrl":"https://doi.org/10.1083/jcb.202510010","url":null,"abstract":"Aging cells functionally decline and accumulate damage through poorly understood mechanisms. In this issue, Antentor et al. (https://doi.org/10.1083/jcb.202412064) find that increased vacuolar pH in older yeast cells slows clathrin-mediated endocytosis. These findings have broad implications in aging-related plasma membrane protein quality control.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"8 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145288398","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}
This perspective traces the ways in which information obtained with different technologies for sample preparation, imaging, and image analysis has interacted with biochemical information and theories of protein polymerization to form an ever deeper understanding of microtubule dynamics. Essential to this progress has been a more accurate knowledge of microtubule structure, especially at the polymer's tip, where subunits are commonly added and removed. I follow the contributions of light microscopy, i.e., bright-field, polarization, differential interference contrast, and dark-field optics, then fluorescence microscopy with either immunolabeling or fluorescent labeling of tubulin itself. I also trace electron microscopy from its use on thin sections of fixed materials through negative staining of isolated polymers, then on to imaging of fast-frozen samples with cryo-electron microscopy and tomography. The results from these observations are combined with data from biochemistry and x-ray crystallography, image analysis, and atomistic molecular dynamic modeling to build what is likely to be an accurate overview of how microtubules polymerize.
{"title":"Understanding microtubule dynamics: The synergy of technology, theory, and experiment.","authors":"J Richard McIntosh","doi":"10.1083/jcb.202505046","DOIUrl":"https://doi.org/10.1083/jcb.202505046","url":null,"abstract":"This perspective traces the ways in which information obtained with different technologies for sample preparation, imaging, and image analysis has interacted with biochemical information and theories of protein polymerization to form an ever deeper understanding of microtubule dynamics. Essential to this progress has been a more accurate knowledge of microtubule structure, especially at the polymer's tip, where subunits are commonly added and removed. I follow the contributions of light microscopy, i.e., bright-field, polarization, differential interference contrast, and dark-field optics, then fluorescence microscopy with either immunolabeling or fluorescent labeling of tubulin itself. I also trace electron microscopy from its use on thin sections of fixed materials through negative staining of isolated polymers, then on to imaging of fast-frozen samples with cryo-electron microscopy and tomography. The results from these observations are combined with data from biochemistry and x-ray crystallography, image analysis, and atomistic molecular dynamic modeling to build what is likely to be an accurate overview of how microtubules polymerize.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"23 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145288397","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}
José Cubillán-Marín,Ulrike Fröhlke,Gala Ramón-Zamorano,Sheila Mainye,Joëlle Paolo Mesén-Ramírez,Guilherme B Farias,Katharina Höhn,Tim-Wolf Gilberger,Richárd Bártfai,Tobias Spielmann
Vesicle adaptors are critical for transport of proteins to the correct cellular destination. In malaria parasites general and specialized organelles depend on faithful protein transport to mediate host cell invasion and for intracellular survival. However, the role of adaptors in the parasite and the comparability of the sorting machinery with model organisms are unclear. Here, we show that AP-1, AP-3, and AP-4 are all important for parasite survival. AP-1 was needed for intracellular growth, biogenesis of specialized invasion organelles, and formation of invasive progeny, while AP-3 and AP-4 were both required for invasion into host cells. AP-1 acted through the multi-ligand receptor sortilin while AP-4 sorted multi-transmembrane proteins. Proxiomes from live cells revealed remarkable similarities of the configuration of the adaptor sorting machinery between the parasite and evolutionarily distant model organisms, but also unconventional features such as tepsin functioning with AP-1 and clathrin with AP-4. This work reveals unexpected exchangeability of key elements in otherwise surprisingly conserved adaptor sorting pathways.
