bluuebunny/crossref_metadata_2025 huggingface dataset:
The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope
Authors: Lewis P. R. , Shute C. C. D.
Published: April 2021

The thiocholine technique for cholinesterase has been successfully adapted for the demonstration of enzyme distribution with the electron microscope. After fixation in buffered glutaraldehyde, thin slices of tissue were taken through a histochemical procedure designed to minimize diffusion artifacts and cytological damage; appropriate areas were dissected out, fixed with osmium tetroxide and embedded in Araldite.

This technique has been used to study the distribution of enzyme in and around known cholinergic neurons in the rat. In diaphragm muscle the intense staining of the motor end plates is confined to the actual synaptic clefts. The distribution of cytoplasmic staining was similar in all three types of cholinergic neurons studied (ventral horn cells from the cervical cord, cells from the dorsal motor nucleus of the vagus, and cells from the hypoglossal nucleus). There was intense staining of the space contained within the individual bilaminar sheets of the rough endoplasmic reticulum and occasionally in areas of the nuclear envelope. Mitochondria, lysosomes, the smooth endoplasmic reticulum and most of the plasma membrane were unstained.

In cholinergic nerve fibres staining was particularly intense at the axonal membrane and absent from the myelin sheath. In several regions of the brain known to receive an afferent cholinergic innervation many of the identifiable synaptic areas were heavily stained. Staining here was usually present round most of the presynaptic process, spreading over part of the postsynaptic process and often penetrating into the actual synaptic space. The mitochondria, microvesicles and the general cytoplasm of the synaptic processes were all unstained. In some areas, such as the hippocampus, there was intense membrane staining of fine cholinergic neuropil.

The enzyme specificity of the technique is not in doubt and evidence is adduced for the view that diffusion artifacts are small. The close correlation between the electron-microscopic results and evidence from physiological and biochemical investigations is discussed.
Evolution of the nucleoli during oogenesis in Xenopus laevis studied by electron microscopy
Authors: Van Gansen P. , Schram A.
Published: April 2021

Xenopus laevis tadpoles and toads were killed at several ages. The structure of the nuclei of the germinal cells has been observed by light and electron microscopes. We distinguish 11 successive stages in nucleolar structure: (1) a single, essentially granular nucleolus in the oogonium (10 μm diameter), (2) a reticulated nucleolus in the leptotene oocyte, (3) fragmentation of this nucleolus into a few smaller nucleoli, (4) multiple tiny nucleoli appearing in the cap of the pachytene oocyte, (5) enrichment in the fibrillar constituent of these intra-cap nucleoli, (6) grouped spherical nucleoli, with well segregated granular and fibrillar constituents, as the disintegration of the cap is going on (diplotene A oocyte, 30 μm diameter), (7) dispersion of those nucleoli in the nuclear sap (diplotene B oocyte, 50 μm diameter), (8) formation of long, ribboned nucleoli with multiple DNA-rich spots (diplotene C oocyte, 100 μm diameter), (9) fragmentation of the nucleolar ribbons into multiple spherical nucleoli with excentric fibrillar core and granular cortex (diplotene D oocyte, 150 μm diameter), (10) multiple purely fibrillar nucleoli (diplotene E oocyte, between 150 and 400 μm diameter), and (11) multiple classical nucleoli with concentric fibrillar core and granular cortex (diplotene F oocyte, between 400 and 1000 μm diameter).

The multiplication of the nucleoli in Xenopus laevis may occur successively (a) by the fragmentation of the single oogonium nucleolus at the leptotene stage, (b) by de novo formation of nucleolar bodies inside the cap at the pachytene stage, and (c) by the growth of those nucleoli lying free in the nucleolar sap at the early diplotene stage. They evolve into nucleolar ribbons which later on fragment into spherical bodies.

Four successive phases during the growth of an oocyte can be distinguished with respect to the ribosomal system. (I) The first phase is characterized by the nucleolar DNA amplification. (II) During the second phase, the multiplication of the nucleoli is going on. Ribosomes are present in the cytoplasm and the rate of cellular growth is very high. (III) During the third phase, the synthesis of rRNAs seem to be repressed while the synthesis of heterogenous small RNAs is going on. Ribosomes are no longer visible in the cytoplasm. The nucleoli are purely fibrillar. The rate of cell growth is lower than in the preceding phase. (IV) During the fourth (= Duryee lampbrush stages 3—6), or vitellogenic phase, rRNAs are actively synthesized and numerous ribosomes appear in the cytoplasm. The nucleoli have the classical structure and the rate of growth is about the same as during phase III.
Biochemical and structural variations in the flight muscle mitochondria of ageing blowflies, Calliphora erythrocephala
Authors: Tribe M. A. , Ashhurst Doreen E.
Published: April 2021

Biochemical investigations with isolated blowfly flight muscle mitochondria show that uncoupling of oxidation and phosphorylation increases with increasing age of the adult fly. Of the two principal substrates used (i.e. a-glycerophosphate and pyruvate), uncoupling is most marked when α-glycerophosphate is used as substrate.

