Analysis of actin and actin-related protein 3 (ARP3) gene expression following induction of hyphal tip formation and apolar growth in Neurospora.

The genes encoding actin and ARP3 in the filamentous fungus Neurospora crassa were cloned and sequenced. The actin structural gene is interrupted by four introns and encodes a polypeptide of 375 amino acids, which shows very high degree of identity with actin from other sources. N. crassa ARP3 is 439 amino acids in length and is 71% to 80% identical to ARP3s from five other organisms, while actin is 40% to 50% identical to these same ARP3s. Transcript levels for actin and ARP3 decrease upon induction of asexual development (i.e. conidiation) and subsequently increase slightly when conidia are being formed. A concentration of filamentous actin is typically seen at sites of growth in eukaryotic organisms and, using indirect immunofluorescence, we showed that filamentous actin is localized primarily to hyphal tips in N. crassa. To determine if the level of actin increases in response to an increase in the number of growth sites and in the area of the growing surface, we used the temperature-sensitive mutants cot-1 and mcb. Growth of the cot-1 and mcb mutants at restrictive temperature induces hyphal tip formation and a loss of growth polarity, respectively. Unexpectedly, almost no increase in actin levels is observed following a > 20-fold increase in the number of hyphal tips or an increase in the area of the growing surface resulting from a loss of growth polarity. The results suggest that the level of actin monomers within N. crassa hyphae is sufficient to accommodate the need for additional actin patches and filaments that arises when the number of hyphal tips and the area of growing surface per unit length of hypha greatly exceeds that in wild-type.