{"title":"Vesicle adaptors in malaria parasites show conservation and flexibility of protein sorting machinery.","authors":"José Cubillán-Marín,Ulrike Fröhlke,Gala Ramón-Zamorano,Sheila Mainye,Joëlle Paolo Mesén-Ramírez,Guilherme B Farias,Katharina Höhn,Tim-Wolf Gilberger,Richárd Bártfai,Tobias Spielmann","doi":"10.1083/jcb.202504062","DOIUrl":"https://doi.org/10.1083/jcb.202504062","url":null,"abstract":"Vesicle adaptors are critical for transport of proteins to the correct cellular destination. In malaria parasites general and specialized organelles depend on faithful protein transport to mediate host cell invasion and for intracellular survival. However, the role of adaptors in the parasite and the comparability of the sorting machinery with model organisms are unclear. Here, we show that AP-1, AP-3, and AP-4 are all important for parasite survival. AP-1 was needed for intracellular growth, biogenesis of specialized invasion organelles, and formation of invasive progeny, while AP-3 and AP-4 were both required for invasion into host cells. AP-1 acted through the multi-ligand receptor sortilin while AP-4 sorted multi-transmembrane proteins. Proxiomes from live cells revealed remarkable similarities of the configuration of the adaptor sorting machinery between the parasite and evolutionarily distant model organisms, but also unconventional features such as tepsin functioning with AP-1 and clathrin with AP-4. This work reveals unexpected exchangeability of key elements in otherwise surprisingly conserved adaptor sorting pathways.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"18 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145283459","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}
Apicomplexan parasites are obligate intracellular pathogens possessing unique organelles but lacking several components of the membrane trafficking machinery conserved in other eukaryotes. While some of these components have been lost during evolution, others remain undetectable by standard bioinformatics approaches. Using a conditional splitCas9 system in Toxoplasma gondii, we previously identified TGGT1_301410, a hypothetical gene conserved among apicomplexans, as a potential trafficking factor. Here, we show that TGGT1_301410 is a distant ortholog of T. gondii tepsin (TgTEP), localized to the trans-Golgi and functioning as an accessory protein of the adaptor protein complex 4 (AP4). We demonstrate that AP4-TgTEP is essential for the actin-dependent transport of vesicles to the plant-like vacuole (PLVAC) and Golgi organization. Notably, our findings reveal that, unlike in metazoans, the AP4 complex in T. gondii utilizes clathrin as a coat protein, a mechanism more closely aligned with that of plants. These results underscore a conserved yet functionally adapted vesicular transport system in Apicomplexa.
顶复合体寄生虫是专性细胞内病原体，具有独特的细胞器，但缺乏其他真核生物中保守的膜运输机制的几个组成部分。虽然其中一些成分在进化过程中丢失了，但其他成分仍然无法通过标准的生物信息学方法检测到。利用弓形虫的条件分裂cas9系统，我们先前确定了TGGT1_301410，一个在顶复合体中保守的假设基因，作为潜在的运输因子。在这里，我们发现TGGT1_301410是弓形虫缩酶（TgTEP）的远缘同源物，定位于反式高尔基体，并作为adaptor protein complex 4 （AP4）的辅助蛋白。我们证明了AP4-TgTEP对于囊泡到植物样液泡（PLVAC）和高尔基组织的动作依赖转运至关重要。值得注意的是，我们的研究结果表明，与后生动物不同，弓形虫的AP4复合体利用网格蛋白作为外壳蛋白，这一机制与植物的机制更接近。这些结果强调了顶复合体中一个保守但功能适应的囊泡运输系统。
{"title":"Tepsin and AP4 mediate transport from the trans-Golgi to the plant-like vacuole in toxoplasma.","authors":"Janessa Grech,Abhishek Prakash Shinde,Javier Periz,Mirko Singer,Simon Gras,Ignasi Forné,Andreas Klingl,Joel B Dacks,Elena Jiménez-Ruiz,Markus Meissner","doi":"10.1083/jcb.202312109","DOIUrl":"https://doi.org/10.1083/jcb.202312109","url":null,"abstract":"Apicomplexan parasites are obligate intracellular pathogens possessing unique organelles but lacking several components of the membrane trafficking machinery conserved in other eukaryotes. While some of these components have been lost during evolution, others remain undetectable by standard bioinformatics approaches. Using a conditional splitCas9 system in Toxoplasma gondii, we previously identified TGGT1_301410, a hypothetical gene conserved among apicomplexans, as a potential trafficking factor. Here, we show that TGGT1_301410 is a distant ortholog of T. gondii tepsin (TgTEP), localized to the trans-Golgi and functioning as an accessory protein of the adaptor protein complex 4 (AP4). We demonstrate that AP4-TgTEP is essential for the actin-dependent transport of vesicles to the plant-like vacuole (PLVAC) and Golgi organization. Notably, our findings reveal that, unlike in metazoans, the AP4 complex in T. gondii utilizes clathrin as a coat protein, a mechanism more closely aligned with that of plants. These results underscore a conserved yet functionally adapted vesicular transport system in Apicomplexa.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"68 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145283458","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}
The oligosaccharide used for protein N-glycosylation in the ER is built as a glycolipid. A recent study by Li, Suzuki, and colleagues (https://doi.org/10.1083/jcb.202501239) identifies a long-sought enzyme that hydrolyzes this lipid as part of a possible homeostatic/quality control mechanism.