Although age-related changes in the structure of the thoracic flight muscle are observed by electron-microscope studies, no obvious degenerative changes take place in either the mitochondrial structure or the myofibrils of old flies. Labelling experiments with [3H]leucine indicate that uncoupling is mainly due to the lack of repair and replacement of mitochondrial proteins. These experiments also show that mitochondria] protein synthesis is important in the maturation of young flies, that is, those between o and 4 days old, but is insignificant in the further increase in mitochondrial size observed in older flies, where mitochondrial fusion appears to be more important.
The termination of centrifugal fibres in the glomerular layer of the olfactory bulb
Authors: Pinching A. J. , Powell T. P. S.
Published: April 2021

The termination of the centrifugal fibres running in the lateral olfactory tract to the glomerular layer of the rat olfactory bulb has been determined with the electron microscope; this has been done with material perfused at various times after section of the lateral olfactory tract, as well as after a combination of this lesion with the long-term degeneration of olfactory nerves. The axon terminals are sparse at the glomerular level, but undergo typical degenerative changes; they are distributed solely in the periglomerular region and intermediate zone. The most common post-synaptic profiles are the processes of periglomerular cells, but a few centrifugal fibres terminate on short-axon, tufted and mitral cell dendrites. Evidence is produced to suggest that the anterior olfactory nucleus does not project as far as the glomerular layer. The findings are discussed in relation to previous studies with normal material and silver degeneration methods on similar experimental material; the functional implications of the centrifugal pathways in the bulb are briefly discussed.
The effect of neuraminidase on the aggregation of bhk21 cells and bhk21 cells transformed by polyoma virus
Authors: Vicker M. G. , Edwards J. G.
Published: April 2021

Bacterial neuraminidases increase the adhesiveness in suspension of freshly trypsinized hamster fibroblasts (BHK21 cells). The effect is seen most clearly when the enzymes are added to pre-aggregated suspensions, but can also be observed when cells in monolayer culture are pre-incubated with neuraminidase, suspended, and then aggregated in its absence. BHK21 cells transformed by polyoma virus respond only very slightly to neuraminidase, which therefore increases the difference in adhesiveness between transformed and untransformed cells. Neuraminidase does not eliminate the difference in adhesiveness between sparsely and densely grown BHK 21 cells.

These findings are consistent both with the lyophobic colloid theory of cell adhesion and with proposals that intercellular adhesion depends on complementary molecules on the surface of interacting cells. The low adhesiveness and small response to neuraminidase of transformed cells, however, are more readily explained in terms of the latter.
Mitosis in heterobasidiomycetous yeasts: I. leucosporidium scottii (candida scottii)
Authors: McCULLY E. Kathleen , Robinow C. F.
Published: April 2021

Leucosporidium scottii, for many years considered to be an asporogenous yeast in the genus Candida, is now known to have a heterobasidiomycetous life-cycle. Although morphologically similar to ascomycetous yeasts such as Saccharomyces cerevisiae, budding cells of L. scottii have a strikingly different type of mitosis. In L. scottii, mitosis is not intranuclear as it is in 5. cerevisiae. Neither does the nucleus constrict and separate into daughter nuclei in the neck region between mother cell and bud.

In L. scottii the chromatin-containing portion of the nucleus moves into the bud before division. There is partial breakdown of the nuclear envelope and the chromatin divides along a spindle apparatus formed inside the bud within remnants of nuclear envelope. A portion of the nucleus containing the nucleolus is left behind in the mother cell and disintegrates there when the envelope breaks down. After division, the nuclear envelope reforms around daughter nuclei and one daughter moves back to the mother cell. These events have been established on the basis of 3 types of observation: on living cells with phase-contrast microscopy; on cells stained for chromatin and studied with light microscopy; and on glutaraldehyde-fixed cells studied with electron microscopy.

