The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis

Ashbya gossypii has been an ideal system to study filamentous hyphal growth. Previously, we identified a link between polarized hyphal growth, the organization of the actin cytoskeleton and endocytosis with our analysis of the A. gossypii Wiskott-Aldrich Syndrome Protein (WASP)-homolog encoded by the AgWAL1 gene. Here, we studied the role of AgSAC6, encoding a fimbrin in polarized hyphal growth and endocytosis, and based on our functional analysis identified genetic interactions between AgSAC6 and AgWAL1. SAC6 mutants show severely reduced polarized growth. This growth phenotype is temperature dependent and sac6 spores do not germinate at elevated temperatures. Spores germinated at 30 °C generate slow growing mycelia without displaying polarity establishment defects at the hyphal tip. Several phenotypic characteristics of sac6 hyphae resemble those found in wal1 mutants. First, tips of sac6 hyphae shifted to 37 °C swell and produce subapical bulges. Second, actin patches are mislocalized subapically. And third, the rate of endocytotic uptake of the vital dye FM4-64 was reduced. This indicates that actin filament bundling, a conserved function of fimbrins, is required for fast polarized hyphal growth, polarity maintenance, and endocytosis in filamentous fungi.     

A Highlights from MBoC Selection: Mobility,Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae of Ashbya gossypii

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.     

Hyphal tip organization inAllomyces arbuscula

Summary Light and electron microscopic observations on vegetative hyphae ofAllomyces arbuscula revealed the specialized organization of the tip. There were some minor differences related to culture conditions, but the main ultrastructural features common to all hyphal tips disclosed a special type of organization distinct from that of other fungi. A crescent-shaped apical zone consisted of vesicles and membrane cisternae embedded in a granular matrix. Vesicles fused with the apical plasmalemma and presumably contributed to its expansion and to wall growth. The apical zone contained few ribosomes and generally no other organelles. Mitochondria were concentrated in the immediate subapical zone and scattered through the remainder of the hyphae, as were microbodies. Microtubules formed an asterlike structure with its center in the apical zone. Proximally of the apex, microtubules were axially oriented. Nuclei occurred only a certain distance from the tip. The elements of the apex may maintain the polarity of the hyphae via a gradient and hold it in a state of vegetative growth.     

A Highlights from MBoC Selection: Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism

Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.     

Fine-structural Correlates of Growth in Hyphae of Ascodesmis sphaerospora

Mycelial mats of Ascodesmis sphaerospora were fixed and embedded for electron microscopy, and thin sections of 1-mm blocks, taken from the 1st to the 7th mm behind the hyphal tips, were cut parallel to the long axis of the hyphae. The hyphal tip region is characterized by an outer zone of electron-transparent vesicles, 500 to 1,000 A in diameter, and is apparently associated with wall elaboration. Immediately behind this region, dense granules become evident along convoluted membrane systems and along the plasma membrane; in the same region are numerous small lomasomes in the lateral wall. As the hypha grows, septa are laid down at 3- to 7-min intervals at a distance of 200 to 250 μ behind the hyphal tip. A cylinder of endoplasmic reticulum is intimately involved in cross-wall deposition from its earliest stages; as the wall grows in, it becomes increasingly constricted in the pore region, finally assuming a torus-like configuration. Woronin bodies are shown to have a crystalline substructure and to originate in pouch-like membrane systems. Cross-walls from a 7- to 13-hr-old mycelium frequently show highly ordered structures in the vicinity of the pore. These structures may appear either as laminar stacks of discs to one side of the pore or as series of stubby concentric rings within the pore area itself. In the latter case, a mass of granular material is frequently seen plugging the pore. Other unusual organelles and inclusions in 7- to 13-hr hyphae are vesicles containing swirls of beaded or dilated membrane, membrane-enclosed rods, and stacks of unit membranes associated with spherical, electron-transparent vesicles.     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.     

Protoplasmic Organization of Hyphal Tips Among Fungi: Vesicles and Spitzenkörper

Hyphal tips of fungi representing Oömycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes were examined by light and electron microscopy and compared with respect to their protoplasmic organization. In all fungi studied, there is a zone at the hyphal apex which is rich in cytoplasmic vesicles but nearly devoid of other cell components. Some vesicle profiles are continuous with the plasma membrane at the apices of these tip-growing cells. The subapical zones of hyphae contain an endomembrane system which includes smooth-surfaced cisternae associated with small clusters of vesicles. The findings are consistent with the hypothesis that vesicles produced by the endomembrane system in the subapical region become concentrated in the apex where they are incorporated at the expanding surface. Septate fungi (Ascomycetes, Basidiomycetes, and Deuteromycetes) have an apical body (Spitzenkörper) which is associated with growing hyphal tips. In electron micrographs of these fungi, an additional specialized region within the accumulation of apical vesicles is shown for the first time. This region corresponds on the bases of distribution among fungi, location in hyphae, size, shape and boundary characteristics to the Spitzenkörper seen by light microscopy. This structure is not universally associated with tip growth, whereas apical vesicles are widespread among tip-growing systems.     

