Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa |
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| Authors: | Diego L. Delgado-Álvarez Olga A. Callejas-Negrete Nicole Gómez Michael Freitag Robert W. Roberson Laurie G. Smith Rosa R. Mouriño-Pérez |
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| Affiliation: | 1. Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, B.C., Mexico;2. Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA;3. Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR, USA;4. School of Life Sciences, Arizona State University, Tempe, AZ, USA;1. Biozentrum, University of Basel, CH-4056 Basel, Switzerland;2. Department of Anatomy and Molecular Embryology, Ruhr-University Bochum, D-44780 Bochum, Germany;3. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland.;1. Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, B.C., Mexico;2. School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO, USA;3. School of Life Sciences, Arizona State University, Tempe, AZ, USA;1. Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 761001, Israel;2. Plant & Microbial Biology, University of California, Berkeley, Berkeley, CA 94720-3102, USA;3. Departamento de Microbiología, CICESE (Centro de Investigación Científica y Educación Superior de Ensenada), Mexico;1. Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, Baja California, Mexico;2. School of Life Sciences, Arizona State University, Tempe, AZ, USA |
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| 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. |
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