Coordinated process of polarized growth in filamentous fungi

Abstract

Filamentous fungi are extremely polarized organisms, exhibiting continuous growth at their hyphal tips. The hyphal form is related to their pathogenicity in animals and plants, and their high secretion ability for biotechnology. Polarized growth requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain the cell polarity. This review summarizes recent findings unraveling the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Recent findings by a super-resolution microscopy indicate that filamentous fungal cells maintain their polarity at the tips by repeating transient assembly and disassembly of polarity sites.

Key words:

• Aspergillus
• polarity
• actin
• microtubule
• exocytosis

Maintaining cell polarity is essential for cells to ensure their proper functions.^1,2) Symmetry breaking is often preceded by cytoskeleton-dependent polarization of certain key proteins as observed in epithelial cells with apical–basal polarity, neuronal differentiation from dendrites to axons, and migrating cells. Filamentous fungi are highly polarized eukaryotic cells, which continuously elongate their hyphae at the tips. In distal parts, hyphae can initiate new sites of polar growth in the process of branch formation. The establishment and maintenance of polar growth is a fascinating question in biology.^3–5) Filamentous fungi are widely used as a model system for the analysis of the relationship between cell polarity and shape. Some filamentous fungi are pathogen to animals and plants. Their filamentous form is important for the invasion into host cells.⁶⁾ Other fungi are useful in biotechnology, such as for enzyme production, and fermentation in food industry due to their high ability of enzyme secretion.^7,8) Thus, the analysis of polarized growth of filamentous fungi can contribute to the medical, agricultural, and biotechnological fields.

The filamentous ascomycete Aspergillus nidulans has been employed worldwide for more than 60 years as a model organism because it is closely related to clinically and economically important Aspergilli, such as Aspergillus fumigatus, Aspergillus oryzae, Aspergillus niger, etc. The most characteristic cell-type of filamentous fungi is the vegetative hyphae, which consist of multinuclear cells compartmentalized by septa. In single-cell yeasts, such as in budding yeast Saccharomyces cerevisiae and in fission yeast Schizosaccharomyces pombe, polarized growth is restricted to specific time point during the cell cycle, whereas in filamentous fungi cell extension is a continuous and indefinite process. Several overviews have summarized different aspects of polarized growth in fungi.^5,9–11) This review will focus mainly on the latest findings on the role of cell cytoskeleton and the mechanism of polarity maintenance during apical membrane extension.

I. The actin cytoskeleton

Actin cytoskeleton plays a central role in cell morphogenesis of filamentous fungi.^12,13) There are three high order F-actin structures with distinct functions: actin rings, patches, and cables. The actin rings in cooperation with myosin II function in septum formation.^14,15) Actin patches are peripheral punctate structures, where the endocytic machinery is probably located.¹⁶⁾ The predominant localization of these patches at subapical regions suggests spatial coupling of apical exocytosis and subapical compensatory endocytosis (Fig. 1(A)),¹⁷⁾ in addition to endocytic recycling of polarized material at the hyphal tip.¹⁸⁾ The mutant phenotype of genes involved in endocytosis indicates that endocytosis is essential.¹⁶⁾

Fig. 1. Scheme of tip growth in A. nidulans hyphae.

Notes: (A) Vesicle trafficking via the actin and microtubule cytoskeleton. Before fusion with the plasma membrane, secretion vesicles accumulate at Spitzenkörper. (B) MT-organizing center (MTOC) at spindle pole bodies and septa. Most MTs are oriented with their dynamic plus-ends towards the hyphal tip in the tip compartment.

Actin cables are linear bundles of short actin filaments nucleated by formins that are present at the apexes of hyphae. The dynamic actin cables are generally difficult to visualize in filamentous fungi. Phalloidin conjugated to fluorescent dyes has been widely used for imaging F-actin in eukaryotic cells, however, it does not work in most filamentous fungi.¹⁹⁾ Recently specific markers, such as Lifeact and tropomyosin, have been developed to visualize actin cables.^15,20–22) Actin cables are present at the apex of hyphae and are thought to serve as tracks for myosin V-dependent secretory vesicle transport to the tip (Fig. 1(A)).^12,14,20) The “basic” growth machinery involved in the formation of actin cables, vesicle transport, and exocytosis, such as formin, the polarisome, myosin V, and the exocyst complex, are relatively conserved among eukaryotic cells and localize at the apex of hyphae.²³⁾ Before fusion with the plasma membrane, the secretion vesicles accumulate at the so-called “Spitzenkörper” at hyphal tips.^24,25) A Spitzenkörper is a special structure in filamentous fungi determining hyphal shape and growth direction.²⁶⁾ Although the exact composition and organization is still not completely understood, one model proposes that the Spitzenkörper acts as a vesicle supply center for growing tips.²⁷⁾