As in L. scottii, breakdown of nuclear envelope during division has been reported in several mycelial basidiomycetes. The sequence of chromatin movement into the bud of L. scottii, division inside the bud, and return of one daughter nucleus to the mother cell, is closely comparable to the movements of chromatin in and out of clamp connexions known to occur in dikaryotic hyphae of Polystictus versicolor. These mitotic similarities are in accord with the taxonomic relationship of L. scottii to the basidiomycetes.

Observations on a cytoplasmic organelle located outside interphase nuclei of L. scottii have convinced us of its considerable importance during mitosis. This structure consists of 2 globular electron-dense ends joined by a bridge-like middle piece. It accompanies the advancing tips of nuclei which enter the bud. After breakdown of the nuclear envelope, the 2 spherical components of this organelle are seen at opposite poles of the mitotic spindle. Therefore we refer to it as the ‘microtubule organizing centre’ (MTOC). We have also speculated about a possible role for the organelle in the regulation of nuclear envelope growth, breakdown, and reformation.
Differences in adhesion to tissue culture plastic of clonally related transformed and control sublines from an epithelial cell strain
Authors: Steele John G. , Dalton B. Ann , Underwood P. Anne , Smith Garry J.
Published: April 2021

Clonally related sublines of the NAL1A lung epithelial cell strain were used in a comparison of the mechanism of attachment and the morphology of control and transformed epithelial cells. The initial attachment and spreading of the control cells on tissue culture plastic was shown to be dependent upon adsorption of serum vitronectin to the substratum. The alphav subunit of the vitronectin receptor was detected in both the control and transformed cells by immunoprecipitation and immunoblot methods. The spontaneously transformed cells differed from the control cells in that, whereas attachment to tissue culture plastic could occur by binding to adsorbed vitronectin, the transformed cells could also become attached, with time, by a vitronectin-independent mechanism. Attachment by this vitronectin-independent reaction was inhibited by the protein synthesis inhibitor cycloheximide and also by the microtubule-disrupting drugs demicolce-mid and nocodazole. The morphologies of attached control and transformed cells cultured on tissue culture plastic were disrupted by treatment with cytochalasin B, demicolcemid or nocodazole, indicating that the shape of these cultured epithelial cells is dependent upon the microtubule system as well as the actin filaments. These results show one important difference between the control and transformed cells, in that the transformed cells can attach to tissue culture plastic by a vitronectin-independent mechanism that involves new protein synthesis by the cell. Another interesting difference is that this vitronectin-independent attachment of the transformed cells was sensitive to inhibition by microtubule-disrupting agents. On the other hand, the attachment of either transformed or control cells to fibronectin- or vitronectin-coated surfaces was not affected by microtubule-disrupting agents.
Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy
Authors: Sabanay Ilana , Arad Talmon , Weiner Stephen , Geiger Benjamin
Published: April 2021

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.
Lucifer yellow and fluorescein isothiocyanate uptake by cells of Morinda citrifolia in suspension cultures is not confined to the endocytotic pathway
Authors: O’DRISCOLL D. , Wilson G. , Steer M. W.
Published: April 2021

On the basis of previous reports that the uptake of Lucifer Yellow-CH (1YCH) is restricted to the endocytotic pathway in plant cells, it was anticipated that the probe would provide an estimate of endocytotic activity in suspension cultures of Morinda citrifolia. The uptake of LYCH and fluorescein isothiocyanate (FITC) have been examined in whole cell and protoplast forms of M. citrifolia. Rapid staining of the general cytoplasm and nucleus was observed with both fluorochrome treatments, while both markers were excluded from the central vacuole. In addition,treatment of cells with Probenecid, an anion transport inhibitor, prevented LYCH from entering cells. It is suggested that LYCH utilizes an anion transport mechanism to cross the plant cell plasma membrane. Furthermore, LYCH and FITC are excluded from the central vacuole due to the absence of such a mechanism on the tonoplast.
Molecular cloning of the human gene, ccg2, that complements the bhk-derived temperature-sensitive cell cycle mutant tsbn63: Identity of ccg2 with the human x chromosomal scar/rps4x gene
Authors: Watanabe Minoru , Furuno Nobuaki , Goebl Mark , Go Mitiko , Miyauchi Kumi , Sekiguchi Takeshi , Basilico Claudio , Nishimito Takeharu
Published: April 2021

A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell Une after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 °C were shifted to 39.5 °C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in Gt phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10−3. On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA.
Induction of partial mitosis in BHK cells by 2-aminopurine
Authors: Andreassen Paul R. , Margolis Robert L.
Published: April 2021