{"title":"A pyrophosphatase that regulates lipid precursors of N-glycosylation.","authors":"Anant K Menon","doi":"10.1083/jcb.202509041","DOIUrl":"https://doi.org/10.1083/jcb.202509041","url":null,"abstract":"The oligosaccharide used for protein N-glycosylation in the ER is built as a glycolipid. A recent study by Li, Suzuki, and colleagues (https://doi.org/10.1083/jcb.202501239) identifies a long-sought enzyme that hydrolyzes this lipid as part of a possible homeostatic/quality control mechanism.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"115 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145261285","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}
Hongli Li,Suyuan Chen,Celien Lismont,Bram Vandewinkel,Mohamed A F Hussein,Cláudio F Costa,Dorien Imberechts,Yiyang Liu,Jorge E Azevedo,Wim Vandenberghe,Steven Verhelst,Hans R Waterham,Pieter Vanden Berghe,Myriam Baes,Marc Fransen
Pexophagy, the selective degradation of peroxisomes, is essential for removing excess or dysfunctional peroxisomes, and its dysregulation has been linked to various diseases. Although optineurin (OPTN), an autophagy receptor involved in mitophagy, aggrephagy, and xenophagy, has also been implicated in pexophagy in HEK-293 cells, the underlying mechanisms remain unclear. Using proximity labeling, we identified PEX14, a peroxisomal membrane protein, as a neighboring partner of OPTN. Microscopy analyses revealed that clustering of peroxisomes with OPTN is a key feature of OPTN-mediated pexophagy. Biochemical studies demonstrated that PEX14 and OPTN interact through their coiled-coil and ubiquitin-binding domains, respectively. Further analyses showed that the C-terminal half of overexpressed OPTN triggers pexophagy, likely by oligomerizing with endogenous OPTN. The colocalization of PEX14-OPTN complexes with LC3, together with the suppression of OPTN-mediated peroxisome degradation by bafilomycin A1, supports a model in which PEX14 acts as a docking site for OPTN on the peroxisomal membrane, enabling the recruitment of the autophagic machinery for OPTN-mediated pexophagy.
{"title":"PEX14 acts as a molecular link between optineurin and the autophagic machinery to induce pexophagy.","authors":"Hongli Li,Suyuan Chen,Celien Lismont,Bram Vandewinkel,Mohamed A F Hussein,Cláudio F Costa,Dorien Imberechts,Yiyang Liu,Jorge E Azevedo,Wim Vandenberghe,Steven Verhelst,Hans R Waterham,Pieter Vanden Berghe,Myriam Baes,Marc Fransen","doi":"10.1083/jcb.202411184","DOIUrl":"https://doi.org/10.1083/jcb.202411184","url":null,"abstract":"Pexophagy, the selective degradation of peroxisomes, is essential for removing excess or dysfunctional peroxisomes, and its dysregulation has been linked to various diseases. Although optineurin (OPTN), an autophagy receptor involved in mitophagy, aggrephagy, and xenophagy, has also been implicated in pexophagy in HEK-293 cells, the underlying mechanisms remain unclear. Using proximity labeling, we identified PEX14, a peroxisomal membrane protein, as a neighboring partner of OPTN. Microscopy analyses revealed that clustering of peroxisomes with OPTN is a key feature of OPTN-mediated pexophagy. Biochemical studies demonstrated that PEX14 and OPTN interact through their coiled-coil and ubiquitin-binding domains, respectively. Further analyses showed that the C-terminal half of overexpressed OPTN triggers pexophagy, likely by oligomerizing with endogenous OPTN. The colocalization of PEX14-OPTN complexes with LC3, together with the suppression of OPTN-mediated peroxisome degradation by bafilomycin A1, supports a model in which PEX14 acts as a docking site for OPTN on the peroxisomal membrane, enabling the recruitment of the autophagic machinery for OPTN-mediated pexophagy.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"8 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145261087","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}
