The fine structure of the centrohelidian Heliozoan Heterophrys marina

Summary The fine structure of Heterophrys marina (Centrohelidia, Heliozoa) is described with special reference to centroplast structure, morphogenesis and behaviour of kinetocysts (= axopodial granules which perform saltatory movement), and formation of organic spicules in a new type of organelle located in the plasma membrane. A low calcium pretreatment and fixation was used to improve preservation of highly labile axopodia which near their distal end contain a single microtubule (MT) only. Two varieties of H. marina with a respective maximum of 6 and 12 MTs per axopodium, and 2 hitherto undescribed species, H. elati and H. multipoda, were found among 9 stocks collected in Europe and North America. In all species only the central 6 MTs of each axoneme originate from a scaffolding layer of electron dense material which surrounds the central granule. Evidence is presented which indicates that in Heterophrys self-linkage is not the only principle of MT pattern generation but that instead precisely localized MT nucleation and specific linkage of MTs within the cortex of the centroplast lead to the MT patterns observed. Prekinetocysts originate from vesicles found in the neighborhood of the dictyosomes. After maturation the kinetocysts become attached to the plasma membrane which seems to play an important role both in selection of particles transported in the axopodia and particle movement as well.The author thanks Anne Vees for excellent technical assistance. This study was sponsored by the Deutsche Forschungsgemeinschaft. Field trips to Naples and Woods Hole were supported by NATO Research Grant No. 657 to Dr. L. E. Roth, Kansas State University, Manhattan, Kansas, and the author.     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

Microtubule organizing centers in plant cells: localization of a 49 kDa protein that is immunologically cross-reactive to a 51 kDa protein from sea urchin centrosomes in synchronized tobacco BY-2 cells

Summary A 49 kDa protein in tobacco BY-2 cells has been found to be cross-reactive with antibodies raised against a 51 kDa protein that was isolated from sea urchin centrosomes and identified as a microtubule-organizing center (MTOC) in animal cells. Tracing the fate of the 49 kDa protein during progression of the cell cycle in highly synchronized tobacco BY-2 cells revealed that this protein was colocalized with plant microtubules (MTs): the location of the 49 kDa protein coincided with preprophase bands (PPBs), mitotic spindles and phragmoplasts. Furthermore, between the M and G₁ phases, the 49 kDa protein was observed in the perinuclear regions, in which the initials of MTs are organizing to form cortical MTs. At the G₁ phase the location of the 49 kDa protein in the cell cortex coincided with that of the cortical MTs. It appeared that the 49 kDa protein in the cell cortex was transported as granules from the perinuclear regions. Thus, it is highly probable that the 49 kDa protein, which reacts with antibodies against the 51 kDa protein in sea urchin centrosomes, plays the role of an MTOC in plant cells. Thus, the mechanisms for organizing MTs in higher organisms appear to share a common protein, even though the organization of MTs is superficially very different in plant and animal cells.Abbreviations DAPI 4,6-diamidino-2-phenyl indole - MT microtubule - MTOC microtubule-organizing center - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PPB preprophase band - SDS sodium dodecylsulfate     

Cytology of lepidoptera VIII. Acetylation of α-tubulin in mitotic and meiotic spindles of two Lepidoptera species,Ephestia kuehniella (Pyralidae) andPieris brassicae (Pieridae)

Summary The microtubular spindle in spermatocytes of Lepidoptera is unconventional in that the bulk of the microtubules (MTs) ends relatively abrupt about halfway between the spindle equator and the centrosomes from late prometaphase through early anaphase. Membranous elements separate the MT ends from the centrosomes. In the present study, the question is addressed whether MTs in meiotic spindles of male Lepidoptera are — as typical of spindle MTs in other systems — highly dynamic or whether they represent a more stable MT population. To this end, primary spermatocytes of two Lepidoptera species,Ephestia kuehniella (Pyralidae) andPieris brassicae (Pieridae), were probed with a widely used antibody, 6–11B-1, directed against acetylated -tubulin. Tubulin acetylation is believed to indicate the presence of long-lived MTs. In late telophase spermatocytes of both species, spindle MTs were highly acetylated. This is in keeping with observations in other systems: MT dynamics decreases towards telophase. The labeling intensity in younger spermatocytes differed, however, between both species. InE. kuehniella only flagella were labeled, whereas inP. brassicae also the kinetochore MTs and small MT arrays around the centrosomes were detected by the antibody against acetylated -tubullin. The findings are compatible with the suggestion that spindle MTs are dynamic in prometaphase to anaphase spermatocytes ofE. kuehniella. In fact, treatment with taxol, a MT-stabilizing drug, leads to high acetylation of -tubulin throughout spindle MTs ofE. kuehniella in this period. Meiotic spindles inP. brassicae are longer by a factor of 1.3 than those ofE. kuehniella. The shorter MTs inE. kuehniella may turnover completely and cannot accumulate acetylated portions, whereas segments of MTs in the longer spindles ofP. brassicae persist and become post-translationally acetylated. Spermatogonial mitosis was also studied in both species and spindle MTs were found highly acetylated throughout mitosis. Thus, mitotic and meiotic spindles in males of Lepidoptera differ with respect to MT turnover.Abbreviations BSA bovine serum albumin - DAPI 4,6-Diamidino-2-phenylindole-2 HC1 - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis (2-ethane sulfonic acid) - MT microtubule     

