Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin

Simple item page
Author(s)Yamashiro, Sawako
Author(s)Shekhar, Shashank
Author(s)Novak, Stefanie M.
Author(s)Biswas, Sudipta
Author(s)Gregorio, Carol C.
Author(s)Fowler, Velia M.
Date Accessioned2025-04-16T19:01:55Z
Date Available2025-04-16T19:01:55Z
Publication Date2025-02-24
DescriptionThis article was originally published in Cytoskeleton. The version of record is available at: https://doi.org/10.1002/cm.22007. © 2025 The Author(s). Cytoskeleton published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
AbstractActin filaments are dynamic polymers whose length depends on regulated monomer association and dissociation at their ends. Actin barbed-end dynamics are relatively better understood, primarily due to the approximately tenfold faster subunit on/off rates at barbed versus pointed ends. We present experimental approaches to selectively assay actin pointed-end regulation using bulk biochemistry, single filament imaging, and live cell microscopy with an emphasis on tropomodulins (Tmods), a conserved family of eukaryotic proteins that specifically cap pointed ends. Average pointed-end assembly/disassembly rates are measured in bulk solution using pyrene-labeled actin and barbed end-capping protein CapZ. Direct rate measurements of individual pointed ends are performed via microfluidic-assisted total internal reflection fluorescence microscopy (mf-TIRF). Actin pointed-end dynamics in living cells are examined in striated muscle cells expressing fluorescent actin, where the regular arrays of 1- to 2-μm-long actin filaments in sarcomeres enable visualization of filament pointed and barbed ends. These assays will also help advance our understanding of other pointed end regulators, including cyclase-associated protein and leiomodins, which have been implicated in filament stabilization, disassembly, and elongation. This work is relevant to the musculoskeletal field, where precise regulation of filament lengths is particularly critical for sarcomere organization and striated muscle contraction.
SponsorThis work was supported by NIH NEI grant R01EY017724 and NHLBI grant R01HL083464 to V.M.F., NIH NHLBI grant R01HL123078 and R01GM120137 to C.G., NIH NIGMS grant R35GM143050 to S.S., and Japan Society for the Promotion of Science KAKENHI Grant Number JP23H04310 and JP24H01944 to S.Y.
CitationYamashiro, S., Shekhar, S., Novak, S.M., Biswas, S., Gregorio, C.C. and Fowler, V.M. (2025), Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin. Cytoskeleton. https://doi.org/10.1002/cm.22007
ISSN1949-3592
URLhttps://udspace.udel.edu/handle/19716/36043
Languageen_US
PublisherCytoskeleton
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
Keywordsactin polymerization
Keywordsleiomodin
Keywordsstriated muscle sarcomeres
Keywordstropomodulin
Keywordstropomyosin
TitleActin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin
TypeArticle
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Actin Filament Pointed Ends Assays for Regulation of Assembly and Disassembly by.pdf
Size:
2.01 MB
Format:
Adobe Portable Document Format
Description:
Main article
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
2.22 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
Open Access Publications - Department of Biological Sciences