bluuebunny/crossref_metadata_2025 huggingface dataset:
Architecture of the microtubule component of mitotic spindles from Dictyostelium discoideum
Authors: Mcintosh J. R. , Roos U.-P. , Neighbors B. , McDonald K. L.
Published: April 2021

Ten mitotic spindles from Dictyostelium discoideum have been studied by electron microscopy of serial sections. We have used computer graphics to track individual microtubules (MTs) in three dimensions and to compare seven spindles at different stages of anaphase and telophase. The central spindle of early anaphase is formed by the interdigitation of two sets of pole-associated MTs. The distribution of MT lengths at this stage is hetero-disperse. During anaphase total MT length decreases by a factor of about 2 as a result of two opposing changes in MT length: the longer MTs that interdigitate become even longer, while the short MTs, including those attached to kineto-chores, become shorter still and decrease in number. The extent of MT interdigitation is less in longer spindles than in short ones. In metaphase and early anaphase, the MTs are not in an ordered arrangement as seen in spindle cross-sections, but as anaphase proceeds the MTs cluster into a square-packed, paracrystalline bundle in which most of the nearest neighbours come from opposite poles. This arrangement and the condensation-like increase in order suggest the existence of specific interactions between antiparallel MTs. A quantitative analysis of MT positions supports this interpretation, but direct evidence for convincing bridges between MTs is lacking. The pole-distal ends of the MTs that interdigitate show an irregular termination (C-shaped ends in transverse view), as is characteristic of MTs that are either adding or losing subunits. Since it is these interdigitating MTs that elongate, and since the shortening MTs show the customary blunt endings, we conclude that subunits add to the interdigitating MTs at their pole-distal ends. This inference, combined with other structural data, suggests that the interdigitating MTs of Dictyostelium are sliding over one another as they polymerize in anaphase. It also suggests a simple model for why the spindle becomes thinner as it elongates. We propose that MT interdigitation defines a region where MTs bind a factor that will associate only with antiparallel MTs. This factor biases the MT assembly equilibrium toward polymer. As the shorter MTs slide out of this region, they lose their polymerization advantage and depolymerize, releasing subunits to contribute to the further elongation of the already longer MTs. The properties of the Dictyostelium spindle are compared with those of both higher and lower eukaryotes.
Ultrastructural features of transneuronal cell degeneration in the olfactory system
Authors: Pinching A. J. , Powell T. P. S.
Published: April 2021

The electron microscope has been used to study transneuronal degenerative changes in the cells of the olfactory bulb of the rabbit and rat following removal of the olfactory mucosa. The principal features of this type of change are cell shrinkage, a concentration of cytoplasmic and nuclear constituents, an alteration in the organization of nucleic acid material, and a swelling of membrane-bound systems; some elements of cellular organization appear unchanged, including synaptic specializations. The changes are described for each part of theneuron.—soma, dendrite, axon and terminal—as well as the variants found in different cells. The degree of morphological alteration appears to be related to the extent of denervation for any individual cell and does not seem to be sequential. These changes are related to findings on altered impregnation properties of the same cells with the Golgi technique and are discussed in the context of the functional alteration produced by deafferentation. A new interpretation of some ‘dark neurons’ is also suggested on the basis of these findings.
Protein composition of nuclear matrix preparations from hela cells: An immunochemical approach
Authors: Verheijen Ron , Kuijpers Helma , Vooijs Peter , Van Venrooij Walther , Ramaekers Frans
Published: April 2021

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with special emphasis on the use of various nucleases and detergents as well as on the ionic strength of the final salt extraction.

The protein composition of the resulting nuclear matrix preparations was analysed by one- and two-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) and heterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to be present in such nuclear matrix preparations. The nature of some other protein components was elucidated using two-dimensional immunoblotting and immunofluorescence. For this purpose mouse monoclonal antibodies to cytokeletal components (vimentin, cytokeratins), small nuclear RNP (70× 103Mr protein of U1-RNP), hnRNP (C1/C2) and the pore-complex lamina (lamins A, B and C) were used next to human autoimmune sera obtained from patients with connective tissue diseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodies enabled us to identify a number of proteins present specifically in the nuclear matrix and to show that part of the cytoβkeletal proteinβ are still present in the isolated structures.
Distribution of chromomeres as a basis of chromosomal coiling
Authors: Sorsa Veikko
Published: April 2021

Periodicity in the distribution of prominent bands was analysed from the light and electron microscopic maps of salivary gland chromosomes of Drosophila melanogaster. The data obtained indicate that a similar distribution of prominent chromomeres in an individual interphase chromatid results in a unilateral accumulation of chromatin at the chromonema stage, if the helical axis of chromonema consists of ∼5-9 interchromomere + chromomere units per turn. Orientation of the largest chromomeres mainly on one lateral half and the smallest chromomeres mainly on the opposite lateral half of the chromonema apparently bends it to form the chromosomal ‘macro’ coil. Thus the increase in DNA content in the chromomeric loops located at specific intervals along the chromatids may have an important role in the evolution of coiling hierarchy in the eukaryotic chromosomes.
Immunocytochemical differentiation of microtubules in the cytoskeleton of Giardia lamblia using monoclonal antibodies to α-tubulin and polyclonal antibodies to associated low molecular weight proteins
Authors: Crossley Richard , Marshall Jonathan , Clark John T. , Holberton David V.
Published: April 2021

In interphase trophozoites of Giardia lamblia, separate populations of microtubules constitute the four parts of the mastigont apparatus: flagella, ventral disc, funis and median body.

