Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents

Summary. Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of β-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-004-0081-3 Correspondence and reprints: Department of Biological Sciences, University of Delaware, Newark, DE 19716, U.S.A. Present address: Paradigm Genetics Inc., Research Triangle Park, North Carolina, U.S.A.     

TAXOL REDUCES THE RATE OF CYTOKINESIS IN PtK1CELLS

Mitotic PtK₁cells were treated both during mid-anaphase and at furrow initiation with the potent microtubule (MT) stabilizing agent, taxol, to determine the role of MTs in the rate of cytokinetic events. Rates of cytokinesis (μm/min) were measured by changes in furrow diameter. Incubation of PtK₁cells during mid-anaphase with 5 μg/ml taxol slows the rate of cytokinesis by an average of 43%. Instead of furrow initiation to midbody formation taking an average of 10.7 min (1.6 μm/min), furrowing to midbody formation was completed in an average of 19.0 min (0.9 μm/min), which does not include the 7-min period between taxol application in mid-anaphase and furrow initiation. Application of 5 μg/ml taxol to cells at furrow initiation had a reduced effect on decreasing the rate of cytokinesis and midbody formation; furrowing to midbody formation took an average of 14.6 min (1.2 μm/min). These data suggest that delays in the rate of cytokinesis is dependent on the mitotic stage at which taxol is applied. Ultrastructural analysis shows that taxol treatment of anaphase cells prevents midbody formation during early G₁, yet MT number and organization in the furrowed region is not significantly altered from untreated cells. There is little change in the organization and amount of contractile ring microfilaments, yet filaments are also found parallel to midbody MTs. Our results may be explained by the fact that taxol tends to stabilize MTs which probably affects the rate at which they depolymerize in the terminal phases of cytokinesis. Reduction in depolymerization rates of a stable population of MTs could serve to regulate the rate of cytokinesis.     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Increased synthetic capacity for metallothionein in cultured liver cells following dimethyl sulfoxide pretreatment

Cultured TRL 1215 cells in log phase of growth were exposed to dimethyl sulfoxide (DMSO; 14-280 mM) followed 48 h later by cadmium (10 micron). Intracellular concentrations of metallothionein (MT) were measured 24 h after cadmium addition. Cadmium alone caused a 10-fold increase in the levels of MT, while DMSO alone had no effect on cellular MT levels. DMSO pretreatment followed by cadmium exposure, however, resulted in MT levels that were elevated by a factor of as much as 25-fold those observed in control cells. Concurrent treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of DMSO pretreatment on cadmium induction of MT, indicating the requirement of DNA synthesis. An enhancement of the cellular accumulation of the metal ion did not account for the increased cadmium-induced MT synthesis in DMSO-pretreated cells as these cells did not show significantly increased uptake of cadmium during the initial period of exposure. DMSO pretreatment enhances cadmium induction of MT synthesis through a mechanism that appears to be dependent on the synthesis of DNA.     

Microtubule plus end‐tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans

Cytoplasmic microtubules (MTs) serve as a rate‐limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end‐tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end‐tracking of CLIPA, the Kinesin‐7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi.     

Disease measurement in a study of apple scab epidemics

The growth and yield increases in red raspberry which followed the repeated application of benomyl were not due to the suppression of pests. Benomyl had little effect on numbers of aphids and nematodes which, anyway, were insufficiently abundant to cause damage. Benomyl did, however, decrease the earthworm population. In the following years, when benomyl was no longer applied, significantly more canes died from midge blight in the plots treated most frequently with benomyl than in the untreated. This was probably a consequence of increased infestation by larvae of the raspberry cane midge (Resseliella theobaldi) in the benomyl-treated plots because benomyl increased the extent of cane splitting and hence the number of egg-laying sites for cane midge.     

