The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells

Immunofuorescence staining with antibodies to tubulin and vimentin and staining with phalloidin have been used to examine the effects of methylmercury on the cytoskeleton of embryonal carcinoma cells in culture. Exposure of embryonal carcinoma cells to methylmercury (0.01 to 10 m) resulted in concentration- and time-dependent disassembly of microtubules in interphase and mitotic cells. These effects were reversible when cultures were washed free of methylmercury. Spindle microtubules were more sensitive than those of interphase cells. Spindle damage resulted in an accumulation of cells in prometaphase/metaphase, which; correlated with a temporary delay in the resumption of normal proliferation rate upon removal of methylmercury. Of the interphase cytoskeletal components, microtubules were the first affected by methylmercury. Vimentin intermediate filaments appeared relatively insensitive to methylmercury, but showed a reorganization secondary to the microtubule disassembly. Actin microfilaments appeared unchanged in cells showing complete absence of microtubules. Our results 1) support previous reports suggesting that microtubules are a primary target of methylmercury, 2) document a differential sensitivity of mitotic and interphase microtubule systems and 3) demonstrate the relative insensitivities of other cytoskeletal components.Abbreviations -MEM alpha minimal essential medium - EC embryonal carcinoma cells - McHg methylmercury - PBS phosphate buffered saline - SB microtubule stabilizing buffer     

Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.     

Association of acetylated microtubules, vimentin intermediate filaments, and MAP 2 during early neural differentiation in EC cell culture

Pluripotent P19 embryonal carcinoma cell cultures can be induced to differentiate into neurons and glial cells by the addition of 10(-6) M retinoic acid. During early neural differentiation, a bundle of colchicine-stable, acetylated microtubules is formed. This acetylated microtubule array apparently extends to form neurites during neurogenesis. In this paper, we analyze changes in vimentin and MAP 2 distributions during neural differentiation with respect to the changes in the acetylated microtubule array. During a brief period early in differentiation, indirect immunofluorescence staining shows the colocalization of colchicine-stable acetylated microtubules, vimentin, and MAP 2. Using acrylamide to disrupt the organization of vimentin intermediate filaments and estramustine to disrupt the binding of MAP 2 to microtubules, we show that acetylated microtubules, MAP 2, and vimentin intermediate filaments are arranged in an interdependent cytoskeletal array. We suggest this array may serve to stabilize processes in neural stem cells, before the final decision to differentiate into neurons or glia is made.     

A fluorometric assay for γ-glutamyl transpeptidase: Demonstration of enzymatic activity in cultured cells of neural origin

A new fluorometric assay was developed for the measurement of -glutamyl transpeptidase (-GTP). The assay utilizes as a substrate the synthetic compound 7--glutamylamido-4-methyl coumarin which is cleaved by -GTP to yield the highly fluorescent product 7-amino-4-methyl coumarin. Optimal excitation and emission wavelengths for the assay are 345 nm and 470 nm, respectively, and the sensitivity of the assay is greatly enhanced by the high-pressure liquid chromatographic separation of the product from the substrate. The assay is minimally 25 times more sensitive than the conventional spectrophotometric assay and permits analysis of as little as 5000 cultured cells of neuronal and glial origin. Analysis of a variety of cultured cells of neuronal and glial origin with this assay suggests that -GTP is largely present in glia and to a lesser extent in neurons.     

The fine structure of neuroglia in the central nervous system of nereid polychaetes

Summary The principal supportive elements of the nereid central nervous system are non-neuronal cells that are referred to as supportive glia. Supportive glial cells form a conspicuous cortex in the nerve cord. The inner region of this cortex consists of closely packed processes and cell bodies of fibrous supportive glial cells that are arranged in concentric layers around the perimeter of the neuropile. The fibrous appearance of the glial cells results from dense bundles of cytoplasmic filaments. Many fibrous glial processes penetrate the neuropile and ramify among the neuronal elements. Larger, irregularly shaped cells are the chief supportive glial elements of the peripheral region of the cortex where they line the stromal sheath (neural lamella) and invest the neuronal perikarya with extensive concentric systems of lamellate processes. These glial cells usually possess a relatively undifferentiated cytoplasm with scattered glycogen granules, but occasionally have a well developed Golgi apparatus, endoplasmic reticulum and densely packed particulate glycogen. The supportive glia exhibits numerous desmosomes as well as 5-layered (tight) and 7-layered (gap) junctions. Interspersed among the supportive glial cells are non-neuronal cells referred to as granulocytes. These cells have abundant large, granular inclusions, electron lucent vesicles, plasmalemmal infoldings and microtubules. The granulocytes may be derived from undifferentiated glial cells or may represent coelomocytes that have invaded the nervous tissue.Supported by USPHS Grants No. NIH 5P01 NS-07512, NIH 2T01 GM-00102, and NB-00840.The author acknowledges the excellent technical assistance of Sarah Wurzelmann and Stanley Brown, and thanks Dr. Berta Scharrer for many stimulating discussions.     