A light and electron microscopic study of a new species of centroheliozoon, Chlamydaster fimbriatus

The centroheliozoon Chlamydaster fimbriatus n.sp. has been studied in culture by light and electron microscopy. The cell body is surrounded by a fimbriated mucus envelope through which axopods extend. Each cell contains two or three contractile vacuoles and an eccentrically located nucleus with a single nucleolus. The cells have typical centroheliozoan characteristics such as a centroplast with a trilamellate disc, mitochondria with ribbon-like cristae, ball-and-cone kinetocysts, and a peripheral lacunar system. The axopodial axonemes possess a single hexagon of six microtubules. The genus is defined to include only centroheliozoa with a mucus coat. C. fimbriatus differs from the only other species recognized in this genus (C. laciniatus and C. sterni) in its size and number of arms. This is the first ultrastructural account of a species of the genus Chlamydaster.     

Changes in leaf water potential and CAM inSempervivum montanum andSedum album in response to water availability in the field

Summary The short term effects of irrigation on diurnal changes in leaf and titratable acidity were examined both inSempervivum montanum and inSedum album, a facultative CAM plant, in the Spanish Pyrenees. InSemperivivum, leaf responded rapidly to irrigation and, in both the control and irrigated plants, increased during the day and decreased during the night and early morning. By contrast, leaf inSedum responded more slowly to irrigation and showed a decrease during the day and an increase in the period between evening and early morning. Under the conditions of the short-term experiments, changes in acid metabolism were not observed in either species following irrigation. The results suggest that transpirational water loss together with redistribution of water within the plant are more important than the osmotic concentration of malic acid in determining leaf in both species and that daytime water loss is greater inSedum than inSempervivum.The effect of long-term water stress on leaf and acid levels was also assessed in both species over a 3-week period. Both leaf and acidification inSempervivum decreased over this time period but could, at least partially, be reversed by irrigation. InSedum, leaf also declined but a more gradual reduction in acidification occurred than inSempervivum. Irrigation inSedum at least partially reversed the decline in leaf but produced a complex pattern of acid metabolism. Nocturnal acidification in the irrigated plants was lower than in the non-irrigated control when preceded by a cool day but showed complete recovery following a hot day. It is suggested inSedum album that C₃ photosynthesis during the preceding light period, as determined by light intensity and leaf temperature, may be important in determining the extent of nocturnal acidification under field conditions.     

Spindle dynamics and arrangement of microtubules

Changes in microtubule (MT) arrangement were studied in endosperm of Haemanthus katherinae. Individual cells were selected in the light microscope and sectioned perpendicular or parallel to the long axis of the spindle. The following data and conclusions were drawn: During anaphase kinetochore fibers (bundles of kinetochore MTs) always intermingle with non-kinetochore (continuous) fibers (bundles of non-kinetochore MTs). The latter often branch and some free ends are present. Often one non-kinetochore fiber is connected with more than one kinetochore fiber, explaining why chromosomes may lose their ability for independent movement. During anaphase kinetochore fibers move to the poles, the number of kinetochore MTs decreases by one-half and the MTs tend to become more splayed out. At the same time the number of MTs between trailing chromosome arms increases, probably representing segments of kinetochore MTs which break during anaphase. The number of non-kinetochore MTs in the equatorial region at anaphase is twice the number of non-kinetochore MTs in metaphase. The above data agree perfectly with those in polarized light and indicate that a simple sliding system does not exist in the spindle of Haemanthus.     

Reorganization of cortical microtubules and cellulose deposition during leaf formation in Graptopetalum paraguayense

