The mitotic apparatus in fungi, Ceratocystis fagacearum and Fusarium oxysporum.

Vegetative nuclei of fungi Ceratocystis fagacearum and Fusarium oxysporum were studied both in the living condition with phase-contrast microscopy and after fixation and staining by HCl-Giemsa, aceto-orcein, and acid fuchsin techniques. Nucleoli, chromosomes, centrioles, spindles, and nuclear envelopes were seen in living hyphae of both fungi. The entire division process occurred within an intact nuclear envelope. Spindles were produced between separating daughter centrioles. At metaphase the chromosomes became attached to the spindle at different points. In F. oxysporum the metaphase chromosomes were clear enough to allow counts to be made, and longitudinal splitting of the chromosomes into chromatids was observed. Anaphase was characterized in both fungi by separation of chromosomes to poles established by the centrioles, and in F. oxysporum anaphase separation of chromosomes was observed in vivo. Continued elongation of the spindles further separated the daughter nuclei. Maturing daughter nuclei of both fungi were quite motile; and in C. fagacearum the centriole preceded the bulk of the nucleus during migration. The above observations on living cells were corroborated by observations on fixed and stained material.

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.

Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments.

The anaphase-telophase spindle usually elongates, and it has been assumed that the spindle pushes the incipient daughter nuclei apart. To test this assumption, we used a laser microbeam to sever the central spindle of the fungus, Fusarium solani, and measured the rate of separation of incipient daughter nuclei. When the microbeam was aimed beside the spindle separation occurred at a rate (8.6 micrometer/min) that did not differ significantly from the rate (7.6 micrometer/m) in unirradiated cells. But when the spindle was irradiated, it broke, and the separation was much faster (22.4 micrometer/min). Irradiation of cytoplasm lateral to one spindle pole resulted in a 1.5 micrometer/min reduction in the rate (6.1 micrometer/min) of separation. From these and other data, we infer that extranuclear forces, presumably involving astral microtubules, pull on the incipient daughter nuclei and that the central spindle limits the separation rate. Astral microtubules are associated with the plasma membrane or, sometimes, with the rough endoplasmic reticulum. Most of the spindle microtubules that are present at metaphase are depolymerized during anaphase and early telophase.

Ultrastructure and time course of mitosis in the fungus Fusarium oxysporum.

Mitosis in Fusarium oxysporum Schlect. was studied by light and electron microscopy. The average times required for the stages of mitosis, as determined from measurements made on living nuclei, were as follows: prophase, 70 sec; metaphase, 120 sec; anaphase, 13 sec; and telophase, 125 sec, for a total of 5.5 min. New postfixation procedures were developed specifically to preserve the fine-structure of the mitotic apparatus. Electron microscopy of mitotic nuclei revealed a fibrillo-granular, extranuclear Spindle Pole Body (SPB) at each pole of the intranuclear, microtubular spindles. Metaphase chromosomes were attached to spindle microtubules via kinetochores, which were found near the spindle poles at telophase. The still-intact, original nuclear envelope constricted around the incipient daughter nuclei during telophase.

Mechanically induced wall appositions of plant cells can prevent penetration by a parasitic fungus.

Localized, paramural wall appositions resembling appositions commonly induced by fungal attack, were induced in kohlrabi (Brassica oleracea L. gongyloides) by mechanical wounding (bending) of the root hairs before the hairs were inoculated with zoospores of a compatible parasitic fungus. The appositions were effective in preventing fungal penetration at the wound sites, which shows that wall appositions can prevent fungal ingress into plant cells.

Laser microsurgery in cell and developmental biology.

New applications of laser microbeam irradiation to cell and developmental biology include a new instrument with a tunable wavelength (217- to 800-nanometer) laser microbeam and a wide range of energies and exposure durations (down to 25 X 10(-12) second). Laser microbeams can be used for microirradiation of selected nucleolar genetic regions and for laser microdissection of mitotic and cytoplasmic organelles. They are also used to disrupt the developing neurosensory appendages of the cricket and the imaginal discs of Drosophila.

A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis.

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Organelle motility within mitotic asters of the fungus Nectria haematococca.

The mitotic asters of the fungus, Nectria haematococca, pull on the spindle pole bodies during anaphase B and help to elongate the central spindle. Because these asters are invisible in vivo, studies of their functions during mitosis have been limited. Invisible asters in other organisms can be studied in vivo because of visible, membranous organelles that are held or transported within them. This is the first report of intra-astral motility of organelles in a fungus, and it lays the foundation for additional studies of aster function in vivo. Using phase-contrast, video-enhanced microscopy, we observed directed motility of mitochondria, small vesicles of various kinds, lipid bodies and, rarely, small vacuoles within the astral region during anaphase B. Both bidirectional motility--toward and away from the spindle pole body--and reversal of direction by an individual organelle were common. Organelles usually did not tend to accumulate either within the aster or near the spindle pole. They were drawn toward the spindle pole body from up to 5.0 microns away. Average velocities were 2.3 to 3.2 microns/s, depending on the organelle and its direction of movement. Transmission electron microscopy revealed apparent cross bridging between astral microtubules and mitochondria, vesicles, endoplasmic reticulum, microbodies, and vacuoles. The antimicrotubule drug, methylbenzimidazole-2-ylcarbamate (MBC), destroyed astral microtubules and virtually eliminated intra-astral motility in vivo, whereas the antiactin drug, cytochalasin E, did not greatly affect the frequency of intra-astral motility episodes. The results suggest a role for astral microtubules in intra-astral motility in this fungus.