Antigenic differences between the tubules have been investigated by light and electron immunocytochemistry after labelling with two monoclonal antibodies to α-tubulin (YL 1/2 and YOL 1/34 clones), and with polyclonal antibodies to Giardia tubule-associated proteins. Both anti-tubulins stained all tubules after isolated structures were fixed in formaldehyde, but different patterns of reactivity were shown by unfixed tubules. YL 1/2 antibodies labelled flagellar axonemes and basal bodies, funis and median body tubules. Disc microtubules were mostly unlabelled, but the antibody bound strongly to the outer edge of the disc where the ends of tubules are embedded. YOL l/34 antibodies stained disc tubules uniformly, and cross-reacted with the median body but not with tubules of axonemes, basal bodies or funis.

Antibodies to giardins 14A and 14B (∼30000 Mr filament-forming proteins) localized these proteins in the microribbons attached to disc microtubules. The median body was also labelled by anti-giardins, indicating an ontogenetic relationship between this organelle and the ventral disc.

A second set of ∼30000 Mr proteins with no immunoreactivity to anti-giardin was found in flagella purified without removing flagellar membranes. These polypeptides were Triton-soluble and therefore probably originated from an extra-axonemal site. A rabbit antiserum to the labile flagellar proteins specifically stained the two ventral flagella, but not the other six flagella on this cell.
Surface glycoproteins of human spermatozoa
Authors: Kallajoki M. , Virtanen I. , Suominen J.
Published: April 2021

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis.

The distribution of the radiolabel in sperm cells was studied by light-microscopic autoradiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region.

Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48000 and 43 000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120000, 105 000, 78000 and 68 000Afr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two-to fivefold and additional 36000 and 20000Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68 000Mr glycoprotein.
Nuclear antigens follow different pathways into daughter nuclei during mitosis in early Drosophila embryos
Authors: Frasch Manfred , Glover David M. , Saumweber Harald
Published: April 2021

In the early embryonic development of Drosophila melanogaster, there is a series of 13 rapid and highly synchronous nuclear divisions. We have used a collection of monoclonal antibodies to follow the re-distribution of nuclear antigens into daughter nuclei at this developmental stage by indirect immunofluorescence microscopy. The antigens fall into several categories in terms of the pathways that are followed at mitosis. At one extreme is a group of antigens that remains continuously associated with the DNA throughout all the mitotic phases. At the other extreme, another group of antigens is excluded from the nucleus at prophase, and does not associate with the nucleus again until late telophase. One antigen, which becomes incorporated into the nucleolus at cellularization after the thirteenth division, becomes associated with the chromosomes during mitosis, but not until anaphase. Several different antibodies stain a diamond-shaped compartment that develops over the spindle at anaphase. The distribution of antigens within this spindle compartment shows some variation: one antigen appears to be present at higher concentrations in the central region of the spindle; others appear in three quite distinct areas corresponding to the positions of the new daughter nuclei and the old parental nucleus. Yet another antibody gives uniform staining of the spindle compartment. This antibody also recognizes a protein present in centrosomes.
Signal transduction in human neutrophil leucocytes: Effects of external Na+ and Ca2+ on cell polarity
Authors: Haston Wendy S. , Shields James M.
Published: April 2021

Stimulation of neutrophil leucocytes with chemotactic factors is known to result in membrane permeability changes, as evidenced by fluxes of Na+ and K+ across the cell membrane together with an increased uptake of Ca2+ from the medium. These fluxes have been implicated in the transduction mechanisms of various responses, including locomotion and subsequent chemotaxis. We have previously reported that exposure of unstimulated, round neutrophils held in suspension, to the chemotactic peptide fMet-Leu-Phe confers morphological polarity on the neutrophils by stimulating waves of contraction, which are also intimately connected with locomotion on an appropriate substratum. As the acquisition of polarity is the important first step in the chemotactic response we have investigated the effects of modifying the external ionic environment and of various ion channel blockers on the polarizing response of neutrophils held in suspension.

