Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.

Kinesin is a force-generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin-deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.     

Effects of exogenous calcium ions on tip growth,intracellular Ca2+ concentration,and actin arrays in hyphae of the fungus Saprolegnia ferax

The maintenance of growth of hyphae of Saprolegnia ferax was dependent on the presence of external Ca²⁺ and the growth rate increased with increased external Ca²⁺ up to 5 × 10⁻² m Ca²⁺. When Ca²⁺ was greater than 5 × 10⁻² m, growth rates decreased. Internal membrane-associated Ca²⁺ was localized with chlortetracycline. Internal Ca²⁺ became depleted in hyphae grown in the absence of Ca²⁺ and was increased in hyphae grown in high concentrations of Ca²⁺, showing that internal Ca²⁺ can be modulated by external Ca²⁺. However, the range of the internal change was not as great as the range of external concentration used, indicating that the hyphae are capable of regulating Ca²⁺ in the presence of a large concentration gradient. In the absence of external Ca²⁺, growth can occur for a limited time through use of internal Ca²⁺. The actin cytoskeleton was altered in hyphae grown in both high and low Ca²⁺. Hyphae grown in 10⁻³ m Ca²⁺ had more actin in their apical network and peripheral plaques of actin were further from the apex than in more slowly growing hyphae in 10⁻¹ m and 0 Ca²⁺. The tips of hyphae growing in low Ca²⁺ also had a tendency to swell, giving these hyphae irregular shapes. Ca²⁺ is known to affect cell wall rigidity and the consistency of actin gels, two factors that can be expected to affect hyphal growth. External Ca²⁺ does play a role in hyphal growth possibly directly by acting on the cell wall and indirectly by altering internal Ca²⁺, thus affecting the actin cytoskeleton and possibly other growth processes.     

Myosin-10 and actin filaments are essential for mitotic spindle function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.     

Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa

Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa. Presence of copper in culture media, but not of pro-oxidants like H2O2 or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid. Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements. Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants. These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements.     

A mutation in the Neurospora crassa actin gene results in multiple defects in tip growth and branching

Actin has a pivotal function in hyphal morphogenesis in filamentous fungi, but it is not certain whether its function is equivalent to that of a morphogen, or if it is simply part of a mechanism that executes orders given by another regulatory entity. To address this question we selected for cytochalasin A resistance and isolated act1, the first actin mutant in Neurospora crassa. This mutant branches apically and shows an altered distribution of actin at the tip. Based on the properties of this mutant, we propose a model of tip growth and branching in which actin effects tip growth by regulating the rate of vesicle flow from proximal to distal regions of a hypha, thereby controlling the tip-high gradient of cytoplasmic calcium. The actin-controlled calcium gradient at the tip is necessary for maintenance of tip growth as well as the dominance of one polarized site at the hyphal tip. The phenotype of act1 indicates that actin controls the balance between lateral and apical branching.     

Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.

The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.     

Lack of the GTPase RHO-4 in Neurospora crassa causes a reduction in numbers and aberrant stabilization of microtubules at hyphal tips

The multinucleate hyphae of the filamentous ascomycete fungus Neurospora crassa grow by polarized hyphal tip extension. Both the actin and microtubule cytoskeleton are required for maximum hyphal extension, in addition to other vital processes. Previously, we have shown that the monomeric GTPase encoded by the N. crassa rho-4 locus is required for actin ring formation during the process of septation; rho-4 mutants lack septa. However, other phenotypic aspects of the rho-4 mutant, such as slow growth and cytoplasmic bleeding, led us to examine the hypothesis that the microtubule (MT) cytoskeleton of the rho-4 mutant was affected in morphology and dynamics. Unlike a wild-type strain, the rho-4 mutant had few MTs and these few MTs originated from nuclear spindle pole bodies. rho-4 mutants and rho-4 strains containing a GTP-locked (activated) rho-4 allele showed a reduction in numbers of cytoplasmic MTs and microtubule stabilization at hyphal tips. Strains containing a GDP-biased (negative) allele of rho-4 showed normal numbers of MTs and minor effects on microtubule stabilization. An examination of nuclear dynamics revealed that rho-4 mutants have large, and often, stretched or broken nuclei. These observations indicate that RHO-4 plays important roles in regulating both the actin and MT cytoskeleton, which are essential for optimal hyphal tip growth and in nuclear distribution and morphology.     

Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa

Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth. This requirement may be met by the localization and/or activation of an apical SNARE-based machinery. We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa. Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants. All surviving transformants grew slowly, conidiated poorly, and were male sterile. In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation. For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores. Those that germinated produced slow-growing hyphae with abnormal branching. The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.     

cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase.

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32 degrees C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-1 by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-1 are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation.     

vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation

con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.     

Sphingolipids are essential for differentiation but not growth in Leishmania

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.     

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.     

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca²⁺ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.     

Nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in Neurospora crassa

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions.     

The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.     

Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.

Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical beta(Heavy)-spectrin (beta(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of beta(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of beta(H) prevents the stable recruitment of alpha-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (alphabeta(H))(2)-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.     

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.     

The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.     

The relation between turgor and tip growth inSaprolegnia ferax: Turgor is necessary,but not sufficient to explain apical extension rates

Linear growth rate ofSaprolegnia was reduced in direct proportion to increased osmotic pressure (II) of the medium, when sorbitol or PEG-400 was used as osmotica. However, increasing medium II reduced hyphal turgor only to a minimum positive level, which was maintained while extension rates continued to decline. TPA, a K⁺-channel agonist effective onSaprolegnia protoplasts, also caused dose-dependent linear growth rate reductions but did not substantially affect turgor. When turgor was compared with linear growth rate in the osmoticum experiments, there was a positive correlation only for hyphae growing faster than 12 μm/min; below this, there was a twofold range in extension rate despite essentially constant turgor. As well, TPA-treatments produced a twofold reduction in hyphal extension rate without substantially affecting turgor. Turgor should be consistent within a coenocyte, and is steady under constant growth conditions. However, under such conditions, we found average variations of fivefold in extension rate between hyphae, and twofold for hyphae over time. These results suggest that turgor is not the prime determinant of tip extension rate, and they are consistent with cytoskeletal regulation of that rate. Linear growth rates ofSaprolegnia colonies were similar on basal medium containing 1% (w/v) glucose, sorbitol, or PEG and only slightly faster than without added carbohydrate. Increasing medium II with glucose also reduced hyphal extension rate.