Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.     

Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa

Calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete Saprolegnia ferax and the ascomycete Neurospora crassa and pseudohyphal Saccharomyces cerevisiae using self-referencing ion-selective probes. S. ferax exhibited well-defined transport zones absent in N. crassa. Ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net Ca2+ flux was either inward (75% of all experiments) or outward. The inward component of the net flux was inhibited by Gd3+, known to inhibit Ca2+ permeable stretch-activated channels. Because the Ca2+ flux is located at the region of maximal hyphal expansion, exocytosis may contribute to Ca2+ efflux, in addition to the stretch-activated channel mediated influx. Maximal inward H+ flux was observed 10-30 microm behind the hyphal tip where peak mitochondria densities taper off at the onset of a vacuolation zone, presumably due to highly localized H+ cotransporter activity. By contrast, N. crassa exhibited no net Ca2+ flux and a consistently inward H+ flux (93% of all experiments) that was homogeneously distributed up to 60 microm behind the hyphal apex. Both hyphal organisms have similar tip morphology and growth rates, and are reported to have tip-high cytosolic Ca2+ gradients associated with growth. Only S. ferax exhibited tip-localized Ca2+ fluxes and a well defined H+ influx zone just behind the tip. Differences in ecological habitats and cytology--S. ferax is an aquatic organism that grows as a migrating plug of cytoplasm while N. crassa is normally terrestrial with a cytoplasm-rich mycelium and highly active cytoplasmic streaming behind the growing margin--may account for the differences in the 'architecture' of ion transport occurring during the process of tip growth. Net Ca2+ efflux and H+ influx of growing S. cerevisiae pseudohyphae were also measured but localization was not possible due to small cell size.     

The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.     

Microtubules are dispensable for the initial pathogenic development but required for long-distance hyphal growth in the corn smut fungus Ustilago maydis

Fungal pathogenicity often involves a yeast-to-hypha transition, but the structural basis for this dimorphism is largely unknown. Here we analyze the role of the cytoskeleton in early steps of pathogenic development in the corn pathogen Ustilago maydis. On the plant yeast-like cells recognize each other, undergo a cell cycle arrest, and form long conjugation hyphae, which fuse and give rise to infectious filaments. F-actin is essential for polarized growth at all these stages and for cell-cell fusion. Furthermore, F-actin participates in pheromone secretion, but not perception. Although U. maydis contains prominent tubulin arrays, microtubules are neither required for cell-cell recognition, nor for cell-cell fusion, and have only minor roles in morphogenesis of yeast-like cells. Without microtubules hyphae are formed, albeit at 60% reduced elongation rates, but they reach only approximately 50 mum in length and the nucleus fails to migrate into the hypha. A similar phenotype is found in dynein mutants that have a nuclear migration defect and stop hyphal elongation at approximately 50 mum. These results demonstrate that microtubules are dispensable for polarized growth during morphological transition, but become essential in long-distance hyphal growth, which is probably due to their role in nuclear migration.     

Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins

The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.     

A small,novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells

Plant cell shapes are defined by their surrounding walls, but microtubules and F-actin both play critical roles in cell morphogenesis by guiding the deposition of wall materials in expanding cells. Leaf epidermal cells have lobed shapes, which are thought to arise through a microtubule-dependent pattern of locally polarized growth. We have isolated a recessive mutation, brk1, which blocks the formation of epidermal cell lobes in the maize leaf. Mutant epidermal cells expand to the same extent as wild-type cells but fail to establish polar growth sites from which lobes arise. In expanding brk1 epidermal cells, microtubule organization differs little from that in wild-type, but localized enrichments of cortical F-actin seen at the tips of emerging lobes in wild-type cells fail to form. These observations suggest a critical role for F-actin in lobe formation and together with additional effects of brk1 on the morphogenesis of stomata and hairs suggest that Brk1 promotes multiple, actin-dependent cell polarization events in the developing leaf epidermis. The Brk1 gene encodes a novel, 8 kD protein that is highly conserved in plants and animals, suggesting that BRK1-related proteins may function in actin-dependent aspects of cell polarization in a wide spectrum of eukaryotic organisms.     

Actin depolymerizing factor is essential for viability in plants, and its phosphoregulation is important for tip growth

Actin depolymerizing factor (ADF)/cofilin is important for regulating actin dynamics, and in plants is thought to be required for tip growth. However, the degree to which ADF is necessary has been elusive because of the presence of multiple ADF isoforms in many plant species. In the moss Physcomitrella patens , ADF is encoded by a single, intronless gene. We used RNA interference to demonstrate that ADF is essential for plant viability. Loss of ADF dramatically alters the organization of the F-actin cytoskeleton, and leads to an inhibition of tip growth. We show that ADF is subject to phosphorylation in vivo , and using complementation studies we show that mutations of the predicted phosphorylation site partially rescue plant viability, but with differential affects on tip growth. Specifically, the unphosphorylatable ADF S6A mutant generates small polarized plants with normal F-actin organization, whereas the phosphomimetic S6D mutant generates small, unpolarized plants with a disorganized F-actin cyotskeleton. These data indicate that phosphoregulation at serine 6 is required for full ADF function in vivo , and, in particular, that the interaction between ADF and actin is important for tip growth.     