Removal and chelation of both Ca2+ and Mg2+ from the external medium did not inhibit the acquisition of polarity and a variety of inorganic Ca2+ channèl blockers together with the organic Ca2+ antagonists, verapamil and D600, were ineffective in inhibiting the response. Replacement of Na+ in the external medium with choline inhibited the polarizing response completely but tetrodotoxin, which blocks fast Na+ channels, and amiloride, which inhibits Na+/K+ exchange, had no effect. Inhibition of the Na+/K+-ATPase with ouabain and also tetraethylammonium ions, which block potassium channels, had no inhibiting effect on polarization. These results indicate that while Ca2+ and Mg2+ are not required in the external medium, Na+ is essential, and therefore Na+/K+ fluxes across the cell membrane play a role in initiating locomotion.
Inositol 1,4,5-trisphosphate and calcium stimulate actin polymerization in Dictyostelium discoideum
Authors: Europe-Finner G. Nicholas , Newell Peter C.
Published: April 2021

The effect of chemoattractants such as cyclic AMP and folate on amoebae of the cellular slime mould Dictyostelium discoideum is to cause a series of rapid intracellular responses. One of the most rapid of these responses is the polymerization of actin associated with the cytoskeleton, an event correlated with pseudopodium formation, which ocurrs within 3—5 s of chemotactic receptor stimulation. We report that this response can be mimicked by addition of 5 μM-inositol 1,4,5-trisphosphate (IP3) or by addition of 100μM-Ca2+ to saponin-permeabilized amoebae.

The data suggest that cytoskeletal actin polymerization occurs in normal cells as a result of IP3 formation in response to cell surface receptor stimulation and the consequent release of Ca2+ from internal stores.
Two-directional pattern of movements on the cell surface of Amoeba proteus
Authors: Grebecki Andrzej
Published: April 2021

Particles of latex, glass and precipitated Alcian Blue were studied cinematographically on the surface of migrating Amoeba proteus and in the surrounding medium. The majority of the attached and all unattached particles flow steadily forward in the direction of the endoplasmic streaming and cell locomotion. Flow on the surface is faster than in suspension. Some particles stuck on the membrane move backwards from the frontal region. This retrograde transport is slower than the anterograde flow, and the rate decreases further when the particles approach cell regions adhering to the substratum, accurately following the pattern of the withdrawal of ectoplasm in the same zone. Both movements coexist in the same region and retrograde particles may pass anterograde ones at a distance less than their diameter. Transition from forward flow to backward transport occurs just behind the frontal cap, where the new ectoplasm is formed. The anterograde movement is interpreted as reflecting the general forward flow of the laterally mobile fluid membrane components, which become added to the frontal surface of the locomoting cell; the retrograde movement as retraction of membrane components that, externally, are linked to the transported material and, on the cytoplasmic side, to the contractile microfilamentous layer, as is postulated for cap formation in tissue cells.
Dissociation of basement membrane protein deposition and cell spreading in virally transformed rat mammary myoepithelial cells
Authors: Warburton Michael J. , Ferns Sharon A. , Kimbell Rosemary , Monaghan Paul
Published: April 2021

A myoepithelial-like cell line (Rama 401), isolated from rat mammary gland, has been transformed with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV). Rama 401-tsRSV cells adopt a spindle morphology and fail to deposit basement membrane proteins when grown at the permissive temperature (35°C). When switched to the non-permissive temperature (41°C), the cells flatten (with a 5-fold increase in area), and deposit an extracellular matrix containing basement membrane proteins. When the cells are switched from 35°C to 41 °C in the presence of monensin (an ionophore that inhibits protein secretion), basement membrane proteins are no longer deposited extracellularly although the cells flatten, their area increasing by ninefold. Cells switched from 35 °C to 41 °C in the presence of cycloheximide still flatten and deposit basement membrane proteins, whereas the morphological change on switching from 41 °C to 35 °C is inhibited by cycloheximide. These experiments indicate that the ability of Rama 401-tsRSV cells to spread on a plastic substratum is not dependent on the de novo synthesis and deposition of basement membrane proteins.
Differential effects of epidermal growth factor (EGF) on cell locomotion and cell proliferation in a cloned human embryonal carcinoma-derived cell line in vitro
Authors: Engström Wilhelm
Published: April 2021

The effects of epidermal growth factor (EGF) on clones from a human embryonal carcinoma-derived cell line (Tera-2) have been studied. Cells were plated at clonal densities, whereafter the effects of serum and EGF on cell locomotion and cell proliferation were examined. The addition of 50ngEGFml−1 resulted in increased migration, as judged by increased colony diameter in the presence of EGF. However, the effect of EGF on cell locomotion was rarely accompanied by any effect on cell proliferation. It was concluded that EGF exerts a preferential effect on cell migration in human embryonal carcinoma cells in vitro.
Inhibition of stenotele differentiation by head tissue in Hydra
Authors: Fujisawa Toshitaka
Published: April 2021

