{"title":"CD8α-CI-M6PR粒子运动测定法,用于研究培养活细胞中CI-M6PR受体的逆行运动。","authors":"Shalini Rawat, Mahak Sharma","doi":"10.21769/BioProtoc.4979","DOIUrl":null,"url":null,"abstract":"<p><p>The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate. Key features • This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells. • The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells. • Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"14 9","pages":"e4979"},"PeriodicalIF":1.0000,"publicationDate":"2024-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082784/pdf/","citationCount":"0","resultStr":"{\"title\":\"CD8α-CI-M6PR Particle Motility Assay to Study the Retrograde Motion of CI-M6PR Receptors in Cultured Living Cells.\",\"authors\":\"Shalini Rawat, Mahak Sharma\",\"doi\":\"10.21769/BioProtoc.4979\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate. Key features • This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells. • The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells. • Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).</p>\",\"PeriodicalId\":93907,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":\"14 9\",\"pages\":\"e4979\"},\"PeriodicalIF\":1.0000,\"publicationDate\":\"2024-05-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082784/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.4979\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.4979","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
CD8α-CI-M6PR Particle Motility Assay to Study the Retrograde Motion of CI-M6PR Receptors in Cultured Living Cells.
The cation-independent mannose 6-phosphate receptors (CI-M6PR) bind newly synthesized mannose 6-phosphate (Man-6-P)-tagged enzymes in the Golgi and transport them to late endosomes/lysosomes, providing them with degradative functions. Following the cargo delivery, empty receptors are recycled via early/recycling endosomes back to the trans-Golgi network (TGN) retrogradely in a dynein-dependent motion. One of the most widely used methods for studying the retrograde trafficking of CI-M6PR involves employing the CD8α-CI-M6PR chimera. This chimera, comprising a CD8 ectodomain fused with the cytoplasmic tail of the CI-M6PR receptor, allows for labeling at the plasma membrane, followed by trafficking only in a retrograde direction. Previous studies utilizing the CD8α-CI-M6PR chimera have focused mainly on colocalization studies with various endocytic markers under steady-state conditions. This protocol extends the application of the CD8α-CI-M6PR chimera to live cell imaging, followed by a quantitative analysis of its motion towards the Golgi. Additionally, we present an approach to quantify parameters such as speed and track lengths associated with the motility of CD8α-CI-M6PR endosomes using the Fiji plugin TrackMate. Key features • This assay is adapted from the methodology by Prof. Matthew Seaman for studying the retrograde trafficking of CI-M6PR by expressing CD8α-CI-M6PR chimera in HeLa cells. • The experiments include live-cell imaging of surface-labeled CD8α-CI-M6PR molecules, followed by a chase in cells. • Allows the monitoring of real-time motion of CD8α-CI-M6PR endosomes and facilitates calculation of kinetic parameters associated with endosome trajectories, e.g., speed and distance (run lengths).