Spitzenkorper localization and intracellular traffic of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa |
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| Authors: | Riquelme Meritxell Bartnicki-García Salomon González-Prieto Juan Manuel Sánchez-León Eddy Verdín-Ramos Jorge A Beltrán-Aguilar Alejandro Freitag Michael |
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| Affiliation: | Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), San Ysidro, CA 92143-0222, USA. riquelme@cicese.mx |
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| Abstract: | The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.Fungal hyphae elongate and branch by a complex process based on polarized secretion. Many studies have investigated the cellular and molecular components involved in shaping fungal cells, but no detailed understanding of the mechanisms that govern and regulate polarized fungal growth has been achieved (4, 25). In the yeast Saccharomyces cerevisiae, many of the main components of the secretory pathway, including some of the enzymes involved in cell wall formation, have been extensively characterized (32). Filamentous fungi encode homologues of some key components known from the yeast secretory pathway, but despite their apparent orthology, relatively little is known about how this pathway is organized to accomplish the highly polarized growth typical of hyphae. There are some differences in cell wall synthesis between filamentous fungi and S. cerevisiae. In hyphae of septate fungi, vesicles and other components accumulate at the apex, as part of the Spitzenkörper (Spk) (14, 22-24, 28). The composition and mode of action of this pleomorphic and dynamic structure have intrigued fungal biologists for many decades.Fungal cells have at least two types of well-defined secretory vesicles (5). It has been suggested that macrovesicles, or conventional secretory vesicles, carry the components of the amorphous phase of the cell wall, in addition to the load of extracellular enzymes (5, 27). There is a large body of evidence characterizing the chitin synthase (CHS)-carrying microvesicles as chitosomes (3, 8, 13, 30). CHS are β-glycosyltransferases that catalyze the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin (47), a major structural polymer of the fungal cell wall (2). Chitin synthesis occurs in highly localized fashion both at the hyphal apices (7) and at nascent septa (29). Chitosomes are the smallest vesicles with the ability to form chitin microfibrils in vitro and have been suggested to carry and transport CHS to the cell surface at the apex of hyphae for cell wall synthesis (13, 37, 48, 55, 56). In recent years, studies on fungal CHS have concentrated mainly on gene identification. Given this wealth of information, we chose CHS as candidate markers to investigate vesicle traffic in fungal hyphae.Fungi have multiple chs genes grouped into two divisions, with seven classes, primarily on the basis of similarities in the primary sequence of the predicted proteins (12, 16, 37, 50). Division I includes classes I, II, and III, which share a catalytic domain surrounded by a hydrophilic N-terminal region and a hydrophobic C-terminal region (12). Division II includes classes IV, V, and VII, all with a catalytic domain preceded by a cytochrome b5-like domain. In addition, classes V and VII contain an N-terminal myosin motor-like domain, suggesting a direct interaction with the actin cytoskeleton (15, 20, 58). Class VI has not been assigned to either division and includes recently identified CHS of unknown function (16). Earlier studies suggest that the various CHS have specific roles in chitin cell wall synthesis that are time or space dependent (60). In contrast to most filamentous fungi, S. cerevisiae (46) and Candida albicans (40) have only three or four CHS isozymes, respectively. S. cerevisiae Chs1p, C. albicans Chs2p, and C. albicans Chs8p belong to class I; S. cerevisiae Chs2p and C. albicans Chs1p belong to class II; and S. cerevisiae Chs3p and C. albicans Chs3p belong to class IV (46). While potential roles in hyphal growth have been suggested for some of the seven CHS classes described in filamentous fungi (9, 64, 65), we lack specific information on the cellular localization and trafficking to their sites of action in regions of active cell wall growth for most of these proteins.The goal of this study was to elucidate the traffic of CHS-containing vesicles en route from their site of genesis to their site of exocytosis in living hyphae of Neurospora crassa. The availability of an almost-complete genome sequence for this fungus allowed the identification of seven open reading frames with high homology to previously described chs genes (10). We chose to trace the intracellular location and secretory paths of CHS-3 and CHS-6. Neurospora CHS-3 belongs to the previously reported class I CHS with known homologues in all fungi tested, including S. cerevisiae Chs1p. In contrast, CHS-6 is a newly identified CHS assigned to class VI, homologous to Aspergillus fumigatus ChsD (39) and Coccidioides posadasii CHS-6 (34) but with no apparent homologues in S. cerevisiae or C. albicans. To trace both proteins, we fused green fluorescent protein (GFP) to the carboxyl terminus of the CHS coding regions and analyzed the fate of the resulting CHS-3-GFP and CHS-6-GFP fusion proteins by high-resolution confocal laser scanning microscopy (CLSM) in living hyphae of N. crassa. |
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