In the regeneration of a shoot from a leaf of the succulent, Graptopetalum paraguayense E. Walther the first new organs are leaf primordia. The original arrangement of cellulose microfibrils and of microtubules (MTs) in the epidermis of the leaf-forming site is one of parallel, straight lines. In the new primordium both structures still have a congruent arrangement but it is roughly in the form of concentric circles that surround the new cylindrical organ. The regions which undergo the greatest shift in orientation (90°) were studied in detail. Departures from the original cellulose alignment are detected in changes in the polarized-light image. Departures from the original cortical MT arrangement are detected using electron microscopy. The over-all reorganization of the MT pattern is followed by the tally of MT profiles, the various regions being studied in two perpendicular planes of section. This corrects for the difference in efficiency in counting transverse versus longitudinal profiles of MTs. Reorientation takes place sporadically, cell by cell, for both the cellulose microfibrils and the MTs, indicating a coordinated reorientation of the two structures. That MTs and cellulose microfibrils reorient jointly in individual cells was shown by reconstruction of the arrays of cortical MTs in paradermal sections of individual cells whose recent change in the orientation of cellulose deposition had been detected with polarized light. Closeness of the two alignments was also indicated by images where the MT and microfibril alignments co-varied within a single cell. The change-over in alignment of the MTs appears to involve stages where arrays of contrasting orientation co-exist to give a criss-cross image. During this critical reorganization, the frequency of the MTs is high. It falls during subsequent enlargement of the organ. It was found that the rearrangement of the cortical MTs to approximate a series of concentric circles on the residual meristem occurred before the emergence of leaf primordia. Through their apparent influence on microfibril alignments, the changes in MT disposition, described here, have the potential to generate major biophysical changes that accompany organogenesis.Abbreviation MT(s) microtubule(s)     

Organization and Control of Microtubule Pattern in Centrohelidan Heliozoa*†

SYNOPSIS. Comparative studies of axopodial microtubule pattern in 10 different centrohelidan Heliozoa belonging to the genera Acanthocystis, Raphidiophrys and Heterophrys suggest that 2 basic principles govern pattern formation in centrohelidan Heliozoa. While the larger “open” arrays with unspecified number of microtubules, e.g. in A. aculeata and R. ambigua, may result from self-linkage of additional microtubules around centroplast-nucleated “starter microtubules,” the smaller “closed” arrays with specified microtubule number, e.g. in A. pectinata and H. marina, favor a template-driven linkage mechanism. The centroplast is a highly complex microtubule organizing center involved in the control of orientation, number, and diameter of the axonemes. Its shell may serve as a surface upon which the microtubule nucleating sites assemble, but how the precise positioning of these sites occurs is still open to debate. Some of the unsolved problems of microtubule pattern formation may be explained by the “linker nucleation hypothesis” which is an extension of the “gradion hypothesis” by Roth et al. It is shown how both the formation of closed arrays and the balanced lateral growth of open arrays may result from linker-induced microtubule nucleation.     

Role of plasma membrane proteins and cell wall in meridional arrangement of cortical microtubules inBoodlea coacta

Summary The effects of 2,6-dichlorobenzonitrile (DCB, an agent which inhibits cellulose synthesis) and cycloheximide (CHI, a known inhibitor of protein synthesis) on the construction and stability of the cortical microtubule (MT) cytoskeleton in two kinds of protoplasts (smaller protoplasts and larger ones) prepared fromBoodlea coacta (Dickie) Murray et De Toni were examined by immunofluorescence microscopy. In smaller protoplasts which develop from released protoplasmic masses in culture media, parental cortical MTs assume a convoluted configuration, but new cortical MTs appear following disassembly of convoluted MTs. New cortical MTs initially have a random arrangement but later, a rough meridional arrangement following development of cell polarity and finally, a high density meridional arrangement. In larger protoplasts which are formed within cell wall cylinders of thalli cut at 500 m length, longitudinally oriented parental cortical MTs are preserved. Each exhibits a curving configuration just after protoplast formation, but a straight configuration after 3 h of culture. In smaller protoplasts, cortical MT orientation changes from random to rough meridional orientation but never to a high density meridional orientation following treatment with 10 M CHI, and MT density decreases after 12 h. However, rough meridional and high density meridional arrangements of MTs ceased to be formed and MT density decreased following treatment with 10 M DCB. In larger protoplasts, high density meridional arrangements of MTs were noted not to be affected by treatment with CHI; instead, they continued to remain oriented meridionally, but the length and density were decreased after treatment with DCB for 3–4 h. After 10 h, the MTs became fragmented and orientation was random. From these findings it is summarized that: (1) There are no putative anchors in the plasma membrane of nascent smaller protoplasts, but the meridional orientation of cortical MTs requires anchors which may be distributed in the plasma membrane following the establishment of cell polarity. (2) Plasma membranes in larger protoplasts contain parental anchors oriented meridionally. Anchors stabilize cortical MTs via their close relation to cell walls (especially to cellulose). Anchors are detached from the plasma membrane when cellulose is not formed. (3) Cellulose regeneration may be indispensable to the formation and stabilization of the MT cytoskeleton inBoodlea.Abbreviations CHI cycloheximide - DCB 2,6-dichlorobenzonitrile - DMSO dimethylsulfoxide - MT microtubule     

Bewegungs- und Teilungsverhalten der Chromatophoren vonEunotia pectinalis var.polyplastidica und andererEunotia-Arten bei der Zellteilung

