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Eukaryotic Cell doi:10.1128/EC.00088-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Spitzenkörper localization and intracellular traffic of GFP-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa

Department of Microbiology. Center for Scientific Research and Higher Education of Ensenada (CICESE). Km 107 Ctra. Tijuana-Ensenada. 22860 Ensenada, Baja California, México; Center for Genomic Biotechnology. Blvd. del Maestro s/n. 88710 Cd. Reynosa, Tamaulipas, México; Department of Biochemistry and Biophysics ALS 2011. Oregon State University, Corvallis, OR 97331-7305, USA

^* To whom correspondence should be addressed. Email: riquelme{at}cicese.mx.

	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 laser scanning confocal microscopy (LSCM). While we found some differences in the overall distribution and appearance of CHS-3-GFP and CHS-6-GFP, most features were similar and 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 (20-40 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 (5-20 µm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching (FRAP) 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 co-localization of CHS-GFP with ER-Golgi 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 secretory pathway.

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