II. Microtubules

Microtubules (MTs) play a crucial role during mitosis, but also have additional functions in interphase of filamentous fungi. They are important for the distribution of nuclei or other organelles and serve as tracks for endosomes and secretory vesicles, thus they are important for rapid hyphal growth.^{4,17,28–30)}

The rather stable minus end of MTs is located at the MT-organizing center (MTOC), whereas the plus end is facing to the cell periphery with alternating growing and shrinking phases. In filamentous fungi, spindle pole bodies serve as MTOCs (Fig. 1(B)).³¹⁾ They contain γ-tubulin, first discovered in A. nidulans, which is required for nucleation of MTs.^31,32) Furthermore, areas close to the septa act as MTOCs in A. nidulans (sMTOCs), however, their composition remains elusive.^33–35) In the tip compartment of A. nidulans, most MTs are oriented with their dynamic plus ends towards the hyphal tip.³⁶⁾ Nuclei migrate probably along MTs until they reach a certain position. The entire hypha looks therefore very organized with equally spaced nuclei.

III. Vesicle transport through the cell cytoskeleton

Two classes of MT-dependent motors, the minus end-directed dynein and the plus end-directed kinesins, are involved in the positioning of organelles and transport of membranes. Whereas genomes of filamentous fungi contain a single dynein motor, they usually encode 10–12 kinesins.³⁷⁾ The function of kinesin-3 and the dynein motor in the transport of early endosomes has been extensively studied.^4,38–40)

The deletion of conventional kinesin (kinesin-1) in different fungi decreased the growth rate, and caused defects in Spitzenkörper stability, protein secretion, and pathogenicity.^41–46) These results suggest a possible conserved role in vesicle transportation similar to higher eukaryotic cells. Secretory vesicles are thought to be transported by kinesin-1 along MTs for long distances toward hyphal tips in filamentous fungi, although the localization of ER and Golgi close to hyphal tips raises questions about the function and cargoes of kinesin-1.^47,48) Therefore, long distance transport of secretion vesicles could be less important and actin-dependent movement could be rather sufficient for polarized growth. Indeed, hyphal extension can occur long time without functional MTs, but is immediately stopped if the integrity of the actin cytoskeleton is disturbed.^13,29) Although the dependency on MT and actin cytoskeleton could be diverse in different fungi, vesicle movement and delivery to the tip plasma membrane likely depends on the cooperation of actin and MT-dependent motors.^14,45,49,50)

IV. Polarity markers

Polar cell extension depends on spatially defined insertion of new materials controlled by polarity marker proteins that localize to the plasma membrane. Polarity markers on the plasma membrane determine the polarity site, where actin cables are organized and secretory vesicles are delivered along the actin cables (Fig. 2(A)). In general, membrane-associated polarity markers are transported via secretory vesicles and supplied to the polarity site by exocytosis. In S. cerevisiae, exocytosis takes place at sites of the Rho GTPase Cdc42, a polarity marker.⁵¹⁾ Two positive feedback loops are thought to contribute to Cdc42 polarization. One pathway involves recruiting GEFs (guanine nucleotide exchange factors) and effector complexes from the cytoplasm in a cytoskeleton-independent manner.⁵²⁾ The other one is a vesicle-recycling feedback loop, where Cdc42 orients actin cables, which in turn deliver Cdc42 as cargo on secretory vesicles.⁵³⁾

Fig. 2. Relationship between polarity marker and exocytosis.

Notes: (A) Polarity markers determine polarity sites through the arrangement of actin cytoskeleton, vesicle transport and exocytosis. Polarity markers are delivered to the polarity site as cargo on secretory vesicles. (B) Active exocytosis may result in dilution or dispersion of the membrane-associated polarity markers.

