In vitro reconstitution of kinesin-based, axonal mRNA transport

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• dc.contributor.author Grawenhoff, Julia
• dc.contributor.author Baumann, Sebastian
• dc.contributor.author Maurer, Sebastian
• dc.date.accessioned 2022-05-30T10:28:37Z
• dc.date.available 2022-05-30T10:28:37Z
• dc.date.issued 2022
• dc.description.abstract Motor protein-driven transport of mRNAs on microtubules and their local translation underlie important neuronal functions such as development, growth cone steering, and synaptic plasticity. While there is abundant data on how membrane-bound cargoes such as vesicles, endosomes, or mitochondria are coupled to motor proteins, surprisingly little is known on the direct interactions of RNA-protein complexes and kinesins or dynein. Provided the potential building blocks are identified, in vitro reconstitutions coupled to Total Internal Reflection Microscopy (TIRF-M) are a powerful and highly sensitive tool to understand how single molecules dynamically interact to assemble into functional complexes. Here we describe how we assemble TIRF-M imaging chambers suitable for the imaging of single protein-RNA complexes. We give advice on optimal sample preparation procedures and explain how a minimal axonal mRNA transport complex can be assembled in vitro. As these assays work at picomolar-range concentrations of proteins and RNAs, they allow the investigation of molecules that cannot be obtained at high concentrations, such as many large or disordered proteins. This now opens the possibility to study how RNA-binding proteins (RBPs), RNAs, and microtubule-associated proteins act together in real-time at single-molecule sensitivity to create cytoplasmic mRNA distributions.
• dc.description.sponsorship This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2017-85361-P], [BFU2014-54278-P], [BFU2015-62550-ERC], by the Juan de la Cierva-Incorporación Program [IJCI-2015-25994], the Human Frontiers in Science Program (HFSP) [RGY0083/2016], the European Research Council (ERC) [H2020-MSCA-IF-2014-659271] and the Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE) [PRE2018-084501]. We further acknowledge support of the Spanish Ministry of Economy and Competitiveness to the EMBL partnership, “Centro de Excelencia Severo Ochoa” [SEV-2012-0208] and [SEV-2015-0533], and the CERCA Programme/Generalitat de Catalunya
• dc.format.mimetype application/pdf
• dc.identifier.citation Grawenhoff J, Baumann S, Maurer SP. In vitro reconstitution of kinesin-based, axonal mRNA transport. Methods Mol Biol. 2022;2431:547-568. DOI:10.1007/978-1-0716-1990-2_29
• dc.identifier.doi http://dx.doi.org/10.1007/978-1-0716-1990-2_29
• dc.identifier.issn 1940-6029
• dc.identifier.uri http://hdl.handle.net/10230/53313
• dc.language.iso eng
• dc.publisher Springer
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/659271
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BFU2014-54278-P
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BFU2015-62550-ERC
• dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/BFU2017-85361-P
• dc.rights © Julia Grawenhoff, Sebastian Baumann, Sebastian P Maurer 2022. This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.other Interaccions RNA-proteïna
• dc.subject.other Cinesiologia
• dc.subject.other Dineïna
• dc.title In vitro reconstitution of kinesin-based, axonal mRNA transport
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion