RT Journal Article
SR Electronic
T1 mNeonGreen-tagged fusion proteins and nanobodies reveal localization of tropomyosin to patches, cables, and contractile actomyosin rings in live yeast cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.05.19.492673
DO 10.1101/2022.05.19.492673
A1 Tomoyuki Hatano
A1 Tzer Chyn Lim
A1 Ingrid Billault-Chaumartin
A1 Anubhav Dhar
A1 Ying Gu
A1 Teresa Massam-Wu
A1 Sushmitha Adishesha
A1 Luke Springall
A1 Lavanya Sivashanmugam
A1 William Scott
A1 Masanori Mishima
A1 Sophie G Martin
A1 Snezhana Oliferenko
A1 Saravanan Palani
A1 Mohan K. Balasubramanian
YR 2022
UL http://biorxiv.org/content/early/2022/05/20/2022.05.19.492673.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.