Effects of phorbol ester on immunoreactive protein kinase C,insulin binding,and glucose uptake in astrocytic glial and neuronal cells from the brain

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [³H]2-deoxy-d-glucose (dGlc) uptake and [¹²⁵I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the and forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effecs of [³H]dGlc uptake and insulin binding in glial cells.     

Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

Distribution of CK2, its substrate MAP1B and phosphatases in neuronal cells

Neuronal morphogenesis depends on the organization of cytoskeletal elements among which microtubules play a very important role. The organization of microtubules is controlled by the presence of microtubule-associated proteins (MAPs), the activity of which is modulated by phosphorylation and dephosphorylation. One of these MAPs is MAP1B, which is very abundant within growing axons of developing neurons where it is found phosphorylated by several protein kinases including CK2. The expression of MAP1B is notably decreased after neuronal maturation in parallel with a change in the localization of the protein, which becomes largely concentrated in neuronal cell bodies and dendrites. Interestingly, MAP1B remains highly phosphorylated at sites targeted by protein kinase CK2 in mature neurons.We have analyzed the expression and localization of CK2 catalytic subunits along neuronal development. CK2 subunit appears early during development whereas CK2 subunit appears within mature neurons at the time of dendrite maturation and synaptogenesis, in parallel with the change in the localization of MAP1B. CK2 subunit is found associated with microtubule preparations obtained from either grey matter or white matter from adult bovine brain, whereas CK2 subunit is highly enriched in microtubules obtained from grey matter. These results lend support to the hypothesis that CK2 subunit is concentrated in neuronal cell bodies and dendrites, where it associates with microtubules, thus contributing to the increased phosphorylation of MAP1B in this localization in mature neurons.     

An argyrophil fibrillar system and amitotic nuclear division in pars intermedia cells of the rainbow trout (Salmo irideus)

Summary Argyrophil fibrils were revealed in pars intermedia chromophobe cells of Salmo irideus. For the light microscopical demonstration of the fibrils a recently developed copper sulphate-silver protein technique for Bouin-fixed hypophyses was used.The fibrils, apparently belonging to one fibrillar system, are found only in limited regions of the cytoplasm. They occur in ring-, loop-, or cracknel shapes in the close vicinity of the rounded nucleus in the chromophobe cell, and continue in linear shape into the cell base or cell apex where they may end near the adjacent basement membrane.Electron microscopically, the fibrils are composed of filaments, 70–90 Å in diameter, which are arranged in parallel. Bundles of filaments are frequently found near the nucleus. In addition to perinuclear filaments, bundles of filaments with tiny offshoots occur in different areas of the cytoplasm, extending sometimes as far as the cell membrane which is located near the basement membrane of the pars intermedia epithelium.In cells with a bi- or trilobular, apparently amitotically dividing, nucleus a fibrillar loop or ring usually surrounds the interlobular part(s) of the nucleus. This relation of the fibrils to the dividing nucleus and their consisting of regularly arranged filaments indicates the significance of the fibrillar system in the chromophobe cell. It is thus suggested that the fibrillar system is involved in the constriction of the nucleus during amitosis.The number of chromophobe cells in which fibrils are light microscopically visible varied greatly among the rainbow trout used.Miss M. C. Wentzel is thanked for her technical assistance, Mr. M. Veenhuis for his advice and excellent help with the preparation of the electron micrographs, and Mr. L. Hoekstra for preparing the final drawings. Mr. N. J. Williams' corrections to the English are gratefully acknowledged.     

Retinal FABP principally localizes to neurons and not to glial cells

The presence of fatty acid-binding protein (FABP) in the embryonic chick retina may be linked to the demand for polyunsaturated fatty acids in this developing neural tissue. There is a decline in the overall level of FABP as the retina matures, suggesting a role for FABP in cellular differentiation. However, this pattern is not present in the chick brain, indicating a unique function for FABP in the retina. Immunohistochemical staining of paraffin sections of chick retina from embryonic day 21 revealed immunopositive photoreceptor inner segments, outer nuclear layer, radial processes in the inner nuclear layer, a subpopulation of cells in the ganglion cell layer, and inner limiting membrane. This pattern suggested that FABP positive cells were photoreceptors, Müller (glial) cells, and possibly ganglion cells. Staining of sections for glutamine synthetase, an enzyme specific for Müller cells, was similar but not identical to the pattern observed with FABP; thus identification of these cells as FABP-positive was not conclusive. However, in retinal cells dissociated from day E14 embryos and cultured for one week, staining with FABP was more intense in the neurons than in the flat cells (presumed to be derived from the Müller cells). Retinal FABP thus appears to be localized predominantly in neurons, and may serve to sequester fatty acids in preparation for neurite outgrowth as the retinal cells differentiate.Abbreviations FABP Fatty Acid-Binding Protein - PUFA Polyunsaturated Fatty Acid     