Stenotele nematocytes in Hydra are differentiated predominantly in the proximal regions and in gradually decreasing numbers in the more distal regions of the body column. To test whether this position effect is directed by an inhibitory signal from head tissue or by a stimulatory signal from foot tissue, head or foot tissue was laterally grafted from one animal to different positions on another animal. Heads grafted to proximal positions strongly inhibited stenotele differentiation, while the foot exhibited no stimulatory effect. In addition, tissue from gastric regions showed intermediate levels of inhibition. Thus, the inhibitory signal appears to be distributed in a gradient along the body column from head to foot. During head regeneration, the inhibitory signal disappeared abruptly from the distal tip and reappeared rapidly. These results suggest that the inhibitory signal is involved in generating the regional pattern of stenotele differentiation. Head tissue from epithelial hydra, which lacks the interstitial cell lineage, also inhibited stenotele differentiation, suggesting that the inhibitory signal is localized in epithelial cells.
Type I collagen permits invasive behaviour by retinal pigmented epithelial cells in vitro
Authors: Docherty R. J. , Forrester J. V. , Lackie J. M.
Published: April 2021

Epithelial cells cultured on type I collagen gels adopt a typical apical-basal polarity and undergo differentiation. We have compared the behaviour of chick embryo retinal pigmented epithelial (RPE) cells on collagen and on plastic with and without gelatin coats. RPE cell proliferation was similar on all three substrata, and post-confluent cultures exhibited multilayering. On plastic and gelatin-coated plastic, dome formation, typical of transporting epithelia, occurred. On type I collagen gels, however, dome formation did not occur, but rather invasion of the gel matrix by cords of epithelial cells took place. In contrast, invasive behaviour of the cells was markedly reduced on type IV coated collagen gels, particularly in the presence of laminin. These results illustrate the prominent role of the extracellular matrix on phenotypic expression by RPE cells and may represent a more general phenomenon.
Gtp Analogues Stimulate Inositol Trisphosphate Formation Transiently In Dictyostelium
Authors: Europe-Finner G. Nicholas , Newell Peter C.
Published: April 2021

Permeabilization of amoebae of Dictyostelium discoideum with saponin was found not to uncouple the chemotactic cell surface cyclic AMP receptors from inositol trisphosphate (IP3) formation, and stimulation of permeabilized amoebae with 50 nM-cyclic AMP produced peaks of IP3 at 5, 15 and 30 s in a manner comparable to that seen previously in non-permeabilized cells. The possible involvement of a GTP-binding protein (G-protein) in this IP3 signal transduction pathway was investigated by studying the effects on such permeabilized amoebae of added GTP and non-hydrolysable GTP analogues. While GDP produced only very minor effects, stimulation of the amoebae (in the absence of added cyclic AMP) with GTP or the non-hydrolysable GTP analogues GTPγS (guanosine 5’-O-(3-thio-triphos-phate)) and Gpp(NH)p (5’-guanylylimido-diphosphate) induced transient formation of IP3 in an oscillatory manner, with peaks similar in magnitude and timing to those elicited by cyclic AMP. A dose-response curve for GTPγS indicated a concentration for half-maximal stimulation of approximately 8 μM. When tested at 300 s after addition of GTPγ, the basal level of IP3 was found to be twofold elevated with shallow (presumably asynchronous) oscillations still just discernible.

The significance of the IP3 oscillations elicited by GTP and its analogues is discussed in relation to the mechanism of signal adaptation and the presumed role of G-proteins.
Direct measurement of cell detachment force on single cells using a new electromechanical method
Authors: Francis G. W. , Fisher L. R. , Gamble R. A. , Gingell D.
Published: April 2021

We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.
Levels of microtubules during the meiotic maturation of the Xenopus oocyte
Authors: Jessus Catherine , Thibier Catherine , Ozon René
Published: April 2021

The total level of tubulin and the ratio of polymeric tubulin to tubulin dimer were measured by a colchicine filter-binding assay during meiotic maturation of the Xenopus oocyte. Although the total level of tubulin remains unchanged (0·12 ± 0·03 μg/oocyte), the level of polymeric tubulin decreases during maturation (25% in prophase oocytes versus 20% in metaphase oocytes). The percentage of polymerized tubulin was estimated after drug (nocodazole and taxol) treatments and cold treatment in prophase and progesterone-matured oocytes; in all cases the microtubules present in mature oocyte are less stable than prophase microtubules. The presence of the nucleus modifies neither the level nor the stability of prophase microtubules. Our quantitative results as well as cytological arguments suggest that full-grown Xenopus oocytes may contain a cortical microtubular array.
Stimulus-response coupling in the contraction of tentacles of the suctorian protozoon Heliophrya erhardi
Authors: Butler R. D. , Mccrohan C. R.
Published: April 2021

