Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

Scale-associated glycoproteins of Scherffelia dubia (Chlorophyta) form high-molecular-weight complexes between the scale layers and the flagellar membrane

Flagellar scales were isolated from the flagellate green alga Scherffelia dubia. The flagellar scales consist mainly of acidic polysaccharides (70%) and glycoproteins (10%), and monosaccharide analyses show that the scales contain high amounts of unusual 2-keto-sugar acids. Approximately, 72 mol% of total carbohydrate is 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methylmanno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of at least 18 different scale-associated proteins (SAPs), ranging in apparent molecular mass from 77 kDa to over 300 kDa. Lectin blot analyses performed in combination with glycosidase treatment, showed that SAPs contained N-glycans of the highmannose type and the hybrid type, as well as a complex type that was not immunologically related to higher-plant complex glycans. Most of the SAPs were present in two or possibly three high-molecular-weight complexes. In these complexes, individual polypeptides are cross-linked by disulfide bridges. A polyclonal antibody was raised against a SAP of 126 kDa (SAP 126), a glycoprotein present in a high-molecular-weight complex. The SAP126 antibody was used to localize the protein between scale layer and flagellar membrane. We suggest that these high-molecular-weight complexes link scales to the flagellar membrane.Abbreviations AAA Aleuria aurantia agglutinin - DSA Datura stramonium agglutinin - DTT dithiothreitol - GNA Galanthus nivalis agglutinin - RCA Ricinus communis agglutinin - SAP Scale-associated protein - TBS Tris-buffered saline Dedicated to Professor Eberhard Schnepf on the occasion of his 65th birthday.This work was supported by the Deutsche Forschungsgemeinschaft and an Alexander von Humboldt Foundation research award to L. Perasso. We thank G. Noat for providing the anti--glucosidase and anti-pectin methyl esterase antibodies.     

The flagellar apparatus ofMesostigma viride (Prasinophyceae): Multilayered structures in a scaly green flagellate

Mesostigma viride Lauterborn (Prasinophyceae) is the first green flagellate found to have multilayered structures (MLS) in its flagellar apparatus. MLS's were previously known from green algae only in charophycean swarmers, linking theCharophyceae to the origin of land plants, whose male gametes (when flagellated) also possess an MLS.M. viride is, therefore, probably more closely related to the origin of theCharophyceae than any other green flagellate that has been thoroughly studied so far. The occurrence of MLS's in green flagellates and apparently in other algae and protozoans suggests that an MLS occurred in an ancient group of flagellates and has survived in various protistan lines, including the line of green algae related to land plants. The occurrence of a synistosome inM. viride and other of its characteristics suggest that it is more closely related toPyramimonas than to other genera of scaly green flagellates.This work was supported by National Science Foundation Grant DEB-78-03554.     

The flagellar apparatus of the scaly green flagellatePyramimonas obovata: Absolute configuration

Summary The flagellar apparatus of the quadriflagellate scaly green algaPyramimonas obovata has been studied in detail and the absolute configuration of the flagellar apparatus has been determined. The flagellar root system is cruciate (4-2-4-2-system). 18 major basal body associated fibrous structures connect the four basal bodies with each other. Each basal body is linked to an adjacent basal body by a unique set of connecting fibres, i.e., the flagellar apparatus does not exhibit 180° rotational symmetry. The flagellar apparatus ofPyramimonas obovata is compared with that of quadriflagellate motile cells of theChlorophyceae sensu Stewart andMattox and the phylogenetic relationships are discussed.     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). II: Cell wall secretion and assembly

Summary Secretion of the cell wall (theca) in the scaly green flagellateScherffelia dubia (Prasinophyceae) has been examined by electron microscopy during cytokinesis. The bi-laminate wall forms by the extracellular amalgamation of two layers of scales produced in the Golgi apparatus (GA). Each mature GA cisterna contains ca. 12,000 scales of two distinct varieties arranged in two layers on the cisternal membrane. GA cisternae undergo turnover and one scale containing cisterna matures from thetransface of each dictyosome every 3–4 minutes. Cisternae then fuse with the plasma membrane at the anterior end of the cell releasing the scales onto the cell surface. The two layers of wall scales integrate on the cell surface in a time-dependent self-assembly process. The first scales deposited commence assembly at the cell posterior and the wall develops anteriorly by edge growth. The daughter cell wall is composed of ca. 1.2 million scales deposited in about 3 hours. Calculations of net membrane flow strongly indicate extensive endocytosis during wall deposition.     

