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Figure 1.7 Arthroconidia. From Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall, London. With permission of Kluwer Academic Publishers.

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Figure 1.7 Arthroconidia. From Ingold and Hudson, The Biology of Fungi (1993), Chapman and Hall, London. With permission of Kluwer Academic Publishers.

1.1.7 Cytoskeleton

Staining of hyphae with anti-tubulin antibody bound to a fluorescent dye, fluorescein isothiocyanate (FITC), showed an extensive array of green-stained, long pipe-like structures called microtubules. These microtubules are composed of the protein tubulin and generally lie parallel to the long axis of hyphae (see Figure 1.8). A cytoskeleton element visualized by staining with rhodamine phalloidin is a microfilament (Figure 1.8) composed of the protein actin (Heath et al., 2000). Cytoskeleton in fungi differs from that of animals in that the fungal cytoskeleton is highly dynamic—assembling and disassembling in response to changing cellular needs. These cytoskeletons have a primary role in the migration and positioning of organelles (Riquelme et al., 1998).

1.1.8 Protein Secretion

The substratum on which the hyphae grow generally contains polymeric compounds such as cellulose, hemicellulose, starch and lignin. The enzymes required for depolymerizing

Figure 1.8 Hypha of Saprolegnia ferax. (A) Differential interference contrast image of tip. (B) Stained with rhodamine phalloidin showing actin concentrated at the tip. (C) and (D) Stained with anti-tubulin to show sub-apical microtubules. Few microtubules extend to the tip. Reprinted from Heath et al. (2000). With permission of Elsevier.

Figure 1.8 Hypha of Saprolegnia ferax. (A) Differential interference contrast image of tip. (B) Stained with rhodamine phalloidin showing actin concentrated at the tip. (C) and (D) Stained with anti-tubulin to show sub-apical microtubules. Few microtubules extend to the tip. Reprinted from Heath et al. (2000). With permission of Elsevier.

these polymers are secreted from the hyphal tips. This was demonstrated by growing a colony of Aspergillus niger sandwiched between two perforated polycarbonate membranes placed on a starch medium (Wösten et al., 1991). The sandwiched fungal colony could be lifted and exposed to labeled compounds, N-acetyl [14C] glucosamine or [35S] sulfate, washed and used for imaging by autoradiography (Figure 1.9) for monitoring, respectively, the site of chitin and new protein synthesis. This technique allowed simultaneous visualization of hyphal growth and the site of secretion of glucoamylase by immunogold labeling as well as monitoring the zone of starch-degrading activity by I2-KI staining. The results showed that cell wall synthesis is limited to the growing edge of hypha whereas protein synthesis occurred throughout the hypha. However, Western blotting showed that secretion of glucoamylase occurred only from the hyphal apices. This suggests that the apical region is porous compared to the rest of the hypha and that breakdown of the polymeric compounds is closely connected with apical growth. The large surface area of hyphae and their active protein secretion are features that are being exploited for production of various

Polycarbonate membrane Agarose layer Sandwiched fungal colony

Agarose layer Polycarbonate membrane Culture medium Petri dish

Polycarbonate membrane Agarose layer Sandwiched fungal colony

Agarose layer Polycarbonate membrane Culture medium Petri dish

Figure 1.9 Protein secretion at growing hyphal apices. Above, diagram of method of culturing Aspergillus niger as sandwiched colony between two perforated polycarbonate membranes placed on starch medium. (A) Diagram of autoradiograph imaging of mycelium for monitoring protein synthesis. (B) Diagram of autoradiograph showing chitin (cell wall) synthesis. (C) Diagram of immunogold labeling showing site of glucoamylase secretion. (D) Diagram of zone of starch degradation by I2-KI staining. Based on Wösten et al. (1991).

Figure 1.9 Protein secretion at growing hyphal apices. Above, diagram of method of culturing Aspergillus niger as sandwiched colony between two perforated polycarbonate membranes placed on starch medium. (A) Diagram of autoradiograph imaging of mycelium for monitoring protein synthesis. (B) Diagram of autoradiograph showing chitin (cell wall) synthesis. (C) Diagram of immunogold labeling showing site of glucoamylase secretion. (D) Diagram of zone of starch degradation by I2-KI staining. Based on Wösten et al. (1991).

enzymes on an industrial scale such as glucoamylase, protease and xylanase for use in the preparation of glucose syrups, the manufacture of cheese and in the paper industries, respectively. It is envisaged that hyphae of Aspergillus sp. will be utilized for production of mammalian antibodies (Ward, 2000).

1.1.9 Nutrient Uptake

In 1974, a miniaturized microelectrode with a very high spatial resolution was used to measure voltage along germinating zygotes of brown algae, growing pollen tubes and root hair cells. It was found that these cells generate a current—a longitudinal pH gradient—in the surrounding medium due to an extracellular flow of a proton (positive charge) that enters the tip and exits from behind (Jaffe and Nuccitelli, 1974). This observation was extended to fungi. Measurement of the pH profile along the hypha of Neurospora showed that the apical zone (200 to 300 ^m) was relatively alkaline and the distal zone relatively acidic (Kropf et al., 1984; Harold, 1999). It was hypothesized that the fungal hypha is electrically polarized. Protons (H+ ions), produced because of oxidative metabolism of glucose, are extruded from the rear region of the hypha, making this region of hypha relatively acidic. The extruded protons re-enter from the apex, making it relatively alkaline. The hypha thus drives a current of protons through itself with an inward flow of protons from the tip and their efflux from the distal region (Figure 1.10).

The hypha secretes a variety of enzymes which breakdown the polymeric constituents of the substratum into simple forms by means of extracellularly secreted enzymes. The entry of protons is coupled to the active co-transport (symport) of ions, sugars and amino acids. The rapid internalization of solubilized nutrients is the basis of the absorptive mode of nutrition of fungi. The spatial separation of H+-pump and nutrient transporters suggest that a hypha is not only cytologically but also physiologically polarized.

Figure 1.10 Diagram of proton pump and symport in hypha. Based on Harold (1999).
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