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mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells
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Hatano, Tomoyuki, Lim, Tzer Chyn, Billault-Chaumartin, Ingrid, Dhar, Anubhav, Gu, Ying, Massam-Wu, Teresa, Scott, William, Adishesha, Sushmitha, Chapa-y-Lazo, Bernardo, Springall, Luke, Sivashanmugam, Lavanya, Mishima, Masanori, Martin, Sophie G., Oliferenko, Snezhana, Palani, Saravanan and Balasubramanian, Mohan K. (2022) mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells. Journal of Cell Science, 135 (18). jcs260288. doi:10.1242/jcs.260288 ISSN 1477-9137.
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Official URL: https://doi.org/10.1242/jcs.260288
Abstract
Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.
Item Type: | Journal Article | ||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School | ||||||
SWORD Depositor: | Library Publications Router | ||||||
Journal or Publication Title: | Journal of Cell Science | ||||||
Publisher: | The Company of Biologists Ltd | ||||||
ISSN: | 1477-9137 | ||||||
Official Date: | 23 September 2022 | ||||||
Dates: |
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Volume: | 135 | ||||||
Number: | 18 | ||||||
Article Number: | jcs260288 | ||||||
DOI: | 10.1242/jcs.260288 | ||||||
Status: | Peer Reviewed | ||||||
Publication Status: | Published | ||||||
Access rights to Published version: | Open Access (Creative Commons) | ||||||
Date of first compliant deposit: | 24 October 2022 | ||||||
Date of first compliant Open Access: | 24 October 2022 | ||||||
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