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     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Reassembly of microtubules in protoplasts ofSaccharomyces cerevisiae after nocodazole-induced depolymerization

Summary By following microtubule neoformation after their complete destruction by nocodazole, we analyzed the pattern of microtubule nucleation in protoplasts ofSaccharomyces cerevisiae. Using immunofluorescence, the drug was shown to induce rapid and complete disassembly of both cytoplasmic and spindle microtubules and to selectively block protoplast nuclear division at a defined stage of the cell cycle. Treated protoplasts placed in a drug-free environment recovered a more abundant microtubular system. The majority of microtubules re-formed at SPBs whereas a minority of free-ended microtubules nucleated in the cytoplasm of the protoplasts without any detectable association with recognizable nucleation sites. Random nucleation of free microtubules might be induced by high amounts of unpolymerized tubulin likely to be present in the protoplasts at the moment of drug release.Abbreviations MT microtubule - NOCO nocodazole - SPBs spindle pole bodies - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumine - sMT spindle microtubule - cMT cytoplasmic microtubule - MTOC microtubule organizing center     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt⁺) cells and induces the release of gametic protoplasts from matingtype minus (mt⁻) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt⁻ cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt⁻ cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10⁻⁸ M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt⁻ cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Light, a nitrogen-depleted medium and cell-cell interaction in the conjugation process of Closterium ehrenbergii Meneghini

The conjugation process of heterothallic Closterium ehrenbergiiMeneghini consisted of the first cell division, cell aggregation,the second cell division, papilla formation and zygospore formation.Light irradiation and the depletion of nitrogen in the culturemedium were required for the first cell division and for cellaggregation, but the second cell division, papilla formationand zygospore formation took place independent of the presenceof light and of the depletion of nitrogen. The presence of twopartner strain cells was indispensable for cell aggregationand papilla formation. The first and second cell divisions andzygospore formation took place in the absence of partner straincelb, but interaction between the two strains was indispensableto induce them. (Received January 27, 1979; )     

The formation and consumption of lipid droplets in the zygote and germinated cells ofClosterium ehrenbergii

The formation and consumption of lipid droplets was observed with an electron microscope in the zygote and the germinated cells of the green alga,Closterium ehrenbergii. The lipid droplets were formed in lysosomal vesicles during zygote maturation following conjugation. In the germinated cells, they were enclosed in ERs and gradually consumed in them. This consumption occurred in the cells at the early stages of expansion. The derivative substances may possibly be used for cell surface expansion.     

Cell walls,plasma membranes,and dictyosomes during zygote maturation ofClosterium ehrenbergii

Summary The cell wall formation and its correlation with the plasma membrane and dictyosome were investigated by an electron microscope in the zygote cells ofClosterium ehrenbergii. During zygote maturation, six wall layers were formed outside the plasma membrane. Wall layer III was the thickest layer and consisted of microfibril bundles. Dictyosomes produced flat vesicles during formation of wall layer III. Hexagonal arrays of rosette particles appeared in the plasma membrane in this period, thus confirming the simultaneous occurrence of flat vesicles and hexagonal particle arrays in the formation of microfibril bundles even at different stages of the life cycle. Wall layer VI was second in thickness and consisted of single microfibrils. Neither flat vesicles nor hexagonal particle arrays were observed during formation of this layer.     

The change of pattern in microfibril arrangement on the inner surface of the cell wall of Closterium acerosum during cell growth

Closterium acerosum (Schrank) Ehrenberg cells cultured on cycles of 16 h light and 8 h dark, undergo cell division synchronously in the dark period. After cell division, the symmetry of the daughter semicells is restored by controlled expansion, the time required for this restoration, 3.5–4 h, being relatively constant. The restoration of the symmetry is achieved by highly oriented surface expansion occurring along the entire length of the new semicell. During early semicell expansion, for about 2.5 h, microfibrils are deposited parallel to one another and transversely to the cell axis on the inner surface of the new wall. Wall microtubules running parallel to the transversely oriented microfibrils are observed during this period. About 2.5 h after septum formation, preceding the cessation of cell elongation, bundles of 7–11 microfibrils running in various directions begin to overlay the parallel-arranged microfibrils already deposited. In the fully elongated cells, no wall microtubules are observed.     

