A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation
Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-04-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/41768 |
| _version_ | 1811199849394077696 |
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| author | Linton M Traub |
| author_facet | Linton M Traub |
| author_sort | Linton M Traub |
| collection | DOAJ |
| description | Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane. |
| first_indexed | 2024-04-12T01:54:17Z |
| format | Article |
| id | doaj.art-e553550a782e4d4d9477e8d3a35ad0e2 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T01:54:17Z |
| publishDate | 2019-04-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-e553550a782e4d4d9477e8d3a35ad0e22022-12-22T03:52:51ZengeLife Sciences Publications LtdeLife2050-084X2019-04-01810.7554/eLife.41768A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiationLinton M Traub0https://orcid.org/0000-0002-1303-0298Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, United StatesBesides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.https://elifesciences.org/articles/41768clathrinAP-2EPS15VHH intrabodyfunction-blockingFCHO1 |
| spellingShingle | Linton M Traub A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation eLife clathrin AP-2 EPS15 VHH intrabody function-blocking FCHO1 |
| title | A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation |
| title_full | A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation |
| title_fullStr | A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation |
| title_full_unstemmed | A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation |
| title_short | A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation |
| title_sort | nanobody based molecular toolkit provides new mechanistic insight into clathrin coat initiation |
| topic | clathrin AP-2 EPS15 VHH intrabody function-blocking FCHO1 |
| url | https://elifesciences.org/articles/41768 |
| work_keys_str_mv | AT lintonmtraub ananobodybasedmoleculartoolkitprovidesnewmechanisticinsightintoclathrincoatinitiation AT lintonmtraub nanobodybasedmoleculartoolkitprovidesnewmechanisticinsightintoclathrincoatinitiation |