Key differences between lateral and apical branching in hyphae of Neurospora crassa

We examined in fine detail growth kinetics and intracellular events during lateral and apical branching in hyphae of Neurospora crassa. By high-resolution video-enhanced light microscopy, we found remarkable differences in the events preceding lateral vs apical branching. While apical branching involved a significant disturbance in the apical growth of the parental hypha, lateral branching occurred without any detectable alterations in the growth of the parental hypha. Prior to the emergence of a lateral branch, an incipient Spitzenk?rper was formed about 12-29 microm behind the apex. Lateral branch formation did not interfere with the elongation rate of the primary hypha, the shape of its apex or the behavior of its Spitzenk?rper. In sharp contrast, apical branching was preceded by marked changes in physiology and morphology of the parental hypha and by a sharp drop in elongation rate. The sequence involved a cytoplasmic contraction, followed by a retraction, dislocation, and disappearance of the Spitzenk?rper; hyphal elongation decreased sharply and a transient phase of isotropic growth caused the hyphal apex to round up. Growth resumed with the formation of two or more apical branches, each one with a Spitzenk?rper formed by gradual condensation of phase-dark material (vesicles) around an invisible nucleation site. The observed dissimilarities between lateral and apical branching suggest that these morphogenetic pathways are triggered differently. Whereas apical branching may be traced to a sudden discrete disruption in cytoplasmic organization (cytoplasmic contraction), the trigger of lateral branching probably stems from the subapical accumulation of wall precursors (presumably vesicles) reaching a critical concentration.     

Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.

BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenk?rper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenk?rper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways.     

The cytoplasmic organization of hyphal tip cells in the fungusAllomyces macrogynus

Summary Light and transmission electron microscopy were used to examine hyphal tip cells of the fungusAllomyces macrogynus (Chytridiomycetes). A well defined apical body, i.e., Spitzenkörper, was observed at the extreme apex of hyphal cells. This distinctive, spherical cytoplasmic region consisted of a granular matrix devoid of ribosomes and most organelles. To our knowledge this is the first report describing such a structure in hyphae of an aseptate fungus. Vesicles (45–65 nm diameter) were concentrated in the peripheral cytoplasm of the apex, while relatively few were observed within the Spitzenkörper. Filasomes, spherical patches of dense fibrillar material containing a microvesicle core, were abundant in the apical regions near the plasma membrane. Microtubules traversed the Spitzenkörper at various angles and were in close association with the plasma membrane. Microfilaments were observed as individual elements in the cytoplasm or were organized into bundles. Individual microfilaments were frequently in close association with the plasma membrane, vesicles and microtubules. In the immediate subapical region mitochondria, multivesicular bodies, microbodies, Golgi equivalents and nuclei were abundant.Abbreviations CW cell wall - F filasome - M mitochondria - N nucleus - PM plasma membrane - TEM transmission electron microscopy     

Microtubules Are required for motility and positioning of vesicles and mitochondria in hyphal tip cells of Allomyces macrogynus

We have used video-enhanced light microscopy and digital image processing to characterize the intracellular motility and positioning of vesicles ( approximately 1-microm diameter) and mitochondria in growing hyphal tip cells of Allomyces macrogynus. These observations were coupled with cytoskeletal inhibitory experiments to define the roles of the microtubule and actin cytoskeletons in organelle translocation and positioning. Vesicles and mitochondria were abundant in apical and subapical hypha regions. Vesicles traveled along paths that were parallel to the longitudinal axis of the cell. Anterograde (i.e., toward the hyphal apex) and retrograde (i.e., away from the hyphal apex) movements of vesicles occurred at average rates of 4.0 and 2.2 microm/s, respectively. Bidirectional travel of vesicles along common paths was noted in the cortical cytoplasm. Mitochondria were aligned mostly parallel to the long axis of the hypha, except those extending into the hyphal apex, which were oriented toward the Spitzenk?rper. In regions of the subapical hypha mitochondria were often restricted to the cortical cytoplasm and nuclei occupied the central cytoplasmic region. Mitochondria displayed rapid anterograde movements reaching speeds of 3.0 microm/s, but primarily maintained a constant position relative to either the advancing cytoplasm or the lateral cell wall. Cytoskeletal disruption experiments showed that the positioning of mitochondria and motility of vesicles and mitochondria were microtubule-based and suggested that the actin cytoskeleton played uncertain roles.     

Live-cell imaging of vegetative hyphal fusion in Neurospora crassa

The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenk?rper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.     

The hyphal tip structure of Basidiobolus sp.: a zygomycete fungus of uncertain phylogeny

To date, among the zygomycete fungi that have been examined, a Spitzenk?rper has not been reported. In this paper, the cytoplasmic order of hyphal tip cells of Basidiobolus sp.,?a zygomycete genus of uncertain phylogeny, has been examined using light microscopy and transmission electron microscopy methods. With phase-contrast light optics, a phase-dark body was observed at the tips of growing hyphae of Basidiobolus sp. The hyphal apex also showed high affinity for FM4-64 labelling resulting in an intense fluorescence signal. The phase-dark inclusion exhibited independent motility within the hyphal apex and its presence and position were correlated to the rate and direction of hyphal growth. The hyphal apex of Basidiobolus sp. did not contain γ-tubulin. Ultrastructural observations revealed a dense cluster of vesicles at the hyphal apex. These results suggest that the growing hypha of Basidiobolus sp. contains a Spitzenk?rper, a character generally attributed to members of the ascomycete and basidiomycete fungi and not to zygomycete fungi.     

