Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, B.C., Mexico.
Fungal Genet Biol. 2010 Jul;47(7):573-86. doi: 10.1016/j.fgb.2010.03.004. Epub 2010 Mar 17.
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.
丝状肌动蛋白（F-actin）在丝状真菌中发挥着至关重要的作用，就像在所有其他真核生物中一样，在细胞生长、细胞内运动和胞质分裂等多种细胞过程中发挥作用。我们通过共聚焦显微镜观察生长中的菌丝，可视化了活 Neurospora crassa 细胞中 F-actin 的组织和动态，这些菌丝表达 GFP 融合蛋白，与肌动蛋白结合蛋白 fimbrin（FIM）和微管蛋白（TPM-1）的同源物、Arp2/3 复合物的一个亚基（ARP-3）以及最近开发的活细胞 F-actin 标记物 Lifeact（酿酒酵母的 ABP140）融合。FIM-GFP、ARP-3-GFP 和 Lifeact-GFP 与质膜下细胞质中的小斑块结合，这些斑块在亚顶环中浓缩，这对于所有三种标记物都是相似的，但在表达 Lifeact-GFP 的菌丝中最宽。这些皮质斑块是短暂存在的，其中一部分在整个菌丝中是运动的，表现出向前和向后的运动。TPM-1-GFP 和 Lifeact-GFP 在菌丝顶端的 Spitzenkörper（Spk）核心内共定位，也在菌丝中的肌动蛋白电缆中观察到。研究的所有 GFP 融合蛋白也在隔膜处短暂定位：Lifeact-GFP 在收缩环形成的早期阶段首先作为一个宽环出现，后来聚集成一个更锐利的环，TPM-1-GFP 在成熟的隔膜中观察到，FIM-GFP/ARP3-GFP 标记的皮质斑块在隔膜两侧形成一个双环。我们的观察表明，分析的每个 N. crassa F-actin 结合蛋白都与不同的 F-actin 结构亚群相关联，这可能反映了它们在 F-actin 组织和动态中的不同作用。此外，Lifeact-GFP 标记了最广泛的 F-actin 结构；它可能是丝状真菌中 F-actin 的通用活细胞标记物。
