The role of GYP-3 in cellular morphogenesis of Neurospora crassa: Analyzing its relationship with the polarisome.

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarity. The polarisome, which in Saccharomyces cerevisiae consists of Spa2, Pea2, Bud6 and Bni1 is described as a small network of functionally related proteins that regulate polarized growth. In yeast Msb3 and Msb4 are considered polarisome components since both proteins interact directly with Spa2 and are involved in Bni1-nucleated actin assembly in vivo. Additionally they regulate exocytosis through their GAP activity towards Sec4 and perhaps other Rab GTPases. In filamentous fungi the role of these proteins has not been investigated, and in the genome of Neurospora crassa only the gene gyp-3 (NCU04514) was found to correlate with MSB3 and MSB4 of S. cerevisiae. Therefore in this work the role of GYP-3 and its relationship with the polarisome in N. crassa was analyzed. The results show that GYP-3 is required for normal colony development and cell morphology since the Δgyp-3 strain displayed a substantial reduction in colony diameter and hyphae showed a distorted morphology expressed as a general pattern of bulging areas in the distal region and hyphae were thinner at the active growing zone. The lack of GYP-3 had no effects on the localization of the polarisome components SPA-2 and BNI-1. Likewise, GYP-3 was not necessary for the normal localization of the F-actin population, however the dynamics of the Spitzenkörper (Spk) and the actin population at the apical region seemed to be destabilized. Additionally, the lack of GYP-3 strongly affects the localization and dynamics of SEC-4; which no longer accumulates at the tip of hyphae. The results presented here strongly suggest that GYP-3 is not part of the polarisome; however it requires the scaffold protein SPA-2 for arriving at the tip of hyphae. Although GYP-3 is not essential for cell survival, it has an important role in maintaining normal cell growth and morphology in N. crassa.