Preparation of polarity-marked microtubules using a plus-end capping DARPin

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  • dc.contributor.author Henkin, Gil
  • dc.contributor.author Brito, Cláudia
  • dc.contributor.author Plückthun, Andreas
  • dc.contributor.author Surrey, Thomas
  • dc.date.accessioned 2025-02-06T08:00:57Z
  • dc.date.available 2025-02-06T08:00:57Z
  • dc.date.issued 2024
  • dc.description.abstract The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell. Fluorescence microscopy-based in vitro assays with purified proteins and stabilized microtubules have been used to characterize polarity-dependent and end-specific behaviors. These assays require ways to visualize the polarity of the microtubules, which has previously been achieved either by the addition of fluorescently tagged motor proteins with known directionality or by fluorescently polarity marking the microtubules themselves. However, classical polarity-marking protocols require a particular chemically modified tubulin and generate microtubules with chemically different plus and minus segments. These chemical differences in the segments may affect the behavior of interacting proteins of interest in an undesirable manner. We present here a new protocol that uses a previously characterized, reversibly binding microtubule plus-end capping protein, a designed ankyrin repeat protein (DARPin), to efficiently produce polarity-marked microtubules with different fluorescently labeled, but otherwise biochemically identical, plus- and minus-end segments. Key features • Produces polarity-marked microtubules with biochemically identical segments • Allows analysis of end-specific and polarity-dependent activities of purified microtubule-associated proteins • Requires purified microtubule plus-end capping DARPin (D1)2 • Concentrations optimized for porcine brain tubulin.
  • dc.description.sponsorship We acknowledge support of the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa (CEX2020‐001049‐S, MCIN/AEI/10.13039/501100011033), and the Generalitat de Catalunya through the CERCA programme. We are grateful to the CRG Core Technologies Programme for their support and assistance. T.S. acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 951430) and from the Spanish Ministry of Science and Innovation (grants PID2019-108415GB-I00/AEI/10.13039/501100011033, and PID2022-142927NB-I00/AEI /10.13039/501100011033/FEDER, UE). C.B. was supported by EMBO long-term fellowship ALTF-883-2020 and Marie Curie fellowship TuRCReg.
  • dc.format.mimetype application/pdf
  • dc.identifier.citation Henkin G, Brito C, Plückthun A, Surrey T. Preparation of polarity-marked microtubules using a plus-end capping DARPin. Bio Protoc. 2024 Nov 20;14(22):e5109. DOI: 10.21769/BioProtoc.5109
  • dc.identifier.doi http://dx.doi.org/10.21769/BioProtoc.5109
  • dc.identifier.issn 2331-8325
  • dc.identifier.uri http://hdl.handle.net/10230/69511
  • dc.language.iso eng
  • dc.publisher Bio-protocol
  • dc.relation.ispartof Bio Protoc. 2024 Nov 20;14(22):e5109
  • dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/951430
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/CEX2020‐001049‐S
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PID2019-108415GB-I00
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PE/PID2022-142927NB-I00
  • dc.rights © 2024 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
  • dc.rights.accessRights info:eu-repo/semantics/openAccess
  • dc.rights.uri http://creativecommons.org/licenses/by/4.0/
  • dc.subject.keyword DARPins
  • dc.subject.keyword Fluorescence microscopy
  • dc.subject.keyword In vitro reconstitution
  • dc.subject.keyword Microtubule polarity
  • dc.subject.keyword Microtubule-associated proteins (MAPs)
  • dc.subject.keyword Microtubules
  • dc.subject.keyword Molecular motors
  • dc.title Preparation of polarity-marked microtubules using a plus-end capping DARPin
  • dc.type info:eu-repo/semantics/article
  • dc.type.version info:eu-repo/semantics/publishedVersion

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