Helical structures in the cytoplasm of differentiating cells ofMicrasterias thomasiana

Summary Helical structures so far not known inMicrasterias are observed in the cytoplasm of differentiating cells ofMicrasterias thomasiana which were pretreated by a special fixation procedure. The structures measure about 45 nm in diameter and are situated in the area of the postmitotic nucleus and its surrounding microtubule system, sometimes close to different kinds of vesicle. Nature and function of the helical structures are still obscure; a relationship to microtubules is discussed.     

Experimental and ultrastructural studies on cell shape formation in the defect mutant cellmicrasterias thomasiana f. uniradiata

Summary Cell development and ultrastructure are studied in the defect mutant cellMicrasterias thomasiana f. uniradiata which lacks cell pattern at one side of the cell.The ultrastructural studies reveal an uneven distribution of vesicles, preponderating at the normally growing side of the cell, as well as the presence of a special kind of dark vesicles.By means of turgor reduction and treatment with chlorotetracycline and cycloheximide some processes involved in cell shape formation are pointed out and are compared with those already described for biradiateMicrasterias cells.It is demonstrated that the asymmetric cell shape of the mutant cell is already determined at the early stage of bulb formation and is due to a unilateral growth during the later stages of development. The asymmetric arrangement of the growth areas during cell development of the mutant is expressed by an asymmetric distribution of primary wall accumulations induced by turgor reduction as well as by the presence of fluorescence zones after treatment with the Ca²⁺ -chelate probe chlorotetracycline at only one side of the cell. Inhibition of protein synthesis by cycloheximide during cell growth of the mutant leads to the formation of a characteristically reduced cell pattern (anuclear type of development) similar to that ofMicrasterias denticulata andMicrasterias thomasiana under the same conditions. Nevertheless, this cell pattern develops at only one side of the cell, indicating that the mutant does not have any information for cell pattern formation at the defective side.     

Cytoskeletal aspects of nuclear migration during tip-growth in the fernAdiantum protonemal cell

Summary In the tip-growing protonemal cell, the nucleus migrates with the tip as it grows, keeping a constant distance between them. Cytoskeletal control of this nuclear migration was analyzed inAdiantum capillus-veneris. Using rhodamine-phalloidin (Rh-Phal), tubulin antibodies and confocal laser scanning microscopy, we found the presence of microtubule (MT) and microfilament (MF) strands connecting the cell nucleus to the cortex of the growing apex. The strands come from the apical end of the spindle-shaped nucleus and run through the endoplasm, arriving at the apical cortex, where a circular arrangement of MTs and MFs is present. Strands of MFs and MTs were also found to emanate from the proximal end of the nucleus and extend towards the cortex of the basal part of the cell. Double staining of MTs and MFs revealed a co-localization of these cytoskeletal elements. When MF strands were disrupted by cytochalasin B (CB), tip-growth ceased and nuclear movement stopped. After the application of colchicine, MT structures disappeared, tip-growth was largely inhibited, and the nucleus moved towards the basal part of the cell. When both CB and colchicine were applied to the cell, no basipetal migration of cell nucleus was observed. These results suggest that the MT strands between the apex and the nucleus may have a role in the anchorage of the cell nucleus to the tip during tip-growth, and that the MF strands may be important for basipetal movement of the nucleus. When the nucleus was dislocated basipetally by centrifugation, cytoskeletal strands between the cell apex and the nucleus were still observed, and by acropetal movement the nucleus resumed its previous position. The acropetal movement of the nucleus was inhibited by the application of both CB and colchicine but not by CB alone nor by colchicine alone, indicating that both cytoskeletal elements are involved in the forward movement of cell nucleus.Abbreviations CB cytochalasin B - DAPI4 6-diamino-2-phenylin-dole - DMSO dimethylsulfoxide - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N,N,N-tetraacetic acid - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule - PMSF phenylmethylsulfonyl fluoride - PSM polyoxyethylene sorbitan monolaurate - Rh-Phal rhodamine-labeled phalloidin     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

A fungal actin-related protein involved in nuclear migration

The ro-4 mutant of the filamentous fungus Neurospora crassa forms distinctive colonies in which hyphae grow into rope-like aggregates. This unusual morphology coincides with a defect in hyphal nuclear migration. The ro-4 gene was cloned from a cosmid library by complementation of the closely linked pab-2 gene. The deduced 380 amino acid protein is most similar to the vertebrate actin-related protein/centractin. The R04 protein is not essential for cell viability, and new strains created by inducing point mutations at the ro-4 locus have a phenotype which is very similar to that of the original mutant. This study provides genetic evidence that an actin-related protein plays a role in nuclear motility. Since nuclear motility is believed to be a microtubule-dependent process, the ro-4 gene product may function as a component of the dynactin complex which activates force generation by cytoplasmic dynein.     