The Aspergillus nidulans Peripheral ER: Disorganization by ER Stress and Persistence during Mitosis

The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of ‘secretory’ ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains ‘loaded’ with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.     

The Spatial Distribution of the Exocyst and Actin Cortical Patches Is Sufficient To Organize Hyphal Tip Growth

In the hyphal tip of Candida albicans we have made detailed quantitative measurements of (i) exocyst components, (ii) Rho1, the regulatory subunit of (1,3)-β-glucan synthase, (iii) Rom2, the specialized guanine-nucleotide exchange factor (GEF) of Rho1, and (iv) actin cortical patches, the sites of endocytosis. We use the resulting data to construct and test a quantitative 3-dimensional model of fungal hyphal growth based on the proposition that vesicles fuse with the hyphal tip at a rate determined by the local density of exocyst components. Enzymes such as (1,3)-β-glucan synthase thus embedded in the plasma membrane continue to synthesize the cell wall until they are removed by endocytosis. The model successfully predicts the shape and dimensions of the hyphae, provided that endocytosis acts to remove cell wall-synthesizing enzymes at the subapical bands of actin patches. Moreover, a key prediction of the model is that the distribution of the synthase is substantially broader than the area occupied by the exocyst. This prediction is borne out by our quantitative measurements. Thus, although the model highlights detailed issues that require further investigation, in general terms the pattern of tip growth of fungal hyphae can be satisfactorily explained by a simple but quantitative model rooted within the known molecular processes of polarized growth. Moreover, the methodology can be readily adapted to model other forms of polarized growth, such as that which occurs in plant pollen tubes.     

The fine structure ofPhycomyces

Summary The cytological organization of the apices of sporangiophores and hyphae ofPhycomyces Blakesleeanus was studied by means of light- and electron microscopy. The sporangiophore apex in growth stage I contains a mass of cytoplasm in which is embedded a cluster of lipid globules. Within the plug several zones are differentiated by the grouping of organelles. These zones are not separated by membranes. The most apical zone is low in nuclei and vesicles but rich in mitochondria and dense bodies. Below this zone lies a compact group containing up to several hundred nuclei. Along the midline of the cell, below these nuclei, lies an ovoid region from which vesicles, nuclei and mitochondria are excluded. In this ovoid exclusion zone lies the cluster of lipid globules mentioned above. Lateral to the exclusion zone (i.e. in the peripheral region of the cell) the cytoplasm is rich in nuclei, mitochondria, dense bodies, and especially in developing autophagic vesicles. Of these vesicles, the most mature are found farthest from the cell apex. The region between the exclusion zone and the upper end of the cell's large central vacuole is occupied largely by mature, swollen autophagic vesicles. In addition to the zonal organization described above, microtubules are found to run along the cylindrical cell's axis at a distance from the cell wall, and extend to the extreme apex of the cell. Similar tubules occur in growing hyphae, together with dense bodies, and the hyphal apex contains non-autophagic vesicles that increase in size with distance from the hyphal tip. The hyphae lack the zonation shown by sporangiophore apices. Perinuclear masses of cisternae are described and related to the dictyosomes of higher plants. The findings are discussed in relation to the function of the apices in tip growth and sporulation.This work was supported in part by a National Science Foundation Graduate Fellowship to the author, and in part by grant No. GB 3241 from the National Science Foundation to ProfessorKenneth V.Thimann.     

