Centrosome and microtubule dynamics in apical cells ofSphacelaria rigidula (Phaeophyceae) treated with nocodazole

Summary Treatment of young thalli ofSphacelaria rigidula with 0.04 g of nocodazole (Nz) per ml for up to 36 h affects microtubules (Mts) only slightly, but blocks a large number of mitotic cells in metaphase, without disruption of the metaphase plate. Higher concentrations of Nz (0.1 g/ml) depolymerize interphase Mts. Only a few perinuclear and some short Mts resist and remain associated with the centrosomes. Fragmented Mts or groups of Mts sometimes remain in the apical dome. After treatment with 0.1 g of Nz per ml, prometaphase cells are blocked at metaphase, while post-metaphase cells become binuclear, due to the failure of cytokinesis. With anticentrin immunofluorescence, a positive centrin signal is always observed in the centrosome area. Centrosome duplication is not affected by Nz, but separation is disturbed. After recovering for 2–4 h, most of the blocked metaphases proceed normally. In such cells duplicated centrosomes are seen in different stages of separation. In some cells independent aster-like microtubule configurations appear in the apical dome, occasionally displaying centrin at their centre. During recovery various configurations of bimitosis or multipolar mitosis were found. The multipolar spindles may share common centrosomes. Up to four centrosomes may accompany each nucleus. In some 24 h treated cells, as well as in cells recovering for 2 h, the centrin-positive structure is rod-like, extending in opposite directions from the usual position to the poles. Electron microscopical examination of thin sections revealed that the growth pattern of the apical cells is disrupted after Nz treatment. The observations show that: (a) the Mt cytoskeleton is involved in maintaining the polarity and growth pattern of apical cells, (b) mitosis is blocked by low concentrations of Nz without significant depolymerization of Mts, (c) the centrosome cycle is independent of the nuclear cycle, (d) centrosome separation and differentiation are disturbed by Nz treatment, (e) during recovery from Nz treatment, centrosomal material that may have separated from the centrosomes, as well as Mt fragments that resisted depolymerization, may operate as Mt nucleation centres.Abbreviations DIC differential interference contrast - EM electron microscope - Mt microtubule - MTOC microtubule-organizing center - Nz nocodazole - NBBC nucleus-basal body connector     

Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast

The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.     

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

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

Mariculture of Laminaria japonica (Laminariales, Phaeophyceae) using protoplast regeneration

The conditions for culture of viable protoplasts from Laminaria japonica were investigated and the regenerative processes were observed in detail. As a result of culturing at four water temperatures (5, 10, 15, and 18°C), we found that low water temperature was better for survival, division and rhizoidal formation of protoplast‐derived cells. Only epidermis‐derived protoplasts developed into normal sporophytes through a direct developmental process. Protoplast‐derived cells divided after 5 days and 2–10 celled germlings formed the first rhizoids after 15 days. Only initial sporophytes with the first rhizoids grew to normal sporophytes with multilayered blades, stipes and holdfasts. When these young sporophytes were transplanted into the sea, they grew to normal fertile sporophytes.     

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.     

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

Summary To assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - DMSO dimethylsulphoxide - EGTA ethyleneglycol-bis-(-aminoethyleter)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleido-benzoyl N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - MT microtubule - PIPES 1,4-piperazine-dietha-nesulphonic acid - PPB pre-prophase band - Rh-ph rhodamine phalloidin     

Evidence for division and polarity in apical cells of bryophytes and pteridophytes

Apical cells on the verge of dividing, or having recently formed a new segment, or actually dividing, are not uncommonly encountered in bryophytes and pteridophytes. This is interpreted as evidence for the classical concept of active apical segmentation in these plants (versus the concept of a quiescent apical cell). In certain species a polarized organization of the cytoplasm of the dividing apical cells is identifiable.     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

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.     

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus)

Summary The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8+1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15–20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells serve as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.     

