Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans

Abstract

The role of actin in apical growth and enzyme secretion in the filamentous fungus Aspery(illuJ nidulans was studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and a t the sites of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence a t the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded apperance. We also studied the effect of CA on the secretion of a-and pgalactosidase. a-Galactosidase is secreted into the culture medium, whereas pgalactosidase remains in the mycelium, with part of its activity bound to the cell wall. When A. nidulans mycelium was incubated in the presence of CA, a reduction in the secretion of a-galactosidase into the culture medium and a decrease in the a-and / % galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion in A. nidulans. Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.

The Tip Growth Apparatus ofAspergillus nidulans

“…At the time of polarity establishment a few patches become associated with the germ tube emergence site ( Figure 1A). In germlings and mature hyphae, they are concentrated near the hyphal tip as was observed previously (Harris et al, 1994;Torralba et al, 1998), but we found that the pattern changes as the germlings grow into mature hyphae. In germlings, the patches are simply concentrated near (but not restricted to) the apex ( Figure 1B).…”

“…In previous studies, these have been difficult to image, but they have been visualized by immunofluorescence (Harris et al, 1994;Araujo-Bazán et al, 2008) and by using a GFP-tropomyosin fusion (Pearson et al, 2004). As reported previously (Harris et al, 1994;Torralba et al, 1998;Cheng et al, 2001), actin was also associated with forming septa (data not shown).…”

“…Cytochalasin A was used at a final concentration of 1.0 g/ml (2.1 M), and latrunculin B was used at a concentration of 40 g/ml (100 M). It is worth noting that we have used a much lower concentration of cytochalasin A than was used by Torralba et al (1998). One of us has found that A. nidulans apparently degrades cytochalasin A such that low concentrations of cytochalasin A inhibit growth, but eventually growth inhibition ends.…”

“…Actin has been visualized in fixed A. nidulans by immunofluorescence microscopy (Harris et al, 1994(Harris et al, , 1997McGoldrick et al, 1995;Torralba et al, 1998;Cheng et al, 2001), but it has not been imaged in living cells. We consequently constructed a strain expressing a fusion of GFP to the N terminus of actin (A. nidulans contains a single actin gene; Fidel et al, 1988).…”

“…Polarized growth in A. nidulans requires actin (Torralba et al, 1998), and its location and dynamics are likely to be a key to tip growth. Actin has been visualized in fixed A. nidulans by immunofluorescence microscopy (Harris et al, 1994(Harris et al, , 1997McGoldrick et al, 1995;Torralba et al, 1998;Cheng et al, 2001), but it has not been imaged in living cells.…”