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.     

Actin and microtubules in neurite initiation: are MAPs the missing link?

During neurite initiation microtubules align to form a tight bundle and actin filaments reorganize to produce a growth cone. The mechanisms that underlie these highly coordinated cytoskeletal rearrangements are not yet fully understood. Recently, various levels of coordination between the actin- and microtubule-based cytoskeletons have been observed during cellular migration and morphogenesis, processes that share some similarities to neurite initiation. Direct, physical association between both cytoskeletons has been suggested, because microtubules often preferentially grow along actin bundles and transiently target actin-rich adhesion complexes. We propose that such physical association might be involved in force-based interactions and spatial organization of the two networks during neurite initiation as well. In addition, many signaling cascades that affect actin filaments are also involved in the regulation of microtubule dynamics, and vice versa. Although several candidates for mediating these effects have been identified in non-neuronal cells, the general mechanism is still poorly understood. In neurons certain plakins and neuron-specific microtubule associated proteins (MAPs), like MAP1B and MAP2, which can bind to both microtubules and F-actin, are promising candidates to play key roles in the specific cytoskeletal rearrangements controlling the transition from an undifferentiated state to neurite-bearing morphology. Here we review the effects of MAPs on microtubules and actin, as well as the coordination of both cytoskeletons during neurite initiation.     

Cytoskeleton in Pollen and Pollen Tubes of Ginkgo biloba L.

The distribution of F-actin and microtubules was investigated in pollen and pollen tubes of Ginkgo biloba L. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. A dense F-actin network was found in hydrated Ginkgo pollen. When Ginkgo pollen was germinating,F-actin mesh was found under the plasma membrane from which the pollen tube would emerge. After pollen germination, F-actin bundles were distributed axially in long pollen tubes of G. biloba. Thick F-actin bundles and network were found in the tip of the Ginkgo pollen tube, which is opposite to the results reported for the pollen tubes of some angiosperms and conifers. In addition, a few circular F-actin bundles were found in Ginkgo pollen tubes. Using immunofluorescence labeling, a dense microtubule network was found in hydrated Ginkgo pollen under confocal microscope. In the Ginkgo pollen tube, the microtubules were distributed along the longitudinal axis and extended to the tip. These results suggest that the cytoskeleton may have an essential role in the germination of Ginkgo pollen and tube growth.     

Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze- substitution of hyphal tip cells

The effects of methyl benzimidazole-2-ylcarbamate (MBC), one of only a few agents that are active against microtubules of fungi, were analyzed at the ultrastructural level in freeze-substituted hyphal tip cells of Fusarium acuminatum. Nontreated and control cells had numerous microtubules throughout. After just 10 min of exposure to MBC, almost no cytoplasmic microtubules were present, except near spindle pole bodies. After 45 min of exposure to MBC, no microtubules were present in hyphal tip cells, but they were present in the relatively quiescent subapical cells. These observations suggested that there are different rates of turnover for cytoplasmic microtubules in apical and subapical cells and for microtubules near spindle pole bodies and that MBC acts by inhibiting microtubules assembly. A statistical analysis of the distribution of intracytoplasmic vesicles in thick sections of cells treated with MBC, D2O or MBC + D2O was obtained by use of a high- voltage electron microscope. More than 50% of the vesicles in the apical 30 micrometers of control cells were found to lie within 2 micrometers of the tip cell apex. MBC treatment caused this vesicle distribution to become uniform, resulting in a substantial increase in the number of vesicles in subapical regions. The reduction in the number of cytoplasmic microtubules, induced by MBC, apparently inhibited intracellular transport of these vesicles and rendered random the longitudinal orientation of mitochondria. In most cases, D2O appeared capable of preventing these MBC-effects through stabilization of microtubules. These observations support the "vesicle hypothesis" of tip growth and establish a transport role for cytoplasmic microtubules in fungal 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.     

Calmodulin concentrates at the apex of growing hyphae and localizes to the Spitzenkörper in Aspergillus nidulans

The calmodulin (CaM) localization pattern in the growing hyphal tip of Aspergillus nidulans was studied with the functional GFP::CaM fusion protein. A faint tip-high gradient of CaM was found in the growing hyphal tip, with CaM highly localized in the region corresponding to the Spitzenk?rper forming a bright granule. The position of highly concentrated CaM in the extreme apex seemed to determine the orientation of the hypha. The normal pattern of CaM localization was also shown to be dependent on the integrated actin cytoskeleton. When the growth of the hyphal tip ceased, CaM failed to localize in the bright granule and was evenly distributed in the hyphal tip. These findings suggest that CaM may play an important role in establishing and maintaining apical organization, morphogenesis, and growth in Aspergillus nidulans.     

Aspergillus myosin-V supports polarized growth in the absence of microtubule-based transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV.     

Role of microtubules in tip growth of fungi

Polarized cell growth is observed ubiquitously in all living organisms. Tip growth of filamentous fungi serves as a typical model for polar growth. It is well known that the actin cytoskeleton plays a central role in cellular growth. In contrast, the role of microtubules in polar growth of fungal tip cells has not been critically addressed. Our recent study, using a green fluorescent protein (GFP)-labeled tubulin-expressing strain of the filamentous fungus Aspergillus nidulans and treatment with an anti-microtubule reagent, revealed that microtubules are essential for rapid hyphal growth. Our results indicated that microtubule organization contributes to continuous tip growth throughout the cell cycle, which in turn enables the maintenance of an appropriate mass of cytoplasm for the multinucleate system. In filamentous fungi, the microtubule is an essential component of the tip growth machinery that enables continuous and rapid growth. Recent research developments are starting to elucidate the components of the tip growth machinery and their functions in many organisms. This recent knowledge, in turn, is starting to enhance the importance of fungal systems as simple model systems to understand the polar growth of cells.     

Critical evaluation of the VSC model for tip growth

The vesicle supply centre (VSC) model (Bartnicki-Garcia et al., 1989) for hyphal tip growth is powerful because it can model diverse developmental morphologies and predicts cellular organization based in current cell biology. It predicts that tip growth results from the random distribution of cell surface synthesizing vesicles from a point in the tip, the VSC, which determines their pattern of impact and fusion at the plasma membrane. We derive equations for tip-high gradients of vesicle fusions, generated by mechanisms not related to a supply centre, which create typical hyphal morphologies. These equations direct the conceptual basis for tip growth to vesicle fusion gradients, presumably mediated by a putative membrane skeleton associated with the plasma membrane. We also show that the organization and behaviour of motile organelles in growing hyphal tips of the oomycete,Saprolegnia ferax, argue against the presence of an apparatus capable of generating the distribution of vesicles postulated by the VSC model. We conclude that the VSC model is unlikely to describe the mechanistic basis of tip growth inS. ferax, and therefore, at best, it is not universally applicable.     

Hyphal tip growth and nuclear migration

Recent molecular and cytological studies have greatly advanced our understanding of hyphal tip growth and nuclear migration in filamentous fungi. Mutants involved in various aspects of hyphal tip growth have been isolated. Genes involved in nuclear migration continue to be identified, including putative regulators. The role of microtubules and microtubule motor proteins in hyphal tip growth has also been studied.     

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenk?rper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenk?rper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.     

Cloning and characterization of the actin gene from Puccinia striiformis f. sp. tritici

The fungus Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Urediniospores are the most common spore type involved in the epidemiology of this disease. Tip growth of germ tubes of germinated urediniospores is a key step during infection of wheat, but few studies have investigated it so far. Recent research has found that actin is closely associated with hyphal tip growth. In this study, we have cloned and obtained the full-length actin cDNA from P. striiformis f. sp. tritici and characterized its expression. Furthermore, actin filament (F-actin) patterns were visualized microscopically during germ tube formation. The most conspicuous actin-containing structures were actin patches. They were mainly concentrated near the hyphal tip and scattered throughout the cortex. By using cytochalasin B, we observed that depolymerization of F-actin greatly reduced the germination rate of urediniospores and disrupted the transport of vesicles to the germ tube tip, indicating that F-actin played a key role in the tip growth of P. striiformis f. sp. tritici. This work helps us to understand the tip growth mechanism of P. striiformis f. sp. tritici, and may provide a theoretical framework for designing novel pesticides.     

Distribution of F-actin and Microtubules in Pollen and Pollen Tube of Lilium davidii

F-actin and microtubules are important components of pollen tube, which have very important function in cytoplasm streaming of pollen tube. The authors observed the distribution of Factin and microtubules in the pollen tube of Lilium davidii Duch. by immunofluorescence technique and confocol laser scanning microscopy, through which some new results were obtained. 1. Chemical fixation could preserve F-actin well in pollen tube, so the relation between F-actin and microtubules could be studied by the methods of chemical fixation and fluorescence labelling in pollen tube. 2. F-actin bundles were absent near the pollen tube tip, while microtubules were abundant and web formed in the pollen tube tip. The authors found that the terminal of microtubules was closely associated with the plasma membrane in the pollen tube tip. 3. Only a few F-actin bundles co-exist with the microtubules in the pollen tube of Lilium davidii. The results provided new evidence for the fimction and relationship between F-actin and microtubules in the pollen tube.