Streblonema (Ectocarpales,Phaeophyceae) infection in the kelp Laminaria saccharina (Laminariales,Phaeophyceae) in the western Baltic

The brown alga Laminaria saccharina is the dominant subtidal macroalga in Kiel Bay, western Baltic. It is infected by the microscopic brown alga, Streblonema aecidioides. Infected thalli may show symptoms of Streblonema disease, i.e. alterations of blade and stipe, ranging from dark spots to heavy deformations and completely crippled thalli. Samples taken from a single locality all year round show that (i) the host population is infected at a high rate of 87±13% (SD), but that (ii) a considerable proportion of thalli containing Streblonema does not show disease symptoms, and that (iii) juvenile hosts, which mainly appear in autumn, are infected at almost the same rate. Thus the infection seems to occur early in the host's life. Juveniles in nature show fewer symptoms of the disease than adults. Two months after infection, oxygen production and growth in laboratory-raised experimentally infected juvenile hosts was not different from uninfected controls. Experimental thalli showed more severe morphological alterations than uninfected controls only four months after infection. Both field and laboratory observations indicate that a lag phase exists between infection and outbreak of the disease.     

Epidermal growth factor (EGF) receptor endocytosis is accompanied by reorganization of microtubule system in HeLa cells

Translocation of endosomes along microtubules (MTs) from the cell periphery toward the juxtranuclear region proximal to MTOC is well established. During this translocation the radial MT system is believed to retain its organization. Here we demonstrate that epidermal growth factor receptor (EGFR) endocytosis in HeLa cells is accompanied by dramatic remodeling of the MT system. Synchronized endocytosis was stimulated by warming the cells after EGF prebinding to EGFR on ice. Soon after that MTs were fully reestablished and EGFR was found in EE aligned along peripheral MTs. By the beginning of EE-to-LE sorting, the number of long MTs decreased and MTs appeared like an entangled meshwork of disorientated fragments and were partially depolymerized. Simultaneously, tubulin staining increased in juxtranuclear region, and at the time of LE-Lys interaction, enlarged EGFR-containing endosomes were localized there. Radial MTs were re-established when EGF-EGFR degradation started in lysosomes. In EGF absence, no alterations occurred upon MTs re-establishment. We conclude that MT remodeling is endocytosis-dependent.     

First record of Asteronema rhodochortonoides (Phaeophyceae) for the Pacific Ocean

Morphological observations of a minute, filamentous, branched brown alga epiphytic on Sargassum thun‐bergii (Mertens ex Roth) Kuntze were made on material collected at Tsuyazaki (33°48′N, 130°27′E), Fukuoka Prefecture, southern Japan. This alga was assignable to Asteronema rhodochortonoides (Børgesen) Möller et Parodi in having stellately arranged chloroplasts with several pyrenoids grouped in the center, predominantly apical growth, narrow filaments, and elliptical or broadly elliptical plurilocular zoidangia that are apically or laterally formed on upright filaments. A comparison of partial nuclear small subunit rDNA sequences between the Japanese material and A. rhodochortonoides from the Canary Islands showed only two or three nucleotide differences. This supports our assignment of the Japanese material to this species as a first report for the Pacific Ocean. In laboratory cultures, zoids released from plurilocular zoidangia developed into plants with morphology similar to the field‐collected plants. This cycle repeated without production of unilocular zoidangia in our cultures.     

Seasonal stability of sulfuric acid accumulation in the Dictyotales (Phaeophyceae)

Concerning the accumulation of S0₄^2‐ in cells, three types of species are known in the Dictyotales: (i) acidic type (high H₂S0₄ accumulation); (ii) high MgS0₄ accumulation type; and (iii) nonacidic type (low S0₄^2‐ accumulation). Seasonal changes of intracellular pH and concentrations of inorganic ions were examined in six dictyotalean species. In acidic species (Dictyopteris prolifera (Okamura) Okamura and Spatoglossum eras‐sum J. Tanaka), intracellular concentrations of S0₄^2‐ and H⁺ estimated by pH were high through all seasons. In high MgS0₄ accumulating species (Dictyotasp. and Padina arborescens Holmes), intracellular concentrations of S0₄^2‐ and Mg²⁺ were high through all seasons. In nonacidic species (Dictyota dichotoma (Hudson) Lamouroux and Dictyopteris undulata Holmes), intracellular concentration of sulfuric acid ion was low all year round.     

