A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.
Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated s...
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Public Library of Science (PLoS)
2011-03-01
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| Series: | PLoS Biology |
| Online Access: | https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1000604&type=printable |
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| author | Marcus J Taylor David Perrais Christien J Merrifield |
| author_facet | Marcus J Taylor David Perrais Christien J Merrifield |
| author_sort | Marcus J Taylor |
| collection | DOAJ |
| description | Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes. |
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| id | doaj-art-9037a24fb8a84cca8f99b71ded2c463e |
| institution | Kabale University |
| issn | 1544-9173 1545-7885 |
| language | English |
| publishDate | 2011-03-01 |
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| series | PLoS Biology |
| spelling | doaj-art-9037a24fb8a84cca8f99b71ded2c463e2025-08-20T03:46:42ZengPublic Library of Science (PLoS)PLoS Biology1544-91731545-78852011-03-0193e100060410.1371/journal.pbio.1000604A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.Marcus J TaylorDavid PerraisChristien J MerrifieldDual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1000604&type=printable |
| spellingShingle | Marcus J Taylor David Perrais Christien J Merrifield A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. PLoS Biology |
| title | A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. |
| title_full | A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. |
| title_fullStr | A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. |
| title_full_unstemmed | A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. |
| title_short | A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis. |
| title_sort | high precision survey of the molecular dynamics of mammalian clathrin mediated endocytosis |
| url | https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.1000604&type=printable |
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