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.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Immunofluorescence microscopy of microtubule arrangement in Closterium acerosum (Schrank) Ehrenberg

The microtubule (MT) arrangement in Closterium acerosum cells was observed by indirect immunofluorescence microscopy both during and following cell division, and during cell expansion without cell division. (During the division period, some cells of this alga divide whereas other cells expand in their middle region without division.) Before septum formation, all cells had a ring-like MT bundle (MT ring) in their middle. Both septum formation and expansion without cell division occurred at the position of this ring. During the periods of division, short, hair-like MTs appeared around the nucleus in some of the cells, in addition to the MT ring. In dividing cells, spindle MTs appeared as the chromosomes were condensed. During the early stages of expansion of the semicells, after cell division, the spindle MTs assumed a radial arrangement, moved, and settled in a position between the daughter chloroplasts. These MTs disappeared about 1.5 h after septum formation. As the new semicells were growing, wall MTs appeared, arranged transversely along the expanding wall. These transverse MTs disappeared gradually 4–5 h after septum formation, and only an MT ring remained near the boundary between the new and old semicells. The MT ring was present until the next cell division or expansion without cell division. During the latter course of development, transverse wall MTs were present only at the band-like expanding region. At the earlier stage of expansion without cell division, the short, hair-like MTs remained around the nucleus, but as time passed, both the hair-like MTs and, somewhat later, the transverse ones disappeared and only the MT rings remained. The remaining MT ring was not always positioned at the boundary between the expanding and the old cell region. The temporal relationships between the changes in MT arrangement, and the orientation and localization of cellulose-microfibril deposition are discussed.Abbreviations DAPI 46-diamino-2-phenylindole - EGTA ethyleneglycol-bis-(-aminoethylether)-N, N, N, N-tetraacetic acid - MT mierotubule - PMSF phenylmethylsulfonyl fruoride     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Changes in microtubule and microfibril arrangement during polarotropism inAdiantum protonemata

Polarotropism was induced inAdiantum (fern) protonemata grown under polarized red light by turning the electrical vector 45 or 70 degrees. One hour after the light treatment, tropic responses became apparent in many cells as a slight distortion of the apical dome. Changes in the position of the circumferentially-arranged cortical microtubule band (Mt-band) (Murataet al., 1987) and the arrangement of microfibrils around the subapical part of protonemata were investigated in relation to the polarotropic responses. Twenty minutes after turning the electrical vector, preceding the morphological change of cell shape, the Mt-band began to change its orientation from perpendicular to oblique to the initial growing axis. After 30 min, the Mt-band changed its orientation further under 45 degrees polarized light, but under light rotated 70 degrees, it began to disappear. In phototropic responses induced by local irradiation of a side of the subapical part of a protonema with a non-polarized red microbeam, the Mt-band on the irradiated side disappeared or became faint within 20 min, but neither disappearance nor a change of orientation of Mts occurred on the non-irradiated side. One hour after turning the electrical vector 45 degrees, in half of the cells tested, the innermost layer of microfibrils in the subapical part of the protonema changed its orientation from perpendicular to oblique to the growing axis, corresponding to the changes in the orientation of the Mt-band. After 2 hr, those changes were obvious in all cells examined. The same basic results on the orientation of microfibrils were obtained with protonemata cultured for 2 hr under 70 degrees polarized light. The role of the Mt-band in tropic responses is discussed.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Cell multiplication and ultrastructure ofMicrasterias denticulata (Desmidiaceae) grown under salt stress