Life history of Chnoospora implexa (Chnoosporaceae, Phaeophyceae) in culture

The life history of the brown alga Chnoospora implexa J. Agardh (Chnoosporaceae, Scytosiphonales) from Japan was studied in laboratory cultures. This species showed a heteromorphic and diphasic life history, alternating between erect gametophytes and discoid sporophytes. The gametophytes were dioecious and produced isogametes. The zygotes developed into sporophytes at 20°C under long‐day conditions, which formed plurilocular zoidangia. Zoids released from the plurilocular zoidangia developed again into sporophytes that always formed plurilocular zoidangia at 20°C and 25°C in long‐day conditions, and mainly unilocular zoidangia at 25°C in short‐day conditions. Zoids released from unilocular zoidangia developed into dioecious gametophytes. At 15°C zygotic erect thalli were formed and were revealed to be diploid by microspectrofluorometric measurements of nuclear DNA contents. The development and reproduction of unfused gametes were similar to those of zygotes. Some strains showed a direct‐type life history; gametophytic thalli were produced, but not via a sporophytic phase.     

Taxonomy and distribution of Sargassum (Phaeophyceae) in the Gulf of Thailand

Ten species of Sargassum (Sargassaceae, Phaeophyceae) were found along the Gulf of Thailand. Morphological characteristics of Sargassum baccularia (Mertens) C.A. Agardh, S. binderi Sonder, S. cinereum J.G. Agardh, S.crassifolium J.G. Agardh, S. longifructum Tseng et Lu, S. oligocystum Montagne, S. polycystum C.A. Agardh, S. siliquosum J.G. Agardh, S. swartzii (Turner) C.A. Agardh and one unidentified species were examined and are described in detail. The most common species were S. polycystum distributed widely in almost all the study sites, S. crassifolium restricted to Prachuap Khirikhan Province, S. longifructum restricted to Chumphon Province, S. siliquosum restricted to Surat Thani Province and one unidentified species restricted to Songkhla Province. Three species (S. cinereum, S. longifructum and S. swartzii) are new records for the algal flora of Thailand. Five species (S. baccularia, S. cinereum, S. longifructum, S. polycystum and the unidentified species) belong to the section Zygocarpicae (J.G. Agardh) Setchell.     

Protoplast isolation,development, and regeneration in different strains ofPilayella littoralis (L.) Kjellm. (Phaeophyceae)

Summary Using an antibody against tyrosinated tubulin and epifluorescence microscopy, mitosis was studied in three different microvessel endothelial cell types recently isolated from bovine corpus luteum. Dividing cells were flat and at certain stages individual microtubules could be followed for considerable lengths. The structure of the spindle apparatus and the course of mitosis were conventional. Microtubule asters were small from prophase until metaphase in all three cell types. However, whereas in two cell types telophase asters remained inconspicuous, prominent asters, of mostly straight microtubules, formed in telophase cells of a third cell type. Thus, aster size is heterogeneous between different endothelial cell types. Large microtubule asters are not regularly found in dividing cultured mammalian cells. The microendothelial cell types present themselves as appropriate systems for spindle research and especially for the study of aster microtubule dynamics and function.     

Verosphacela silvae sp. nov. (Onslowiaceae, Phaeophyceae) from the Mediterranean Sea

We describe Verosphacela silvae sp. nov., from the Mediterranean Sea. It consists of horizontal filaments living on the lower face of the red alga Peyssonnelia rubra (Greville) J. Agardh, from which erect filaments up to 1.5 mm high rise and grow upright after passing through the thallus of the supporting species. There are both horizontal and erect filaments growing by apical cells. In the subapical cells, 1–2 longitudinal divisions occur (more frequently in the erect filaments) but no secondary transverse divisions occur. Erect filaments bear lateral propagules on a stalk of one to three (rarely more) cells. Propagules, with neither apical cells nor arms, consist of seven cells. Zoidangia are borne at the apex of erect laterals. The new species differs from V. ebrachia Henry mainly in habit, propagules and zoidangia. In addition, distinct from V. ebrachia, filaments of V. silvae never penetrate between the cuticle and the cell wall of the supporting alga. Moreover, propagules of V. silvae consist of seven cells, whereas those of V. ebrachia consist of 9–13 cells, and zoidangia are terminal on laterals in V. silvae, whereas in V. ebrachia they are sessile on both axes and laterals.