Parker, F., Chuntharpursat-Bon, E., Molloy, J.E. orcid.org/0000-0002-8307-2450 et al. (1 more author) (2024) Using FRET to Determine How Myo10 Responds to Force in Filopodia. In: Mechanobiology. International Symposium of Mechanobiology, 06-09 Nov 2022, Sydney, Australia. https://www.springer.com/series/835 . Springer International Publishing , pp. 67-77. ISBN 9783031539046
Abstract
Myosin 10 (Myo10) is an actin-based molecular motor that is essential for filopodia formation and likely senses tension through interactions with integrins in filopodial tips. It possesses a single α-helical (SAH) domain at the end of its canonical lever, which amplifies the movement of the motor. We have shown the SAH domain can contribute to lever function and possesses the properties of a constant force spring. Here we investigate whether the SAH domain plays a role in tension sensing and whether it becomes extended under load at the filopodial tip. Previously, we found that removing the entire SAH domain and short anti-parallel coiled coil (CC) region at the C-terminal end of the SAH does not prevent Myo10 from moving to filopodial tips in cells. Exploiting this, we generated recombinant forms of Myo10, in which a tension-sensing module (TSMod), comprising a FRET-pair YPet and mCherry separated by a linker sequence of amino acids was then inserted between the Myo10 motor and tail domains, so as to replace the SAH domain and CC region. The linker sequence comprised either a portion of the native SAH domain, or control sequences that were either short (x1: stiff) or long (x5: flexible) repeats of “GPGGA”. As additional controls we also placed the TSMod construct at the N-terminus, where it should not experience force. Our FRET measurements indicate that the SAH domain of Myo10 may become extended at when the protein is stalled at the filopodial tips, so the SAH domain may therefore act as a force sensor.
Metadata
Item Type: | Proceedings Paper |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2024. This is an open access conference paper under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Myo10; Myosin; Filopodia; FRET |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cell Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 20 Jun 2024 14:05 |
Last Modified: | 20 Jun 2024 14:05 |
Published Version: | http://dx.doi.org/10.1007/978-3-031-45379-3_4 |
Status: | Published |
Publisher: | Springer International Publishing |
Series Name: | https://www.springer.com/series/835 |
Identification Number: | 10.1007/978-3-031-45379-3_4 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:213670 |