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.     

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.     

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     

Temporal and spatial changes of cellulose synthesis inClosterium acerosum (Schrank) Ehrenberg during cell growth

The amount and distribution of wall microfibril synthesis were investigated in the cell-division cycle ofClosterium acerosum. Electron-microscopic examination and a methylation analysis of alkali-extracted wall fragments showed that alkali-extracted wall was mainly composed of microfibrils and that the microfibrils ofC. acerosum were 4-linked glucans, i.e., cellulose. Cellulose synthesis was measured as incorporation of¹⁴C, fed to cells as NaHCO₃, into extracted wall fragments. Extensive cellulose synthesis was coincident with septum formation, continued for more than 6 h and then ceased. It was found by microautoradiography that cellulose synthesis after cell division was essentially restricted to the expanding new semicells. Such a restricted distribution of cellulose synthesis was maintained for more than 6 h after septum formation, i.e., for more than 2 h after the cessation of expansion; afterwards, cellulose synthesis in some, but not all, cells became extended to the old semicells, and then ceased. Considerable cellulose synthesis also took place in the band-like expanding part of non-divided cells, indicating that cell division was not necessarily required for the induction of cellulose synthesis and the latter was coupled with cell expansion. Extension of cellulose synthesis to old semicells was brought about in divided cells by treatment with 3 mM colchicine, 28 M vinblastine, 50 M isopropyl-N-phenylcarbamate or 1 M isopropyl-N(3-chlorophenyl)carbamate, indicating that microtubules are involved in the limitation of cellulose synthesis to the new semicells.Abbreviations CIPC isopropyl-N(3-chlorophenyl)carbamate - DPO 2,5-diphenyloxazole - IPC isopropyl-N-phenylcarbamate     

Changes in microfibril arrangement on the inner surface of the epidermal cell walls in the epicotyl of Vigna angularis ohwi et ohashi during cell growth

Throughout the entire period of cell growth, the microfibrils on the inner surface of the outer tangential walls of the epidermal cells of Vigna angularis epicotyls are running parallel to one another and their orientation differs from cell to cell. Although transverse, oblique and longitudinal microfibrils can be observed irrespective of cell age, the frequency distribution of microfibril orientation changes with age. In young cells, transversely oriented microfibrils predominate. In cells of medium age, which are still undergoing elongation, transverse, oblique and longitudinal microfibrils are present in quite similar frequencies. In old, non-growing cells, longitudinally oriented microfibrils are predominent. A decrease in the relative frequency of transversely oriented microfibrils with cell age was also observed in the radial epidermal walls.     

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     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.     

Microtubules do not control microfibril orientation in a helicoidal cell wall

Summary The secondary cell wall layer of the young root hair ofEquisetum hyemale (L) has a helicoidal texture. The cortical microtubules in these hairs maintain an axial alignment while microfibrils are being deposited with a different orientation in each subsequent layer. The role of cortical microtubules in microfibril orientation is disputed.I gratefully acknowledge the support of Professor Dr. M. M. A.Sassen and the technical assistance of M.Wolters-Arts.     

The role of microtubules during the cytokinetic events of cell wall ontogenesis and papilla development inCarteria crucifera

Summary An ultrastructural study of cytokinesis, cell wall ontogenesis, and papilla development/form inCarteria crucifera Korsh. andChloromonas rosae Ettl was undertaken. After typical phycoplast-mediated cytokinesis, wall ontogenesis begins at the level of Golgi apparatus activation and secretion to the outside of the daughter cells of fibrillar wall precursors which self assemble into the typical chlamydomonad wall (sensuRoberts 1974). As wall ontogenesis approaches the flagellar region of the cell, several precisely timed events occur: flagellar apparatus formation, flagellar emergence, protoplasmic extension in the future papilla area underlined by series of parallel aligned microtubules, wall formation (at least the W₂–W₆ layers), retraction of the protoplasmic extension and loss of underlying microtubules, and final wall modification (gap filling by W₁ material) to yield the characteristic wall papilla. The transient cytoplasmic extensions mimic the shape of the future wall papilla and are maintained, at least inCarteria, by underlying microtubules. Structural and developmental properties of the papilla are characterized and phylogenetic implications are discussed.This research was supported by National Science Foundation Grant DEB 78-0554.     