As a consequence of exocytosis at the polarity site, the added membrane may result in dilution or dispersion of the membrane-associated polarity markers (Fig. 2(B)).⁵⁴⁾ Recent mathematical models predict that, under certain simulated conditions with highly active exocytosis (8–32 times faster than in budding yeast), membrane insertion via exocytosis can almost eradicate concentrated Cdc42 polarity complexes.⁵⁵⁾ The dilution of polarity marker due to exocytic membrane insertion has been shown to be essential for effective chemotropism in yeast.⁵⁶⁾

Rho type GTPases, Cdc42, and Rac1 (ModA and RacA), have also been found in A. nidulans.⁵⁷⁾ Both Cdc42 and Rac1 share at least one overlapping function that is required for polarity establishment. The combination of Δcdc42 with Δrac1 appeared synthetically lethal in A. nidulans.⁵⁷⁾ Besides the Rho GTPases, the so-called “cell-end markers” (TeaA and TeaR) function as polarity markers. The cell-end markers localize at the tip of the hyphae and control growth direction (Fig. 3(A)).⁵⁸⁾ TeaA, is delivered specifically to the apex by growing MTs, and is anchored to the membrane by direct interaction with another cell-end marker at the plasma membrane, TeaR.^58,59) TeaR has CaaX motif for post-translational lipid modification and anchors to the cell membrane. The interaction of TeaA and TeaR at the apical membrane initiates the recruitment of additional downstream components including the formin SepA, which polymerizes actin cables for targeted cargo delivery.⁶⁰⁾ Deletion of cell-end markers results in highly curved or zigzagged instead of straight hyphae.⁵⁸⁾ Structurally straight MTs growing to hyphal tips transmit positional information to cell-end markers at hyphal tips. This mechanism is important for the maintenance of polarity sites at hyphal tips and growth direction.⁵⁾ In addition, a functional connection between TeaA and a MT polymerase AlpA is required for the proper regulation of MT growth at hyphal tips and focusing of polarity.^11,61)

Fig. 3. Transient polarity model. (adapted from [63]).

Notes: (A) Scheme of cell-end markers polarization and vesicle transport through actin and MT cytoskeleton. (B) Timelapse PALM of TeaR show the appearance of a new cluster (t = 5 s), a translational movement (t = 10 s) and a spreading of the signal along the plasma membrane. (C) Scheme of transient polarity model.

In A. nidulans, hyphal tips extend with speeds of 0.3–1.0 μm/min, requiring a high frequency of exocytosis events (400–2000 vesicles/min). Hyphal elongation in A. nidulans is approximately 10–30 times faster than bud formation in budding yeast, indicating more active vesicle transport and exocytosis.^29,54,62) Although thousands of exocytosis events take place at the apex every minute, which may displace membrane-associated polarity markers from the growing tip (Fig. 2(B)), filamentous fungi are able to maintain their polarity sites. How can filamentous fungi maintain the accumulation of polarity markers in the presence of rapid insertions of membranes via highly active exocytosis?

V. Transient polarity assembly in active exocytosis

Our recent study shows that polarity sites visualized by the membrane-associated cell-end marker TeaR are highly dynamic at growing hyphal tips.⁶³⁾ A MT grows towards the hyphal tip, pauses in close contact with the apical membrane, and then undergoes a catastrophe resulting in retraction. The TeaR signal accumulates at the hyphal tip membrane, whenever a MT plus-end touches the tip membrane. The TeaR fluorescence decreases immediately after the MT starts to shrink. Colocalization studies further support the notion that TeaR clusters represent zones of exocytosis and extension of the apical membrane.

The localization of TeaR cluster at hyphal tips has been analyzed by super-resolution microscopy PALM (photoactivation localization microscopy).⁶³⁾ The resolution of conventional light microscopy techniques is limited around 250 nm due to light diffraction. Super-resolution microscopy techniques, such as STED, SIM, STORM, PALM, etc., have overcome the diffraction limit, resulting in lateral image resolution as high as 20 nm and providing a powerful tool for the investigation of protein localization in high detail.^64,65) The PALM imaging clearly shows TeaR clusters near the apex of the cell and also along the plasma membrane (Fig. 3(B)). The average size of TeaR clusters is approximately 120 nm. Quantified analysis using the PALM data suggests an estimated 20 TeaR proteins per cluster on average. The timelapse PALM reveals many abrupt appearances and disappearances of clusters near the plasma membrane. In some instances, a cluster appears, moves along the plasma membrane, mostly towards the apex, spreads along the membrane and then disappears (Fig. 3(B)). Overlay of the first and the last frame shows a small membrane growth. These images are most likely to indicate a fusion-like morphological change of TeaR clusters followed by membrane extension, where a small patch of membrane is added via exocytosis, and the TeaR cluster gets quickly dispersed over the membrane.

