Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis. V. The cytoskeleton and the effects of microtubule poisons

Summary InStephanoeca diplocostata microtubules are located in four positions namely: within the flagellar axoneme; just beneath the plasmalemma; associated with the silica deposition vesicles (SDVs) during early stages of costal strip deposition; and in the mitotic spindle. At the anterior end of the cell the 50–60 peripheral microtubules, which are organized more or less parallel to the long axis of the cell, converge around the base of the emergent flagellum. A short second flagellar base is positioned between the nucleus and the base of the emergent flagellum. Developing costal strips are located individually within SDVs in the peripheral cytoplasm. During the early stages of silica deposition each SDV is curved and subtended longitudinally on its concave side by two microtubules. When a costal strip has achieved sufficient rigidity to withstand bending the SDV-associated microtubules are depolymerized. Treatment of exponentially growing cells with sublethal concentrations of microtubule poisons, such as colchicine, podophyllotoxin, griseofulvin andVinca alkaloids depresses growth. Treatment with these drugs also affects the length and morphology of developing costal strips perhaps by interfering with the shaping and supporting functions of SDV-associated microtubules. Instead of being long and crescentic with a standard radius of curvature, costal strips of treated cells are usually short and misshapen, with irregular bends. After drug treatment, juveniles produced as a result of cell division do not develop flagella but can still assemble a lorica although it is usually misshapen. The role of microtubules and microfilaments in lorica production is discussed.     

嘉兰栽培及其秋水仙碱含量测定

嘉兰原属热带性地区的植物,但从云南南部引种到广州地区种植。表现生长发育良好,且其秋水仙碱含量高达0.40—0.45％。是一种有推广种植价植的药用植物。     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Ultrastructural effects of colchicine,vinblastine, and taxol in drug-sensitive and multidrug-resistant J 774.2 cells

Summary Cell lines derived from the murine macrophage-like cell J 774.2 are resistant to the cytotoxic effects of colchicine, vinblastine, and taxol. These multidrug-resistant (MDR) cells overproduce a family of 130–150 kDa P-glycoproteins (P-gp) associated with the plasma membrane region and display other typical features of the MDR phenotype. Ultrastructural analysis of drug-treated cells indicated that although hallmark structural effects engendered by each drug at efficacious doses were profound in the drug-sensitive J 774.2 cells, they were not evident in the similarly treated MDR cell lines. Thus, MDR phenotypic expression involved maintaining drug levels at subthreshold values so as to preclude the advent of these morphologic changes, and allowed vital tubulin-associated cellular processes, including replication, to occur. Using a polyclonal antibody specific for the P-gp, electron microscopic immunocytochemical evidence is presented for substantial association of P-gp with the plasma membrane/cell surface in the resistant cells which was not demonstrable in the drug-sensitive J 774.2 cells. This key cell surface localization of P-gp is germane to the postulated transport and related mechanisms whereby P-gp may play a pivotal role in endowing cells with multidrug resistance.     

A new approach to chromosome doubling for haploid rice plants

Summary Rice nodal segments from three flowering haploids were excised and treated for different lengths of time with 0.3% or 0.4% colchicine (dissolved in 2% DMSO) in an attempt to induce fertile seeds. A combination of higher colchicine concentration and longer hours of treatment reduced the survival rate of treated segments, but more fertile plants were transformed. Pooled data showed that of the 842 segments used, 42.2% survived the treatment and sprouted, but only 31.9% were successfully established and grown to maturity. Among the 269 mature plants, 29,4% produced fertile seeds (panicles) with an average of 146.2 seeds per diploidized plant.     

嘉兰块茎形成过程中物质变化的研究

嘉兰(Gloriosa superba L.)系百合科草本植物,其块茎含有秋水仙碱。本文研究块茎形成规律及其形成过程中物质变化。研究结果表明:块茎播后,新生的块茎在生长初期和后期生长速度都较慢,中期生长速度最快,也是嘉兰块茎产量形成的主要时期。块茎中营养物质主要是淀粉。新生块茎中淀粉含量,是随着新块茎的生长,其含量逐渐增高。叶片的光合速率和叶面积,从播后逐渐增加,开花前达到高峰,花后又逐渐降低。开花前如能增加光合叶面积,可为后期块茎生长提供较多物质。块茎中秋水仙碱含量是随着块茎的成熟与淀粉含量的渐增而增加,至收获期达到高峰。这一结果表明:收获未充分成熟的块茎,会降低秋水仙碱含量。     

Effect of colchicine on lysosomal structures in maturation-ameloblasts of the rat incisor

Summary The lysosomal systems in maturation-ameloblasts affected by colchicine were examined using trimetaphosphatase cytochemistry. Demineralized segments of rat incisor were incubated for trimetaphosphatase. At all time intervals, lysosomal structures exhibited reduced enzyme reactivity and were clustered in the Golgi region of the cell. Both ruffle-ended and smooth-ended ameloblasts maintained essentially normal morphology up to 4 h after colchicine injection, except for some migration of organelles. After 8 h, the ruffled border was markedly modified and the associated dense granular material was no longer present. Changes in the lysosomal system and ruffled border indicate interference by colchicine with a putative resorptive function of the maturation-ameloblasts.     

Fine-structural localization of neuropeptide tyrosine (NPY)-like immunoreactivity in the neuronal somata of colchicine-pretreated celiac ganglia of rats

Summary In colchicine-pretreated cells of sympathetic ganglia, intensely NPY-immunoreactive material was localized within vacuoles and vesicles of the disorganized, widely dispersed Golgi apparatus. Intensely positive large granular vesicles, which are known to be one of major storage sites of various peptides in the autonomic nerve endings, were essentially unobserved in the perikaryal cytoplasm. The present finding provides evidence that one pool of NPY-like immunoreactivity is localized in the Golgi apparatus of colchicine-pretreated as well as normal sympathetic ganglion cells. It is also clear that visualization of NPY-immunoreactive somata by colchicine-pretreatment in the sympathetic ganglia is due to the accumulation of the neuropeptide in the disorganized Golgi stacks instead of increased amount of the large granular vesicles containing NPY.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.