Aberrant microtubule organization can result in genetic abnormalities in protoplast cultures of Vicia hajastana Grossh

Protoplast cultures of Vicia hajastana have a high division frequency. However, 20–40% of the microcolonies fail to develop beyond the 20-30-cell stage. Aneuploids and polyploids were found in early divisions and persisted in older cultures. The resulting protoplast-derived suspension culture differed karyologically from the original culture. Karyokinesis and cytokinesis were studied using simultaneous staining of microtubules (MT) by immunofluorescence, DNA by Hoechst 33258 (2-[2-(4-hydroxyphenyl)-6-benzimidazoyl]-6-[1-methyl-4-piperazyl]benzimidazole) and cell walls by Calcofluor. Freshly prepared protoplasts showed mitoses and high frequencies of binucleate cells, which probably resulted mainly from failure of cytokinesis. In early divisions, many mitoses showed metaphase chromosomes with kinetochore MT but lacking polar MT. These aberrant mitoses probably accounted for an increase in hyperploid cells observed in protoplast cultures. Multipolar spindles, which gave rise to hypoploid cells, were also seen in the early divisions. Telophase abnormalities included dislocated phragmoplasts and incomplete formation of cross walls. Many divisions resulted in daughter nuclei of unequal size. Unequal segregation of chromosomes was detected by cytofluorimetric measurements of telophase nuclei stained with Hoechst. After 5 d of culture, 91% of the divisions with incomplete cross walls also contained different-size nuclei; conversely, 78% of the divisions with fully formed cross walls contained nuclei of equal size. The malfunctioning of spindles and phragmoplasts in the same cells indicates a functional interdependence of the different MT configurations in mitosis. During the first 24 h of culture, a high frequency of abnormalities was found in spindles, cross-wall formation and chromosome segregation; this was reduced substantially in the cells undergoing first division by 48 h. The data indicate that it may be possible to manipulate the frequency of abnormalities by controlling the onset of the first division in protoplast cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MT microtubule(s) - PB prophase band(s) - PNF perinuclear fluorescence - PPB pre-prophase band     

Microtubule organization during the development of the mitotic apparatus in cultured mesophyll protoplasts of higher plants – an immunofluorescence microscopic study

Microtubule organization in the course of the interphase-mitosis transition, karyokinesis and cytokinesis in cultured mesophyll protoplasts of Medicago sativa L. cv. Regen S and Nicotiana tabacum L. cv. Wisconsin 38 was investigated during the early culture stages. A prominent circular band of microtubules, the prophase band of microtubules (PB) was seen in the cell cortex during prophase in most cells. The band appeared during very early prophase and disappeared during late prophase. Other prophase microtubule arrays were nuclear-envelope associated microtubule fluorescence (perinuclear fluorescence, PNF) and groups of microtubules radiating from the nuclear envelope into the cytoplasm. These radiating microtubutes (perinuclear radiating microtubules, PNR) were particularly long and prominent in the PB plane appearing to link the PNF with the PB network. The possible functions of the PNR are discussed. The role of the PB is discussed in the light of the unorganized nature of these cells.     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

Aluminium effects on microtubule organization in dividing root-tip cells of Triticum turgidum. I. Mitotic cells

The effects of aluminium (Al) on dividing root-tip cells of Triticum turgidum were investigated with tubulin immunolabelling and electron microscopy. Aluminium affects the mechanisms controlling the organization of microtubule (MT) cytoskeleton, as well as tubulin polymerization, and induces the following aberrations in mitotic cells. (1) It delays the MT disassembly during mitosis, resulting in the persistence of preprophase MT bands in the late prophase cells, the presence of prophase spindles in prometaphase cells, and a disturbance in the shortening of kinetochore MT bundles in anaphase cells. (2) It interferes with the self-organization process of MTs into bipolar systems, inhibiting the formation of prophase and metaphase spindles. (3) Aluminium induces the formation of atypical MT arrays, which in the immunofluorescent specimens appear as ring-like tubulin aggregations in the cortical cytoplasm of the preprophase/prophase cells and as endoplasmic tubulin bundles in prophase and metaphase/anaphase cells; abnormal preprophase MT bands are assembled, consisting of atypical cortical and endoplasmic MT bundles, the latter clearly lining the nuclear envelope on the preprophase MT band plane. (4) It disorders the chromosome movements carried out by the mitotic spindle. In addition, after prolonged Al treatments chromatin condensation is inhibited. The outcome is greatly disturbed organization and function of the mitotic apparatus, as well as inhibition of cells from entering mitosis. This study shows that the MT cytoskeleton is a target site of Al toxicity in mitotic root-tip cells of T. turgidum . The possible mechanisms by which Al exerts its toxicity on MT organization and function are discussed.     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Loss of microtubular orientation and impaired development of prophase bands upon inhibition of RNA synthesis in root meristem cells

Synchronous cells in Allium cepa L. root meristems were treated with 3deoxyadenosine (cordycepin, an inhibitor of RNA synthesis) when roughly half of the population had reached G₂ phase (fast cells) while the other half was still in late S (slow cells). Since this drug also blocks DNA replication, slow cells remained in S while the treatment was continued.Indirect immunodetection of tubulin showed that a band typical of prophase formed in some cells with a portion of their nuclear DNA unreplicated. Most of the cells arrested in late S which had not developed a prophase band had a diminished number of cortical microtubules. Changes in their orientation (from transversal to oblique or longitudinal) occurred in roughly a cycle time (28 h).In the presence of 3deoxyadenosine, fast cells which had reached G₂ at the start of the treatment proceeded to prophase and remained there. Mature prophase bands were also formed in these cells but eventually they disoriented and, finally, disappeared. The data suggest that microtubular orientation in the meristematic cells depends on long-lived RNA species.Abbreviations 3AdR 3deoxyadenosine - MT microtubule - PB tubulin band typical of prophase     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