Induction of multiple germ tubes in Neurospora crassa by antitubulin agents

The antitubulin fungicide benomyl suppressed the linear growth of Neurospora crassa wild type strain St. Lawrence 74 at micromolar concentrations. The rate of germination of macroconidia was not affected. Macroconidia exposed to 1.7 microM benomyl for 5 h formed multiple germ tubes. When germlings incubated for 4 h were exposed to 1.7 microM benomyl for 3 h, their germ tube stopped growing, swelled and emitted several branches. Normal linear growth was restored after removal of the fungicide. Linear growth of N. crassa was resistant up to 16 microM nocodazole. This drug induced multipolar germination at 8 microM, and griseofulvin only at 140 microM. The microtubule (MT) cytoskeleton of N. crassa could be revealed by indirect immunofluorescence with the monoclonal antibody YOL 1/34 directed against yeast alpha-tubulin. We detected no striking effects of the benomyl treatments on MT organization. The MT-stabilizing agents deuterium oxide (D2O) and cAMP have no antagonistic effects on the benomyl-induced multipolar germination. The positioning of nuclei and mitochondria was determined from the DAPI and Rhodamine 123 fluorescence patterns, respectively. Benomyl inhibited nuclear migration into multiple germ tubes. Quantitative scanning cytophotometry revealed a peak in the intensity of the mitochondria-associated Rhodamine 123 fluorescence near the apex of untreated germlings. This peak disappeared in multiple germ tubes. Benomyl-resistant mutant bml 511 (r), mutated in its beta-tubulin gene, germinated normally in the presence of the fungicide. This strongly suggests that multiple germ tube formation was due to the effect of benomyl on beta-tubulin. Benomyl-resistant strain 74-3, constructed by reintroducing the cloned mutant N. crassa beta-tubulin gene into the cells by transformation, displayed a partial resistance to benomyl with respect to multipolar germination. Its rate of germination was slow (50% germination reached after 4 h at 37 degrees C as compared to 2.5 h for the wild type). In contrast to N. crassa, the other ascomycete Aspergillus nidulans is nocodazole-sensitive (linear growth suppressed at 1.6 microM). It did not respond to the MT inhibitors benomyl and nocodazole with respect to the pattern of germ tube emergence. Our results suggest that microtubule or membrane beta-tubulin is involved in the maintenance of developmental polarity during germ tube emergence and growth of N. crassa.     

The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.     

Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors

Summary Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the quickstaining method, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the methacrylate method, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.Abbreviations CB cytochalasin B - CD cytochalasin D - DMSO dimethyl sulfoxide - FA formaldehyde - GA glutaraldehyde - LAT-A latrunculin A - LAT-B latrunculin B - MFs microfilaments - MT microtubule Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

The effect of benomyl on growth and ultrastructure of two isolates of Phytophthora infestans from Egypt

The effect of benomyl as a fungicide on the growth rate and ultrastructure of two isolates (P1319 and P623) of Phytophthora infestans is compared. Benomyl caused a concentration-dependent inhibition of the mycelial growth of both isolates. The isolate P1319 was found to be more sensitive to benomyl than the isolate P623. Ultarstructural studies confirmed these observations. The hyphae of isolate P1319 subjected to 100 and 500 ppm benomyl showed more severe changes in the cytoplasm than those of isolate P623. An increase in lipid bodies and vacuoles in the hyphal cytoplasm was the characteristic phenomenon after treatment with benomyl, particularly at a concentration of 500 ppm.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Effects of aldicarb and benomyl on nematodes, vesicular arbuscular mycorrhizas and the growth and yield of forage maize

The effects of aldicarb and benomyl on plant-parasitic nematodes, vesicular arbuscular mycorrhiza and the growth of forage maize were measured in 1980—1982 in two field experiments at Woburn, Bedfordshire and in a pot experiment using loamy sand soil from the field site. The most numerous migratory nematode, Tylenchorhynchus dubius increased three to four-fold during each season in untreated soil and was effectively controlled by aldicarb. Pratylenchus species were fewer but equally well controlled. The cereal cyst-nematode (Heterodera avenae), a serious maize pathogen in Northern France, was relatively scarce in untreated roots and was further decreased by aldicarb treatment; post-harvest H. avenae egg numbers were not affected by treatments; they declined equally because maize is such a poor host. Significant yield benefits (up to 37%) followed aldicarb treatment and were ascribed to nematode control in the absence of attribution to insect or other pests. Benomyl did not increase yields nor did it significantly affect the incidence of mycorrhiza. The results confirm that considerable losses of forage maize can be caused by nematodes in light soil and that aldicarb is an effective nematicide even at the rate of 1·7 kg a.i./ha.     