Joseph O Magliozzi,Lucas A Runyan,Adah Welsh,Shae B Padrick,Bruce L Goode
In this study, we show that two fungal proteins, Aip5 (related to vertebrate SH3BGRL) and Bud6, directly interact to form a novel "composite nucleator," in which the pointed end of a nascent actin seed is bound by the thioredoxin-related domain of Aip5. The Aip5-Bud6 complex assembles F-actin seeds with free barbed ends and recruits formins to processively elongate and protect these ends from capping protein. The nucleation activities of Aip5 and Bud6 are critical for maintaining proper thickness of actin cable bundles in vivo, which prevents premature cable detachment from the bud neck and secretory traffic defects, as revealed by live imaging. In vitro single-molecule imaging reveals that after actin nucleation, Aip5 remains associated with a pointed end of the filament, and in vivo Aip5 puncta are observed directionally streaming inward from polarity sites by actin cable retrograde flow. Our findings expand the known diversity of actin nucleation mechanisms and reveal that Aip5 functions as a pointed-end capper in vivo.
{"title":"Aip5 forms a \"composite\" actin nucleator with Bud6 and caps pointed ends of actin filaments.","authors":"Joseph O Magliozzi,Lucas A Runyan,Adah Welsh,Shae B Padrick,Bruce L Goode","doi":"10.1083/jcb.202505039","DOIUrl":"https://doi.org/10.1083/jcb.202505039","url":null,"abstract":"In this study, we show that two fungal proteins, Aip5 (related to vertebrate SH3BGRL) and Bud6, directly interact to form a novel \"composite nucleator,\" in which the pointed end of a nascent actin seed is bound by the thioredoxin-related domain of Aip5. The Aip5-Bud6 complex assembles F-actin seeds with free barbed ends and recruits formins to processively elongate and protect these ends from capping protein. The nucleation activities of Aip5 and Bud6 are critical for maintaining proper thickness of actin cable bundles in vivo, which prevents premature cable detachment from the bud neck and secretory traffic defects, as revealed by live imaging. In vitro single-molecule imaging reveals that after actin nucleation, Aip5 remains associated with a pointed end of the filament, and in vivo Aip5 puncta are observed directionally streaming inward from polarity sites by actin cable retrograde flow. Our findings expand the known diversity of actin nucleation mechanisms and reveal that Aip5 functions as a pointed-end capper in vivo.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"9 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246970","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}
Kevin A Swift,Iryna Pustova,William Kasberg,Jenna Bowman,Krithi Gopinath,Erin Voss,Hayden Nelson,Anjon Audhya
Protein trafficking within the endosomal system involves several distinct membrane remodeling events, including those with opposing orientations that lead to the production of intraluminal vesicles (ILVs) and recycling tubules. Components of the endosomal sorting complex required for transport (ESCRT) machinery have been implicated in both pathways, although few studies have directly examined their native dynamics in mammalian cells. Here, we demonstrate that the endogenous ESCRT-III subunit Ist1 exists in at least two different pools on endosomes. High-speed, live-cell imaging further showed that one pool of Ist1 forms transiently on endosomes, while the other is relatively stable. However, upon growth factor stimulation, the stable pool of Ist1 becomes more mobile, and the transient pool accumulates more rapidly on endosomes. Our data indicate that ESCRT-III dynamics are distinct from that of other ESCRT complexes and additionally suggest an intrinsic amount of time is required for ESCRT-mediated ILV biogenesis, irrespective of environmental conditions.