A model for the pattern of deposition of microfibrils in the cell wall of Glaucocystis

Freeze-fracturing of Glaucocystis nostochinearum Itzigsohn cells during cell-wall microfibril deposition indicates that unidirectionally polarized microfibril ends are localized in a zone of synthesis covering about 30% of the sarface area of the plasma membrane. Within this zone there are about 6 microfibril ends/m² cell surface. It is proposed that microfibrils are generated by the passage of their tips over the cell surface and that the pattern of microfibril organization at the poles of the cells, in which microfibrils of alternate layers are interconnected at 3 rotation centres, results directly from the pattern of this translation of microfibril tips. In a model of the deposition pattern it is proposed that the zone of synthesis may split into 3 sub-zones as the poles are approached, each sub-zone being responsible for the generation of one rotation centre. It is demonstrated that the microfibrillar component of the entire wall could be generated by the steady translation of the microfibril tips (at which synthesis is presumed to occur) over the cell surface at a rate of 0.25–0.5 m min^-1. Microcinematography indicates that the protoplast rotates during cell-wall deposition, and it is proposed that this rotation may play a role in the generation of the microfibril deposition pattern.     

Effects of colchicine and amiprophos-methyl on microfibril arrangement and cell shape inAdiantum protonemal cells

Summary 5 mM colchicine and 1 g/ml amiprophos-methyl, known antimicrotubule agents, were applied to fernAdiantum protonemata under red light. Both drugs caused microtubule disruption and subsequent apical swelling of protonemal cells after certain lag periods. While the lag periods for the onset of microtubule disruption after application of the two drugs were different (within 15 minutes in amiprophos-methyl, 1 hour in colchicine), the lag periods of apical swelling after microtubule disruption were nearly the same (approx. 70 minutes). The results suggest that the apical swelling is a consequence of microtubule disruption.In cells examined 1 hour after microtubule disruption by either drug, the microfibril arrangement of the innermost layer of the cell wall was random at the tip, transverse in the subapical region, and roughly longitudinal in the cylindrical region. This pattern of microfibrils was similar to that of untreated cells in which the microtubules show a similar arrangement (Murata and Wada 1989). Surprisingly, even after approx. 4 hours of microtubule disruption, when apical swelling had occurred in most cells, the pattern of microfibril deposition was not altered. The role of microtubules in oriented microfibril deposition and the mechanism of control of cell shape are discussed.Abbreviations APM amiprophos-methyl - DMSO dimethylsulfoxide - MT(s) microtubule(s) - PBS phosphate buffered saline     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Ionic currents flow throughMicrasterias andClosterium cells during expansion of the primary cell wall

The results of studies of Micrasterias rotata (Grev.) Ralfs, M. thomasiana Archer (biradiate and uniradiate forms) and Closterium sp. using one- and two-dimensional vibrating probes show that transcellular ionic currents are detectable only around cells undergoing expansion of the primary cell wall (half-cell); current enters local regions of expansion and exits over both the rigid surface of the secondary wall and regions of the primary wall where hardening of the wall prevents further expansion. Current densities remain at steady levels until expansion stops with maturation of the primary wall, whereupon currents are no longer detectable. The temporal and spatial correlation between the currents and regions of wall expansion is particularly evident because morphogenesis of the half-cell is a determinate process. Measurements of inward currents ranged from 0.1 to 5.4 A · cm^–2, and outward currents ranged from-0.05 to -1.5 A · cm^–2 measured at 18 from the cell surface. The results of ion substitution and channel-blocker studies indicate that the currents may be carried at least in part by Ca²⁺, Cl^–, H⁺ and K⁺ ions. The possible role of a Ca²⁺ influx during tip growth in desmids is discussed.This work was conducted at the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA. Dr. Lionel F. Jaffe, Director of the Facility, and Dr. Jeremy D. PickettHeaps, University of Colorado, Boulder, USA, provided valuable guidance and support, and gave unstinting encouragement during these studies. Dr. Franklin M. Harold provided support for the writing of this paper during C.L.T.'s postdoctoral year at the National Jewish Center for Immunology and Respiratory Research, Denver. Mr. Alan Shipley and Mr. Steve Dixon provided talented technical assistance. C.L.T. is grateful for support received from a National Institutes of Health Pre-doctoral Training Grant in the Department of Molecular, Cellular and Developmental Biology, University of Colorado. The work was supported by N.I.H. grants 5 P41 RR01395 and 3 P41 RR01395-02S1 (to L.F.J.), National Science Foundation grants No. BSR 82 14199 and PCM 83 09331 (to J.P.-H.), and No. DCB 86 18694 (to F.M.H.).     