The protein kinase inhibitor 2-aminopurine (2-AP) inhibits a subset of mitotic events in BHK cells. In the presence of the drug, these cells form a bipolar spindle in mitosis, but chromatin fails to generate functioning chromosomes. Cells in 2-AP progress through a partial mitosis, in which there is no observable metaphase, anaphase or telophase events. After 12 h of exposure to 2-AP the chromatin in mitotic cells fails to condense into discrete chromosomes, and is displaced by the spindle to form ‘binucleate’ cells and cells containing abnormally shaped nuclei in the subsequent interphase. Other mitotic modifications of nuclei, such as nucleolar and nuclear lamina disassembly, occur normally. Centromeres in these nuclei do not become engaged in the spindle, but instead show either no association or a lateral arrangement around the spindle. Cells treated with 2-AP are not arrested in mitosis. Therefore, mitotic exit is not inhibited by the failure of these cells to progress through the latter stages of mitosis. Further, nocodazole-arrested cells quickly exit mitotic arrest when 2-AP is added. We conclude that 2-AP interferes with a specific subset of mitotic events, and that it allows cells to overcome checkpoints that require spindle function for mitotic progression.
Wild type and tailless CD8 display similar interaction with microfilaments during capping
Authors: André Pascale , Gabert Jean , Benoliel Anne Marie , Capo Christian , Boyer Claudec , Schmitt-Verhulst Anne Marie , Malissen Bernard , Bongrand Pierre
Published: April 2021

We examined the influence of the intracytoplasmic region of CD8 a on capping and interaction with microfilaments. We used cell clones obtained by transfecting a CD4+ T-cell hybridoma with (a) T-cell receptor (TCR) α and β chains from a cytolytic clone and (b) CD8a genes that were either native or modified by extensive deletion of the intracytoplasmic region or replacement of the transmembrane and intracytoplasmic domains with those of a class I major histocompatibility complex gene (Letourneur et al. (1990). Proc. natn. Acad. Sci. U.S.A. 87, 2339–2343). Different cell surface structures were cross-linked with anti-T-cell receptor, anti-CD8 or anti-class I monoclonal antibodies and anti-immunoglobulin (Fab’)2. Double labeling and quantitative image analysis were combined to monitor fluorescence anisotropy and correlation between different markers. Microfilaments displayed maximal polarization within two minutes. The correlation between these structures and surface markers was then maximal and started decreasing, whereas the redistribution of surface markers remained stable or continued. Furthermore, wild type and altered CD8□ exhibited similar ability to be capped and to induce co-capping of TCR and MHC (major histocompatibility complex) class I: the fraction of cell surface label redistributed into a localized cap ranged between 40% and 80%. Finally, cytochalasin D dramatically decreased CD8 capping in all tested clones.

It is concluded that the transmembrane and/or intracellular domains of CD8 molecules are able to drive the extensive redistributions of membrane structures and cytoskeletal elements that are triggered by CD8 cross-linking.
The epidermal growth factor-induced migration of rat liver epithelial cells is associated with a transient inhibition of DNA synthesis
Authors: Geimer Pia , Bade Ernesto G.
Published: April 2021

Epidermal growth factor (EGF) is a potent mitogen for most cultured cells and has previously been shown to induce the migration of rat liver epithelial cells. We have now demonstrated that under migration-inducing conditions EGF does not stimulate cell proliferation, but causes instead a transient inhibition of DNA synthesis. Analysis at the singlecell level by [3H]thymidine autoradiography indicated that in 40-50% of the EGF-treated cell population the entry into S phase is delayed. The simultaneous demonstration of migration tracks by laminin immunofluorescence revealed that the transient inhibition of DNA synthesis is not restricted to the migratory cells. The effect is also observed with the stationary subpopulation and appears, therefore, to be independent of the induction of migration. The independence of both processes was further supported by showing that induction of migration by EGF proceeds undisturbed in cells blocked in S phase by aphidicolin.

These results indicated that for rat liver epithelial cells the induction of migration by EGF has priority over cell proliferation. The data also emphasize the need for a time-course analysis when studying factors that stimulate or inhibit DNA synthesis or cell proliferation.
Circadian rhythmicity in the activities of adenylate cyclase and phosphodiesterase in synchronously dividing and stationary-phase cultures of the achlorophyllous ZC mutant of Euglena gracilis
Authors: Tong Jian , Carre Isabelle A. , Edmunds Leland N.
Published: April 2021