The flagellar apparatus and cytoskeleton of the dinoflagellates

Summary Modern microscopical approaches have allowed more accurate investigations of the three-dimensional nature of the dinoflagellate flagellar apparatus (FA) and several other cytoskeletal protein complexes. Our presentation overviews the nature of the dinoflagellate FA and cytoskeleton in a number of taxa and compares them with those of other protists. As with other protists, the FA of the dinoflagellates can be characterized by the presence of fibrous and microtubular components. Our studies and others indicate that the dinoflagellate FA can be expected to possess a striated fibrous root on the basal body of the transverse flagellum and a multimembered microtubular root on the basal body of the longitudinal flagellum. Two other features that appear widespread in the group are the transverse striated root associated microtubule (tsrm) and the transverse microtubular root (tmr). The tsrm extends at least half the length of the transverse striated root while the tmr extends from the transverse basal body toward the exit aperture of the transverse flagellum. In most cases, the tmr gives rise to several cytoplasmic microtubules at a right angle. The apparent conserved nature of these roots leads us to the conclusion that the dinoflagellate FA can be compared to the FA of the cryptomonads, chrysophytes, and the ciliates for phylogenetic purposes. Of these groups, the chrysophytes possess an FA with the most structures in common with the dinoflagellates. Our immunomicroscopical investigations of the microtubular, actin and centrin components of the dinoflagellate cytoskeleton point to the comparative usefulness of these cytological features.Abbreviations aptb apical transverse microtubular band - FA flagellar apparatus - Imr longitudinal microtubular root - mls multilayered structure - tmr transverse microtubular root - tmre transverse microtubular root extension - tsr transverse striated fibrous root - tsrm transverse striated root associated microtubule     

Transformation and development of the flagellar apparatus ofCryptomonas ovata (Cryptophyceae) during cell division

Summary InCryptomonas ovata, long, dorsal flagella are produced which transform during the following cell division into short, ventral flagella. At division there is a reorientation in cell polarity, and the parental basal apparatus, which comprises the basal bodies and associated roots, is distributed to the daughter cells via a complex sequence of events. Flagellar apparatus development includes the transformation of a four-stranded microtubular root into a mature root of different structure and function. Each newly formed basal body nucleates new microtubular roots, but receives a striated fibrous root from a parental basal body. The striated roots are originally produced on the transforming basal body and are transferred to the new basal bodies at each successive division. The development of the asymmetric flagellar apparatus throughout the cell cycle is described.     

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculata with monoclonal anti-p210

Summary.  Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3–4 μm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly. Received February 18, 2002; accepted May 17, 2002; published on line October 31, 2002 RID="*" ID="*" Correspondence and reprints: Botanisches Institut, Universit?t zu K?ln, Gyrhofstrasse 15, 50931 K?ln, Federal Republic of Germany     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus

Summary The effects of colchicine on ultrastructure of the lactating mammary cell in the rat and goat were studied by electron microscopy. Changes in tissue of the rat were examined over time (1, 2 and 4 h). The goat gland was evaluated by comparing ultrastructure of tissue at the time of maximum milk flow suppression induced by the drug with that of untreated tissue. Colchicine produced notable changes in the tissue of both species: 1) the secretion of lipid droplets and Golgi vesicle contents (exocytosis) was inhibited and the droplets and vesicles became randomly distributed throughout the cell, 2) the Golgi apparatus was significantly reduced in size, 3) casein and lipid continued to be synthesized as evidenced by greater numbers of secretory vesicles and increased sizes of casein micelles and lipid droplets, 4) secretory vesicles showed a propensity to cluster around lipid droplets, 5) isolated microtubules were found occasionally in the control tissue, ordinarily in the vicinity of the Golgi apparatus, but rarely in the colchicine-treated tissue. These observations indicate that colchicine has two effects leading to suppression of exocytosis in the mammary cell: one involves early interference with capacity of secretory vesicle membranes to fuse and a further effect, related to higher concentrations of colchicine, causes intracellular disorganization and loss of polarity. Microtubules were not seen as directly involved in the mechanisms of exocytosis. The secretion of milk fat globules is coupled to exocytosis and thereby is also inhibited by colchicine.Supported in part by grant HL 03622 of the U.S. Public Health Service     

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary. A naturally occurring unusual amino acid, hypusine [N ^ɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.     

