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 interr upted by four introns and encodes a polypeptide of 375 amino acids, wh ich 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% identica l to ARP3s from five other organisms, while actin is 40% to 50% identi cal to these same ARP3s. Transcript levels for actin and ARP3 decrease upon induction of asexual development (i.e. conidiation) and subseque ntly increase slightly when conidia are being formed. A concentration of filamentous actin is typically seen at sites of growth in eukaryoti c organisms and, using indirect immunofluorescence, we showed that fil amentous actin is localized primarily to hyphal tips in N. crassa. To determine if the level of actin increases in response to an increase i n the number of growth sites and in the area of the growing surface, w e used the temperature-sensitive mutants cot-1 and mcb. Growth of the cot-l and mcb mutants at restrictive temperature induces hyphal tip fo rmation and a loss of growth polarity, respectively. Unexpectedly, alm ost no increase in actin levels is observed following a > 20-fold incr ease in the number of hyphal tips or an increase in the area of the gr owing surface resulting from a loss of growth polarity. The results su ggest that the level of actin monomers within N. crassa hyphae is suff icient to accommodate the need for additional actin patches and filame nts 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.