AN ULTRASTRUCTURAL BASIS FOR HYPHAL TIP GROWTH IN PYTHIUM ULTIMUM

Hyphae of the fungus Pythium ultimum extend by tip growth. The use of surface markers demonstrates that cell expansion is limited to the curved portion of the hyphal apex. Growing and non-growing regions are reflected in internal organization as detected by light and electron microscopy. The young hypha consists of three regions: an apical zone, a subapical zone and a zone of vacuolation. The apical zone is characterized by an accumulation of cytoplasmic vesicles, often to the exclusion of other organelles and ribosomes. Vesicle membranes are occasionally continuous with plasma membrane. The subapical zone is non-vacuolate and rich in a variety of protoplasmic components. Dictyosomes are positioned adjacent to endoplasmic reticulum or nuclear envelope, and vesicles occur at the peripheries of dictyosomes. A pattern of secretory vesicle formation by dictyosomes is described which accounts for the formation of hyphal tip vesicles. Farther from the hyphal apex the subapical zone merges into the zone of vacuolation. As hyphae age vacuolation increases, lipid accumulations appear, and the proportional volume of cytoplasm is reduced accordingly. The findings are integrated into a general hypothesis to explain the genesis and participation of cell components involved directly in hyphal tip growth: Membrane material from the endoplasmic reticulum is transferred to dictyosome cisternae by blebbing; cisternal membranes are transformed from ER-like to plasma membrane-like during cisternal maturation; secretory vesicles released from dictyosomes migrate to the hyphal apex, fuse with the plasma membrane, and liberate their contents into the wall region. This allows a plasma membrane increase at the hyphal apex equal to the membrane surface of the incorporated vesicles as well as a contribution of the vesicle contents to surface expansion.     

The Neurospora crassa exocyst complex tethers Spitzenk?rper vesicles to the apical plasma membrane during polarized growth

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.     

Golgi organization and the apical extension of fungal hyphae: an essential relationship

The Golgi apparatus performs crucial functions in the sorting and processing of proteins destined for secretion from eukaryotic cells. In filamentous fungi, organization of the Golgi apparatus reflects the unique challenges brought about by the highly polarized nature of hyphal growth. Recent results show that Golgi compartments are spatially segregated within hyphal tip cells in a manner that depends upon the integrity of the cytoskeleton. Moreover, loss of normal Golgi organization stops polarized hyphal extension and triggers de‐polarization of the hyphal tip. These results emphasize the point that a spatially organized and dynamic Golgi apparatus represents an adaptation that is as important for hyphal extension as is the presence of a Spitzenkörper. In addition, they also identify regulatory mechanisms that could enable controlled de‐polarization of hyphae during development or infection‐related morphogenesis.     