Key factors in the adenosine 3’,5-cyclic monophosphate (cyclic AMP) metabolic pathway are two enzymes responsible for its generation and degradation, namely, adenylate cyclase (AC) and phosphodiesterase (PDE). In LD: 12,12 (12 h light, 12 h dark), these enzymes were found to undergo bimodal, circadian variation of activity in both dividing and nondividing cultures of the photosynthesis-deficient, achlorophyllous ZC mutant of Euglena gracilis Klebs (Z). Maximal AC activity occurred 2 h after the onset of the light interval (CT 02) and at the beginning of darkness (CT 12-14); these times corresponded to the acrophase profile for the rhythmic changes in cyclic AMP content that have been previously reported. The activity of PDE also exhibited a daily oscillation, but with an inverse phase pattern. Both the AC and PDE activity rhythms persisted after the cultures were transferred from LD: 12,12 to constant darkness. The activity of AC was activated significantly in vivo by forskolin at the trough phase (CT 20), while that of PDE was inhibited by 3-isobutyl-l-methyl-xanthine (IBMX) at its peak phase. These results indicate that the rhythms of both AC and PDE may be the main factors generating the circadian oscillations of cyclic AMP content in Euglena, which appear to be under control of an endogenous pacemaker.
Isolation of a sea urchin egg kinesin-related protein using peptide antibodies
Authors: Cole Douglas G. , Cande W. Zacheus , Baskin Ronald J. , Skoufias Dimitrios A. , Hogan Christopher J. , Scholey Jonathan M.
Published: April 2021

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 ×10a that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.
Salt-stable interaction of the amino-terminal head region of vimentin with the α-helical rod domain of cytoplasmic intermediate filament proteins and its relevance to protofilament structure and filament formation and stability
Authors: Traub P. , Scherbarth A. , Wiegers W. , Shoeman R. L.
Published: April 2021

Previous studies have shown that the non-a-helical, amino-terminal head region of vimentin is essential for the formation and stability of vimentin intermediate filaments (IFs). In order to specify its target site on companion protein subunits, it was cut off from vimentin at amino acid position 96 with lysine-specific endoproteinase and allowed to react with intact vimentin and other IF proteins. In solution of high salt concentration (500 mM KCl), the isolated polypeptide (vim NT) showed a high affinity for all cytoplasmic IF proteins tested, but not for nuclear lamins. Employing limited digestion of the IF proteins with different proteinases, the binding site was shown to reside in their a-helical rod domains. Other polypeptides possessing a-helical regions with the potential to form coiled-coil structures like tropomyosin and myosin subfragment 2 did not react with vim NT. The binding to IF proteins was strongly inhibited by phosphorylation of vim NT and totally abolished in the presence of 200 mM arginine hydrochloride, whereas the same concentration of lysine hydrochloride was ineffective. Limited chymotryptic digestion of vim NT produced polypeptides that were unable to react with the a-helical region of vimentin at high salt concentration. Consistent with these observations, vim NT strongly inhibited filament formation in vitro from protofilamentous vimentin. A 14-mer oligo peptide comprising the amino acids 3 to 16 of the amino terminus also inhibited filament formation, though to a lesser extent. Conversely, vim NT and, with a lower efficiency, the 14-mer oligopeptide also severely affected the structure of preformed vimentin filaments by unraveling them. Phosphorylated vim NT was considerably less active in this respect. Further digestion of the rod domain of vimentin with chymotrypsin yielded 17.4 and 21 kDa polypeptides, which were tentatively characterized as originating from the carboxy- and amino-terminal half of the rod domain, respectively. Both formed salt-stable complexes with vim NT, the smaller polypeptide with a higher efficiency than the larger one. These results suggest that the staggered, antiparallel arrangement of the two coiled-coils in the protofilaments of IF proteins is, at least in part, determined by the twofold, symmetrical association of the amino-terminal head regions of one coiled-coil rope structure with the carboxy-terminal halves of the a-helical rod domains of the other coiled-coil and that similar interactions occur during filament assembly and in the intact filament.
α-Actinin and vinculin in human neutrophils: reorganization during adhesion and relation to the actin network
Authors: Yürüker Banu , Niggli Verena
Published: April 2021

We have studied the reorganization of vinculin and α-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent Factin-rich large pseudopods. α-Actinin became enriched during cell adhesion in retraction fibers and, in 40-50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of α-Actinin at a certain stage.