An autolysin dissolves the inner cell wall layer of the algaPediastrum (Hydrodictyaceae)

Summary An autolysin produced by young colonies ofPediastrum frees them from the vesicle in which they are formed within 12 hours of release of zoospores from the parent cell. The polysaccharide vesicle is derived from the inner wall layer of the parent cell. Refrigeration delays vesicle disintegration; boiling stops it completely. A purified, lyophilized extract of the vesicle fluid added to boiled vesicled colonies removes the vesicle in 2 hours with the release of reducing sugars and polysaccharides.Biogel P2 and P10 chromatography of the products following incubation of the enzyme preparation and wall showed no more than 1% oligosaccharides; the remaining carbohydrates had a molecular weight of several thousand daltons. Analyses of isolated vesicle wall material (70–85% of the dry weight) showed mannose accounting for approximately 50% of the dry weight, with none of the other neutral sugars present (fucose, xylose, galactose and glucose) representing more than 3%. Uronic acids account for 20–25% of the wall weight, and proteins less than 2%. Pediastrum colonies are thus freed from the vesicles in which they are formed by the action of an autolysin they produce. The autolysin acts on the vesicle wall material to generate reducing sugars and cause it to disintegrate into its constituent polysaccharides.     

Cortical microtubules and microfibril deposition in the cell wall of root hairs ofEquisetum hyemale

Summary The cell wall of root hairs ofEquisetum hyemale is shown to be composed of three different cell wall textures. The growing cell wall at the tip of the hair is composed of a dispersed texture of microfibrils, which continues along the outside of the whole hair. With increasing distance from the tip an increasing number of helicoidally arranged lamellae is deposited. These findings correspond with the observed isotropism of young hairs in polarized light.Hairs of approximately 4 days old become positive birefringent, indicating that longitudinally oriented layers prevail over layers with a transverse direction. This phenomenon starts at the base of the hair. Full-grown hairs are positive birefringent up to the tip and concordantly show a thick additional inner cell wall layer which forms a helical pattern the length of the hair, with a mean microfibril angle of 25 with the cell axis.Cortical microtubules, subjacent to the dispersed, the helicoidal and the helical wall texture are axially aligned and, thus, not in coalignment with the last deposited microfibrils.Coated and smooth vesicles are present in the cortical cytoplasm of both growing and full-grown hairs. Electron-dense profiles (20 nm in diameter), surrounded by a halo (of 50 nm) were observed on the wall-plasmalemma interface in full-grown hairs only. A relation of these structures with microfibril deposition could not be demonstrated. They might represent channels transporting material to the wall, which, in full-grown hairs, is heavily impregnated with a tawny brown substance.The general hypothesis that cortical microtubule orientation directs microfibril deposition is disputed.     