These results propose a “transient polarity model” (Fig. 3(C)).⁶³⁾ In this model, the interaction between the MT plus-end, where TeaA is located, and TeaR at the apical membrane initiates the recruitment of other polarity markers, resulting in the assembly of TeaR polarity site (Fig. 3(C, i)). The accumulated cell-end markers induce actin polymerization followed by active exocytosis (ii). Newly synthesized TeaR is delivered to the tip membrane on secretory vesicles through MT and actin cables. The plasma membrane extends locally at the site of vesicle fusion (iii) and, subsequently, the TeaR polarity site is dispersed or displaced along the membrane (iv). Once the polarity site is disassembled, however, next MT comes to the tip and gathers TeaR floating in the membrane through the interaction with TeaA at the MT plus-end and the cycle starts over (i′). This model is qualitatively reproduced by computer simulation that includes the plasma membrane, exocytic and endocytic vesicles, MTs, actin, and TeaR.⁶³⁾ Moreover, simultaneous visualization of actin cables and MTs suggests the temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons, as shown in the model.²²⁾

This transient polarity model provides an explanation for maintenance of the polarized growth in filamentous fungi. In contrast to the static polarity site picture, the polarity site is transiently established by MTs that transport cell-end marker proteins to the apical membrane. The polarity sites are localized near the apex of the hyphae because MT plus-ends tend to reach there before the catastrophe.⁶¹⁾ When MT plus-ends come in contact with the apex, downstream reactions are triggered locally. The transfer of membrane via exocytosis depletes active binding proteins for secretory vesicles, dilutes the material at the site, and leads to disassembly of polarity. Using the residual TeaR as a guide, the polarity site is rapidly reassembled by the arrival of a new MT carrying the next batch of cell-end marker proteins at a slightly displaced location. In line with this model, recent work on another filamentous fungus, Neurospora crassa, reports “bursts of exocytosis” events at different positions within the apical membrane rather than a persistent exocytosis site.⁶⁶⁾ The subtle change in TeaR accumulation sites can result in a change in the overall growth direction. This mechanism of short bursts of cell extension also provides more opportunities for the cell to adjust growth directions and allows quicker responses to environmental signals, such as chemotropism, or to avoid obstacles.

VI. Coordinated cycles of cell growth

The transient polarity model suggests a coordinated process by several steps and cycles (or oscillations without a fixed period). Coordinated cycles or oscillations of exocytosis associated with cell growth may be a conserved phenomenon shared among various organisms, including filamentous fungi,⁶⁷⁾ mammalian cells,⁶⁸⁾ fission yeast,⁶⁹⁾ and plant root hairs⁷⁰⁾ and pollen tubes.⁷¹⁾ The intracellular Ca²⁺ level is known to regulate actin assembly and vesicle fusion.^72,73) Plant cells show an oscillation in the Ca²⁺ concentrations at the cell tip, which is correlated with the change in growth rate. In mammalian cells, oscillations of cortical actin and Ca²⁺ concentrations correlate with oscillations of vesicle secretion.⁶⁸⁾ Ca²⁺ oscillations have also been observed at hyphal tips in filamentous fungi, suggesting that the oscillations are related to the growth rate.⁷⁴⁾ The Ca²⁺ oscillation is likely to play an important role in regulating actin polymerization and triggering the synchronized fusion of accumulated vesicles within a local region in the tip growth of filamentous fungi as well.

These cycles of cell growth suggest a transient waiting time of secretory vesicles before exocytosis. Polarity markers, exocyst complex, and SNARE proteins, which are required for exocytosis, could be diluted locally on the plasma membrane after the exocytosis. The transient waiting time until an upcoming exocytosis event may allow these components to rearrange at the next polarity sites. Traffic lights indicating “go” and “stop” for secretory vesicles may be necessary to guarantee robust polarized growth. In addition, the stepwise growth allows cells to respond more quickly and often to internal and external cues such as chemical or mechanical environmental signals.

Disclosure statement

No potential conflict of interest was reported by the author.

Funding

The work was supported by the DFG [TA819/2-1]; JSPS KAKENHI [grant number 15K18663].