Disruption of the actin network with cytochalasin D revealed a differential interaction of α-Actinin and vinculin with the actin network. α-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10−5 M of the drug. Vinculin organization in contrast was not affected by up to 10−6 M cytochalasin. At 10−5 M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas α-Actinin may structure the actin network in retraction fibers and in less adherent pseudopods.
Localised application of an activating signal to a cell: experimental use of fibronectin bound to beads and the implications for mechanisms of adhesion
Authors: Curtis A. S. G. , Mcgrath Mike , Gasmi Lakdar
Published: April 2021

Small beads derivatised with fibronectin or with bovine serum albumin are allowed to attach to BHK cells in suspension at low ratios of beads to cells. In this way populations of cells bearing predominantly one bead per cell can be prepared. We show that the attachment of one bead per cell affects the adhesion and spreading of that cell on substrata, raising adhesion and increasing spreading if the signal molecule is fibronectin, decreasing these quantities if the bead bears BSA. The experiments are conducted in the absence of other sources of exogenous fibronectin and in some cases in the additional absence of endogenous sources. The effects are especially marked if the substratum is adsorbed haemoglobin on which control cells show little attachment or spreading. We further show by interference reflection microscopy and by scanning electron microscopy that the beads are found on the non-adhering side (uppermost or outer) of the cell when fibronectin-bearing beads are used, presumably because fibronectin will not attach to haemoglobin. The increased adhesion and spreading found in such cases must be attributed to an activation produced by the bead, which spreads to other parts of the cell and which activates a fibronectin-independent mode of adhesion.
Redistribution and differential extraction of soluble proteins in permeabilized cultured cells Implications for immunofluorescence microscopy
Authors: Melan Melissa A. , Sluder Greenfield
Published: April 2021

Immunofluorescence microscopy is widely used to characterize the cellular distribution of both soluble and structural proteins. Control experiments generally address only the specificity of the antibodies used. The permeabilization/fixation conditions used to prepare cells for antibody application are assumed to preserve faithfully the in vivo distibutions of the protein(s) being examined. We systematically tested the extent to which soluble proteins are redistributed into inappropriate locations and are differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application. We separately introduce six soluble FITC-coiyugated proteins of different net charges and sizes into living cultured cells. The labeled proteins do not adhere to the external surfaces of living cells and are evenly distributed throughout the cytoplasm with the larger proteins being excluded from the nucleus. The cells are then prepared as if for immunofluorescence using several conditions that encompass many of the methods commonly used for this purpose. Cells permeabilized with 0.1-0.2% Triton X100 before fixation with 3.7% paraformaldehyde show a striking localization of all but one of the test proteins to the nucleus and/or nucleoli of 60-80% of labeled cells. Punctate cytoplasmic labeling and cytoskeletal-like arrays of labeled protein are also observed. Extraction with 1% detergent prior to fixation removes most but not always all of the exogenous proteins from the cell remnants. Permeabilization of cells with 0.1% detergent after paraformaldehyde fixation leaves a reticular, uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Direct fixation/permeabilization with −20°C methanol largely preserves the in vivo distributions of fluorescent proteins with some preferential localization of these proteins to nuclei, nucleoli and the perinuclear region. These results show that misleading apparent localizations of soluble proteins can result from their redistribution and/or differential extraction during the preparation of cells for primary antibody application.
Different patterns of rDNA organization at interphase in nuclei of wheat and rye
Authors: Leitch A. R. , Öller W. Mosg , Shi M. , Heslop-Harrison J. S.
Published: April 2021

The physical location of the rDNA repeating units (25 S, 18 S and 5.8 S rRNA genes and the intergenic spacer sequences) was investigated in rye (Secale cereale L.) and wheat (Triticum aestivum L.) root tip meristematic cells by in situ hybridization using light and electron microscopy. The rDNA sequences are organized differently in the two related and intercrossable species. In rye (2n=14, one pair of chromosomes with nucleolar organizing regions, NORs), two condensed blocks of rDNA-containing chromatin occurred in each interphase nucleus. The blocks were associated with the periphery of nucleoli and a single-labelled, decondensed rDNA fibre extended into the nucleolus from the block. We term this expression pattern terminal decondensation. In wheat (2n=6× =42, five pairs of chromosomes with NORs), inactive condensed labelled chromatin was found unassociated with nucleoli. Active NORs had some condensed rDNA associated with the nucleolar periphery, but, in contrast to rye, condensed rDNA was also found within the nucleolus. The condensed labelled rDNA in wheat nucleoli was visible as fluorescent foci in the light microscope and labelled condensed chromatin in the electron microscope. Its absence in rye shows that condensed rDNA need not be present in active plant nucleoli. Diffuse labelled sites of rDNA, likely to represent actively transcribed rDNA, were found in both rye and wheat. Active rDNA loci in wheat have many expressed segments separated by unexpressed, condensed, rDNA - fragmented decondensation - while each locus in rye has a single, unexpressed perinucleolar condensed block of rRNA genes. Thus the positions of actively transcribed genes within the tandem arrays of rDNA at each locus are fundamentally different in the two cereals. The NOR chromosome appeared to extend through the nucleolus, and active rDNA sequences did not loop out from chromatin into the nucleolus as is frequently described in nucleolar models.
A novel structure associated with a lampbrush chromosome in the chicken, Gallus domesticus
Authors: Solovei Irina , Gaginskaya Elena , Allen Terry , Macgregor Herbert
Published: April 2021