Experiments were carried out to determine the role of the plasma membrane in the control of tentacle contraction in Heliophrya erhardi. Intracellular recordings gave membrane potentials between −20 and −30 mV. In a Ca2+-containing medium mechanical stimulation induced tentacle contraction but no associated electrical events were recorded. Intracellular stimulation with 50 nA, 100 ms hyperpolarizing current induced contraction, but no significant changes in membrane potential, whereas up to 100 nA, 100 ms depolarizing current had no effect. In a Ca2+-free medium neither mechanical stimulation nor electrical stimulation induced contraction. Extracellular stimulation of 15 V, 100 ms induced a Ca2+-dependent, unilateral (anodal) contraction response with a threshold of 5× 10−9M-Ca2+. At concentrations above this neither latency to contraction nor contraction time showed significant variation. In a standard concentration of 10−4M-Ca2+ the sensitivity to extracellular stimulation was increased and latency to contraction was reduced in the presence of a phorbol ester (TPA), which mimics the second messenger diacylglycerol in stimulating the activity of protein kinase C. It is suggested that control of tentacle contraction is unlikely to be mediated by stimulus-activated ion channels in the plasma membrane, and the possibility that the polyphosphoinositide signalling pathway is involved is discussed.
Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell
Authors: Kimata Isao , Tanabe Kazuyuki
Published: April 2021

Monoclonal antibodies against Toxoplasma gondii were prepared to characterize antigens of the parasite. Immunoperoxidase staining of parasites fixed with paraformaldehyde and glutaraldehyde (PFAGA) followed by Triton X-100 treatment showed that the antibody of clone 1–63 recognized an antigen located in the anterior part of the parasite. When analysed by SDS-PAGE and immunoblotting, the antigen migrated in a 66 ×103Mr region. The parasite antigen diminished greatly in parasites after invasion of host cells, but reappeared around a time when intracellular T. gondii multiplied. Immunodetection on PFAGA-fixed T. g’ondn-infected cells, whose membranes were permeabilized by freeze-thawing in the presence of 5 % glycerol, demonstrated that, immediately after parasite invasion, the 1–63 antibody-reactive antigen appeared to become associated with the parasitophorous vacuole (PV) membrane, that had been formed mainly by invagination of the host-cell plasma membrane so as to surround an invading parasite. The antigen remained associated with the PV membrane for some time, but disappeared later when the PV increased in size after the parasites had multiplied several times. These results were strengthened by immunoelectron microscopic observations: the antigen that had been localized at the anterior part of the parasite before invasion appeared in an area of the host cell cytoplasm around the tips of penetrating parasites and, thereafter, extended throughout the surface of the PV membrane when parasites completed invasion. Thus, it appears that the I-63-reactive antigen is secreted by T. gondii upon invasion of the host cell and becomes associated with the PV membrane shortly after invasion.
Direction of sliding and relative sliding velocities within trypsinized sperm axonemes of Gallus domesticus
Authors: Woolley David M. , Brammall Andrew
Published: April 2021

Trypsin digestion of demembranated fowl spermatozoa caused a longitudinal splitting of the distal part of the axoneme. The resulting strands, consisting of groups of doublet microtubules, formed left-handed helices. On the evidence of electron micrographs, the digestion had caused the loss of dynein arms from the outer row; it is assumed that the doublets remained linked together by dynein arms of the inner row. When such helices were mechanically detached from the proximal flagellum and reactivated with adenosine triphosphate, they lengthened in an orderly way by inter-doublet sliding. All the doublets of the axoneme could be reactivated and in all instances the direction of sliding was the same as that reported for the cilia of Tetrahymena. Within the groups of doublets the measured inter-doublet displacements were generally similar, suggesting that the rates of sliding had been equivalent. These findings are contrasted with the differential pattern of activation that is assumed to occur in vivo.
Allelic antigen and membrane-anchor epitopes of Paramecium Primaurelia surface antigens
Authors: Capdeville Yvonne , Caron Francois , Antony Claude , Deregnaucourt Christiane , Keller Anne-Marie
Published: April 2021

Paramecium aurelia can express a repertoire of surface antigens (SAgs) according to culture conditions. These high Mr proteins are anchored in the plasma membrane by a glycolipid, and they can be isolated in two different forms, an amphiphilic membrane-bound form (mSAg) and a hydrophilic soluble form (sSAg). Endogenous or exogenous phospholipase C can convert mSAg to sSAg with unmasking of a carbohydrate antigenic determinant similar to that found in the soluble form of Trypanosoma variant surface glycoproteins and called the cross-reacting determinant. By immunizing mice with cilia from strain 156 of P. primaurelia expressing the G SAg, we obtained six monoclonal antibodies against the 156G SAg, which could be classified into two groups. Y4 and Y8, representative of each group, have been characterized by checking their reactivity in situ and in vivo towards a series of allelic G and D SAgs in P. primaurelia and the 5IB SAg in P. tetraurelia.

