untitled

membrane. It then seems to be internalized by an

active process, because it cannot passively cross the inner

leaf of the plasma membrane. Similar hyphae treated

with a metabolic inhibitor (sodium azide) showed no

evidence of dye uptake.

In several instances (Fig. 3.17) a satellite

Spitzenkörper was seen to originate at some distance

behind the hyphal tip and it moved towards the tip,

ultimately fusing with the main Spitzenkörper.

Although none of these lines of evidence provides

definitive proof of the existence of an endosomal

system in filamentous fungi, the evidence is at least

compatible with an endosomal system in which vesicle

trafficking between different organelles provides a

mechanism for recycling of membranes and their

contents between different subcellular compartments.

On this basis, a hypothetical model of the organiza-

tion of a vesicle-trafficking network, including the

possible involvement of endosomes, has been pro-

posed (Fig. 3.18). This is, however, an area of contro-

versy because Torralba & Heath (2002) did not find

evidence of endocytosis in Neurospora crassahyphae.

Studies with mutants may be needed to resolve this

issue. Many proteins associated with endocytosis

in Saccharomyces cerevisiae have homologs in the

Neurosporagenome sequence and could be candidates

for mutational studies to test whether filamentous

fungi have an equivalent endosomal system (Read &

Kalkman 2003).

The cytoskeleton and molecular motors

The cytoskeleton plays a major role in the internal

organization of eukaryotic cells, providing a dynamic

structural framework for transporting organelles, for

cytoplasmic streaming, and for chromosome separation

during cell division. The three main elements of

the cytoskeleton are: (i) microtubules, consisting of

polymers of tubulin proteins, (ii) microfilaments,

consisting of the contractile protein actin, and (iii) inter-

mediate filaments which provide tensile strength.

The distribution of the cytoskeletal components can be

visualized in living hyphae by using fluorescent vital

dyes (Fig. 3.19). These elements are considered to play

a major role in coordinating hyphal tip growth (Heath

1994).

In electron micrographs of hyphae, the micro-

tubules are seen as long, straight tubules, about 25 nm

diameter, occurring either singly or as parallel arrays.

They are seen mainly in the peripheral regions of the

hypha but can also extend up to the membrane at the

extreme tip (Fig. 3.4). Microtubules are also seen to be

closely associated with membrane-bound organelles

(e.g. Fig. 3.20), indicating that they could provide a

tramline system for organellar movement. Consistent

with this, the benzimidazole fungicides that have

been used widely to control plant pathogens (Chapter

17) exert their antifungal action by binding to fungal

microtubules, and this causes hyphae to stop growing.

The benzimidazole compounds similarly block nuclear

division by binding to the spindle microtubules.

Microtubules are dynamic cellular components.

They depolymerize in response to treatments such as

cold shock, and conversely they can be stabilized by

compounds such as taxol, the toxin from yew trees

(which is also toxic to humans). In normally growing

cells the microtubules are thought to be continuously

degraded and reformed. Consistent with this, micro-

tubules can polymerize by self-assembly in cell-free

systems. This is a two-stage process: in the first stage,

a molecule of the protein αα-tubulincombines with a

62 CHAPTER 3

Fig. 3.15(a,b) Hyphal tips of Neurospora crassatreated

with two steryl dyes to detect putative endocytosis. Both

steryl dyes, FM4-64 and FM1-43, became internalized,

and hyphae treated with both dyes continued to grow

during confocal laser imaging, as shown by the images

taken at 30-second intervals. (Courtesy of N.D. Read;

from Fisher-Parton et al. 2000.)

(a)

(b)