Calcium as a branching signal in Neurospora crassa

J L Reissig, S G Kinney
1983 Journal of Bacteriology  
The divalent cation ionophore A23187 was found to induce apical branching in Neurospora crassa. Optimal effects were obtained by treatment with 0.1 mM ionophore for 30 min. Branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. Calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. The branching response was
more » ... hing response was markedly reduced in the presence of 10 to 30 mM cyclic AMP or derivatives thereof. In molds, mycelial morphology is determined by the allocation of branching events. Branching can take place either apically, i.e., by splitting of the growing apex into two or more apices, or laterally, i.e., by the emergence of new apices along the lateral hyphal walls. The final configuration of the fungal colony, and hence its ecological strategy, is determined by the distribution of branching events in time and space. As a preliminary to understanding how this process of morphogenesis is programmed, we have investigated the biochemical nature of the signals which trigger branching in Neurospora crassa. The study of profusely branched (i.e., colonial) mutants of Neurospora spp. has provided evidence for the involvement of cyclic AMP in the regulation of branching, the colonial phenotype being dependent on a low intracellular level of cyclic AMP (5, 10, (14) (15) (16) 20) . Because of the known instances of the antagonistic regulatory role of Ca2' and cyclic AMP (1, 13) and as a corollary to the above observations, it occurred to us that Ca2+ might stimulate branching. The methodology to test this hypothesis was facilitated by the availability of A23187, an ionophore capable of raising the cytosolic Ca2+ concentration in a variety of organisms (12). It was found that A23187 (henceforth called the ionophore) induces profuse branching in Neurospora spp. The present paper documents and extends observations reported in a preliminary publication (J. L. Reissig, J. Cell Biol. 75:30a, 1977). It implicates Ca2+ as the ion involved in the branching signal and demonstrates the antagonistic action of cyclic AMP. MATERIALS AND METHODS Two strains of N. crassa were used: St. L. 74A (a wild type, FGSC 262) and the adenylate cyclaset Present address: deficient strain Crisp-1, allele B123 (FGSC 488). The wild type was used for most experiments; its use is implied unless otherwise indicated. We used Vogel N medium (21) plus 2% sucrose, modified by replacement of ammonium nitrate by an equimolar amount of potassium nitrate. This modification was found to reduce hyphal crowding and thus facilitate scoring. The Ca2+-deficient medium had no added CaCl2 and 1/10 the standard MgCl2; all its ingredients (with the exception of MgCl2 and the trace element solution) were passed through a Chelex-100 column to free them from contaminating Ca2+. The solid medium contained 1.5% Bacto-Agar (Difco Laboratories). The experimental cultures were grown first in liquid and then on solid medium. For the first stage, 105 to 106 conidia from a 1-to 2-week-old slant were inoculated into 10 ml of medium in a petri dish and incubated overnight at 25°C. These cultures could be stored for up to 4 days at 5°C before use as inocula for the next stage; however, longer storage led to an amplification of the branching response such that the induced branches became too numerous to count. The solid medium was prepared by pouring 7 ml into a petri dish (8 cm in diameter) and cutting out slabs, which were placed on microscope slides. One of these thin slabs was usually superposed on top of a second slab to prevent drying, but only the top slab was used for photomicroscopy. For the second stage of growth, the slabs were inoculated by fishing out mycelial clusters from the liquid culture (which should not have reached confluence) and streaking them on the slabs so as to cause the hyphae to join into a single elongated bundle. Upon incubation at 25°C in a damp chamber, hyphae grew out of the bundles roughly at right angles, and 2 h after inoculation the cultures were ready for treatment. Although the liquid cultures were kept sterile, no such precautions were needed for the solid cultures. For the standard treatment, a stock solution of ionophore (10 mM in dimethyl sulfoxide), kept frozen, anhydrous, and protected from light, was diluted by mixing 10 RI1 with 1 ml of medium in a test tube while vigorously agitating with the aid of a vibrating mixer. Drops of the resulting mixture (which, being supersat-1397 on May 9, 2020 by guest
doi:10.1128/jb.154.3.1397-1402.1983 fatcat:qbj7mtuqivgtdfvzc6qh4n2usq