The cell characters of two Helioflagellates related to the Centrohelidian lineage:Dimorpha andTetradimorpha

Characters of the two HelioflagellatesDimorpha andTetradimorpha were compared to other Heliozoa particularly the Centrohelidia and to the helioflagellateCiliophrys. They both have a condensed central MTOC from which originate the axopodial MTs as in the Centrohelidia. The axopodia are contractile and participate in both prey capture and slow movements of the cell. InDimorpha the MTs of the axopodial cytoskeleton are arranged in quincunx resulting in a quadrangular arrangement or in a squared packed arrays. This type of arrangement differs markedly from that of other centrohelidian Heliozoa where MTs are assembled in triangles and hexagons or in irregular adjacent hexagons. InTetradimorpha the MTs are irregularly arranged, although they are linked together and triangular figures are visible. This latter arrangement could be considered as an incomplete and primitive system. The kinetocysts ofDimorpha andTetradimorpha are highly structured like most of the Centrohelidia but the central body is composed of encased cylinders similar to that ofClathrulina. Mitochondria have contorted cristae inTetradimorpha and vermicular ones inDimorpha thus they differ from the plate-like cristae of the centrohelidians. They do not have any mastigonemes on their flagella.These two helioflagellates have in common with centrohelidian heliozoa: a central MTOC, a close packing arrangement of MTs in axopods and highly structured kinetocysts. They might be considered as different levels or branches in the evolution of this lineage. They are different from the actinophryidian Heliozoa and from the helioflagellateCiliophrys in respect of the characters cited above. Their study does not reveal any characters common to their supposed ancestors the chrysomonad flagellates except the orthogonal pair of kinetosomes.     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

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 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.     

Tobacco protoplasts differentiate into elongate cells without net microtubule depolymerization

Summary Microtubules (MTs) are important for plant cell morphogenesis because they influence the deposition of cell plate and wall components. It has been observed that tobacco protoplasts contain a disordered MT array in the cortex. Following several days in culture, these protoplasts become elongate cells with an orderly cortical MT array. The transformation of the MT array may occur by net depolymerization of the disordered MTs and repolymerization of MTs into an ordered array, or by movement of the array as an integral unit. To experimentally distinguish between these two possibilities, the drug taxol was used to stabilize MTs. Protoplasts derived from suspension cultured tobacco,Nicotiana tabacum, were grown in a medium containing the two plant hormones -naphthaleneacetic acid and benzyladenine, in the presence or absence of 10M taxol. Changes in cell size and shape were quantified using a video image analysis system. Cell elongation had begun within 48h of protoplast conversion, in both treatments, and continued for 7 days. Immunolocalization of tubulin showed that, in the majority of cells, MTs were disorganized immediately following protoplast conversion. After elongation, the MT arrays were observed to have reoriented to an ordered state. Taxol-treated protoplasts were found to elongate faster and to a greater extent than the non-treated controls. Additionally, the cortical array of taxol-treated protoplasts reorganized more quickly. These data indicate that the net depolymerization of disordered cortical MTs is not necessarily required for the differentiation of a protoplast into an elongate cell.Abbreviations APM amiprophosmethyl - BSA bovine serum albumin - DIC differential interference contrast - DTT dithiothreitol - EGTA ethylenegrycol-bis-(-aminoethyl ether)N,N,N,N-tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FMS Fukuda, Murashige, and Skoog - MS Murashige and Skoog - MT(s) microtubule(s) - PBS phosphate buffered saline - PIPES piperazine-N,N-bis (2-ethanesulfonic acid, 1.5 sodium) - PM plasma membrane - Tris Tris(hydroxymethyl)amino-methane     

Pre- and postmitotic reorientation of microtubule arrays in youngSphagnum leaflets: Transitional stages and initiation sites

Summary The microtubules (MTs) of developingSphagnum leaflets rearrange from the interphase array into the preprophase band without obvious participation of definite initiation sites. At late prophase, additional MTs appear along the nuclear envelope, with the same orientation as in the peripherally situated preprophase band. Spindle formation begins along the nuclear envelope; spindle MTs run perpendicular to preprophase band MTs and converge in several focus points with indistinct polar bodies. After cytokinesis, most spindle and phragmoplast MTs disappear. Interphase MTs reappear at first along the central part of the new cell wall, in a region which was occupied before by the initial phragmoplast; their orientation is perpendicular to the phragmoplast MTs. Also here, distinct MT organizing centers could not be observed. Then the MT spread out over the cell periphery. The observations suggest that diffuse MT organizing zones rather than definite MT organizing centers play a role in the rearrangement of the different MT arrays during the cell cycle.     

Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

Summary The kinetics of MT reorientation in primary roots ofZea mays cv. Merit, were examined 15,30,45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4–5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10^–9 to 10^–6M IAA. IAA treatment at 10^–8M or less showed no major or consistent changes but 10^–7 M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10^–5M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether) N,N,NN-tetraacetic acid - MTs cortical microtubules - QC quiescent center - MES/TRIS 2-(N-morpholino)ethanesulfonic acid/tris(hydroxymethyl)aminomethane - IAA indole-3-acetic acid - PBS phosphate buffered saline - PHEMD [60 mM Pipes (piperazine-diethanesulfonic acid), 25 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 10 mM EGTA, 2mM MgCl₂ pH7.0 adjusted with NaOH] containing 5% dimethyl sulfoxide     

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     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

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     

Formation of spindle fibers,kinetochore orientation,and behavior of the nuclear envelope during mitosis in endosperm

The formation of kinetochore (chromosomal) and continuous fibers, and the behavior of the nuclear envelope (NE) was described in studies combining light and electron microscopy. Microtubules (MTs) push and pull the NE which becomes progressively weaker before breaking. It breaks to a certain extent due to mechanical pressure. Clear zone MTs penetrate into the nuclear area as dense bundles and form continuous fibers. These MTs also attach to some kinetochores during this process. Some kinetochore fibers seem to be formed by the kinetochores themselves which are also responsible for further development and changes of kinetochore fibers. Formation of kinetochore fibers is asynchronous for different chromosomes and even for two sister kinetochores. Often temporary faulty connections between different kinetochores or the polar regions are formed which usually break in later stages. This results in movements of chromosomes toward the poles and across the spindle during prometaphase. The NE, whose fine structure has been described, breaks into small pieces which often persist to the next mitosis. Old pieces of NE are utilized in the formation of new NE at telophase. Several problems concerning the mechanism of chromosome movements, visibility of the NE, etc., have also been discussed.     

Influence of the herbicide oryzalin on cytoskeleton and growth ofFunaria hygrometrica protonemata

Summary Protonemata ofFunaria hygrometrica were exposed to the herbicidal MT inhibitor oryzalin. A reduction of the growth rate together with a disturbance of oriented polar growth is observed. Both effects are reversible. Visualization of MT by IFT reveals differential sensitivities of MT. At lower concentrations (10^–6 M) only the cytoplasmic MT are depolymerized causing impairment of the migration of the nucleus and the transport of the plastids. Close association of MT with the surface of the plastids is demonstrated. At higher concentrations of oryzalin spindle and phragmoplast MT are affected as well. They are found in unusual orientations and display a variety of aberrant forms like multipolar spindles or the occurrence of several mini-spindles within one cell. The mode of action of oryzalin is discussed and the necessity of a continuous network of cytoplasmic MT between nucleus and growing tip for the maintenance of polar growth is emphasized.Abbreviations CIPC isopropylN-3-chlorophenyl carbamate - DAPI Diamidino-2-phenylindol - DMSO dimethyl sulfoxide - EGTA ethy-leneglycol-tetraacetic acid - FITC fluorescein isothiocyanate - IFT indirect immunofluorescence technique - IPC isopropylN-phenylcar-bamate - MSB microtubule stabilizing buffer - MT microtubule - MTOC microtubule organizing centre - Ph phragmoplast - PIPES piperazine-diethane sulfonic acid - S spindle Dedicated to Prof. Dr. K. E.Wohlfarth-Bottermann on the occasion of his 65th birthday.     

Microtubule organization in cultured soybean and black spruce cells: Interphase —mitosis transition and spindle morphology

Summary Microtubule (MT) distribution during the cell cycle, especially spindle organization, has been investigated using immunofluorescence light microscopy in cultured cells of two higher plant species, soybean (angiosperm) and black spruce (gymnosperm). In soybean, the prophase and metaphase spindles were different in morphology and structure. The prophase spindle covering the nucleus was barrel-shaped and MTs extended between poles. The metaphase spindle consisted mainly of short MT bundles on either side of the chromosome mass. During prometaphase, the polarity and shape of the prophase spindle disappeared, suggesting that the metaphase spindle is newly formed in prometaphase and not derived from the prophase spindle. A striking feature of MT organization in black spruce was sharply defined poles during prometaphase and anaphase. They were located close to the cell edge, suggesting that a structure in the cytoplasm or associated with the plasma membrane is responsible for their formation. In black spruce the metaphase spindle was long with pointed poles and MT fir tree structures. In contrast, the metaphase spindle of soybean was short with very broad poles and lacked MT fir trees. These results suggest that MT fir tree structure may not be necessary for a functional spindle.     

