6.7 Wall synthesis and remodelling
Glucan and chitin cell wall components are synthesised vectorially on the plasma membrane and extruded into the space external to the plasma membrane during their synthesis. Wall glycoproteins progress through the endoplasmic reticulum-Golgi secretory pathways to be secreted into the same wall space where they are integrated into the cell wall structure. The different components of the cell wall being cross linked in situ by cell-wall-associated glycosylhydrolases and glycosyltransferases (Fig. 3).
During active hyphal growth all of this activity is concentrated at the hyphal tip. Autoradiographic studies indicate that all synthesis of chitin and glucans takes place within 10 µm of the apex of the hypha of Neurospora crassa. The tip is highly plastic as the wall is laid down, but as walls mature they become more rigid. The rigidity is provided by cross-linking of polymers, thickening of fibrils and the deposition of materials in the interfibrillar matrix. The process is highly polarised, and reliant on maintenance of a positive turgor pressure within the cytoplasm (Bartnicki-Garcia et al., 2000). Apical growth of the hypha requires long-distance transport between the subapical part and the apex of the tip cell. We have discussed some of the mechanisms that may be involved in Chapter 5 (CLICK HERE to view the page).
It is becoming increasingly evident that microtubule-based motors deliver vesicles containing enzymes and substrates over long distances to the hyphal tip (Bartnicki-Garcia, 2006; Riquelme et al., 2007). In addition, hyphal growth is accompanied by the secretion of exoenzymes that participate in both lysis and synthesis of fungal cell wall components which make the wall in apical regions flexible. Extension of the hypha can then occur as both turgor and cytoskeleton-based cytoplasmic expansion push the cytoplasm against the flexible apical wall. A recent diagrammatic compilation of these events is shown in Fig. 4 (taken from Steinberg, 2007). This, together with the discussion in Chapter 5 (CLICK HERE to view the page) should provide a sufficiently detailed view of current knowledge of fungal extension growth.
|Fig. 4. Cartoon representation of an overall molecular model of hyphal growth. The key feature of hyphal apical growth is rapid movement towards the apex of all the materials needed to create new wall, new membranes and new cytoplasmic components. Most of these materials are exported in vesicles by the endoplasmic reticulum (ER) and Golgi organelles, the vesicles being delivered to the apical vesicle cluster (called the Spitzenkörper; see Section 5.15 and Fig. 5.5) along microtubules powered by motor proteins of the kinesin and dynein families (see Section 5.12 and Fig. 5.8). The Spitzenkörper organises the final distribution of microvesicles along actin microfilaments to the plasma membrane at the extending tip. Vesicle fusion with the membrane is enabled by t-SNARE and v-SNARE proteins. Sterol-rich ‘lipid rafts’ at the hyphal tip could provide domains for apical proteins like signalling and binding complexes and might facilitate endocytosis. Endocytosis at the hyphal tip is dependent upon actin patches where myosin-1 polymerises actin into filaments that take the endocytotic vesicles away from the membrane. The extreme apex of hyphal tips undergoes extensive exocytosis, which is mainly devoted to synthesis of wall polymers outside the membrane and wall construction and maturation (Sections 6.3 and 6.4 and Figs 6.2 and 6.3). Endocytosis features in the flanking regions of the hyphal tip, and this both recycles membrane components (originally delivered as exocytotic vesicles) and imports nutrients; both of which are transported to the endomembrane system for sorting and appropriate use (see Sections 5.10 and 5.12 and Fig. 5.5). This figure also shows that (potentially many) subterminal hyphal cells contribute to the apical migration of resources; (streams of) vesicles, (trains of rapidly moving) vacuoles (Section 5.12) and mitochondria are all transported towards the apex and this transport extends through hyphal septa. Also note that the position of nuclear division spindles is probably specified by interaction between astral microtubules and membrane-bound dynein-dynactin complexes (Figs 5.10 and 5.11), and septal positioning is associated with rings of actin microfilaments (Section 5.17). Remember: this IS a cartoon, no attempt is made to portray relative scale or relative timing (some structures, like division spindles) are more transient than others (like the Spitzenkörper). Also, everything happens, quickly; in the text (Sections 5.12 and 5.15) we show that 38,000 vesicles have to fuse with the apical membrane each minute (that’s over 600 every second) to support extension of each hyphal tip of Neurospora crassa when it is growing at its maximum rate. See text of Chapters 5 and 6 for complete explanation, and refer to Steinberg (2007) and Rittenour, Si & Harris (2009).|
We do not wish to over-emphasise apical wall growth here, because it is not the end of the story for the fungal wall. During the past several years a clear picture has emerged for the cell wall of yeasts and filamentous fungi alike being an extremely dynamic construction with its components maintained in continual balance as wall enzymes repair and remodel the original wall. Cell wall damage in budding yeast, Saccharomyces cerevisiae, triggers a salvage mechanism called the cell-wall-integrity pathway consisting of at least 18 cell-wall-maintenance genes controlled by a single transcription factor and a specific signal transduction pathway. Sequences belonging to this integrity pathway are conserved in several yeasts and filamentous fungi (Smits et al., 1999; de Nobel, van den Ende & Klis, 2000; Bowman & Free, 2006; Klis et al., 2006).