Summary The correlation between the start of chromatophore division and cell division is very different in various species ofEunotia. In some species the chromatophore division occurs before, in others after cell division. Eunotia pectinalis var.polyplastidica, with eight chromatophores per cell, represents an extreme type of behaviour in so far as two of the four plastids in each daughter cell prior to their division are shifted to the new hypovalve while the other two rest in situ. There occur two patterns of distribution of the four plastids, and that in the ratio 11, whereas the theoretically possible third pattern is never realized. The cause of that phenomenon is discussed. The division of the four plastids, in the meantime grown to full size, is performed not before they have reached their definitive equilibrium position at the epivalve or, respectively, at the hypovalve in twos. InEunotia pectinalis var.polyplastidica, by its mode of chromatophore division, the constant dissymmetric (right-left-handed) arrangement of the growing chromatophores, established inEunotia arcus, is not to be expected and is in fact not realized. InEunotia lunaris, however, the shift of the daughter chromatophores, in relation to the dorsiventrality of the cell, shows not only the same kind of dissymmetry as inEunotia arcus but also the same direction of shifting.The four and four chromatophores inEunotia pectinalis var.polyplastidica correspond to the two chromatophore plates in other species but the are not comparable with the numerous little chromatophore discs of Diatomaceae and other species the number of which decreases with the reduction of cell size.Cells ofEunotia pectinalis var.polyplastidica are capable to move by raphe action.

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Herrn Dr.Franz Berger zum 70. Geburtstag gewidmet.     

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.     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

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)     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

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     

Microtubule organization in root cortical cells ofHyacinthus orientalis

Summary Overall cellular arrangement of cortical microtubules (MTs) is studied by reconstruction of MT images on serial thin sections. The mature root cortex ofHyacinthus orientalis L. cv. Delft blue is composed of elongate, highly vacuolate nondividing parenchyma cells. In longitudinal sections in these cells, MTs generally form parallel arrays at oblique angles to longitudinal cell axes. These MTs extend towards the transverse face of the cell where they appear in localized parallel arrays as well as in crisscross patterns. Repeated observations of oblique parallel arrays of MTs along the length of the cell and the continuity of MT bundles in serial sections suggest that MTs form a single helix in the cell. MTs in neighboring cells appear in sections either as parallel or as herringbone patterns, suggesting that the MT helices in these cells may spiral in the same or the opposite directions.Abbreviations MT Microtubule - MF microfibil - EM electron microscopy     

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

Summary The Host Factor required for in vitro coliphage Q RNA replication, a heat-stable RNA binding protein present in uninfectedEscherichia coli, has been detected by both immunological and functional tests inAcinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa andPseudomonas putida. It was not detectable by these criteria inBacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata orSaccharomyces cerevisiae. InEscherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demostrated here that this association is specific for the 30S ribosomal subunit.     

Isolation of Axopodial Axonemes from Mass-cultured Echinosphaerium nucleofilum1

The heliozoan Echinosphaerium nucleofilum produced about 75 times 10³ floating cells per 19-cm culture dish per day when fed the green flagellate Chlorogonium elongatum. This method yields enough cells for usable quantities of subcellular fractions. Heliozoa were lysed in a detergent mixture containing stabilizing reagents, and axonemal bundles of axopodial microtubules were isolated from the lysate by differential centrifugation. Polyacrylamide gel electrophoresis in sodium dodecylsulfate showed two prominent bands tentatively designated alpha- and beta-tubulin. Apparent molecular weights were 51.8 times 10³ and 48.1 times 10³, respectively. As assayed by electron microscopy of negatively stained whole mounts, the microtubule bundles splintered readily, although glycerol tended to inhibit this fraying. Intermicrotubule bridges could be observed in some axonemal splinters.     

Microtubule formation on the nuclear surface during meiosis inAcetabularia acetabulum

Summary Microtubules (MT) are a feature of all eukaryotic cells. However, they have not been observed in the cytoplasm of the vegetative phase ofAcetabularia acetabulum. Previous investigators have reported that, in the propagative phase, MTs function as anchors in the transport of secondary nuclei to the cap. They also form elaborate arrays around nuclei during cyst formation. The life history ofA. acetabulum is marked by changes in chromatin, the nucleolus, and the perinuclear cytoplasm. In this study light microscopical features of the nucleolus and changes in chromatin, labelled with anti-histon antibodies, were used to define the developmental stages. Anti-tubulin antibodies have been used to trace the origin and development of MTs, MTs are formed on the surface of the primary nucleus. They are organized first into short thick sticks and then later elongate into thinner strands which enclose the nucleus in a dense network. Following these events on the surface of the nucleus, the spindle develops inside the nuclear membrane which remains intact throughout the mitotic division.