Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis VI. Effects of silica replenishment on silica impoverished cells

Summary Stephanoeca diplocostata has a facultative requirement for silica in that silica starvation does not inhibit growth as measured by increase in cell numbers. In spite of the absence of a lorica silica impoverished protoplasts still divide in the characteristic tectiform manner and a juvenile protoplast, when released from the parent cell, still extends its lorica assembling tentacles despite the absence of costal strips with which to produce a lorica. Replenishment of silica to silica starved cells in mid to late exponential phase cultures results in a decrease in the growth rate but at the same time silica is taken up and utilised for the deposition of costal strips. Mature costal strips are extruded and accumulated in bundles of 5–8 on the surface of the protoplast but are not passed to the top of the collar as would be expected in silica enriched loricate cells. Eventually silica replenished protoplasts use the bundles of costal strips to assemble loricae for themselves. In early exponential phase cultures naked protoplasts are capable of division whilst at the same time depositing costal strips in preparation for subsequent lorica assembly. An undamaged protoplast deprived of its lorica by ultrasonic treatment also ultimately replaces the lost lorica. The manner in which the tectiform mode of costal strip accumulation and lorica assembly is modified to allow a cell to produce its own lorica is discussed.Abbrevations SDV silica deposition vesicle     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata Ellis

Summary Cell division inStephanoeca diplocostata follows the accumulation of a large number of costal strips in horizontal bundles at the top of the parent collar. Prior to nuclear division the flagellum is lost and the protoplast is large and rectangular. Nuclear division takes place whilst the protoplast undergoes vigorous metabolic movements and subsequent cytokinesis is achieved by equatorial constriction. The anterior of the two daughter protoplasts is the juvenile and is inverted with respect to the sister which remains attached to the parent lorica. The two protoplasts are joined by a cytoplasmic strand that consists of two threads both of which are initially attached to the daughter protoplasts at one side of the collar. Cell separation involves elongation of the strand and after each thread has broken contact with one of the daughter cells the two threads slide over each other until the juvenile is released. The juvenile takes the accumulation of supernumerary strips as it leaves the parent lorica and after release of the juvenile the strips are mobilised to form a new lorica. The collar tentacles of the parent are thought to play a significant role in the movement of strips during division and certain selected tentacles on the juvenile are associated with lorica assembly. Cell separation takes between 9–12 minutes and lorica assembly by the juvenile 2–3 minutes.     

Developmental studies on the loricate choanoflagellate Stephanoeca diplocostata Ellis. III. Growth and turnover of silica,preliminary observations

Growth, turnover of silica and lorica morphology of Stephanoeca diplocostata Ellis have been investigated in batch cultures at 20 °C. Mean cell doubling times for separate experiments ranged from 9.1–13.8 h. During exponential growth, uptake of reactive silicate progressed steadily and throughout this phase the average amount of biogenic silicon per cell was 2.1 pg. Once growth declined, net dissolution of silica from loricae became apparent and progressed steadily throughout the stationary and death phases. The minor difference in solubility between loricae of living cells and costal strips cleaned with acid indicates that even if an organic component is associated with the silica of costal strips it does not inhibit silica dissolution. The first effects of silica dissolution on cells as revealed by electron microscopy are limited to corrosion on the surfaces and the hollowing out of the centres of costal strips but ultimately a consistent pattern of lorica disintegration occurs.     

The effects of diazepam on mitosis and the microtubule cytoskeleton II. Observations on newt epithelial and PtK1 cells

Summary The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton were examined using live and fixed PtK₁ and newt (Taricha granulosa) epithelial lung cells. DZP treatment caused rapid shortening of spindle MTs at prometaphase and metaphase, inducing movement of the poles together while chromosome oscillations continued. DZP treatment slowed the rate of anaphase A but did not detectably affect anaphase B, cell cleavage or interphase cells. Our results suggest that DZP inhibits mitosis by affecting prometaphase and metaphase MTs. Its action is not equivalent to that of common anti-MT drugs, since only a small subpopulation of MTs are significantly susceptible. Likewise, its effects are not equivalent to those generated by metabolic inhibitors. The related benzodiazepines, medazepam and oxazepam, induce effects equivalent to those of DZP.     

Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

The effect of S-100a and S-100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.     