Interneuronal and glial-neuronal gap junctions in the lamina ganglionaris of the compound eye of the housefly,Musca domestics

Summary The cell-body layer of the lamina ganglionaris of the housefly, Musca domestica, contains the perikarya of five types of monopolar interneuron (L1–L5) along with their enveloping neuroglia (Strausfeld 1971). We confirm previous reports (Trujillo-Cenóz 1965; Boschek 1971) that monopolar cell bodies in the lamina form three structural classes: Class I, Class II, and midget monopolar cells. Class-I cells (L1 and L2) have large (8–15 m) often crescentshaped cell bodies, much perinuclear cytoplasm and deep glial invaginations. Class-II cells (L3 and L4) have smaller perikarya (4–8 m) with little perinuclear cytoplasm and no glial invaginations. The midget monopolar cell (L5) resides at the base of the cell-body layer and has a cubshaped cell body. Though embedded within a reticulum of satellite glia, the L1–L4 monopolar perikarya and their immediately proximal neurites frequently appose each other directly. Typical arthropod (-type) gap junctions are routinely observed at these interfaces. These junctions can span up to 0.8 m with an intercellular space of 2–4 nm. The surrounding nonspecialized interspace is 12–20 nm. Freezefracture replicas of monopolar appositions confirm the presence of -type gap junctions, i.e., circular plaques (0.15–0.7 m diam.) of large (10–15 nm) E-face particles. Gap junctions are present between Class I somata and their proximal neurites, between Class I and Class II somata and proximal neurites, and between Class II somata. Intercartridge coupling may exist between such monopolar somata. The cell body and proximal neurite of L5 were not examined. We also find that Class I and Class II somata are extensively linked to their satellite glia via gap junctions. The gap width and nonjunctional interspace between neuron and glia are the same as those found between neurons. The particular arrangement and morphology of lamina monopolar neurons suggest that coupling or low resistance pathways between functionally distinct neurons and between neuron and glia are probably related to the metabolic requirements of the nuclear layer and may play a role in wide field signal averaging and light adaptation.     

Cell cycle,morphogenesis, and ultrastructure in the pseudoheliozoan clathrulina elegans

Summary The development of the sessile pseudoheliozoan Clathrulina elegans Cienkowski (Desmothoraca, Rhizopoda, Protozoa) was followed by light and electron microscopy. The cell cycle comprises a heliozoan-like stage sitting in an organic perforated capsule, an amoeboid resp. biflagellate stage ensuring dispersal, and an encysted stage. Special reference is given to the morphogenetic processes during the flagellate-to-heliozoan transformation. The differentiation of the stalk is described in detail and a hypothesis of the morphogenesis of the capsule is presented. Granules in the filopods displaying a jerky movement, are identified as a hitherto underscribed type of haptocyst-like organelles. These kinetocysts are shown to be a useful marker for studying the turnover of the plasma membrane at organelle level. Evidence is presented that in the flagellate stage a surplus of plasma membrane is stored in numerous lacunae thus facilitating the rapid transformation into the heliozoan stage with its comparatively large surface area. The morphogenetic significance of microtubules during the formation of the stalk, the latter being an asymmetric extracellular secretion product, is elucidated. The role of microtubule nucleation sites, the precisely timed assembly and disassembly of microtubules and other factors which probably control development are discussed to some extent.The author thanks Mrs. G. Borges and Mrs. B. Gauss for excellent technical assistance. This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Dynamics of the cytoskeleton of epidermal cells in situ and in culture

Summary The cytoskeleton of primary tissue-culture cells from the epidermis of Xenopus laevis tadpoles was investigated by phase-contrast, immunofluorescence, and electron microscopy. The connection between the arrangement of different types of filaments and the mechanical properties of the epidermis is discussed. The bilayered epidermis attains stability from thick bundles of tonofilaments interconnecting the basal desmosomes. Twisting of tonofilaments around each other can explain the occurrence of elastic filamentous curls forming a meshwork braced between rows of small desmosomes in the apical region of the epidermis. Actin is arranged as a diffuse meshwork and sometimes forms bundles intermingling with tonofilament bundles. Surface membranes and rows of small desmosomes are delineated by actin and contain -actinin. Actin raises the tension for rounding and spreading of cells. Microtubules stabilize already well-developed lamellae.     

Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells

Summary Treatment with lysine prior to fixation of tobacco BY-2 cells with formaldehyde improved the preservation of actin filaments in the cells and enabled us to observe both networks of actin filaments and microtubules in the same cells. By using this method, we observed that (1) actin filaments were present in the preprophase band; (2) the actin filaments in the preprophase band and phragmoplast were runnig in the same direction as the microtubules in their respective structures; (3) a cortical network of actin filaments was present throughout all stages of cell cycle.The present method did not preserve the cortical actin filaments in interphase cells. The procedure for staining microtubules destroyed them.Abbreviations EGTA Ethyleneglycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF Phenylmethylsulfonyl fluoride - TLCK Na-p-tosyl-L-lysine chloromethyl ketone     

Immunocytochemistry and ultrastructure of the neuropil located ventral to the rat supraoptic nucleus

Summary The neuropil located ventral to the SON was investigated by the use of immunoperoxidase staining for neurophysins, oxytocin and vasopressin, and electron miroscopy. The study was performed in six groups of rats: 1) control; 2) infusion of isotonic saline into the CSF; 3) infusion of hypertonic saline into the CSF; 4) drinking hypertonic saline for 4 days; 5) same as group 4 but injection of colchicine into the CSF on second day of dehydration; 6) salt loading for 3 months. In the control rats the ventral neuropil contained a few immunoreactive processes, the general morphology of which was completely different from that of the neurosecretory axons emerging from the SON at its dorsal aspect. In rats of groups 3 to 6 the ventral processes (VP) became loaded with neurosecretory granules, whereas the perikarya and axons were depleted. Based on their general morphology and reactivity pattern it is suggested that the VP are dendrites. Most of these dendrites were embedded in a glial cushion formed by the processes of a particular type of marginal glia. Some of these dendrites enveloped an arteriole penetrating the optic tract. All VP were rich in synaptic contacts. The possibility that the VP of neurosecretory cells may be functionally related to the subarachnoid CSF and the arteriolar blood flow is discussed.Supported by Grant RS-82-18 from Direccíon de Investigaciones, Universidad Austral de Chile     

The cytoskeleton of Cobaea seed hairs:

The cell wall of Cobaea scandens seed hairs developed in a characteristic sequence, with the deposition of a cellulose thread onto a pectic swelling layer was the final event. The cellulose thread was intracellularly accompanied by a band of 10–18 microtubules. During the formation of the swelling layer the microtubules were homogeneously distributed; they ran circumferentially normal to the cell axis. When cellulose-thread formation started, the microtubules became arranged in a helical band. The density of the microtubules varied during the different phases of development. The highest density was observed before cellulosethread formation and ranged from 6–15 m·m^-2. The length of the microtubules, 20–30 m, was determined by direct measurements, as well as estimated from the total microtubular length in a given area and the counted free ends. With the indirect immunofluorescence technique the microtubules of the band stained inhomogeneously. Those which were located at the edges of the band fluoresced more intensely than those of the central part. Attempts to visualize actin filaments in the hair cells with rhodaminyl-conjugated phalloidin resulted in a homogeneous staining of the area of the microtubular band, indicating that actin filaments may be present in this region. Though, in thin sections and dry-cleaved cells, filamentous structures were observed between the microtubules, caution is expressed that the observed fluorescence was, indeed, due to actin filaments. The role of the filamentous structures is discussed with respect to formation and maintenance of the microtubular band. Microtubules apparently did not cross coated pits which were visualized in the plasma membrane through the dry-cleaving technique.Abbreviations IFT indirect immunofluorescence technique - RP rhodaminyl-conjugated phalloidin - SEM scanning electron microscopy     

Improved recovery of (1→3),(1→4)-β-D-glucan synthase activity from Golgi apparatus ofZea mays (L.) using differential flotation centrifugation

Summary The synthesis of (13), (14)--D-glucan (MG) is associated with the Golgi apparatus of maize. Identification of in vitro reaction products by enzymic hydrolysis and separation of diagnostic oligosaccharides by HPLC was used as a specific assay for MG synthase activity. Large quantities of highly enriched membrane are needed to study the enzyme components of MG synthesis. We directly obtained highly enriched Golgi apparatus in a single flotation centrifugation, without the necessity of an initial downward centrifugation. This new procedure has improved the yield of Golgi apparatus, and has improved recovery of MG synthase activity. The substrate in glucan synthase reactions is UDP-Glc, but UDP-Glc is also a substrate in many other reactions, including the production of simple glucosides. In addition, much of the labeled Glc from UDP-Glc is broken down to Glc-1-P and Glc under MG synthase reaction conditions. We have explored some inhibitors of phosphatase, phosphorylase, phosphodiesterase, and glucosidase activities in order to minimize these competing reactions and increase the activity of MG synthase.Abbreviations BSA bovine serum albumin - DTT dithiothreitol - MG (13), (14)--D-glucan - PAD pulsed amperometric detector     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element