Key result
mNeonGreen-tagged tropomyosin and camelid-nanobodies reveal tropomyosin localization to contractile actomyosin rings in live cells.
Why the study?
In vivo investigations of tropomyosin have been limited, largely because suboptimal live cell imaging tools are currently available in many organisms.
The development of mNG-tagged tropomyosin and specific nanobodies provides powerful tools for visualizing actin cytoskeletal dynamics in live cells.
Limited in vivo tropomyosin data due to imaging constraints; leaves open need for advanced live-cell tools in cytoskeletal research.
Tropomyosins are structurally conserved α-helical coiled-coil dimeric 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 in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. 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 in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. 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 actin cables in vivo .
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Hatano et al. (2022) studied this question. mNeonGreen-tagged tropomyosin and camelid-nanobodies was evaluated. mNeonGreen-tagged tropomyosin and camelid-nanobodies revealed the localization of tropomyosin to patch-like structures, cables, and contractile actomyosin rings in live yeast and mammalian cells.
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