Conidiogenous loss of structuro-functional polarity in the hyphal tips of Sclerotinia fructigena

Ultrastructural depolarization in the conidiogenously induced hyphal tips is initiated by Spitzenk?rper (apical body) disintegration, followed by spreading of wall-associated vesicles and migration of mitochondria and lipid granules into the exclusion zone of the vegetative apices.     

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.     

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenk?rper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.     

p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.     

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.     

Neurospora crassa ro-10 and ro-11 genes encode novel proteins required for nuclear distribution

Movement and distribution of nuclei in fungi have been shown to be dependent on cytoplasmic microtubules and the microtubule-associated motor cytoplasmic dynein. We have isolated hundreds of Neurospora crassa mutants, known as ropy, that are defective in nuclear distribution. Three of the ro genes, ro-1, ro-3 and ro-4, have been shown to encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. In this report, we describe the isolation and initial characterization of two additional ro genes, ro-10 and ro-11. ro-10 and ro-11 are non-essential genes that encode novel 24 kDa and 75 kDa proteins respectively. Both ro-10 and ro-11 mutants retain the ability to generate long cytoplasmic microtubule tracks, suggesting that the nuclear distribution defect is not caused by a gross defect in the microtubule cytoskeleton. RO10, as well as RO4 (actin-related protein ARP1, the most abundant subunit of dynactin), appears to be required for the stability of RO3 (p150Glued), the largest subunit of dynactin. We propose that ro-10 mutants lack proper nuclear distribution, because RO10 is either a subunit of dynactin and required for dynactin activity or essential for assembly of the dynactin complex. ro-11 mutations have no effect on RO1 or RO3 levels and have only a very slight effect on the localization pattern of cytoplasmic dynein and dynactin. The role of RO11 in the movement and distribution of nuclei in N. crassa hyphae remains unknown.     

Microscopic analysis of Neurospora ropy mutants defective in nuclear distribution.

Movement and distribution of nuclei in fungi has been shown to be dependent on microtubules and the microtubule-associated motor cytoplasmic dynein. Neurospora crassa mutants known as ropy are defective in nuclear distribution. We have shown that three of the ro genes, ro-1, ro-3, and ro-4, encode subunits of either cytoplasmic dynein or the dynein activator complex, dynactin. Three other ro genes, ro-7, ro-10, and ro-11, are required for the integrity or localization of cytoplasmic dynein or dynactin. In this report, we describe a microscopic analysis of N. crassa ro mutants. Our results reveal that defects in germination of conidia, placement of septa, and mitochondrial morphology are typical of ro mutants. Two classes of cytoplasmic microtubules are identified in wild-type and ro mutants. One class of microtubules has no obvious association with nuclei while the other class of microtubules connects spindle pole bodies of adjacent nuclei. The possible role of internuclear microtubule tracts in the movement and distribution of nuclei is discussed.     

Aggregation and collapse in a mechanical model of fungal tip growth

 Interconnected hyphal tubes form the mycelia of a fungal colony. The growth of the colony results from the elongation and branching of these single hyphae. The material being incorporated into the extending hyphal wall is supplied by vesicles which are formed further back in the hyphal tip. Such wall-destined vesicles appear conspicuously concentrated in the interior of the hypha, just before the hyphal apex, in the form of an apical body or Spitzenk?rper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenk?rper and the transport of vesicles, but as yet there is no postulated mechanism for this. We propose a mechanism by which forces generated by the cytoskeleton are responsible for biasing the movement of vesicles. A mathematical model is derived where the cytoskeleton is described as a viscoelastic fluid. Viscoelastic forces are coupled to the conservation equation governing the vesicle dynamics, by weighting the diffusion of vesicles via the strain tensor. The model displays collapse and aggregation patterns in one and two dimensions. These are interpreted in terms of the formation of the Spitzenk?rper and the initiation of apical branching. Received: 16 September 1996 / Revised version: 20 July 1998     

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.     

Apical growth and mitosis are independent processes in Aspergillus nidulans

Summary. It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.Present address: Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

The mating projections of Saccharomyces cerevisiae and Candida albicans show key characteristics of hyphal growth

Fungi can grow in a variety of growth forms: yeast, pseudohyphae and hyphae. The human fungal pathogen Candida albicans can grow in all three of these forms. In this fungus, hyphal growth is distinguished by the presence of a Spitzenk?rper-like structure at the hyphal tip and a band of septin bars around the base of newly evaginated germ tubes. The budding yeast Saccharomyces cerevisiae grows as yeast and pseudohyphae, but is not normally considered to show hyphal growth. We show here that in mating projections of both C. albicans and S. cerevisiae a Spitzenk?rper-like structure is present at the growing tip and a band of septin bars is present at the base. Furthermore, in S. cerevisiae mating projections, Spa2 and Bni1 form a cap to the 3-dimensional ball of FM4-64 staining, exactly as previously observed in C. albicans hyphae, suggesting that the putative Spitzenk?rper may be a distinct structure from the polarisome. Taken together this work shows that mating projections of both S. cerevisiae and C. albicans show the key characteristics of hyphal growth.     

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.