Spitzenk?rper,Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.Polarized growth of fungi requires that a supply of secretory vesicles is delivered along cytoskeletal tracks to the site of cell expansion (for reviews, see references 13, 29, 30, and 31). Fusion of these membrane-bound vesicles with the plasma membrane allows the necessary expansion of the plasma membrane and releases the enzymes and raw materials for the synthesis of new cell wall material and the remodeling necessary to allow this newly synthesized material to be inserted into the existing cell wall. The process of polarized growth has been extensively studied in the budding yeast Saccharomyces cerevisiae and provides a model for studying the process in other fungi (for a review, see reference 20). Post-Golgi vesicles travel to sites of polarized growth along actin cables (23). Actin cables are nucleated at sites of polarized growth by the formin Bni1 facilitated by a multiprotein complex called the polarisome, which consists of Spa2, Bud6, and Pea1(5, 22, 24, 27). The motive force for vesicle transport is provided by Myo2, a class V myosin, complexed to its regulatory light chain Mlc1 (22, 26). At the plasma membrane, secretory vesicles dock with a second multiprotein complex called the exocyst before fusion with the plasma membrane (14, 15, 32, 33), mediated by v-SNARES on the vesicle and t-SNARES on the membrane. The exocyst is an octomeric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (21). It is thought that Sec3 and a fraction of the Exo70 pool are localized at sites of polarized growth independently of the actin cytoskeleton (3, 6). The other exocyst subunits and the remainder of the Exo70 pool are thought to be transported to sites of polarized growth on secretory vesicles, where together with Sec3 and Exo70 they form the exocyst complex (3). Secretory vesicles exit the Golgi apparatus, travel toward sites of polarized growth, and dock with the exocyst by use of the Rab-type GTPase Sec4 in its GTP-bound form, which is activated by its GEF, Sec2 (12, 19, 35, 36). In the S. cerevisiae cell cycle, polarized growth is initially directed toward the bud tip in young buds (17). Growth subsequently becomes isotropic in larger buds before being directed toward the mother bud neck during cytokinesis at the end of the cell cycle. Accordingly, polarisome and exocyst components localize to the tips of young buds (7, 27, 28).The rate of hyphal tip extension is much greater than that of the growth of a yeast or pseudohyphal bud. In rich yeast extract-peptone-dextrose (YEPD) medium, Candida albicans hyphae extend at the rate of 0.25 μm min⁻¹, compared to 0.0625 μm min⁻¹ in yeast buds and 0.125 μm min⁻¹ in pseudohyphal cells (P. Sudbery unpublished observations). In hyphae of filamentous fungi, a structure called a Spitzenkörper is present at the tip, which is rich in secretory vesicles (8, 9, 11, 29, 34). It is believed that the Spitzenkörper acts as a vesicle supply center (VSC) (1). This model proposes that the Spitzenkörper is maintained at a fixed distance from the hyphal tip. Vesicles radiate out in equal directions to fuse with the plasma membrane, so that more vesicles per unit area fuse with the hyphal tip itself than with other parts of the hyphae. Mathematical modeling shows that this explains the distinctive shape of hyphal tips.In order to investigate the mechanism of polarized growth in the hyphae of Candida albicans, we previously determined the localization of Mlc1-yellow fluorescent protein (YFP) and the polarisome components Bud6-YFP and Spa2-YFP (4). We found that in hyphae, polarisome components localized to a surface crescent, as they did in young yeast buds and the tips of elongated pseudohyphal buds. However, in hyphae Mlc1-YFP localized to a bright spot, which at least in some hyphae was clearly inside the tip, rather than at the surface, and which appeared spherical in three-dimensional reconstructions. We concluded that this represented a Spitzenkörper. In some hyphae Mlc1-YFP also localized to a surface crescent, similar to the pattern displayed by polarisome components. This observation suggested that the Spitzenkörper and polarisome were separate structures, both of which were present at hyphal tips, but that only the polarisome was present at the bud tips of pseudohyphae and yeast. Moreover, the dual localization of Mlc1-YFP to a crescent and a spot suggested that Mlc1 may be present in both structures.While S. cerevisiae has proved to be an excellent model to investigate the molecular genetics of polarized growth, it is less optimal to study the spatial organization of the molecular components because polarized growth of the bud is restricted to a short period after bud emergence when the nascent bud is small. Thus, there has been little effort to investigate the fine detail of the spatial organization of the different components of the polarization machinery beyond noting that they localize to sites of polarized growth. In this study we exploited the opportunities afforded by the continuous polarized growth of C. albicans hyphae to clarify the relationship between the Spitzenkörper, polarisome, and exocyst, which cooperate to mediate the extreme polarized growth of hyphae. We show that the vesicle-associated marker Sec4 also localizes to a Spitzenkörper-like structure, confirming the existence of a vesicle-rich area corresponding to a Spitzenkörper at the hyphal tip. We show that exocyst components such as Sec3, Sec6, Sec8, Exo70, and Exo84 localize to a surface crescent, so the exocyst, like the polarisome, is also a spatially separate structure from the Spitzenkörper. We used three independent strategies to investigate the dynamic properties of these structures. Fluorescence recovery after photobleaching (FRAP) was used to measure the rate at which new proteins arrived at the tip. Fluorescence loss in photobleaching (FLIP) was used to measure the rate at which proteins exited the tip. Cytochalasin A was used to disrupt actin cables, allowing the persistence of proteins at the tip to be measured after the supply of new proteins was blocked. In each case we found that Spitzenkörper components Sec4, Sec2, and Mlc1 were highly dynamic, while the polarisome component Spa2 was stable. Intriguingly, exocyst components showed intermediate dynamic properties, suggesting that they are delivered to the tip on vesicles but that not all vesicles carry a complement of exocyst components. We suggest that these data are consistent with a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.     

The ultrastructure of the growing regions of aerial hyphae ofRhizopus sexualis (Smith) callen

Summary The fine structure of the growing region of the aerial hyphae ofRhizopus sexualis is described. The protoplasmic components are organised in zones. At the hyphal tip there are abundant cytoplasmic vesicles and few ribosomes. Mitochondria, ribosomes, and endoplasmic reticulum are present in the sub-apical region. Nuclei occur in the posterior subapical region, some have been observed containing microtubules and bearing long projections. Microtubules have been observed extending for some distance through the cytoplasm.     

Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinA^rigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.     

In Candida albicans hyphae,Sec2p is physically associated with SEC2 mRNA on secretory vesicles

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans‐Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.     

Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

Fluorescent proteins illuminate the structure and function of the hyphal tip apparatus

Fungal hyphae show extreme polarized growth at the tip. Electron microscope studies have revealed a apical body called the Spitzenk?rper that is thought to drive polarized growth. Studies of polarized growth in S. cerevisiae have identified the protein components of the polarized growth machinery, that are conserved in other fungi. Fusion of these proteins to GFP and its variants has for the first time allowed the localization of these proteins in real time to the hyphal tip without the need for drastic fixation procedures. Such studies showed that vesicle-associated proteins localize to the Spitzenk?rper and identified a second compartment located at the tip surface composed of exocyst and other proteins that mediate the fusion of secretory vesicles with the plasma membrane.