G. Turian

Conidiation in Neurospora

ConclusionsThus, from the gene to the character, and back from the typical character (standard conidium) to the gene, we have traced a few of the biosynthetic pathways which must obligatorily be triggered (switched on or off) to effect the transition from the relatively undifferentiated vegetative hyphae through the fertile, conidiogenous hyphae to the differentiated cells, the conidia. There is good promise that further, deeper studies will finally revealNeurospora to have one of the most highly integrated control systems for eucaryotic sporogenesis when considered as a model of cell differentiation.

Identification of a calcium- and phospholipid-dependent protein kinase (protein kinase C) in Neurospora crassa

Abstract Protein kinase C was partially purified by DEAE-cellulose chromatography from the soluble fraction of mycelium of Neurospora crassa. In the presence of calcium phosphatidylserine was found to be the best activator among the various lipids tested. Diacylglycerol, under our experimental conditions, affected neither the K m of the enzyme for calcium nor the basal or maximum enzymatic activity. Two endogenous substrates were copurified with the enzyme and were better phosphate acceptor than other exogenous basic or acidic proteins tested. The largest phosphorylated band observed on sodium dodecylsulfate-polyacrylamide gel had an apparent molecular weight of 85 000 daltons. The M r of the other was smaller than 14 000.

Conidiation ofNeurospora crassa in submerged culture without mycelial phase

SummaryConidia ofNeurospora crassa shaken in liquid cultures at 46°C for 15 h and then shifted-down to 25°C germinate directly into conidiophores producing new conidia (macroconidia).

Hyphal tip organization inAllomyces arbuscula

SummaryLight and electron microscopic observations on vegetative hyphae ofAllomyces arbuscula revealed the specialized organization of the tip. There were some minor differences related to culture conditions, but the main ultrastructural features common to all hyphal tips disclosed a special type of organization distinct from that of other fungi. A crescent-shaped apical zone consisted of vesicles and membrane cisternae embedded in a granular matrix. Vesicles fused with the apical plasmalemma and presumably contributed to its expansion and to wall growth. The apical zone contained few ribosomes and generally no other organelles. Mitochondria were concentrated in the immediate subapical zone and scattered through the remainder of the hyphae, as were microbodies. Microtubules formed an asterlike structure with its center in the apical zone. Proximally of the apex, microtubules were axially oriented. Nuclei occurred only a certain distance from the tip. The elements of the apex may maintain the polarity of the hyphae via a gradient and hold it in a state of vegetative growth.

Purification and identification of Calmodulin from Neurospora crassa

The calcium-dependent regulatory protein calmodulin first described [ 1,2] as an activator of the CAMP-phosphodiesterase (PDE) in the bovine brain has been found in a variety of eucaryotic organisms. Calmodulin isolated from some fungi [3-61 and plants [7-91 seems to be similar to mammalian calmodulin [lo]. 1 mM 2-mercaptoethanol, pH 7.0) was used at 1 ml/g mycelia wet wt. The homogenate was filtered through a single layer of nylon cloth, the remaining fragments of mycelia were recovered and retreated in half a volume of extraction buffer. This homogenate was pooled with the first filtrate. The pooled material was centrifuged at 48 000 X g for 30 min in the fixedangle rotor SS-34 with a Sorvall RC-SB centrifuge. All steps were carried out at 4°C. Calmodulin has not yet been reported in Ascomycetes and calmodulin-like activity could not be detected in the yeast Saccharomyces cerevisiae [5]. Here, we describe the use of an affinity chromatography column with an immobilized chlorpromazine derivative [ 1 l] which enabled us to isolate Neurospora crassa calmodulin. It was identified on the basis of its electrophoretic properties, its activation of bovine heart PDE and its absorption spectrum. The resulting supernatant was mixed with DEAESephacel and filtered on a sintered glass funnel by gravity. The filtrate was passed again on the resin and recovered by suction. The bulk of loaded proteins was released by subsequent washings with 100 mM NaCl in the extraction buffer. Further washings with 500 mM NaCl in the same buffer released another mixture of proteins (-15% of the loaded proteins) containing calmodulin. CaClz was added to the eluate to a final concentration of 5 mM. The suspension was clarified by centrifugation at 48 000 X g for 30 min. 2. Materials and methods

Calcium, calmodulin-dependent protein phosphorylation in Neurospora crassa

A calcium, calmodulin‐dependent protein kinase activity has been partially purified by calmodulin‐Sepharose affinity chromatography from the soluble fraction of Neurospora crassa. The phosphorylated peptide has an apparent molecular mass on SDS‐polyacrylamide gel of 47 kDa. The apparent half maximal phosphorylation is obtained after 1.5 min at 30° C in the presence of calcium and calmodulin. The apparent half maximal activation of the phosphorylation is obtained at 1 μM calcium, and 0.1 or 0.2 μM calmodulin from bovine brain or Neurospora, respectively. The 32P incorporation is enhanced about 10‐fold by calmodulin.

Microcyclic microconidiation in Neurospora crassa

Microcyclic microconidiation (microconidiation without mycelial phase) could be obtained in Neurospora crassa by treating the microconidia of the fluffy mutant at 46°C for 24–48 h on phosphate buffer and transferring them to 25°C for 96 h. The successive stages of this shortened differentiation are described with both optical and electron microscopy. Microcyclic microconidiation appears to depend upon an arrest of apical growth followed by a lateral differentiation of microconidiogenous phialides. The most striking ultrastructural changes in heat-treated microconidia are their numerous vacuoles with dense inclusions which disappear during germination. This type of microcycle could also be obtained using wild-type microconidia selectively produced by an iodoacetate treatment.

Ultrastructural study of microcyclic macroconidiation in Neurospora crassa.

Heat-shock of macroconidia of Neurospora crassa at 46°C followed by shift-down to 25°C determines premature conidiogenesis.The nuclei and cytoplasm of heat-treated, swollen conidia contain spots of a dense material especially concentrated around the nucleolus in short time treated ones. In the first proconidium apically budding on the enlarged tip of the premature conidiophore, small vesicles are peripherally spread. A few such vesicles are later seen lining the initially simple septum separating the proconidial units into conidia. The doubling of this interconidial septum is surface viewn as a thick annulus. Disarticulation of the conidial units intervenes along a septal furrow of electroluscent material. Interconidial continuity through the septal pores is transiently insured by a connective which is ruptured for final liberation of the conidia.

Ultrastructure ofAspergillus nidulans conidia and conidial lomasomes

SummaryLomasomes in the conidia ofAspergillus nidulans can be divided into at least two distinct structures. The first is a twice double membrane bound core of cytoplasmic origin. The outermost membrane of the lomasome becomes incorporated into the plasmalemma as it migrates to rest next to the cell wall. The second lomasome structure appears to be a triangle shaped series of tubules arranged in a parallel fashion. The wide end next to the cell wall connected to the plasmalemma and the opposite end to an element of the endoplasmic reticulum. The term membranosome has been coined to designate this lomasome structure with its function of plasmalemma extension. Various structures of the conidium such as wall, endoplasmic reticulum and the cytoplasmic matrix undergo changes from the conidial chain stage to the free or resting conidial stage. This suggests that after conidiation and before the resting stage, the conidium continues to mature.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.