The overexpression of GMAP-210 blocks anterograde and retrograde transport between the ER and the Golgi apparatus

Golgi Microtubule-Associated Protein (GMAP)-210 is a peripheral coiled-coil protein associated with the cis -Golgi network that interacts with microtubule minus ends. GMAP-210 overexpression has previously been shown to perturb the microtubule network and to induce a dramatic enlargement and fragmentation of the Golgi apparatus (Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM. J Cell Biol 1999; 145: 83–98). We now report that overexpressing GMAP-210 blocks the anterograde transport of both a soluble form of alkaline phosphatase and the hemagglutinin protein of influenza virus, an integral membrane protein, between the endoplasmic reticulum and the cis /medial (mannosidase II-positive) Golgi compartment. Retrograde transport of the Shiga toxin B-subunit is also blocked between the Golgi apparatus and the endoplasmic reticulum. As a consequence, the B-subunit accumulates in compartments positive for GMAP-210. Ultrastructural analysis revealed that, under these conditions, the Golgi complex is totally disassembled and Golgi proteins as well as proteins of the intermediate compartment are found in vesicle clusters distributed throughout the cell. The role of GMAP-210 on membrane processes at the interface between the endoplasmic reticulum and the Golgi apparatus is discussed in the light of the property of this protein to bind CGN membranes and microtubules.     

Blood amino acids concentration during insulin induced hypoglycemia in rats: the role of alanine and glutamine in glucose recovery

Summary. Our purpose was to determine the blood amino acid concentration during insulin induced hypoglycemia (IIH) and examine if the administration of alanine or glutamine could help glycemia recovery in fasted rats. IIH was obtained by an intraperitoneal injection of regular insulin (1.0 U/kg). The blood levels of the majority of amino acids, including alanine and glutamine were decreased (P < 0.05) during IIH and this change correlates well with the duration than the intensity of hypoglycemia. On the other hand, the oral and intraperitoneal administration of alanine (100 mg/kg) or glutamine (100 mg/kg) accelerates glucose recovery. This effect was partly at least consequence of the increased capacity of the livers from IIH group to produce glucose from alanine and glutamine. It was concluded that the blood amino acids availability during IIH, particularly alanine and glutamine, play a pivotal role in recovery from hypoglycemia.     

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.     

Observations on the relationship between the extracellular changes of taurine and glutamate during cortical spreading depression, during ischemia, and within the area surrounding a thrombotic infarct

Summary. Taurine and glutamate were monitored by microdialysis technique during various cerebral insults: a. Application of K⁺ triggered a cortical spreading depression (CSD). Taurine and glutamate increased concomitantly but recovery of glutamate was faster than that of taurine. b. Application of NMDA induced also CSD but only taurine increased. c. Induction of an infarct triggered repetitive CSDs. Taurine increased rapidly whereas glutamate rose slowly starting with some delay. d. After induction of ischemia, taurine and glutamate increased after onset of depolarisation. The increase of glutamate occurred late after a small, transient increase in parallel with the depolarisation. These data suggest a close functional relationship between the changes of both amino acids. Therefore, they should be monitored together especially in clinical settings: during excitation, only taurine will increase; during overexcitation, taurine will also increase but to a higher maximum followed by a moderate rise of glutamate; after energy failure, taurine will accumulate to its highest level followed by a continuous rise of glutamate. Received January 25, 2000/Accepted January 31, 2000     

Expression profiles of the genes associated with metabolism and transport of amino acids and their derivatives in rat liver regeneration

Summary. Amino acids (AA) are components of protein and precursors of many important biological molecules. To address effects of the genes associated with metabolism and transport of AA and their derivatives during rat liver regeneration (LR), we firstly obtained the above genes by collecting databases data and retrieving related thesis, and then analyzed their expression profiles during LR using Rat Genome 230 2.0 array. The LR-associated genes were identified by comparing the gene expression difference between partial hepatectomy (PH) and sham-operation (SO) rat livers. It was approved that 134 genes associated with metabolism of AA and their derivatives and 26 genes involved in transport of them were LR-associated. The initially and totally expressing number of these genes occurring in initial phase of LR (0.5–4 h after PH), G0/G1 (4–6 h after PH), cell proliferation (6–66 h after PH), cell differentiation and structure-function reconstruction of liver tissue (72–168 h after PH) were respectively 76, 17, 79, 5 and 162, 89, 564, 195, illustrating that these LR-associated genes were initially expressed mainly in initial stage, and functioned in different phases. Frequencies of up-regulation and down-regulation of them being separately 564 and 357 demonstrated that genes up-regulated outnumbered those down-regulated. Categorization of their expression patterns into 22 types implied the diversity of cell physiological and biochemical activities. According to expression changes and patterns of the above-mentioned genes in LR, it was presumed that histidine biosynthesis in the metaphase and anaphase, valine metabolism in the anaphase, and metabolism of glutamate, glutamine, asparate, asparagine, methionine, alanine, leucine and aromatic amino acid almost were enhanced in the whole LR; as for amino acid derivatives, transport of neutral amino acids, urea, γ-aminobutyric acid, betaine and taurine, metabolism of dopamine, heme, S-adenosylmethionine, thyroxine, and biosynthesis of hydroxyproline, nitric oxide, orinithine, polyamine, carnitine, selenocysteine were augmented during the entire liver restoration. Above results showed that metabolism and transport of AA and their derivates were necessary in liver regeneration. Authors’ address: Prof. Dr. C. S. Xu, College of Life Science, No. 46, Jianshe RD, Henan, Xinxiang 453007, China     