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)     

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     

Amiprophos-methyl (APM): A rapid,reversible, anti-microtuble agent for plant cell cultures

Summary In plant cell suspension cultures sensitive to the herbicide amiprophos-methyl (APM), 1 to 3 M APM completely depolymerized both cortical and mitotic microtubule (MT) arrays in 1 hour. In comparison, a 2 hour application of 3 mM colchicine had no effect on MT arrays. Recovery from APM treatment occurred as early as 5 minutes after removal of APM. Short, cortical MTs were visible in 3 hours and complete MT arrays were found within 22 hours after drug removal.Sensitivity to APM-induced MT depolymerization varied according to species but was increased or decreased by varying the mitotic rate in cultures. The results indicated APM sensitivity was related to lowered stability of MT arrays in rapidly cycling cells. APM treatment may help distinguish stabilized cortical MTs in elongating cells and nonstabilized cortical MTs in rapidly dividing cells.Abbreviations MT microtubule - APM amiprophos-methyl - DMSO dimethyl sulfoxide - PBS phosphate buffered saline     

Role of the microtubule cytoskeleton in alternating changes in cellulose-microfibril orientation in the coenocytic green alga,Chaetomorpha moniligera

The functions of the microtubule (MT) cytoskeleton in changing the orientation of microfibrils (MFs) in the cell walls of the coenocytic green alga Chaetomorpha moniligera Kjellman were investigated by electron microscopy. The cortical MT cytoskeleton in Chaetomorpha was comprised of longitudinally oriented MTs. Cellulose MFs, however, alternately changed their orientation longitudinally and transversely to form crisscross MF textures. Microtubules were parallel to longitudinally oriented MFs but never to those that were transversely oriented. The average density of MTs during the formation of longitudinally oriented MFs was 216 per 50 m of wall and that of transversely oriented MFs 170/50 m. To determine exactly the MT-density dependency of each MF orientation, changes in MF orientation were examined by changing MT density after treating and removing amiprophos-methyl (APM). Microtubules were reduced in number by a half (100/50 m) after 2 h and by 3/4 (50/50 m) after 3 h of treatment with APM (3 mM). This reduction was caused by the disappearance of alternating MTs. Microtubules retained this density (50/ 50 m) up to 6 h, and then gradually disappeared within 24 h. Microfibril orientation in the innermost cell wall was transverse after treatment with APM for 2 h but was helicoidal after 6 h. Polymerization of MTs occurred in the longitudinal direction following the removal of APM after treatment for 48 h. Microtubule density rose to about 100/50 m and 200/50 m after 6 h and 24 h, respectively. The orientation of MTs changed from helicoidal to transverse and transverse to longitudinal after 6 h and 24 h, respectively. When APM was removed prior to formation of the helicoidal texture, longitudinally oriented MFs appeared within 6 h. There is thus an alternating cycle of formation of longitudinally and transversely oriented MFs within a 12-h period. Formation of transversely oriented MFs as a result of APM treatment started in the middle of a cell as hoops which then extended in the apical and basal directions. Formation of longitudinally oriented MFs as a result of the removal of APM started from the apical end and proceeded toward the base. It follows from these results that: (1) the point of formation of longitudinally oriented MFs differs from that for transversely oriented MFs, (2) MF orientation in each case depends on a separately functioning mechanism, (3) MT density changes rhythmically to trigger a switch for crisscross orientation of MFs.Abbreviations APM amiprophos-methyl - MF microfibril - MT microtubule - TC terminal complex We thank Dr. K. Okuda for making helpful discussion and Miss. T. Matsuki for assistance with replica preparation.     

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.     

Acetylation of α-tubulin in male meiotic spindles ofPyrrhocoris apterus,an insect with holokinetic chromosomes

Summary Kinetochore structure was examined in metaphase spermatogonia and primary spermatocytes of the red firebug,Pyrrhocoris apterus (Pyrrhocoridae, Hemiptera). Chromosome spreads were analysed using light microscopy and serial sections through spindles were studied using electron microscopy. Mitotic chromosomes were rod-shaped bodies and did not possess primary constrictions. Trilaminar kinetochores occurred throughout about 72% of the chromosomal length. Numerous microtubules (MTs) were connected with the outer plates of the kinetochores and interactions between MTs and the remainder of the chromosomal surface were rare. The bivalents formed dumbbell-shaped bodies in metaphase I spermatocytes. At that stage, MTs were found in contact with the entire poleward surface of the chromosomes. Distinct kinetochore material was, however, not detectable and some MTs penetrated deeply into the chromatin. Mitotic and meiotic chromosomes ofP. apterus are holokinetic and consequently the number of kinetochore MTs is expected to be relatively high. In the second part of the study, the question whether holokinetic chromosomes affect spindle MT dynamics is addressed. To this end, primary spermatocytes ofP. apterus were labelled with a widely used antibody, 6-11B-1, directed against acetylated -tubulin. The acetylation of -tubulin is believed to indicate the presence of long-lived MTs. MT bundles were labelled in metaphase and anaphase I spindles, while prophase and prometaphase I spermatocytes did not contain acetylated MTs. MTs in early and mid telophase spindles were not acetylated. Only late telophase I spindles possessed small amounts of acetylated -tubulin. The acetylated MT bundles of metaphase and anaphase I spindles probably represent kinetochore MTs stabilized by their association with the holokinetic chromosomes at one end and the spindle poles at the opposite end.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole · 2HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis(2-ethane sulfonic acid) - MT microtubule     

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.