The monoclonal Y4 recognizes a conformational determinant, accessible in vivo and common to all the G SAgs. Thus, Y4 defines a G locus-specific epitope that corresponds to a conserved region inside a polymorphic domain. The monoclonal Y8 recognizes two homologous determinants whose detection depends on the presence or absence of the SAg membrane-anchor, and which are mutually exclusive: one is found in the reduced soluble form of all the SAgs and other surface proteins, the cross-reacting glycoproteins (CRGs); the other occurs in the unreduced membrane-bound form of the G SAgs. Thus, Y8 enables us to demonstrate that the membrane-anchor of Paramecium SAgs contains an additional hidden determinant close to the crossreacting determinant and to discriminate between the membrane-bound and soluble form of SAgs.

The in situ organization of the 156G SAg molecules is also discussed on the basis of immunogold labelling obtained using Y4 and a polyclonal antiserum.
Genetic control of plasma membrane adhesion and fusion in Chlamydomonas gametes
Authors: Forest Charlene L.
Published: April 2021

The final stages of gamete interaction in Chlamydomonas reinhardtii occur between the specialized plasma membrane organelles (mating structures) of opposite mating-type cells. A number of mutants affecting these interactions have been obtained in several laboratories; all have been defined as fusion-defective. This paper demonstrates that there are at least two genetically definable stages of mating-structure interaction: (1) the recognition/adhesion of mating structures, and (2) gamete fusion. The possibility that these two functions, the adhesion and subsequent fusion of mating structures, may be mating-type specific is raised, since of the seven independently isolated mutants analysed, all mutants defective in fusion are of mating-type (—), and only mating type (+) mutants show defects in adhesion.
Vimentin intermediate filaments in fish melanophores
Authors: Gyoeva F. K. , Leonova E. V. , Rodionov V. I. , Gelfand V. I.
Published: April 2021

The distribution and chemical composition of intermediate filaments in cultured melanophores of two teleost species -Gymnocorymbus ternetzi and Pterophyllum scalare -were studied by immunofluorescence staining and immunoblotting techniques. The immunofluorescence staining of the melanophores with monoclonal and polyclonal antibodies to the intermediate filament protein vimentin revealed a system of fibrils radiating from the cell centre. These fibrils were resistant to 0·6M-KCl and nocodazole treatments as has been found in other cell types. Transmission electron microscopy confirmed the presence of intermediate filaments in melanophores. Immunoblotting experiments showed the presence of the intermediate filament protein vimentin in melanophore lysates. Therefore, teleost melanophores possess a developed radial system of vimentin intermediate filaments.
Mutant RAS gene induces elevated levels of inositol tris-and hexakisphosphates in Dictyostelium
Authors: Europe-finner G. N. , Ludérus M. E. E. , Small N. V. , driel R. Van , Reymond C. D. , Firtel R. A. , Newell P. C.
Published: April 2021

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium dis-coideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris-and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Glyl2) or a mutant form of the gene (ras-Thrl2). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Glyl2 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thrl2 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three-to fivefold in the ras-Thrl2 strain compared to the strain transformed with ras-Glyl2, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thrl2-transformed cells.

We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.
Chromosome segregation from cell hybrids IV. Movement and position of segregant set chromosomes in early-phase interspecific cell hybrids
Authors: Zelesco Paula A. , graves Jennifer A. Marshall
Published: April 2021

We searched for evidence of aberrant movement or position of segregant set chromosomes in C-banded and G-11-banded early-phase hamster–mouse and hamster–human cell hybrids that had been prepared with minimal disruption. No evidence was obtained for an increased frequency of multipolar mitosis, delayed or precocious meta-phase congression or anaphase segregation, or for exclusion of chromosomes from the daughter nuclei. However, in hamster-human hybrids, segregant set (human) chromosomes were observed to assume a central position within a ring of hamster chromosomes on the metaphase plate. Such non-random positioning may imply that the centromeres of segregant chromosomes make aberrant, or simply less efficient, attachments to the spindle in hybrid cells. This aberrant position may perhaps result indirectly in chromosome loss by interfering with the normal processes of replication, repair or transcription.
Inositol phosphates, G-proteins and RAS genes involved in chemotactic signal transduction of Dictyostelium
Authors: Newell P. C. , Europe-Finner G. N. , Small N. V. , Liu G.
Published: April 2021

Chemotaxis requires some form of signal transduction system (or systems) to convert extracellular messages into intracellular actions. In the amoebal organism Dictyostelium discoideum two pathways of signal transduction have been found to operate, one involves adenylate cyclase for signal relay, and the other, which forms the subject of this review, involves inositol phosphates (Fig. 1).
Characterization of a 125K glycoprotein associated with bovine epithelial desmosomes
Authors: Jones Jonathan C. R.
Published: April 2021