Cells ofMicrasterias denticulata Bréb. were kept in nutrient solution of high osmolality (salt stress) for four weeks. In a special cell multiplication test it was established that cell division is gradually inhibited at increasing salt concentrations and totally arrested at the highest concentration (26 mosm/kg). Recovery studies proved that even cells from the highest concentration range start dividing immediately after being placed in aqua bidest. thus indicating the full reversibility of the inhibiting effect. — Cells of the highest concentration range show marked ultrastructural changes. Besides an enormous accumulation of starch and oil bodies and a condensed appearance of the ground plasma, a reduction of mitochondria, ER and the Golgi-system is found. The most striking effect occurs on the vacuolar system which appears extremely reduced and condensed. The cell wall is thickened by the formation of an additional cell wall layer with a spongy electron microscopical appearance. Through the cell wall many droplets of a probably fat-like substance are excreted. — In summary, salt stress induces growth-inhibited akinete cells in the sense ofFritsch; these can be reactivated by decreasing the salt concentration. The salt-induced akinete state seems to be an ecological adaption to unfavourable conditions rather than a degeneration of the cells.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.23. 12. 1988     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20–40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MT microtubule(s) - PB prophase band(s) - PNF perinuclear fluorescence - PPB pre-prophase band     

The changes of nuclear position and distribution of circumferentially aligned cortical microtubules during the progression of cell cycle inAdiantum protonemata

The intracellular positions of the nucleus and of cortical, circumferentially aligned microtubules (CCAM) in filamentous, single-celled protonemata ofAdiantum capillus-veneris were determined throughout the cell cycle in the dark. When apical growth continued at G₁ phase, the nucleus migrated keeping a constant distance from the tip. When the apical growth stopped at late S or G₂ phase, the nucleus stopped moving forward and then slightly moved backward to the site of cytokinesis. The CCAM were found only in the dome of protonemal tip when growing under continuous red light; they increased in number after dark incubation for 12 hr and then decreased after 20th hr in the dark. The CCAM were usually observed in the region between the nucleus and the tip at 28 hr in the dark. They were located around the nuclear region at pre-prophase and prophase, but then totally disappeared at metaphase and thereafter.     

Number of dictyosomes in a single cell of the green algaMicrasterias

The numbers of dictyosomes in cells ofM. crux-melitensis andM. pinnatifida were counted at various stages in the cell cycle. Dictyosomes synchronously doubled in number by dividing at the premitotic stage and then were separated into two groups by the septum, thus reducing the dictyosomal number to the ordinal number in each new cell. The number remained the same throughout the cell cycle until the next premitotic stage.     

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of tadpole-shaped cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.     

Patterns of microtubule reappearance in root cells ofVigna sinensis recovering from a colchicine treatment

Summary The reticulum of paracrystalline tubulin strands, which is assembled in meristematic root cells ofVigna sinensis treated with a 0.08% colchicine solution, disaggregates and microtubules (Mts) reappear after a 10–14 h recovery of the seedlings from the drug. In recovering interphase cells, Mts reappear in the cortical cytoplasm. Initially, they are short and aligned in different directions but finally they elongate and usually become oriented transversely to the long cell axis.A single or a pair of preprophase Mt bands (PMBs) is organized in cells enclosing one or more nuclei. Simultaneously, Mts traverse the perinuclear cytoplasm. In recovering C-mitotic cells, Mt bundles emerge from the kinetochores. Initially, they exhibit diverse orientations. Afterwards, the C-chromosomes are aligned on ametaphase plate via kinetochore Mt bundles, which become parallel to one another. As time passes non-kinetochore Mts appear among the chromosomes and anaphase proceeds. In recovering cytokinetic cells, normal, abnormally curved or branched phragmoplasts are organized. The latter arise between the nuclei of multinucleate telophase cells or between the lobes of forming polyploid nuclei. In cells which were blocked at an advanced cytokinetic stage by colchicine, phragmoplasts return to the margins of the incomplete cell walls.The observations presented here suggest that in recovering colchicine-treated root cells the Mts and the tubulin reticulum are interchangeable. Although Mts appear in cytoplasmic sites where they are expected to be nucleated, the pattern of Mt reformation differs from that operating in normal and to a smaller extent from that functioning in cells recovering from other anti-Mt drugs.     

Control of shape and pattern during the assembly of a large microtubule bundle

Microtubules are packed and linked together in a well defined hexagonal arrangement in the cytopharyngeal microtubule bundles of the ciliate Nassula. Early stages in the morphogenesis of these bundles have been examined. Elements which nucleate assembly of bundle microtubules are apparently closely associated before tubule assembly commences. These nucleating elements seem to be bound together in highly ordered arrays to form microtubule-nucleating-templetes. Each array of elements is attached to the proximal end of a basal body and appears to establish the pattern of tubule packing and cross-sectional shape of a tubule bundle. A self-assembly procedure which accounts for the anisometric growth and shaping of a template and its microtubule bundle is proposed.