Functional stratification of the Spitzenkörper of Neurospora crassa

Abstract

SummaryGS-1 (ncu04189) is a protein required for the synthesis of b-1,3-glucan in Neurospora crassa. As chitin, b-1,3-glucan is a morphogenetically relevant component of the fungal cell wall. Previously, we showed that chitin synthases are delivered to the growing hyphal tip of N. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the Spitzenkörper (Spk). Tagged with the green fluorescent protein (GFP), GS-1 accumulated in the hyphal apex forming a dynamic and pleomorphic ring-like structure ('Spitzenring') that corresponded to the Spk outer macrovesicular stratum and surrounded the inner core of chitin synthase-containing microvesicles. TIRF microscopy revealed that GS-1-GFP reached the hyphal apex as a population of heterogeneous-size particles that moved along defined paths. On sucrose density gradients, GS-1-associated particles mainly sedimented in a high density range 1.1272-1.2124 g ml -1. Clearly, GS-1 and chitin synthases of N. crassa are contained in two different types of secretory vesicles that accumulate in different strata of the Spk, a differentiation presumably related to the spatial control of cell-wall synthesis.

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

“…Even though PMA-1-GFP was found in cell fractions of a density corresponding to that of ER and macrovesicles (35), it did not accumulate at the Spitzenkörper, where there is the highest concentration of secretory vesicles according to electron micrographs of hyphal tips of N. crassa (36). Instead, PMA-1-GFP localizes in subapical regions and is somehow directly delivered to the PM.…”

“…S4A and B), corresponding to 1.155 and 1.158 g/ml. This indicated that PMA-1 travels in high-density vesicles and is found at the PM (34,35).…”

Neurospora crassa NKIN2, a Kinesin-3 Motor, Transports Early Endosomes and Is Required for Polarized Growth

“…Standard PCR and cloning procedures were used to fuse green fluorescent protein (GFP) and mCherry fluorescent protein (mChFP) N terminally using plasmids pCCG::N-GFP and pJV16-N, respectively, for targeted integration into the his-3 locus of N. crassa strain FGSC 9717 (33,34). The full-length 5.77-kb open reading frame (ORF) of nkin2 (kinesin-3; NCU06733) was amplified by PCR from FGSC 9717 genomic DNA using primers Nkin2_PacI_fwd (5=-CTTAATTAACAT GGCTCCAGGCATGGC-3=) and Nkin2_XbaI_rev (5=-GCTCTAGATCA AAACCTCAACTGACTG-3=), which included PacI and XbaI restriction sites (which are underlined).…”

Fungal Morphogenesis

“…This generally correlates with an increase in the rate of apical extension (Horio and Oakley 2005), as well as the appearance of a Spitzenkörper (apical body). The latter structure is typically located proximal to the hyphal tip (Girbardt 1957;Sudbery 2011a), largely composed of vesicles of different sizes, microfilaments, and ribosomes (Harris et al 2005;Verdin et al 2009). An extensive body of data support the view that Spitzenkörper dynamics determine the shape of the hyphal tip, dictate the orientation of hyphal extension (BartnickiGarcia et al 1995;Riquelme 2013), and help maximize rates of apical extension (Kohli et al 2008).…”