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Scientific article
Open access
English

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Published inJournal of cell science, vol. 135, no. 18, jcs260288
Publication date2022-09-15
First online date2022-09-23
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.

eng
Keywords
  • Actin
  • Live imaging
  • Tropomyosin
  • MNeonGreen
  • Nanobody
  • Cytokinesis
Affiliation Not a UNIGE publication
Funding
  • UK Research and Innovation - Probing the Role of Actin Arginylation Through Synthetic Biology [BB/S003789/1]
  • UK Research and Innovation - Understanding mechanisms of cellular geometry scaling [BB/T000481/1]
  • European Research Council - [AdG-Actomyosin Ring]
  • Wellcome Trust - [WT101885MA]
  • University of Warwick -
  • Biotechnology and Biological Sciences Research Council - [BB/T000481/1]
  • DBT/Wellcome Trust India Alliance - [IA/I/21/1/505633]
  • Science and Engineering Research Board - [SRG/2021/001600]
  • Swiss National Science Foundation - Mechanisms of cell polarization initiation [176396]
Citation (ISO format)
HATANO, Tomoyuki et al. mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells. In: Journal of cell science, 2022, vol. 135, n° 18, p. jcs260288. doi: 10.1242/jcs.260288
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Article (Published version)
Identifiers
ISSN of the journal0021-9533
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