Effect of antibiotics on cell proliferation in the Closterium peracerosum-strigosum-littorale complex (Charophyceae, Chlorophyta)

The effects of several antibiotics on the proliferation of cells of the Closterium peracerosum-strigosum-littorale complex, a unicellular charophycean alga, were examined. When cells were cultured on solid medium containing hygromycin B and phleomycin the proliferation of cells was inhibited at low concentrations of these antibiotics, with a minimum inhibitory concentration of 5.0 and 0.2 μg/mL, respectively. By contrast, kanamycin sulfate was less effective at concentrations up to 50 μg/mL. When cells were incubated in liquid medium containing hygromycin B and phleomycin, cell proliferation was severely inhibited at concentrations of 5.0 and 0.01 μg/mL, respectively. It is concluded that hygromycin B and phleomycin are highly effective for inhibiting the proliferation of C. psl. complex both on solid and in liquid medium and thus are useful for the selection of the cells transformed by selectable marker genes. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Dissection of conjugants and mating type plus and minus cells in selfing clones of the isogamous green alga Closterium ehrenbergii

In the sexual reproduction of the green alga Closterium ehrenbergii, two sexually competent cells that are morphologically indistinguishable from the vegetative cells first come close to each other to form a sexually interacting pair. Each then divides into two gametangial cells. Isogamous conjugation occurs between nonsister gametangial cells of the two resulting pairs. With unusual selfing clones derived from a certain cross of heterothallic strains, we dissected apart a pair of gametangial cells that had already been united together by a delicate transparent tube, into which each gametangial cell was going to develop its conjugation papilla. In spite of such a degree of differentiation, when each was cultured in fresh medium, individual gametangial cells could dedifferentiate into vegetative cells and form subclones. By crossing such subclones with standard stable heterothallic mating-type strains, we show that each selfing clone of this alga actually produces both stable mt ⁺ and stable mt ^- cells, in addition to unstable mt ^- cells with selfing potency, during its mitotic vegetative growth. Although the selfing in C. ehrenbergii studied here differs in certain points from true homothallism, the results of the present study provide insight into how homothallism might have evolved from heterothallism.     

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.     

Isolation of myosin XI genes from the Closterium peracerosum-strigosum-littorale complex and analysis of their expression during sexual reproduction

Myosins comprise a large superfamily of molecular motors that generate mechanical force in ATP-dependent interactions with actin filaments. On the basis of their conserved motor domain sequences, myosins can be divided into at least 17 classes, 3 of which (VIII, XI, XIII) are found in plants. Although full sequences of myosins are available from several species of green plants, little is known about the functions of these proteins. Additionally, sequence information for algal myosin is incomplete, and little attention has been given to the molecular evolution of myosin from green plants. In the present study, the Closterium peracerosum-strigosum-littorale complex was used as a model system for investigating a unicellular basal charophycean alga. This organism has been well studied with respect to sexual reproduction between its two mating types. Three types of partial sequences belonging to class XI myosins were obtained using degenerate primers designed to amplify motor domain sequences. Real-time polymerase chain reaction analysis of the respective myosin genes during various stages of the algal life cycle showed that one of the genes was more highly expressed during sexual reproduction, and that expression was cell-cycle-dependent in vegetatively grown cells.     