We discuss the dynamic nature of the fungal cell wall elsewhere in this text in discussions of:
- hyphal and spore differentiation (CLICK HERE to view the page in Chapter 9),
- hyphal branching (CLICK HERE to view the page in Chapter 4),
- septation (CLICK HERE to view the page in Chapter 4; and CLICK HERE to view the page in Chapter 5),
- hyphal anastomosis (CLICK HERE to view the page in Chapter 5).
All of these processes require that wall synthesis is restarted within a mature wall at a very closely-controlled place and at a particular time. Another relevant circumstance to be aware of is that two hyphal branches that have to be joined together will synthesise a joint wall, and the resultant join can be stronger than the original hyphal walls (CLICK HERE to view the page in Chapter 12).
All such remodelling depends on the coordinated activity of several glycoproteins already present within the wall structure. The ‘wall-associated enzymes’ involved include chitinases, glucanases and peptidases (Cohen-Kupiec & Chet, 1998; Adams, 2004; Seidl, 2008); enzymes that hydrolyse and breakdown cell wall components, as well as glycosyltransferases which are involved in the synthesis and cross-linking of wall polymers. The enzymatic activities of these proteins must be properly controlled so that the mature cell wall gains enough elasticity to allow the new growth while maintaining sufficient strength to avoid cell lysis. Several wall-associated enzymes have been identified in both S. cerevisiae and A. fumigatus and shown to be involved in cell wall remodelling (Bowman & Free, 2006).
In addition to these instances of remodelling, there are many observations of secondary hyphal walls being synthesised as internal thickenings mostly made up of thick fibrils. These are probably glucans accumulated as intermediate to long-term nutritional reserves. This occurs during the final stages of maturation of the fruit body when its nutritional support is completely endogenous, requiring no external sources of nitrogen or carbon. During fruit body development of Schizophyllum commune, an alkali-insoluble cell wall component, which was called R-glucan, was the main fraction of the wall to be broken down. R-glucan contained both β1,6 and β1,3 linkages, and was distinct from S-glucan which was alkali-soluble and constituted the bulk of the cell wall material left after mobilisation of the R-glucan.
Studies of the mobilisation process indicated that cell wall degradation correlated with cap development and was controlled by changes in the level of a specific R-glucanase enzyme. It seems that while glucose remains available in the medium carbohydrate is temporarily stored in the form of R-glucan in the walls of mycelial and fruit body hyphae. During this phase of net R-glucan synthesis the R-glucanase is repressed by glucose in the medium, but when this is exhausted the repression is lifted, R-glucanase is synthesised and by breaking down the R-glucan it provides substrate(s) specifically required for fruit body development (Bartnicki-Garcia, 1999).
The story is slightly different in Coprinopsis cinerea. In this organism glycogen seems to serve a similar sort of function to the R-glucan of Schizophyllum commune during fruit body development (glycogen is an α1,4 /α1,6 linked glucan (see Breakdown of polysaccharide: starch and glycogen section of Chapter 10, CLICK HERE to view the page); the reason may be that fruit bodies of C. cinerea develop much more rapidly than those of S. commune and the glycogen represents a much more efficient transient reserve that enables large quantities of sugar to be rapidly translocated through the fruit body with no disturbance to solute balance. Glycogen is involved in various aspects of vegetative morphogenesis in C. cinerea (Waters et al., 1975b; Jirjis & Moore, 1976), but so are wall glucans.
Mycelium of C. cinerea forms multicellular sclerotia, about 250 µm diameter, as resistant survival structures which pass through a period of dormancy before utilising their accumulated reserves to ‘germinate’ by producing a fresh mycelium. Glycogen is synthesised and accumulated in young sclerotia, but is not the long term storage product. For long term storage much of the carbohydrate is converted into a form of secondary wall material, probably glucan. Cells in the central bulk of the sclerotium may become extremely thick-walled, the primary walls being thickened on their inner surfaces by loosely-woven and very large fibrils, the development of which coincides with the gradual disappearance of glycogen from the cells (Waters et al., 1972, 1975a & b).
What we mean by ‘thick walled’ is illustrated in the electronmicrographs of Figs 5 to 9. Secondary wall is external to the plasma membrane but internal to primary wall. The illustrations in Figs 7 to 9 make it clear that the secondary walls can come to make up a very considerable proportion of the cell volume. This inevitably constricts the protoplasm to a correspondingly smaller central lumen, but the indications are that these cells remain alive; being effectively dormant until the sclerotium is presented with amenable growth conditions (Erental, Dickman & Yarden, 2008).
|Figs 5 to 9. A selection of transmission electronmicrographs (TEMs) of submerged mycelium and sclerotia of Coprinopsis cinerea showing the two main types of thick walled hyphal cells; rind cells with dense, pigmented secondary walls of heavily-melanised glucan on outer and side walls which make up a plate-like layer of protective rind which resists environmental extremes, and the secondary walls of medullary cells which are uniformly thickened around the cell with large, branched fibres of glucan. This image, Fig. 5, shows a TEM of typical young vegetative hyphae from the submerged mycelium of Coprinopsis cinerea. Hyphae shown in longitudinal (top) and transverse sections. Note the thin (primary) hyphal walls that characterise this undifferentiated tissue. Key: n = nucleus, nc = nucleolus, nm = nuclear membrane, mi = mitochondrion, v = vacuole, er = endoplasmic reticulum, gly = glycogen granules. Electronmicrograph by Henry Waters.|
|Fig. 6. Secondarily-thickened walls of rind cells on the outside of sclerotia of C. cinerea. These secondary walls provide a protective layer and are heavily melanised. Electronmicrograph by Henry Waters.||Fig. 7. Secondarily-thickened walls of the central (medulla) region of a young sclerotium showing a cell with a secondarily-thickened wall in transverse section alongside many normal cells. This secondary wall is not melanised; it is constructed of thick fibres of glucan and is eventually recycled to provide carbohydrate resources when the sclerotium germinates. The granules (labelled gly) are accumulations of glycogen, which forms a short-term carbohydrate resource. Accumulation of glycogen occurs before formation of the glucan fibres and as the fibres are formed the glycogen content declines. Note the dolipore septum (ds) at top left. Electronmicrograph by Henry Waters.|
|Fig. 8. Medullary cells with secondarily-thickened wall shown in longitudinal section, illustrating that the thickening (and consequent constriction of the cell lumen) is fairly uniform over the length of the hypha, and that the thickening can cross and involve the dolipore septa. Electronmicrograph by Henry Waters.|
|Fig. 9. Magnified images of a longitudinal section of a secondarily-thickened wall showing its fibrillar structure. The region of secondarily-thickened (glucan) fibres highlighted in the left hand image is shown at extreme magnification on the right. Electronmicrographs by Henry Waters.|
Dormant sclerotia may survive for several years, being protected by a rind composed of tightly-packed hyphal tips which develop another form of secondary thickening which is a heavily-melanised thickened wall that forms an impervious surface layer (Fig. 6).
Melanin is a dark coloured pigment (almost black at high concentration) which protects the hyphae and spores when it is cross linked into the cell wall structure; so completely interlinked, in fact, that it is possible in the laboratory to digest away all the other wall components to leave ‘melanin ghosts’ of the entire original cell wall (Dadachova et al., 2008). Melanin is extremely resistant to chemical and enzymic attack, and contributes to virulence in many pathogenic fungi (for example Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii) by reducing the susceptibility of melanised fungi to host defences and drugs (Taborda et al., 2008). Fungi with melanised walls are also resistant to electromagnetic and ionising radiations; the pigment seems to provide both physical shielding (from UV light) and quenching of cytotoxic free radicals (caused by ionising radiations).
Other cell wall pigments, the carotenoids (Chapter 10, CLICK HERE to view page), also protect against UV radiation. In general, mutants of the entomopathogenic fungus Metarhizium anisopliae with white conidia are more sensitive to UV radiation than mutants with purple conidia, which were more sensitive than mutants with yellow conidia, which in turn were more sensitive than the green wild strain (Braga et al., 2006). Wall pigments have a function; they’re not just pretty colours.
Updated December 17, 2016