Mitosis in filamentous fungi: how we got where we are.

This review traces the principal advances in the study of mitosis in filamentous fungi from its beginnings near the end of the 19(th) century to the present day. Meiosis and mitosis had been accurately described and illustrated by the second decade of the present century and were known to closely resemble nuclear divisions in higher eukaryotes. This information was effectively lost in the mid-1950s, and the essential features of mitosis were then rediscovered from about the mid-1960s to the mid-1970s. Interest in the forces that separate chromatids and spindle poles during fungal mitosis followed closely on the heels of detailed descriptions of the mitotic apparatus in vivo and ultrastructurally during this and the following decade. About the same time, fundamental studies of the structure of fungal chromatin and biochemical characterization of fungal tubulin were being carried out. These cytological and biochemical studies set the stage for a surge of renewed interest in fungal mitosis that was issued in by the age of molecular biology. Filamentous fungi have provided model studies of the cytology and genetics of mitosis, including important advances in the study of mitotic forces, microtubule-associated motor proteins, and mitotic regulatory mechanisms.

The ultrastructure of mitosis in Plasmodiophora brassicae (Plasmodiophorales).

Mitosis was examined in plasmodia of Plasmodiophora brassicae within artifically inoculated cabbage roots, using light- and electron microscopy. Mitotic nuclear divisions are characterized by a persistent nucleolus, bipolar centrioles paired end-to-end, densely staining chromatin, and a complex array of membranes that surround and ramify through the spindle. Chromatin begins to condense in prophase, and is aligned at metaphase in a reticulate plate on the nuclear equator. The chromatin is not resolvable into distinct chromosomes at metaphase, and a chromosome count is not possible. Large amounts of membrane cisternae within the spindle are most clearly visible at metaphase, and apparently represent the remains of the nuclear envelope. The nuclear envelope is disrupted during prometaphase and may become entangled in the spindle when centriolar microtubules enter the nucleus. Several concentric sheets of perinuclear endoplasmic reticulum surround the spindle and give the mitotic nucleus the superficial appearance of having an intact nuclear envelope. This interpretation of the identity of nucleus-associated membranes differs from those previously reported for other protists, including members of the Plasmodiophorales.

Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study.

Rat kangaroo kidney epithelium (PtK2) cells develop prominent asters and spindles during anaphase B of mitosis. It has been shown that severing the spindle at early anaphase B in living PtK1 cells results in a dramatic increase in the rate of pole-pole separation. This result suggested that the asters pull on the spindle poles, putting tension on the spindle, while the spindle acts as a governor, limiting the rate of pole separation. To further test these inferences, we used a UV-laser microbeam to damage one of the two asters in living PtK2 cells at early anaphase B and monitored the effects on individual spindle pole movements, pole-pole separation rates and astral microtubules (MTs). Irradiation at the estimated position of a centrosome greatly reduced its array of astral MTs and nearly stopped the movement of the irradiated pole, whereas the sister pole retained its normal array of astral MTs and actually accelerated. Control irradiations, either close to the estimated position of the centrosome or beside the spindle at the equator, had little or no effect on either spindle pole movements or astral MTs. These results support the inferences that during anaphase B in living PtK cells, the central spindle is under tension generated by pulling forces in the asters (presumably MT-mediated) and that the spindle generates counterforces that limit the rate of pole separation. The results also suggest that the central spindle in living PtK cells may be able to generate a pushing force.

Calcium Transport in Protoplasts Isolated from ml-o Barley Isolines Resistant and Susceptible to Powdery Mildew.

Free cytoplasmic calcium has been postulated to play a role in preventing powdery mildew in a series of homozygous ml-o mutants of barley, Hordeum vulgare L. Protoplasts isolated from 7-day-old plants of the ml-o resistant-susceptible (R-S) barley isolines, Riso 5678/3(*) x Carlsberg II R and S, were used to test for differences in fluxes of Ca(2+) across the plasmalemma. Greater influx or lesser efflux might account for a higher free cytosolic Ca(2+) postulated to exist in ml-o R mutants. Uniform patterns of uptake were maintained for 3 hours from solutions of 0.2 and 2 millimolar Ca(2+). Washout curves of (45)Ca(2+) from R and S protoplasts revealed three compartments-presumed to represent release from the vacuole, organelles, and the cytoplasm (which included bound as well as free Ca(2+)). Uptake and washout did not differ between isolines. On the basis of recent determinations of submicromolar levels of free cytoplasmic Ca(2+) and our initial rates of (45)Ca-labeled Ca(2+) uptake, we show that measurement of the unidirectional influx of Ca(2+) across the plasmalemma is not feasible because the specific activity of the pool of free cytoplasmic calcium increases almost instantaneously to a level that would result in a significant, but unknown, efflux of label. Similarly, measurement of the efflux of Ca(2+) across the plasmalemma is not possible since the activity of the pool of free cytoplasmic calcium is a factor of 350 smaller than the most rapid component of the washout experiment. This pool of cytoplasmic free Ca(2+) will wash out too rapidly and be too small to detect under the conditions of these experiments.

Cell wall appositions and plant disease resistance: Acoustic microscopy of papillae that block fungal ingress.

Plant cells react to localized stress by forming wall appositions outside their protoplasts on the inner surface of their cellulose walls. For many years it has been inferred that appositions elicited by encroaching fungi, termed "papillae," may subsequently also deter them and thus represent a disease-resistance mechanism. Recently, it has been shown that preformed, oversized papillae, experimentally produced in coleoptile cells of compatible barley, Hordeum vulgare, can completely prevent direct entry of Erysiphe graminis f. sp. hordei that ordinarily penetrates and causes disease. To discover how these papillae may function, acoustic microscopy was used to contrast their in vivo elastic properties with those of ineffective normal papillae and contiguous cell wall. Raster and line scans showed intense acoustic activity at sites of preformed papillae; scans in selected focal planes identified this activity with the papillae, not with subtending cell wall. Minimal acoustic activity was found in normal papillae. It is suggested that some wall appositions could serve in disease resistance as viscoelastic barriers to mechanical forces exerted by the special penetration structures of advancing pathogenic fungi.

A cytoplasmic dynein required for mitotic aster formation in vivo.

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo.

The existence, structural basis and function of astral forces that are active during anaphase B in the fungus, Nectria haematococca, were revealed by experiments performed on living cells. When one of the two asters of a mitotic apparatus was damaged, the entire mitotic apparatus migrated rapidly in the direction of the opposing astral forces, showing that the force that accelerated spindle pole body separation in earlier experiments is located in the asters. When a strong solution of the antimicrotubule drug, MBC, was applied at anaphase A, tubulin immunocytochemistry showed that both astral and spindle microtubules were destroyed completely in less than a minute. As a result, separation of the spindle pole bodies during anaphase B almost stopped. By contrast, disrupting only the spindle microtubules with a laser microbeam increased the rate of spindle pole body separation more than fourfold. Taken together, these two experiments show that the astral forces are microtubule-dependent. When only one of the two or three bundles of spindle microtubules was broken at very early anaphase B, most such diminished spindles elongated at a normal rate, whereas others elongated at an increased rate. This result suggests that only a critical mass or number of spindle microtubules needs be present for the rate of spindle elongation to be fully governed, and that astral forces can accelerate the elongation of a weakened or diminished spindle.

Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration.

Cytoplasmic dynein is a microtubule-associated motor protein with several putative subcellular functions. Sequencing of the gene (DHC1) for cytoplasmic dynein heavy chain of the filamentous ascomycete, Nectria haematococca, revealed a 4,349-codon open reading frame (interrupted by two introns) with four highly conserved P-loop motifs, typical of cytoplasmic dynein heavy chains. The predicted amino acid sequence is 78.0% identical to the cytoplasmic dynein heavy chain of Neurospora crassa, 70.2% identical to that of Aspergillus nidulans and 24.8% identical to that of Saccharomyces cerevisiae. The genomic copy of DHC1 in N. haematococca wild-type strain T213 was disrupted by inserting a selectable marker into the central motor domain. Mutants grew at 33% of the wild-type rate, forming dense compact colonies composed of spiral and highly branched hyphae. Major cytological phenotypes included (1) absence of aster-like arrays of cytoplasmic microtubules focused at the spindle pole bodies of post-mitotic and interphase nuclei, (2) limited post-mitotic nuclear migration, (3) lack of spindle pole body motility at interphase, (4) failure of spindle pole bodies to anchor interphase nuclei, (5) nonuniform distribution of interphase nuclei and (6) small or ephemeral Spitzenkörper at the apices of hyphal tip cells. Microtubule distribution in the apical region of tip cells of the mutant was essentially normal. The nonuniform distribution of nuclei in hyphae resulted primarily from a lack of both post-mitotic nuclear migration and anchoring of interphase nuclei by the spindle pole bodies. The results support the hypothesis that DHC1 is required for the motility and functions of spindle pole bodies, normal secretory vesicle transport to the hyphal apex and normal hyphal tip cell morphogenesis.

Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser.

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wavelength of 1.06 micron. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.