Synthesis of the inner cell wall layer of the chlamydomonad flagellate,Gloeomonas kupfferi

Summary An antibody to the inner wall layer ofGloeomonas kupfferi was isolated and used in a developmental analysis of cell wall processing, secretion and extracellular assembly. The focus of the processing of this matrix layer is the endomembrane system, in particular the Golgi apparatus (GA) and contractile vacuole (CV). During interphase, inner wall materials are processed in the GA, packaged in trans face vesicles and transported to the CV, the final internal depository of wall precursors until release to the cell surface. During cell division, significant changes occur in the inner wall layer processing. Early on in cytokinesis, the GA does not label with our antibody, suggesting that other wall layers are being processed. In later stages of cytokinesis, the GA changes in morphology and begins to produce inner wall layer materials. These wall precursors are shuttled to the CV where they are released around the daughter cell protoplasts. The first wall layer that is formed around daughter cells is the crystalline median wall layer. Once assembled, the inner wall layer condenses upon the crystalline layer and grows in size.     

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.     

The microtubule cytoskeleton in developing cysts of the green algaAcetabularia: Involvement in cell wall differentiation

Summary Cysts of the green algaAcetabularia develop a unique lid structure to enable the release of gametes. This lid is separated from the rest of the thick cellulose cell wall by a circular fault line formed within the fibrillar texture of the wall. By immunofluorescence microscopy, we show that, prior to the first division of the single cyst nucleus, the radially symmetrical, perinuclear microtubule system which is a remnant carried over from previous developmental stages of cyst morphogenesis transforms into a circular microtubule band (CMB) around the nucleus. This band consisting of only a few bundled microtubules beneath the plasma membrane encircles the cyst nucleus at a distance of 75 to 100m. In a previous fine structural study, a lid-forming apparatus (LFA) was described as a circular band of rod-like structures in the plane of the plasma membrane, demarcating the contour of the future lid. Both the CMB and the LFA are superimposed on the rim of the lid. We therefore propose that the microtubule band is a component of the LFA identical with the rod-like structures. Formation of the CMB and, hence, lid formation are blocked by the microtubule-specific herbicide Oryzalin but not by the actin filament-disrupting inhibitor cytochalasin D. Upon recovery from Oryzalin treatment, the nuclei but not the prospective sites of the CMBs serve as nucleation centers, indicating that the CMB is not formed by a pre-existing template in the plasma membrane. This suggests that the dynamic behavior of the microtubules within the perinuclear microtubule cytoskeleton gives rise to the CMB. Since the stage of CMB assembly marks the beginning of cell wall formation, it is proposed that the CMB determines the position of the lid by spatially controlling cell wall deposition. On the basis of current hypotheses, two scenarios for the role of the LFA/CMB in lid formation are discussed.Abbreviations CMB circular microtubule band - EGTA ethylene glycol bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - LFA lid-forming-apparatus - MAP microtubule-associated protein - MT microtubule - MTOC microtubuleorganizing center Dedicated to the memory of Professor Oswald Kiermayer     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Re-formation of microtubules in Closterium ehrenbergii Meneghini after cold-induced depolymerization

Immunofluorescence microscopy was used to examine the re-formation of microtubules (MT), after cold-induced depolymerization, in Closterium ehrenbergii. The C. ehrenbergii cells undergo cell division followed by semicell expansion in the dark period of daily light-dark cycles. Five types of MTs, namely the MT ring, hair-like MTs around the nuclei, spindle MTs, radially arranged MTs and transverse wall MTs, appeared and disappeared sequentially during and following cell division. The wall MTs were distributed transversely only in the expanding new semicells. When cells were chilled in ice water, wall MTs in expanding cells were fragmented, and then disappeared as did the other types of MTs, within 5 min. When cells were warmed at 20°C after 2 h chilling, wall MTs and the other types of MTs re-formed. At the early stage of wall-MT re-formation in expanding cells, small, star-like MTs were formed, and then randomly oriented MTs developed in both the expanding new and the old semicells. The MT ring was also re-formed at the boundary between the new and old semicells. There were no obvious MT-organizing centers in the random arrangement. As time passed, the randomly oriented wall MTs in the old semicells disappeared and those in the expanding new semicells gradually assumed a transverse orientation. These results indicate that wall MTs can be rearranged transversely after they have been re-formed and that nucleation of wall MTs is separable from the mechanism for ordering them.Abbreviations MT(s) microtubule(s) - MTOC(s) microtubule-organizing center(s)