mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells
| Autor: | Tomoyuki Hatano, Tzer Chyn Lim, Ingrid Billault-Chaumartin, Anubhav Dhar, Ying Gu, Teresa Massam-Wu, William Scott, Sushmitha Adishesha, Bernardo Chapa-y-Lazo, Luke Springall, Lavanya Sivashanmugam, Masanori Mishima, Sophie G. Martin, Snezhana Oliferenko, Saravanan Palani, Mohan K. Balasubramanian |
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| Rok vydání: | 2022 |
| Předmět: |
Mammals
Actin Cytoskeleton/metabolism Actins/metabolism Actomyosin/metabolism Animals Cell Cycle Proteins/metabolism Cytokinesis Fluorescent Dyes/metabolism Mammals/metabolism Protein Isoforms/metabolism Saccharomyces cerevisiae/metabolism Schizosaccharomyces/metabolism Schizosaccharomyces pombe Proteins/metabolism Single-Domain Antibodies/metabolism Tropomyosin/genetics Tropomyosin/metabolism Actin Live imaging Tropomyosin mNeonGreen Nanobody Cytokinesis Cell Cycle Proteins Cell Biology Actomyosin Saccharomyces cerevisiae Single-Domain Antibodies Actins Actin Cytoskeleton Ecology Evolution & Ethology Schizosaccharomyces Cell Cycle & Chromosomes Protein Isoforms Schizosaccharomyces pombe Proteins Fluorescent Dyes |
| Zdroj: | Journal of cell science, vol. 135, no. 18, pp. jcs260288 |
| ISSN: | 1477-9137 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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