The relationship between albumin, other plasma proteins and variables, and age in the acute phase response after liver resection in man

Summary. A large series of plasma albumin (ALB, g/dl) and simultaneous blood and clinical measurements were prospectively performed on 92 liver resection patients, and processed to assess the correlations between ALB, other plasma proteins, additional variables and clinical events. The measurements were performed preoperatively and at postoperative day 1, 3 and 7 in all patients, and subsequently only in those who developed complications or died. In patients who recovered normally ALB was 4.3 ± 0.4 g/dl (mean ± SD) preoperatively, 3.7 ± 0.7 at day 1 and 3, and 3.9 ± 0.4 at day 7. In patients with complications its decrease was more prolonged. In non-survivors it was 3.4 ± 0.4 preoperatively, 3.0 ± 0.4 at day 1, and then decreased further. Regression analysis showed direct correlations between ALB and pseudo-cholinesterase (CHE, U/l, nv 5300-13000), cholesterol (CHOL, mg/dl), iron binding capacity (IBC, mg/dl), prothrombin activity (PA, % of standard reference) and fibrinogen, an inverse correlation with blood urea nitrogen (BUN, mg/dl) for any given creatinine level (CREAT, mg/dl), and weaker direct correlations with hematocrit, other variables and dose of exogenous albumin. An inverse relationship found between ALB and age (AGE, years) became postoperatively (POSTOP) also a function of outcome, showing larger age-related decreases in ALB associated with complications (COMPL: sepsis, liver insufficiency) or death (DEATH). Main overall correlations: CHE = 287.4(2.014)^ALB, r = 0.73; CHOL = 16.5(1.610)^ALB (1.001)^ALKPH, r = 0.71; IBC = 68.6(1.391)^ALB, r = 0.64; PA = 13.8 + 16.0(ALB), r = 0.51; BUN = 21.3 + 20.2(CREAT) – 6.2(ALB), r = 0.91; ALB = 5.0–0.013(AGE) – {0.5 + 0.003(AGE) _COMPL + 0.012(AGE) _DEATH } _POSTOP , r = 0.74 [p < 0.001 for each regression and each coefficient; ALKPH = alkaline phosphatase, U/l, nv 98-279, independent determinant of CHOL; discontinuous variables in italics label the change in regression slope or intercept associated with the corresponding condition]. These results suggest that altered albumin synthesis (or altered synthesis unable to compensate for albumin loss, catabolism or redistribution) is an important determinant of hypoalbuminemia after hepatectomy. The correlations with age and postoperative outcome support the concept that hypoalbuminemia is a marker of pathophysiologic frailty associated with increasing age, and amplified by the challenges of postoperative illness.     

The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus

In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.     

The rates of deceleration of nuclear and organellar DNA syntheses differ in the progenitor cells of the apical meristems during carrot somatic embryogenesis

 The synthesis of DNA in nuclei and organellar nucleoids at the various stages of somatic embryogenesis in carrot (Daucus carota L. cv. Kurodagosun) was analyzed using anti-5-bromo-2′-deoxyuridine (BrdU) immunofluorescence microscopy. The active syntheses of both nuclear and organellar DNA started in the cells forming the embryo proper 3 d after the initiation of embryogenesis, but not in cells forming suspensor-like cell aggregates. In the early globular embryo, active DNA syntheses were continuously observed in the whole embryo proper, except for the progenitor cells of the root apical meristem (RAM) and shoot apical meristem (SAM). These were recognized as slowly cycling cells with a non-BrdU-labelled nucleus and strongly BrdU-labelled organellar nucleoids. At the heart- and torpedo-shaped embryo stages, both nuclear and organellar DNA syntheses were inactive in the presumptive RAM and SAM. Thus, slowing down of organellar DNA synthesis is not coupled with, but is later than, that of nuclear DNA synthesis in the progenitor cells of the embryonic RAM and SAM. These findings clearly indicate that the timing of DNA synthesis is similar in the progenitor cells of both the RAM and SAM in the early stages of somatic embryogenesis. Received: 18 January 2000 / Accepted: 2 March 2000