Prophase bands of microtubules occur in protoplast cultures of Vicia hajastana Grossh

Circumnuclear bands of microtubules (MT) have been found in the prophase of mitoses in cultured protoplasts of Vicia hajastana. The timing of the appearance and disappearance of the prophase band of MT (PB) relative to the stage of mitosis was studied using simultaneous staining of MT by immunofluorescence and DNA by Hoechst 33258. These protoplasts regenerate into unorganized tissue. Pre-prophase bands of MT have previously been found only in highly organized tissues of higher plants. The role of PB in cell division is discussed.Abbreviations MT microtubule(s) - PB prophase band(s) - FPB pre-prophase band(s) - PNF perinuclear fluorescence     

Development-specific association of amyloplasts with microtubules in scale cells of Narcissus tazetta

Summary. Narcissus tazetta is one of the major geophyte crops worldwide, but little is known about its cell biology. The narcissus storage organ was studied by monitoring scale cell biology during the growth stage and dormancy, and it was found that amyloplasts gradually increased in size and reached a maximum at dormancy. In parallel, microtubules changed their organisation: during the growth phase (February to March) they were oblique; during April and May, microtubules formed a network with round “holes”; by late June and the beginning of July, when dormancy started, they were organised in parallel arrays. The holes formed in the microtubule array corresponded to amyloplasts. A closer look showed that during a short time window, while the plants were preparing for dormancy, the microtubules surrounded the amyloplasts. In vitro reconfirmation of this phenomenon was obtained when fluorescent bovine brain microtubules enwrapped isolated amyloplasts that had been purified between April and July but not those purified between January and March. Interestingly, protease treatment of amyloplasts did not completely prevent binding of microtubules, which suggests the existence of a protease-resistant factor that docks microtubules to the outer membrane of amyloplasts. Correspondence and reprints: Department of Ornamental Horticulture, Volcani Center, Bet Dagan 50250, Israel.     

Abundance of microtubules in preprophase bands of some Triticum species

Root tip procambial cells of Triticum speltoides, T. tauschii, T. turgidum and T. aestivum have been investigated ultrastructurally for the detection of preprophase microtubule bands (PMBs) and to estimate the number of microtubules comprising the bands. The species selected are phylogenetically related but differ in the ploidy level. It was found that all species develop well-defined PMBs prior to mitosis. Estimations of microtubule abundance in the PMBs was carried out in midpreprophase cells, a stage judged by a feature of the nucleus in which electron-transparent canals are formed around the initial condensations of the chromatin material and the nucleoli. Triticum speltoides bears the smaller average number of microtubules per PMB and T. aestivum the greater. The results indicate that the increase follows the upgrade of the number of chromosome sets. It is suggested that the average number of microtubules of PMBs is related to the ploidy level.Abbreviation PMB preprophase microtubule band     

Re-organization of microtubules during preprophase band development inAdiantum protonemata

Summary Microtubule organization during preprophase band development was investigated using immunofluorescence microscopy in filamentous protonemal cells (approx. 600 m in length, 20 m in width) ofAdiantum capillus-veneris L. Protonemata pre-cultured under red light were transferred to continuous blue light or total darkness to induce synchronous cell division. Preprophase bands were found under both light conditions. In an early stage of development, the preprophase band which is transverse to the cell axis overlapped with an interphase cortical array of microtubules which is random or parallel to the cell axis. The interphase cortical array disappeared thereafter. While the width of the preprophase band became narrow during development under dark conditions, under blue light conditions it did not.Spatial and temporal aspects of the disappearance of the interphase cortical array of microtubules were also investigated. The interphase cortical array began to disappear at nearly the same time as the beginning of preprophase band formation. Under blue light, the disruption of cortical microtubules started at approx. 150 m from the tip (approx. 120 m from the nucleus), and spread toward the tip as far as the nuclear region and toward the base to an area approx. 300–400 m from the tip. Cortical microtubules remained in the basal part of the protonema. The pattern of disappearance between the tip and nucleus could not be determined. Under dark conditions, the pattern of the disappearance of cortical microtubules was somewhat different in many cells from that encountered with exposure to blue light. Microtubules first re-oriented from longitudinal to transverse, and then gradually disappeared. In some cells, the pattern of disappearance was similar to that observed under blue light.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - ICM interphase cortical microtubules - PBS phosphate buffered saline - PPB preprophase band - MT microtubule