The role of +TIPs in directional tip expansion

Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron‐like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth – the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end‐tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.     

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.     

Creation of a non-mycorrhizal control for a bioassay of AM effectiveness

γ -irradiation of soil by 10 and 3 kGy, and the use of a myc⁻ mutant. The methods were examined on clay and loam. Two management histories were included with both soils to study the ability of the methods to differentiate AM effectiveness. For each soil type, two pot experiments were conducted in field soil, one to investigate the effects of the methods on soil nutrient status, and the other to study the effects on mycorrhization and plant response. The test plants, flax (Linum usitatissimum) and pea (Pisum sativum) myc⁺ and myc⁻ mutants, were grown in 1-l pots for 4 weeks in a growth chamber. To test the ability of the bioassay to reflect differences in AM effectiveness in the field, the mutants and benomyl were also studied in the field from which the loam for the pot experiments was obtained. The bioassay accurately represented the situation in the field and the use of benomyl appeared to be the most appropriate method currently available. The advantages were the ability to use a test plant responsive to AM, the use of less elevated nutrient concentrations than with irradiation, and thus the possibility to use untreated soil as the mycorrhizal treatment. The pea mutants proved unresponsive to AM, and reinoculation to irradiated soil resulted in only half the colonization rate in untreated soil. Benomyl may, however, lead to an underestimation of AM effectiveness because the control is not totally non-mycorrhizal. Its use also carries with it health and environmental risks. Received: 9 February 1999 / Accepted: 27 September 1999     

Depletion of Aspergillus nidulans cotA causes a severe polarity defect which is not suppressed by the nuclear migration mutation nudA2

The Aspergillus nidulans homologue of Neurospora crassa cot-1, cotA, encoding a member of the NDR protein kinase family, has been cloned and expressed under the control of the conditional alcA promoter. Depletion of CotA by repression of the alcA promoter led to a severe growth defect accompanied by loss of polarity. Germlings show greatly enlarged volume of the spores and hyphae, accompanied by an increase in number of nuclei per compartment, though the nucleus/volume ratio is not significantly altered. The depleted CotA phenotype was not suppressed by a nuclear migration mutation nudA2. Double mutants showed an additive, defective phenotype, unlike the suppression of the cot-1 ts mutation by ropy mutations seen in N. crassa, suggesting a different relationship between nuclear migration and the cot signalling pathway in A. nidulans. A functional CotA–GFP fusion protein was found in punctate regions of fluorescence similar to the distribution reported for human NDR2, and as a cap at the hyphal tip.     

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.     

Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomyceteSchizophyllum commune

Summary The effects of nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some nocodazole-treated hyphae. After 2 h growth in the presence of nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.Abbreviations BFA brefeldin A - EM electron microscopy - ER endoplasmic reticulum - IIF indirect immunofluorescence - MBC methylbenzimidazole-2-ylcarbamate - MT microtubule - MVB multivesicular body - RER rough endoplasmic reticulum     

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.     

Dynamics of mitochondria during the <Emphasis Type="Italic">Neurospora crassa</Emphasis> tip growth

Tip growth of the mycelial fungus N. crassa vegetative hyphae is realized owing to the combined activities of tens of the cells and diverse intracellular structures, such as microvesicles, microtubules, microfilaments, mitochondria, etc. Using a vital mitochondrial probe Mitotracker Red (10 μM, 10 min) we have found that the same mitochondria can move hundreds of microns along the hyphae within several hours. Analysis of the mitochondria distribution along 100 μm of the tips in intact hyphae as well as in the isolated apical fragments has shown that the congregation of mitochondria in the growing tips can correlate with the rate of elongation. These data together with the earlier electrophysiological estimations of the membrane potential gradients along the hyphal tips suggest that the electrical gradients along the hyphal apical part can be involved in the regulation of the energy supply of the tip growth.