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.
N1 - Funding Information:
This work was supported by Grants from UC-MEXUS/Consejo Nacional de Ciencia y Tecnología to L.G.S and R.R.M.P., the American Cancer Society (RSG-08-030-01-CCG) to M.F., and ASU (LMS9271) to R.W.R. We thank the Fungal Genetics Stock Center for strains. We gratefully acknowledge Carolyn Rasmussen for her advice and for stimulating discussions. We also thank to Douglas Daniels for the training and use of the TIRFM at ASU and we are grateful for the support of Luis Vidali at UM.
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
SN - 1087-1845
VL - 47
SP - 573
EP - 586
JO - Fungal Genetics and Biology
JF - Fungal Genetics and Biology
IS - 7
ER -