Microtubule patterns during meiosis in two higher plant species

Summary A monoclonal antibody to higher plant tubulin was used to trace microtubule (MT) structures by immunofluorescence throughout mitosis and meiosis in two angiosperms,Lycopersicon esculentum andOrnithogalum virens. Root tip cells showed stage specific MT patterns typical of higher plant cells. These included parallel cortical interphase arrays oriented perpendicular to the long axis of the cell, preprophase band MTs in late interphase through prophase, barrelshaped spindles, and finally phragmoplasts. Pollen mother cell divisions exhibited randomly oriented cortical MT arrays in prophase I, pointed spindles during karyokinesis, and elongate phragmoplasts. A preprophase band was not observed in either meiotic division. MT initiation sites were seen as broad zones associated with the nuclear envelope.     

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     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

Somatic hybridization of amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) by electrofusion

Summary Intraspecific somatic hybridization between amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) has been carried out following electrofusion of protoplasts. In initial analytical electrofusion experiments (1 mm electrode separation) optimal fusion conditions were determined by changing the fusion medium (addition of Ca and/or spermine) and the electrical parameters. Subsequently, in large scale experiments, cell suspension protoplasts of aec-1, a variant resistant to AEC, were fused with the same type of protoplasts of 5mt-26 or 5mt-27, both variants resistant to 5MT and cross-resistant to 3 FT. After an extensive selection procedure only somatic hybrid lines of aec-1 + 5mt-26 were obtained. The resistance traits of aec-1 and 5mt-26 were expressed fully, indicating that the variant characters involved are transmitted dominantly. Quantitative examination of the free amino acid content revealed characteristics of both the parental cell lines in most of the somatic hybrids. However, initially selected double resistant colonies from fusions of aec-1 + 5mt-27 lines appeared not to be somatic hybrids.Abbreviations AEC S-aminoethylcysteine - 3FT 3-fluorotyrosine - 5MT 5-methyltryptophan     

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.     

Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Summary Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.Contribution from the Department of Agronomy and published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2730. This research was supported in part by the USDA/SEA Competitive Research Grant 5901-0410-9-0334-0, USDA/ SEA-CSU Cooperative Research Grant 12-14-5001-265 and Colorado State University Hatch Project. This paper was presented partly at the Fourth International Barley Genetics Symposium, Edinburgh, Scotland, July 22–29, 1981     

Multinucleate nature,and mating by use of isozyme analysis inPoria cocos

Double staining study of nuclei and cell walls inPoria cocos indicated that the hyphal cells were multinucleate and had no clamp connections. Isozyme analysis of alcohol dehydrogenase (ADH) in 52 natural isolates revealed that there were three types of banding patterns: type I, five bands; type II, one slow band; type III, one fast band. Regenerants expressing type-II or type-III ADH-isozyme pattern were obtained from type-I isolates via protoplast manipulation. When the type-II regenerants were mated with the type-III regenerants, hyphae of type-I phenotype appeared. These data indicated that these type-II and type-III regenerants derived from protoplasts of the type-I isolates were primary hyphae. These primary hyphal cells were also multinucleate. Inter-strain mating ofP. cocos was performed and confirmed by ADH-isozyme analysis. Confronting cultures of a type-III regenerant derived from protoplasts of a type-I isolate and a type-II regenerant derived from a type-II isolate resulted in type-I hyphae.     

Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.Abbreviation PBP penicillin-binding protein     

Distribution and function of actin in the developing stomatal complex of winter rye (Secale cereale cv. Puma)

Summary The dynamics of actin distribution during stomatal complex formation in leaves of winter rye was examined by means of immunofluorescence microscopy of epidermal sheets. This method results in actin localization patterns that are the same as those seen with rhodamine-phalloidin staining, but are more stable. During stomatal development MFs are extensively rearranged, and most of the time the orientation or placement of MFs is distinctly different from that of MTs, the exception being co-localization of MTs and MFs in phragmoplasts. Although MFs show an orientation similar to that of MTs in interphase guard mother cells, no banding of MFs into anything resembling the interphase MT band is observed. From prophase to telophase, a distinct, dense concentration of MFs is found in subsidiary cell mother cells (SMCs) between the nucleus and the region of the cell cortex facing the guard mother cell. Cytochalasin B treatment causes incorrect positioning of the SMC nucleus/daughter nuclei and abarrent placement and orientation of the new cell wall that forms the boundary of the subsidiary cell at cytokinesis. These results suggest that MFs are involved in maintaining the SMC nucleus in its correct position and the SMC spindle in the correct orientation relative to the division site previously delineated by the preprophase band. Because these MFs thus appear to assure that the SMC phragmoplast begins to form in the correct orientation near the division site to which it needs to grow, we suggest that MFs are involved in control of correct placement and orientation of the new cell wall of the subsidiary cell.Abbreviations CB cytochalasin B - DIC differential interference contrast - DMSO dimethylsulfoxide - MBS m-maleimidobenzoyl-N-hydroxylsuccinimide ester - MF microfilament - MT microtubule - PBS phosphate buffered saline - SMC subsidiary cell mother cell Dedicated to the memory of Professor Oswald Kiermayer     

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

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

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell