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

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

doi:10.1242/jcs.260288

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

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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5 Citations (Scopus)

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.

Original language	English
Article number	jcs260288
Journal	Journal of cell science
Volume	135
Issue number	18
DOIs	https://doi.org/10.1242/jcs.260288
Publication status	Published - 15 Sept 2022

Keywords

Actin
Live imaging
mNeonGreen, Nanobody, Cytokinesis
Tropomyosin

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10.1242/jcs.260288

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Hatano, T., Lim, T. C., Billault-Chaumartin, I., Dhar, A., Gu, Y., Massam-Wu, T., Scott, W., Adishesha, S., Chapa-Y-Lazo, B., Springall, L., Sivashanmugam, L., Mishima, M., Martin, S. G., Oliferenko, S., Palani, S., & Balasubramanian, M. K. (2022). mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells. Journal of cell science, 135(18), Article jcs260288. https://doi.org/10.1242/jcs.260288

@article{ed412609f3d54f919a04cfd38c28b047,

title = "mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells",

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.",

keywords = "Actin, Live imaging, mNeonGreen, Nanobody, Cytokinesis, Tropomyosin",

author = "Tomoyuki Hatano and Lim, {Tzer Chyn} and Ingrid Billault-Chaumartin and Anubhav Dhar and Ying Gu and Teresa Massam-Wu and William Scott and Sushmitha Adishesha and Bernardo Chapa-Y-Lazo and Luke Springall and Lavanya Sivashanmugam and Masanori Mishima and Martin, {Sophie G.} and Snezhana Oliferenko and Saravanan Palani and Balasubramanian, {Mohan K.}",

note = "Funding Information: This work was supported by the Wellcome Trust (grants 203276/Z/16/Z and WT101885MA to M.K.B. and grant 220790/Z/20/Z to S.O.), the Schweizerischer Nationalfonds zur F{\"o}rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; grant 310030B_176396 to S.G.M.), the European Research Council (grant AdG-Actomyosin Ring to M.K.B. and grant CoG Cell Fusion to S.G.M.), the Biotechnology and Biological Sciences Research Council (grant BB/ S003789/1 to M.M. and grant BB/T000481/1 to S.O.), The Wellcome Trust DBT Funding Information: This work was supported by the Wellcome Trust (grants 203276/Z/16/Z and WT101885MA to M.K.B. and grant 220790/Z/20/Z to S.O.), the Schweizerischer Nationalfonds zur F{\"o}rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; grant 310030B_176396 to S.G.M.), the European Research Council (grant AdG-Actomyosin Ring to M.K.B. and grant CoG Cell Fusion to S.G.M.), the Biotechnology and Biological Sciences Research Council (grant BB/ S003789/1 to M.M. and grant BB/T000481/1 to S.O.), The Wellcome Trust DBT India Alliance (grant IA/I/21/1/505633 to S.P.), the DST Science and Engineering Research Board (SERB; grant SRG/2021/001600 to S.P.) and a GATE fellowship (to A.D.). Open Access funding provided by University of Warwick. Deposited in PMC for immediate release. Funding Information: India Alliance (grant IA/I/21/1/505633 to S.P.), the DST Science and Engineering Research Board (SERB; grant SRG/2021/001600 to S.P.) and a GATE fellowship (to A.D.). Open Access funding provided by University of Warwick. Deposited in PMC for immediate release. Publisher Copyright: {\textcopyright} 2022 Company of Biologists Ltd. All rights reserved.",

year = "2022",

month = sep,

day = "15",

doi = "10.1242/jcs.260288",

language = "English",

volume = "135",

journal = "Journal of cell science",

issn = "1477-9137",

publisher = "The Company of Biologists",

number = "18",

}

Hatano, T, Lim, TC, Billault-Chaumartin, I, Dhar, A, Gu, Y, Massam-Wu, T, Scott, W, Adishesha, S, Chapa-Y-Lazo, B, Springall, L, Sivashanmugam, L, Mishima, M, Martin, SG, Oliferenko, S, Palani, S & Balasubramanian, MK 2022, 'mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells', Journal of cell science, vol. 135, no. 18, jcs260288. https://doi.org/10.1242/jcs.260288

TY - JOUR

T1 - mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

AU - Hatano, Tomoyuki

AU - Lim, Tzer Chyn

AU - Billault-Chaumartin, Ingrid

AU - Dhar, Anubhav

AU - Gu, Ying

AU - Massam-Wu, Teresa

AU - Scott, William

AU - Adishesha, Sushmitha

AU - Chapa-Y-Lazo, Bernardo

AU - Springall, Luke

AU - Sivashanmugam, Lavanya

AU - Mishima, Masanori

AU - Martin, Sophie G.

AU - Oliferenko, Snezhana

AU - Palani, Saravanan

AU - Balasubramanian, Mohan K.

N1 - Funding Information: This work was supported by the Wellcome Trust (grants 203276/Z/16/Z and WT101885MA to M.K.B. and grant 220790/Z/20/Z to S.O.), the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; grant 310030B_176396 to S.G.M.), the European Research Council (grant AdG-Actomyosin Ring to M.K.B. and grant CoG Cell Fusion to S.G.M.), the Biotechnology and Biological Sciences Research Council (grant BB/ S003789/1 to M.M. and grant BB/T000481/1 to S.O.), The Wellcome Trust DBT Funding Information: This work was supported by the Wellcome Trust (grants 203276/Z/16/Z and WT101885MA to M.K.B. and grant 220790/Z/20/Z to S.O.), the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; grant 310030B_176396 to S.G.M.), the European Research Council (grant AdG-Actomyosin Ring to M.K.B. and grant CoG Cell Fusion to S.G.M.), the Biotechnology and Biological Sciences Research Council (grant BB/ S003789/1 to M.M. and grant BB/T000481/1 to S.O.), The Wellcome Trust DBT India Alliance (grant IA/I/21/1/505633 to S.P.), the DST Science and Engineering Research Board (SERB; grant SRG/2021/001600 to S.P.) and a GATE fellowship (to A.D.). Open Access funding provided by University of Warwick. Deposited in PMC for immediate release. Funding Information: India Alliance (grant IA/I/21/1/505633 to S.P.), the DST Science and Engineering Research Board (SERB; grant SRG/2021/001600 to S.P.) and a GATE fellowship (to A.D.). Open Access funding provided by University of Warwick. Deposited in PMC for immediate release. Publisher Copyright: © 2022 Company of Biologists Ltd. All rights reserved.

PY - 2022/9/15

Y1 - 2022/9/15

N2 - 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.

AB - 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.

KW - Actin

KW - Live imaging

KW - mNeonGreen, Nanobody, Cytokinesis

KW - Tropomyosin

UR - http://www.scopus.com/inward/record.url?scp=85138399264&partnerID=8YFLogxK

U2 - 10.1242/jcs.260288

DO - 10.1242/jcs.260288

M3 - Article

C2 - 36148799

AN - SCOPUS:85138399264

SN - 1477-9137

VL - 135

JO - Journal of cell science

JF - Journal of cell science

IS - 18

M1 - jcs260288

ER -

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

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