An analysis of the concanavalinA binding polypeptide components of bovine tongue epithelial desmosomes reveals that in addition to the known desmosomal glycoproteins of 100/115K (the ‘desmocollins’), 140K and 160/165K (*desmoglein 1’) there is an uncharacterized glycoprotein of 125K (K = Mr× 10−3). This latter polypeptide is immunologically distinct from known desmosomal glycoproteins, as determined by Western immunoblotting, but is recognized by an antibody preparation directed against the epithelial cell adhesion molecule E-cadherin. Moreover, the cadherin antibodies recognize a polypeptide present in bovine muzzle desmosomes that co-migrates with the 125K glycoprotein component of bovine tongue epithelial desmosomes. Upon treatment of bovine tongue desmosomes with a solution containing 9·5 M-urea, the 125K polypeptide becomes enriched in a urea-insoluble, membrane-enriched pelletable desmosomal fraction. Cadherin antibodies and antibodies directed against the 100/115K and 160/165K desmosomal glycoproteins generate similar immunofluorescence staining patterns in cryostat sections of bovine tongue epithelium. However, immunoelectron microscopic analysis of bovine tongue epithelium reveals that cadherin antibodies recognize components located both along the intercellular region of the desmosome and along non-desmosomal cell surfaces whereas antibodies directed against the 100/115K and the 160/165K desmosomal glycoproteins bind specifically to desmosomes. These results suggest that a cadherin-like glycoprotein component may play a role in the adhesive properties of the desmosomes of stratified squamous epithelia.
Cytoskeletal involvement in the sequential capping of rat thymocyte surface glycoproteins
Authors: Turner Christopher E. , Newton Mary R. , Shotton David M.
Published: April 2021

The independent capping of the three major rat thymocyte glycoproteins, the leucocyte-common (L-C) antigen, the leucocyte sialoglycoprotein (LSGP) and Thy-1, was investigated using specific monoclonal antibodies. The capping of each antigen did not require redistribution of the other major surface glycoproteins, and was acΛcompanied by a partial co-capping of the cytoΛskeletal proteins fodrin and actin, but not of tubulin. A study of the ability of a cell that already possesses one glycoprotein cap to cap a second different glycoprotein showed that this was possΛible in all cases to varying degrees, the second cap always forming at the same position on the cell surface as the first. Colchicine failed to perturb this observed sequential capping polarity, indiΛcating that microtubules did not direct this second capping event.
Tubulin gene expression in the Chlamydomonas reinhardtii cell cycle: Elimination of environmentally induced artifacts and the measurement of tubulin mrna levels
Authors: Nicholl Desmond S. T. , Schloss Jeffery A. , John Peter C. L.
Published: April 2021

To investigate the involvement of tubulin gene expression in controlling cell division events in Chlamydomonas reinhardtii we have measured tubulin mRNA levels during the cell cycle under different environmental conditions. In C. reinhardtii cells grown under the synchronizing conditions of 14 h of light followed by 10 h of darkness, mRNAs for tubulin and associated flagellar proteins were found to accumulate periodically with a peak just prior to cell division. This was not seen when previously synchronized cells were transferred to constant environmental conditions in a turbidostat, suggesting that dramatic changes in tubulin mRNA levels are not required for successful completion of the cell cycle. A hypothesis to explain the patterns of tubulin mRNA accumulation found under different environmental conditions is presented.
Exocytosis, endocytosis and membrane recycling in Tetrahymena thermophila
Authors: Tiedtke Arno , Hünseler Peter , Florin-Christensen Jorge , Florin-Christensen Monica
Published: April 2021

Mutant and wild-type cell lines of Tetrahymena thermophila were used to investigate a possible connection between acid hydrolase secretion and the major processes through which membranes are recycled in this ciliated protozoon. These processes consist of food vacuole formation (endocytosis), and food vacuole egestion and muco-cyst release (both exocytosis). We have found that a mutant (MS-1, sec−) blocked in hydrolase secretion is not blocked in either food vacuole formation or egestion and that it has normal mucocyst exocytosis. Another line of experiments with wild-type cells showed also that hydrolase secretion and endocytosis are independent of each other. Thus, sucrose (0·1 M) did not interfere with hydrolase secretion, but blocked food vacuole formation. Furthermore, release of acid hydrolases was selectively stimulated by dibucaine without any effect on food vacuole egestion. Finally, exocytosis of mucocysts could occur without simultaneous release of acid hydrolases, as when cells were exposed to 0’15M-NaCl, which evokes a massive secretory response of mucocysts. Our results demonstrate that formation and egestion of food vacuoles and exocytosis of mucocysts are unrelated to secretion of acid hydrolases. Furthermore, they suggest that secretion of acid hydrolases is not a secondary effect of membrane recycling through these processes.
Insemination of the archegonium and fertilization in Taxus baccata L
Authors: Pennell Roger I. , Bell Peter R.
Published: April 2021