{"title":"Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes.","authors":"Kevin A Swift,Iryna Pustova,William Kasberg,Jenna Bowman,Krithi Gopinath,Erin Voss,Hayden Nelson,Anjon Audhya","doi":"10.1083/jcb.202407013","DOIUrl":"https://doi.org/10.1083/jcb.202407013","url":null,"abstract":"Protein trafficking within the endosomal system involves several distinct membrane remodeling events, including those with opposing orientations that lead to the production of intraluminal vesicles (ILVs) and recycling tubules. Components of the endosomal sorting complex required for transport (ESCRT) machinery have been implicated in both pathways, although few studies have directly examined their native dynamics in mammalian cells. Here, we demonstrate that the endogenous ESCRT-III subunit Ist1 exists in at least two different pools on endosomes. High-speed, live-cell imaging further showed that one pool of Ist1 forms transiently on endosomes, while the other is relatively stable. However, upon growth factor stimulation, the stable pool of Ist1 becomes more mobile, and the transient pool accumulates more rapidly on endosomes. Our data indicate that ESCRT-III dynamics are distinct from that of other ESCRT complexes and additionally suggest an intrinsic amount of time is required for ESCRT-mediated ILV biogenesis, irrespective of environmental conditions.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"1 1","pages":""},"PeriodicalIF":7.8,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246595","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 : 2025-10-06Epub Date: 2025-08-28DOI: 10.1083/jcb.202410210
Kylie Deng, Kitty Sun, Nicole Hallahan, Wan Jun Gan, Michelle Cielesh, Baharak Mahyad, Melkam A Kebede, Mark Larance, Peter Thorn
Insulin granule fusion in pancreatic β cells localizes to where they contact the ECM of the islet capillaries. The mechanism(s) underpinning localization are unclear. Using glucose or high K+ stimulation or the global uncaging of Ca2+, we show granule fusion consistently focused to the β cell-ECM interface, suggesting a specific localization mechanism. We tested for the involvement of liprin-α1, a scaffold protein enriched at the β cell-ECM interface. Liprin-α1 knockdown did not affect high K+-stimulated insulin secretion but did impair localization of exocytosis. Liprin-α1 knockdown impaired glucose-induced insulin secretion with evidence that the C-terminal of liprin-α1 positions liprin-α1 in clusters at the β cell-ECM interface. Liprin-α1 cluster size and number are regulated by glucose, and exocytosis is spatially coupled with the clusters. Immunoprecipitation and mass spectrometry characterized a liprin-α1 interactome, including β2-syntrophin, an insulin granule-linked protein. We conclude that liprin-α1 is part of a complex that is regulated by glucose and locally targets insulin granules to the β cell-ECM interface.
{"title":"Submembrane liprin-α1 clusters spatially localize insulin granule fusion.","authors":"Kylie Deng, Kitty Sun, Nicole Hallahan, Wan Jun Gan, Michelle Cielesh, Baharak Mahyad, Melkam A Kebede, Mark Larance, Peter Thorn","doi":"10.1083/jcb.202410210","DOIUrl":"https://doi.org/10.1083/jcb.202410210","url":null,"abstract":"<p><p>Insulin granule fusion in pancreatic β cells localizes to where they contact the ECM of the islet capillaries. The mechanism(s) underpinning localization are unclear. Using glucose or high K+ stimulation or the global uncaging of Ca2+, we show granule fusion consistently focused to the β cell-ECM interface, suggesting a specific localization mechanism. We tested for the involvement of liprin-α1, a scaffold protein enriched at the β cell-ECM interface. Liprin-α1 knockdown did not affect high K+-stimulated insulin secretion but did impair localization of exocytosis. Liprin-α1 knockdown impaired glucose-induced insulin secretion with evidence that the C-terminal of liprin-α1 positions liprin-α1 in clusters at the β cell-ECM interface. Liprin-α1 cluster size and number are regulated by glucose, and exocytosis is spatially coupled with the clusters. Immunoprecipitation and mass spectrometry characterized a liprin-α1 interactome, including β2-syntrophin, an insulin granule-linked protein. We conclude that liprin-α1 is part of a complex that is regulated by glucose and locally targets insulin granules to the β cell-ECM interface.</p>","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":"224 10","pages":""},"PeriodicalIF":6.4,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12393827/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955505","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}
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