Effects of altered gravity on the actin and microtubule cytoskeleton of human SH-SY5Y neuroblastoma cells

Summary. Human SH-SY5Y neuroblastoma cells were used to study the effects of altered gravity on the actin and microtubule cytoskeleton dynamics. A cholinergic stimulation of the cells during a 6 min period of changing gravity (3 parabolas) resulted in an enhanced actin-driven protrusion of evoked lamellipodia. Likewise, the spontaneous protrusive activity of nonactivated cells was promoted during exposure to changing gravity (6 up to 31 parabolas). Ground-based experiments revealed a similar enhancement of the spontaneous and evoked lamellar protrusive activity when the cells were kept at 2 g hypergravity for at least 6 min. This gravity response was independent of the direction of the acceleration vector in respect to the cells. Exposure of the cells to “simulated weightlessness” (clinorotation) had no obvious influence on this type of lamellar actin cytoskeleton dynamics. A 20 min exposure of the cells to simulated weightlessness or to changing gravity (6 to 31 parabolas) – but not to 2 g (hypergravity, centrifugation) – resulted in an altered arrangement of microtubules indicated by bending, turning, and loop formation. A similar altered arrangement was shown by microtubules which had polymerized into lamellipodia after release from a taxol block at simulated weightlessness (clinorotation) or during changing gravity (5 parabolas). Our data suggest that in human SH-SY5Y neuroblastoma cells, microgravity affects the dynamics and spatial arrangement of microtubules but has no influence on the Rac-controlled lamellar actin cytoskeleton dynamics and cell spreading. The latter, however, seems to be promoted at hypergravity. Correspondence and reprints: Cell and Developmental Neurobiology, Institute of Zoology, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Federal Republic of Germany.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

Effects of microtubule agents on the spatial and electrical properties of the plasma membrane inChara corallina

The freshwater algaChara corallina Klein ex Willd., em. R.D.W. (=C. australis R.Br.) develops alternating outward (acid) and inward (alkaline) current areas on its surface when illuminated. Exposure of cells to vinblastine, colchicine, or oryzalin caused a reduction in and a shifting of this extracellular current pattern. Removal of these agents from the bathing media resulted in regeneration of the initial current profile. Because these agents all affect tubulin, microtubules may be responsible for orchestrating the transmembrane currents responsible for the acid and alkaline banding phenomenon. Analysis of the membrane potential showed a fast depolarization after vinblastine exposure; however, analysis of the current-voltage curve did not show a change in membrane conductance. A 30-min colchicine treatment decreased the conductance of the plasma membrane with either an hyperor a depolarization in the membrane potential. In contrast, although a 9-h exposure to oryzalin caused a major reduction in the extra-cellular current pattern, only minor changes were observed in the membrane potential and conductance. However, in the presence of oryzalin, the time constants in the light response of the membrane potential increased over this 9-h period. Collectively, these results implicate an involvement of microtubules in spatial control of plasma-membrane transport events inC. corallina. This research was supported by National Science Foundation grant DCB-88-16077.     

Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes

The behavior and role of the microtubule (MT) and actin-myosin components of the cytoskeleton during pollen tube growth in two species of Pinus were studied using anti--tubulin, rhodamine-phalloidin, anti-myosin, and the appropriate inhibitors. Within germinated pollen tubes MTs were arranged obliquely or transversely, but in elongated tubes they were arranged along the tube's long axis. MTs were localized in the tube tip region, excluding the basal part. Altered growth was found in pollen tubes treated with colchicine; the tips of many pollen tubes incubated in the liquid medium were branched and/or rounded, and those in the agar medium were divided into many branches. Both the branching and the rounding were considered to be caused by the disturbance of polarizing growth of the tube due to MT disorganization with colchicine treatment. Actin filaments (F-actin) were found in the major parts of many pollen tubes along their long axis, excluding the tip region. In a few tubes, however, F-actin was distributed throughout the tube. The areas in the pollen tube containing F-actin were filled with abundant cytoplasmic granules, but the areas without F-actin had very few granules. The tube nucleus, which migrated from the grain area into the tube, was closely associated with F-actin. Germination of pollen grains treated with cytochalasin B was little affected, but further tube elongation was inhibited. Myosin was identified on cytoplasmic granules and to a lesser extent on the tube nucleus in the pollen tubes. Several granules were attached to the nuclear envelope. Tube growth was completely inhibited by N-ethylmaleimide treatment. In generative cells that were retained in the pollen grain, both MT and F-actin networks were observed. Myosin was localized on the cytoplasmic granules but not on the cell surface. In conclusion, it was shown that actin-myosin and MTs were present in gymnospermous Pinus pollen tubes and it is suggested that the former contributed to outgrowth of the tubes and the latter contributed to polarized growth. Several differences in the behavior of cytoskeletal elements in generative cells compared to angiosperms were revealed and are discussed.     

Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts

Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.     

药用类短命植物新疆阿魏花蜜腺的发育解剖学研究

类短命植物新疆阿魏(Ferula sinkiangensts K.M.Shen),是新疆独具特色的药用蜜源植物,其花蜜腺位于下位子房的花盘之上,由子房上部的表层细胞特化而形成的,属下位子房上的盘状蜜腺.但由于其花柱短,基部呈圆锥状,蜜腺分布于盘状结构表面除花柱外的区域,因而可以认为是花柱基部类蜜腺的一种扩展和特化,属于从子房蜜腺向花柱蜜腺过渡的类型.在蜜腺下方分布有大量的子房维管束,其泌蜜途径可能为:前蜜汁经共质体或非共质体途径到产蜜组织中,加工后再经共质体途径运进气孔下方的特殊细胞中,再分泌到孔下室,经气孔泌出.其花蜜腺在发育过程中液泡化动态明显,PAS反应测试细胞具阳性物质,淀粉粒积累动态较明显.     

The effect of ruthenium red on the assembly and disassembly of microtubules and on rapid axonal transport

The assembly of microtubules was found to decrease in proportion to the amount of added ruthenium red, indicating a high affinity of ruthenium red for the microtubule system. An equimolar amount of ruthenium red per tubulin dimer inhibited the microtubule assembly completely and disassembled existing microtubules. Binding of ruthenium red to tubulin is accompanied by a shift in the absorption maximum from 535 to 538 nm. The binding is very strong, as shown by the finding that ruthenium red could not be displaced from tubulin by gel chromatography on Sephadex, or by the addition of Ca²⁺ or Mg²⁺. The binding of ruthenium red to tubulin did not affect the single colchicine site, nor the Mg²⁺ site(s), as shown by use of Mn²⁺ as an EPR probe. Ruthenium red also interfered with microtubules in an intact cell system, as it inhibited rapid axonal transport in the frog sciatic nerve, measured by the accumulation of [³H]leucine-labelled proteins in front of a ligature.     

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.     

Reprogramming of cells following wounding in pea (Pisum sativum L.) roots. II. The effects of caffeine and colchicine on the development of new vascular elements

Summary Interference with the normal progression of the cell cycle by the drugs caffeine and colchicine does not prevent parenchyma cells in the cortex of pea roots from being reprogrammed to become tracheary and sieve elements following severance of the vascular cylinder of the root. The pattern of secondary wall deposition of the newly differentiated tracheary elements is highly aberrant in the presence of colchicine but is of normal appearance in the caffeinetreated roots. In each case, the new sieve elements have sieve plates and lateral sieve areas with callose deposits. Induction and redifferentiation are achieved in the absence of cell division and microtubules in colchicine-treated roots.Bi- and multi-nucleate cells are produced by both drugs. Microtubules are still present in the caffeine-treated roots but cell plate formation is inhibited. The partitioning of the multinucleate cortical cells by the interdigitation of free-growing walls between the nuclei occurs in the presence of caffeine but not colchicine.     

The effects of colchicine and vincristine on the concentrations of glucose and related metabolites in goat's milk

The effects of colchicine and vincristine on the concentration of glucose and its metabolites in milk were studied following intramammary injection of the alkaloids into one mammary gland of lactating goats. Both alkaloids decreased the rate of milk secretion from the treated gland and produced similar changes in the concentrations of various metabolites in milk. The concentrations of glucose, UDP-galactose, galactose, pyruvate and lactate increased, while those of glucose 6-phosphate and phosphoenolpyruvate decreased in milk from treated glands. The rate of milk secretion from the untreated gland increased, along with the concentrations of glucose in the milk, The changes in the concentrations of the metabolites in milk are discussed in relation to possible biochemical occuring in the mammary gland during the suppression of milk secretion by the alkaloids. It is suggested that, before alkaloid treatment, the rate of milk secretion was limited by intracellular glucose supply.     

Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomyceteSchizophyllum commune

Summary The effects of nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some nocodazole-treated hyphae. After 2 h growth in the presence of nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.Abbreviations BFA brefeldin A - EM electron microscopy - ER endoplasmic reticulum - IIF indirect immunofluorescence - MBC methylbenzimidazole-2-ylcarbamate - MT microtubule - MVB multivesicular body - RER rough endoplasmic reticulum