A study of fertilization in Taxus baccata in the electron microscope has revealed novel features. Insemination of the archegonium is facilitated by local perforation of the wall of the young pollen tube. Digestion of the wall begins before the pollen tube pierces the megaspore membrane but is not completed until its tip makes contact with the neck cells of the archegonium. As soon as a pore is formed a single sperm nucleus and some cytoplasm of the male gametophyte enter the archegonium. Which of the paired sperm nuclei move from the pollen tube into the archegonium appears to be a matter of chance. Close apposition of sperm nucleus and egg nucleus is followed by the formation of numerous points of contact between the two. The membranes fuse at these points and pores are rapidly formed. The progressive enlargement of these pores ultimately eliminates any partitions and yields the zygotic nucleus. There is a possibility that, as in some other gymnosperms, the plastids and mitochondria of the zygote come in part from the male gametophyte, but whether from the remains of the spermatogenous cell cytoplasm or from the pollen tube lumen is not clear.
Freeze-etched pollen walls of Artemisia pycnocephala and Lilium humboldtii
Authors: Southworth D. , Branton D.
Published: April 2021

Mature pollen was examined by freeze-etching. The fracture and etch faces of Artemisia ektexine are unstructured and homogeneous. The endexine fracture face contains particles in paired rows which correspond to the endexine lamellae with a central white line seen in osmium stained thin sections. The intine fracture face in Artemisia is coarsely pitted and has very long fibrils. Small vesicles are observed on both sides of the plasmalemma. The fracture face of Lilium ektexine has radiating fibrils which end in a regular array near the surface; the etch face on the surface is smooth. In thin sections the staining of the Lilium ektexine differs from that of the core, giving evidence that the smooth ektexine surface is chemically distinct from the fibrillar core. The intine in Lilium shows no fibrils. Vesicles near the plasmalemma appear to be fusing with the plasmalemma creating smooth depressions in the particulate plasmalemma fracture face. The plasmalemma in both species is beset with particles in regular arrays.
Silver staining of ribosomal proteins
Authors: Smith J. W. , Stuart R.J.
Published: April 2021

The effects of staining several tissues with silver nitrate were studied in the electron microscope. Tissues were fixed in 2 % glutaraldehyde buffered to pH 7·3 and containing 0·22 M sucrose, some being post-osmicated. Staining was effected by immersion of Araldite sections in unbuffered 5 % silver nitrate for 30 min.

Increase in electron density was restricted to all nucleic acid-containing structures except mitochondrial DNA which is probably not associated with histones. Treatment of fixed tissues with cold 10% perchloric acid for 24 h, which extracts 85 % of the total cell RNA, abolished silver staining within the nucleolus but did not affect that of cytoplasmic or mitochondrial ribo8omes. Incubation of isolated, formalin-fixed liver cells with DNase I did not abolish nuclear staining. This evidence suggests that silver nitrate stains selectively the proteins associated with nucleic acids: the abolition of nucleolar staining by perchloric acid is not at present understood but may be due to some difference in relationship of protein to RNA in this, compared with other situations.

Silver staining indicates that the loci occupied by RNA-associated proteins differ in size and number in different types of ribosomes. Cytoplasmic ribosomes probably contain 5 loci of 4-6 nm diameter, 2 being situated in the 60-s subunit and 3 in the 40-s subunit. Mitochondrial ribosomes appear to contain 2 smaller loci. In the nucleolus the majority of the ribosomes constituting the granulosa contain 2 unequal loci of about 4 and 6 nm respectively, while the appear ance of the pars fibrosa is compatible with the view that it consists of a network of long, extended RNA molecules with which small 2’5-nm protein loci are associated.
Ultrastructure of the fungal nucleus: II. the kinetochore equivalent (kce)*
Authors: Girbardt M.
Published: April 2021

The electron-dense material which regularly occurs at the poles of the nuclei of certain fungi during division has been studied in hyphal interphase nuclei of 4 basidio- and 3 ascomycetes. The shape of this material varies with the species and the nuclear age and may be that of a sphere or a dumbbell. In some instances paired diskoidal structures are found in the place of the globular elements of the dumbbell configuration. The organelle in question lies in an invagination of the nuclear envelope preferentially at the forward pointing pole of the nucleus. Its size (0·1–o·5 /<m) is correlated with the size of the nucleus. In Polystictus versicolor the diameter of both globular elements of the polar organelle increases during initiation of the division process.

The structure is identical with the fungal ‘centriole’ of light-microscopical studies but lacks the characteristic organization of a true centriole. It is suggested that the polar organelle is the equivalent of a kinetochore because it maintains continuous and direct contact with the nuclear envelope (on its cytoplasmic aspect), seems to be connected with the karyoplasm, exhibits oscillating movements, is continuous with microtubules during division and resembles in its fine structure the kinetochores of the chromosomes of higher plants and animals. In addition to its function as an organelle of nuclear motility it also seems to play a part in the initiation of DNA synthesis and nuclear division. If this is correct one would expect to find the genome of the fungal nucleus having the form of a unitary, coherent compound structure (chain) extending from or incorporating a single, solitary kinetochore.