Ras GAP regulation of actin cytoskeleton and hyphal polarity in Aspergillus nidulans

Departamento de Bioquímica. Facultad de Ciencias, Universidad de la República. Iguá 4225 Montevideo CP11400, Uruguay; Institut de Génétique et Microbiologie. Université de Paris-Sud, Centre Universitaire d'Orsay. 91405 Orsay, France; Department of Microbiology, Imperial College London. Flowers Building, Armstrong Road. London SW7 2AZ; Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain

^* To whom correspondence should be addressed. Email: penalva{at}cib.csic.es.

	Abstract

Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenic analyses strongly indicate that GapA regulates one or more ‘true’ Ras. A gapA strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapA strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild-type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germtube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapA disrupts the normal coupling of isotropic growth, polarity establishment and mitosis, resulting in a highly heterogeneous cell population including malformed germlings and a class of giant cells with no germtubes and a multitude of nuclei. Unlike the wild-type, gapA conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been described in strains carrying a rasA dominant-active allele, indicating that GapA downregulates RasA. gapA cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapA cells. As GapA-GFP shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis.