5.16 Cell biology of the hyphal apex
Extension of the cell wall at the hyphal apex is the most striking characteristic of the fungi but the problem is to understand how wall extension can be achieved without jeopardising the integrity of the existing hypha. It is quite clear that new wall material must be delivered to the apex and inserted into pre-existing wall (note that the phrase ‘new wall material’ should be interpreted to include everything that is necessary to extend the tip: membrane, periplasmic materials and all wall layers). The models which have been suggested over the years to account for this process differ in how they account for this being achieved, but underlying them all is the recognition that the act of inserting new wall material could itself weaken the wall. The potential validity of the models must therefore be judged not only on how they provide for wall synthesis, but on how they safeguard the integrity of the hypha whilst wall synthesis is in progress.
The problem is that a fungal hypha is part of a closed hydraulic system which is under pressure. The osmotic influx of water, due to the difference in water activity between the inside and the outside of the semipermeable plasma membrane, attempts to increase the cytoplasmic volume but is counteracted by the wall pressure due to the mechanical strength of the wall outside the plasma membrane. The difference between these two forces is the turgor pressure which is the resultant ‘inflation pressure’ which keeps the hypha inflated. An interesting thing about pressure is that in a closed vessel the pressure is the same over the whole of the inside wall surface. This applies whatever the shape of the vessel; irrespective of shape, the wall pressure is uniform.
In the context of the hypha, IF the mechanical force which the wall can exert is equal to or greater than the force exerted by turgor, then the wall will remain intact. However, if turgor exceeds the breaking strain of the wall at any point then the wall will rupture (possibly explosively), and the cell will die. The problem we face in understanding apical hyphal extension is that the structure of the wall needs to be weakened to allow insertion of new wall material to the continuously elongating tip. How can that be achieved without exploding the tip?
In the true fungi, turgor is regulated to contribute to tip extension by driving the tip forward and shaping it by plastic deformation of the newly-synthesised wall. We emphasise ‘in the true fungi’ here because members of the Oomycota (specifically Achlya bisexualis and Saprolegnia ferax) can grow in the absence of turgor, so their hyphal systems are obviously very different from those of true fungi. A. bisexualis and S. ferax do not regulate turgor, their response to the addition of nutrients which raise the osmotic pressure of the medium is to produce a more plastic wall and continue to grow, and near-normal hyphae are formed by S. ferax in the absence of a measurable turgor pressure. In many respects, hyphal extension in Oomycota is similar to pseudopod extension in animal cells, in that polymerisation of the actin cytoskeletal network at the apex of the hypha plays an indispensable role in the absence of turgor, actin polymerisation becoming the main driving force for extension. The most effective model for these ‘almost-fungi’ envisages that hyphal tip expansion in Oomycota is regulated (restrained under normal turgor pressure and protruded under low turgor) by a peripheral network of F-actin-rich components of the cytoskeleton that is attached to the plasmalemma and the cell wall by integrin-containing linkages (an integrin is an ‘integral membrane protein’ that is permanently attached to the plasma membrane).
This model is not obviously applicable to the true fungi if it is interpreted as postulating involvement of the actin cytoskeleton in order to drive tip extrusion. This is unnecessary because the true fungi can use the hydrostatic pressure of their regulated turgor to ‘inflate’ a plastic hyphal tip and drive tip extrusion that way. However, a model involving the actin cytoskeleton could solve the potential problem that the extending hyphal tip might not be strong enough to resist turgor during the process of wall synthesis.
The cytoskeleton, both microfilaments and microtubules, is involved in the control of polarity in hyphal extension of true fungi (Sudbery, 2008; Rittenour, Si & Harris, 2009), so an actin cytoskeleton with firm attachments through the membrane to the growing wall does appear to be involved at both the tip and the septum regions of wall synthesis. In the case of septum formation, mechanical resistance to turgor does not arise, so an obvious interpretation of the actin network is that it functions in transport, directing wall components to the growing sites for terminal exocytosis.
Actin microfilaments are tension elements and actin filaments anchored to the wall by integrin-like molecules through the membrane close to extension sites could certainly serve to direct microvesicles to their target AND provide tension anchors to supplement the mechanical strength of the synthesis-weakened, plastic cell wall. By being involved in both the directional control of the precursors and in the mechanical support of the weakened wall such structures would be ideally placed to serve also as sensors of the local mechanical strength of the wall and thereby act as regulators of the amount of new synthesis required to restore wall integrity. This idea is also attractive because it can be readily appreciated how it might have arisen as an evolutionary development of some ancestral version of the ‘cytoskeletal extrusion’ mechanism which is used in present day Oomycota. So, it’s likely that the F-actin cytoskeleton at the hyphal apex:
- is anchored through the membrane to the wall at its growing points;
- thereby reinforces the wall so that its components can be partially disassembled for new material to be inserted;
- directs precursors to those points;
- acts as a strain gauge, adjusting the traffic of precursors in step with local mechanical requirements.
Importantly, the contribution made by microtubular components of the cytoskeleton to apical extension do not seem to be as crucial as the contribution made by F-actin. Apical extension of Schizophyllum commune hyphae continued for several hours after drug-induced disruption of cytoplasmic microtubules.
Two major models have been proposed to explain the mechanism of hyphal tip extension in mycelial fungi, both envisage the tip wall being enlarged as the result of fusion with the plasma membrane of vesicles carrying precursors and enzymes, particularly chitin synthase (so the vesicles are called chitosomes), so externalising their contents.
1. In the hyphoid model the rate of addition to any part of the wall depends on its distance from an autonomously moving vesicle supply centre (VSC) which is presumed to be a representation of the Spitzenkörper. The model originates from an interpretation of the particular shape of the hyphal tip as a ‘hyphoid’ curve (as opposed to being hyperbolic or hemispherical). The hyphoid equation was then elaborated into a mathematical model which assumes that wall-building vesicles are distributed from the VSC, this being an organiser from which vesicles move radially to the hyphal surface in all directions at random. Fusion of vesicles with the plasma membrane externalises their content of lytic enzyme (endoglucanase, perhaps chitinase) and:
- hydrolyses structural glucan molecules in the existing wall,
- mechanical stretching pulls the broken molecules apart,
- then resynthesis occurs either by insertion of oligoglucan or by synthetic extension of the divided molecule.
The resynthesised molecules have the same mechanical strength as before, but have been lengthened and the tip has grown. Forward migration of the VSC generates the hyphoid shape. Computer modelling suggests that the position and movement of the VSC determines the morphology of the fungal cell wall. The model both mimics observations made on living hyphae and predicts observations which were subsequently confirmed, making the hyphoid model and its VSC concept a very plausible hypothesis to explain hyphal morphogenesis (Bartnicki-Garcia et al., 1989).
2. The steady state or ‘soft spot’ model assumes that turgor pressure stretches the wall at the hyphal tip where it is still plastic and, in addition, that the synthesis vesicles fuse with the membrane only if they reach parts of the wall that are sufficiently new to be still plastic. If these conditions are met, new wall will be incorporated preferentially into the most recently synthesised wall and this cooperative insertion of newer wall into new wall is the steady state synthesis to which the name of the model refers.
Turgor stretches the new, plastic wall, which thins it but then it is rethickened and restored by vesicular exocytosis of proteins and polysaccharides as wall precursors. The preferential targeting of these vesicles to the most recently synthesised wall means that, once established, the growing point will be maintained (steady state, again). Pre-softening to generate the plastic stretching/thinning in the first place occurs through endolytic cleavage (by chitinase?) though in this model such an enzyme activity is employed only briefly to initiate the growth mode but not to sustain it. Stretching of the wall and addition of new wall material from the cytoplasmic side occur maximally at the extreme tip. Newly added wall components are chitin and β(1→3)-glucan molecules. With time, these two polymers interact to form covalent linkages and to cross link with proteins.
At the extreme tip the wall is minimally cross linked and supposed to be most plastic. Subapically, wall added at the apex becomes stretched and partially cross linked while new wall material is added from the inside to maintain wall thickness. Wall material at the outside is always the oldest. Cross linking increases progressively from the tip and as ‘wall hardening’ proceeds the wall hardly yields to turgor pressure and stretching, and synthetic activity declines. If the tip ceases to extend for any reason (e.g. change in turgor) the steady state breaks down, newer wall is not added to new wall and cross linking between the wall polymers spreads into the apical dome and over the whole apex. From this stopped state, a fresh round of endolytic cleavage would be necessary to restart tip extension (Wessels, 1993).
3. We said there are two models, but we have to join in with all this modelling and suggest our own consensus model of tip extension (see Chapter 2, sections 2.2.4 and 2.2.5, in Moore (1998) for a full discussion). In this we associate chitin synthase activity with stretch receptors, involve membrane architectural proteins, and recognise the contribution of vesicle gradients, combined with other components of the two prime models. Our consensus model has the following features:
- noncovalent interactions occur between mannoproteins and other wall components;
- the initial network is consolidated by formation of covalent cross linkages among the wall polymers;
- cytoplasmic vesicles and vacuoles are assumed to be crucial to the extension of hyphal apices and to be responsible for delivering the enzymes and substrates needed for wall construction;
- the actin cytoskeleton is assumed to be involved directly in hyphal tip extension in a number of ways (in animal cells focal contacts are specialised membrane domains where bundles of actin filaments extend from the intracellular cytoskeleton through the membrane and anchor the cell to its substratum).
This consensus model of tip extension assumes that similar structures to animal focal contacts are connected to the fungal wall. In the fungal cell we anticipate that intracellular cytoskeletal elements could penetrate the membrane to anchor the wall, providing additional tensile strength while the membrane proteins modify the wall structure. These (integrin-mediated?) attachments to the extracellular wall matrix at the hyphal apex, which we will call ‘wall contacts’ would permit the actin cytoskeleton to be directly linked, through the plasma membrane, to components of the wall. Such wall contacts might also be specific for different wall components or even fragments of wall components. Wall contacts are anticipated to function as:
- additional mechanical support to the wall (and via spectrins to the membrane) to compensate for loss of wall strength due to enzymatic cleavage of wall components as new wall materials are incorporated;
- detectors of mechanical strain which might then regulate enzyme activity or modulate the flow of vesicles to the local region;
- feedback detectors of the progress of wall synthesis. In this respect it should be noted that, again in animal systems, interaction between integrins and the extracellular matrix generates signals which cause phosphorylation of intracellular signal transduction pathways. This is known as ‘outside-in signalling’. Such a mechanism at the fungal wall would enable the progress of wall synthesis to be reported to the synthesis machinery allowing it to modulate vesicle supply in quantity and/or kind;
- vesicle traffic directors and regulators; close to the membrane the actin filaments will be directing vesicles with great specificity, in response to the outside-in signalling information; more distant filaments will be marshalling and collecting vesicles into the supply pathways which result in the required overall vesicle fusion gradient. In most, if not all, cases vesicle supply will be channelled through a vesicle supply centre/Spitzenkörper structure.
Evident in all of these models is the proposition that hyphal extension is supported by the continuous forward flow of constructional materials generated within the cytoplasm behind the tip. Much of this flow is in the form of cargo within vesicles derived from the endoplasmic reticulum and processed through Golgi dictyosomes before being transported towards the extending apex.
The vesicles are readily seen in longitudinal sections of fixed hyphae by electron microscopy and consist of two main size classes, the smallest ranging from 20 to 80 nm and the largest from 80 to 200 nm in diameter. The smaller group is readily purified from hyphae and includes the chitosomes that contain chitin synthase. When incubated in UDP and magnesium ions, chitosomes extracted from true fungi produce chitin microfibrils in vitro that are identical to those produced in vivo; so they are clearly essential to creation of the chitinous part of the wall.
Fusion of the vesicles with the membrane at the hyphal apex releases the biosynthetic machinery for wall assembly and also adds new membrane to the growing hypha; synthesis of hyphal wall and plasma membrane are therefore coordinated. Extracellular enzyme secretion into the environment to catalyse the degradation of complex polymers in the substratum, e.g. lignocellulose and polypeptide, is also maximal at the hyphal tip. Potentially, the highly polarised vesicular pathway not only supports rapid hyphal extension, but also acts as a transport mechanism for extracellular enzymes, integrating forward exploration of the substratum with immediate exploitation.
We have already mentioned the astonishing fact that 38 000 vesicles have to fuse with the apical membrane each minute to support hyphal extension in Neurospora crassa when it is growing at its maximum rate. This is a simple calculation based on the observed size (volume) of vesicles in the hypha, and the observed increase in apex volume per minute as the hypha grows.
And that’s the point: the hypha grows. So obviously, the motors and cytoskeletal systems we have described above have no difficulty in supplying materials to the hyphal apex at this sort of rate. Clearly, when we say that materials are transported along microfilament and microtubule tracks, you should understand that to mean that materials are speedily transported. In addition, motile vacuolar systems have been observed in a range of different fungi, as extensive ‘trains’ of vacuoles (very much larger than the apical vesicles) quickly moving along hyphae and tip cells, which could easily supply the vesicular contents used for apical extension growth. So there is an extremely lively forward flow of materials towards the hyphal apex.
Possibly related to organelle movements and the supply of vesicles to the tip is the ‘pulsed growth’ of hyphal tips of several different species of fungi recorded using video analysis and image enhancement. In all fungi tested, the hyphal elongation rate fluctuated continuously with more or less regular intervals of fast and slow growth, which was interpreted as fluctuations in the overall rate of secretory vesicle delivery or discharge at the hyphal apex being reflected in fluctuations of hyphal elongation rate. If this is the case then it will influence our understanding of the mechanism of vesicle supply.
However, a problem with digital video recording is that the pixel (picture element) structure of the electronically-observed image can impose pulsations upon smooth movements. As the edge of the moving object moves from one pixel to the next there is a defined time interval during which no observation is possible, yet the eye, and computer-aided image enhancements, can either compensate for this or amplify it depending on circumstances which have nothing to do with the moving object itself. This effect can readily generate pulsations where they do not exist. A simple test of the validity of pulsations in video measurements (compare recordings made at different optical magnifications) does not seem to have been applied to these observations of fungal growth, but as stepwise changes in elongation rate of hyphae of the bacterium Streptomyces have been recorded using photographic methods not prone to this particular artefact, the phenomenon could happen generally in filamentous systems, so cyclical variations in vesicle delivery may cause pulsations in apex extension rate.
In most, but not all, filamentous fungi the electron dense body we have described as the Spitzenkörper is present in the cytoplasm just distal to the growing apex. This body is composed of a complex of vesicles, and controls both the direction of growth of the hypha and its rate of extension. Satellite Spitzenkörpers separate from the main body and migrate to the lateral cell wall just before new hyphal branches appear. Observations of this sort led to the concept of the Spitzenkörper as a vesicle supply centre (VSC), which acts as the focus from which apical vesicles migrate towards the apical membrane and wall. In this model, vesicles are first delivered to the Spitzenkörper and then they are ‘sprayed’ forwards equally in all directions. A computer model based on this concept generates a tube with a tapering tip very similar to the apex of a growing hypha.
The computer model also shows that the direction of growth can be altered by moving the vesicle supply centre, mimicking the movements observed of the Spitzenkörper during changes in the direction of hyphal growth. The Spitzenkörper is therefore thought to play a critical role in controlling the mechanism of polarised growth of true fungi; not only hyphal extension rate and direction of growth, but also the generation of lateral branches. It seems that the Spitzenkörper is anchored in position behind the growing apex by cytoskeletal elements, most likely F-actin. F-actin is usually found located at the growing tip in the form of a fibrillar network radiating into the cytoplasm from the extreme apex as slender cables, although the majority of actin detectable in the light microscope by immunofluorescence is in the form of ‘actin plaques’ (localised patches of actin) within the first 10 to 12 μm of the hyphal tip. In serial sectioned electron microscope images, these F-actin plaques/patches correspond in position to bodies called filasomes; which are generally spherical, 100-300 nm in diameter and consist of a single microvesicle (35-70 nm diameter) surrounded by fine filaments that contain actin. Filasomes are found adjacent to newly formed glucan fibrils in protoplasts that are regenerating their walls, so the overall interpretation is that a filasome is one of the F-actin plaque/patch structures appearing in the cytoplasm at sites where the cell wall is formed.
The Spitzenkörper also seems to be dependent on the microtubules; experimental interference with dynein, dynactin or microtubules causes eccentric wall deposition and a meandering and more copiously-branched mycelium, suggesting that the dynein/dynactin/microtubule system also positions and maybe stabilises the Spitzenkörper. Remember that this is the system thought to be responsible for nuclear migration toward the growing tip. Elongation of the hyphal tip generally occurs before nuclear migration and the simplest explanation for their tip-ward migration is that nuclei are attached to membrane-anchored dynein and dynactin through astral microtubules from their SPBs (Fig. 12). This assumes that the dynein motors exert a pulling force on the microtubules that position the nuclei. Dependence of Spitzenkörper behaviour on the same system suggests close integration of the transport of all organelles, vacuoles, vesicles and cytoplasmic components that have to migrate forwards as the hyphal apex extends (Xiang & Morris, 1999; Steinberg 2007a).
Despite the evidence that the Spitzenkörper is a crucial component of tip extension in true fungi it is not the only way in which polarised filamentous extension can occur. Hyphal growth in Oomycota, for example Saprolegnia ferax, and other polarised systems, for example pollen tubes, lack a Spitzenkörper. However, all of these filamentous systems, including pollen tubes, seem to share one feature, namely the presence of an apical gradient of Ca2+ at the apex, the highest concentration being immediately distal to the tip. The maintenance of such a gradient is thought to be due to the presence of Ca2+ ion channels in the apical membrane that allow calcium ions to flow down a concentration gradient from the outside to the inside of the cell. The concentration of Ca2+ in eukaryotic cells is highly regulated and maintained at low levels in the cytoplasm in two ways:
- calcium-pumping ATPases located in the plasma membrane pump calcium out of the cell,
- calcium-pumping ATPases located in the vacuolar membranes promote sequestration and storage of the ion within hyphal vacuoles.
Although all the roles and functions of a calcium gradient in the hypha have yet to be established, Ca2+ ions are known to regulate the assembly of the cytoskeleton and to aid vesicle fusion with membranes, so the presence of a ‘tip-high calcium gradient’ is likely to be an important factor in establishing and maintaining apical polarity in the hypha.
Updated January 18, 2017