Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa

Diego L. Delgado-Álvarez, Olga A. Callejas-Negrete, Nicole Gómez, Michael Freitag, Robert Roberson, Laurie G. Smith, Rosa R. Mouriño-Pérez

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Original languageEnglish (US)
Pages (from-to)573-586
Number of pages14
JournalFungal Genetics and Biology
Volume47
Issue number7
DOIs
StatePublished - Jul 2010

Fingerprint

Neurospora crassa
Actins
Hyphae
Actin-Related Protein 2-3 Complex
Microfilament Proteins
Cell Growth Processes
Fungi
Tropomyosin
Cytokinesis
Eukaryota
Confocal Microscopy
Saccharomyces cerevisiae
Cytoplasm

Keywords

  • Actin
  • Arp2/3 complex
  • Fimbrin
  • Lifeact
  • Neurospora crassa
  • Tropomyosin

ASJC Scopus subject areas

  • Genetics
  • Microbiology

Cite this

Delgado-Álvarez, D. L., Callejas-Negrete, O. A., Gómez, N., Freitag, M., Roberson, R., Smith, L. G., & Mouriño-Pérez, R. R. (2010). Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa. Fungal Genetics and Biology, 47(7), 573-586. https://doi.org/10.1016/j.fgb.2010.03.004

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa. / Delgado-Álvarez, Diego L.; Callejas-Negrete, Olga A.; Gómez, Nicole; Freitag, Michael; Roberson, Robert; Smith, Laurie G.; Mouriño-Pérez, Rosa R.

In: Fungal Genetics and Biology, Vol. 47, No. 7, 07.2010, p. 573-586.

Research output: Contribution to journalArticle

Delgado-Álvarez, DL, Callejas-Negrete, OA, Gómez, N, Freitag, M, Roberson, R, Smith, LG & Mouriño-Pérez, RR 2010, 'Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa', Fungal Genetics and Biology, vol. 47, no. 7, pp. 573-586. https://doi.org/10.1016/j.fgb.2010.03.004
Delgado-Álvarez, Diego L. ; Callejas-Negrete, Olga A. ; Gómez, Nicole ; Freitag, Michael ; Roberson, Robert ; Smith, Laurie G. ; Mouriño-Pérez, Rosa R. / Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa. In: Fungal Genetics and Biology. 2010 ; Vol. 47, No. 7. pp. 573-586.
@article{f0d213b1975a47879a9553c8db7d2c49,
title = "Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa",
abstract = "Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenk{\"o}rper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.",
keywords = "Actin, Arp2/3 complex, Fimbrin, Lifeact, Neurospora crassa, Tropomyosin",
author = "Delgado-{\'A}lvarez, {Diego L.} and Callejas-Negrete, {Olga A.} and Nicole G{\'o}mez and Michael Freitag and Robert Roberson and Smith, {Laurie G.} and Mouri{\~n}o-P{\'e}rez, {Rosa R.}",
year = "2010",
month = "7",
doi = "10.1016/j.fgb.2010.03.004",
language = "English (US)",
volume = "47",
pages = "573--586",
journal = "Fungal Genetics and Biology",
issn = "1087-1845",
publisher = "Academic Press Inc.",
number = "7",

}

TY - JOUR

T1 - Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa

AU - Delgado-Álvarez, Diego L.

AU - Callejas-Negrete, Olga A.

AU - Gómez, Nicole

AU - Freitag, Michael

AU - Roberson, Robert

AU - Smith, Laurie G.

AU - Mouriño-Pérez, Rosa R.

PY - 2010/7

Y1 - 2010/7

N2 - Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

AB - Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

KW - Actin

KW - Arp2/3 complex

KW - Fimbrin

KW - Lifeact

KW - Neurospora crassa

KW - Tropomyosin

UR - http://www.scopus.com/inward/record.url?scp=77953534441&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953534441&partnerID=8YFLogxK

U2 - 10.1016/j.fgb.2010.03.004

DO - 10.1016/j.fgb.2010.03.004

M3 - Article

C2 - 20302965

AN - SCOPUS:77953534441

VL - 47

SP - 573

EP - 586

JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

SN - 1087-1845

IS - 7

ER -