At a site near the end of the short arms of lampbrush bivalent 2 in the chicken (Gallus domesticus) there is always a marker structure that appears in the phasecontrast light microscope as a solid object with diffuse edges measuring about 4 μm across. When examined by transmission electron microscopy in thin section, this object appears as a loose bundle of fibres. In some preparations individual fibres appear 15–16 nm thick, smooth in outline and solid in cross-section. In other preparations they are 32-38 nm thick, rougher in outline and ring-like in cross-section. High-resolution scanning electron micrographs of the chromosome 2 marker show it to be a loose bundle of spaghetti-like fibres that is quite unlike anything previously seen on a lampbrush chromosome of any organism. As with the sectioned material, fibres in some preparations were smooth and 15-16 nm in diameter, whereas those in others were more knobbly and about 35 nm thick. The fibres appear to branch and in some cases it is clear that the daughter strands of a branch have the same dimensions as the parent strand. Free ends are rare. Total length of fibre material present at one marker locus is estimated to be between 500 and 2000 μm. Similar structures are not present on the lampbrush chromosomes of quail, wood pigeon or chaffinch.

The nature of this fibrous marker, referred to in this paper as the “spaghetti marker”, is discussed in relation to lampbrush chromosome function and to events that take place during the lampbrush phase of oogenesis in chicken. Evidence is discussed in relation to the possibility that the chromosome 2 marker represents a novel form of nuclear RNP or the specific association of some structural protein with one chromosome locus.
Co-expression of insulin and somatostatin genes in pancreatic endocrine cells selected for their high level of insulin gene transcription
Authors: Saulnier-Michel Catherine , Fromont-Racine Micheline , Pictet Raymond
Published: April 2021

RW cells are pancreatic endocrine RIN cells that have been stably transfected with a chimeric gene that places the expression of the dominant selection gpt gene under the control of the insulin gene regulatory sequences. These RW cells were examined for hormone content using immunocytochemistry. This analysis shows that: first, there are cells that are negative for insulin although they were cultured under selective pressure. Second, there is a higher proportion of somatostatin-producing cells than in the parental RIN cells; these somatostatin cells form two populations: one of cells containing only somatostatin and, surprisingly, one made of cells containing both insulin and somatostatin. Thus: (1) expression of the transfected and endogenous insulin regulatory sequences is not regulated in a coordinate fashion; (2) the presence of both hormones in the same cell suggests that the regulation of the expression of insulin and somatostatin genes and the differentiation pathway of the two respective cell types may be closely related.
Evidence for a non-tubulin spindle matrix and for spindle components immunologically related to tektin filaments
Authors: Steffen Walter , Linck Richard W.
Published: April 2021

Tektins were originally described as a set of three filamentous proteins (tektin A, B and C) associated with the walls of axonemal microtubules of sea urchin sperm. Using affinity-purified polyclonal antibodies raised against tektins of two sea urchin species, Lytechinus pictus and Strongylocentrotus purpuratus, we looked for tektin-like components in microtubule systems other than axonemes. By immunofluorescence microscopy we observed labeling of meiotic spindles in eggs of the surf clam Spisula solidissima and in several mammalian cell lines. In Spisula eggs the tektin-like antigens were still associated with the spindles after about 95% of the tubulin had been removed via a calcium/cold treatment. In pig kidney epithelial cells the tektin-like antigen appeared to be associated with bundles of calcium-stable spindle microtubules. By SDS-PAGE immunoblot the affinity-purified anti-tektins recognized several polypeptides in tubulin-depleted spindle remnants of Spisula eggs: A ˜52 kDa, 1 M KCl-resistant component was identified by the antibody raised against tektin C from S. purpuratus, a ˜48 kDa component was recognized by the antibody specific for tektin A from L. pictus, and three polypeptide bands (˜64 kDa, ˜100 kDa and >200 kDa) were detected by the antibody specific for tektin C from L. pictus. Only the latter antibody, however, stained Spisula spindles by immunofluorescence microscopy. We further report that the sensitivity of antibody recognition of proteins on immunoblots is dependent on the purity of sodium dodecyl sulfate.
Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum
Authors: King Karen L. , Stewart Murray , Roberts Thomas M. , Seavy Margaret
Published: April 2021

Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated α- and β-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that α- MSP is 142 Da larger and 0.6 pH unit more basic than β-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but α-MSP formed filaments at lower solvent concentration than β-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.
Initiation of HeLa cell adhesion to collagen is dependent upon collagen receptor upregulation, segregation to the basal plasma membrane, clustering and binding to the cytoskeleton
Authors: Lu Michael L. , McCarron Richard J. , Jacobson Bruce S.
Published: April 2021

It was recently reported that HeLa cells have three Arg-Gly-Asp-dependent collagen receptors that do not appear to be in the integrin family of extracellular matrix receptors and bind to either type I or IV collagen or to type I gelatin. It was our goal to determine how these receptors function in HeLa cell-substratum adhesion. We report here that the sequence of events by which the receptors mediate adhesion to collagen or gelatin is: (1) induction of cell attachment by specific collagen receptor-substratum interactions with culture dishes covalently coated with either type I collagen or gelatin - attachment is inhibited by soluble gelatin; (2) stabilization of attachment by exocytotic upregulation of the receptors to the basal plasma membrane, which was demonstrated by analyzing, during cell adhesion, the redistribution of the collagen receptors among the apical plasma membrane exposed to the culture medium, the basal plasma membrane contacting the culture dish, and an intracellular pool of plasma membrane vesicles; (3) the initiation of cell spreading by receptor clustering and cytoskeletal association. Cell spreading is a threshold effect with regard to the surface concentration of gelatin, indicating that collagen receptor clustering is a precondition to the onset of spreading. Observations consistent with this interpretation of the threshold effect are that cells attach but spread more slowly on a substratum that retards receptor clustering, and that collagen receptors, when viewed by immunofluorescence microscopy, form a punctate pattern of fluorescence in the basal plasma membrane during cell spreading. It is also shown that more collagen receptors co-isolate with nondenaturing detergent-stable cytoskeletal preparations after the collagen receptors have been either clustered by antibodies or gelatin in solution, or by a collagen matrix. This indicates that clustering drives the receptors to bind to the cytoskeleton and is a necessary step in the transition from cell attachment to cell spreading.
HeLa cells extend and internalize pseudopodia during active invasion by Trypanosoma cruzi trypomastigotes
Authors: Schenkman Sergio , Mortara Renato A.
Published: April 2021

We show here that HeLa cell microfilaments can be stained by phalloidin at the sites of invasion of Trypanosoma cruzi trypomastigotes, one of the infective stages of this protozoan parasite. Concurrently, a projection of the HeLa cell plasmalemma encircles invading parasites. This plasmalemma projection is further internalized and entire membrane protrusions containing parasites are found within cytoplasmic vacuoles of the host cell. Neither the microfilament staining around invading parasites nor the plasmalemma extension is inhibited by cytochalasin D, a drug that is unable to prevent trypomastigote entry into HeLa cells. The internalization of the membrane expansion, however, is blocked by the drug. These novel observations indicate that although the driving force for T. cruzi penetration comes from the parasite, the cortical target cytoskeleton of the target cell is concomitantly modified. The molecular characterization of this phenomenon may provide a new insight into the understanding of the mechanisms involved in the active penetration of T. cruzi into mammalian cells.
The macrophage capacity for phagocytosis
Authors: Cannon Gregory J. , Swanson Joel A.
Published: April 2021

Murine bone marrow-derived macrophages, which measure 13.8 ± 2.3 tun diameter in suspension, can ingest IgG-opsonized latex beads greater than 20 μm diameter. A precise assay has allowed the determination of the phagocytic capacity, and of physiological parameters that limit that capacity. Ingestion of beads larger than 15 μn diameter required IgG-opsonization, and took 30 minutes to reach completion. Despite the dependence on Fc-receptors for phagocytosis of larger beads, cells reached their limit before all cell surface Fc-receptors were occupied. The maximal membrane surface area after frustrated phagocytosis of opsonized coverslips was similar to the membrane surface area required to engulf particles at the limiting diameter, indicating that the capacity was independent of particle shape. Vacuolation of the lysosomal compartment with sucrose, which expanded endocytic compartments, lowered the phagocytic capacity. This decrease was reversed when sucrose vacuoles were collapsed by incubation of cells with invertase. These experiments indicate that the phagocytic capacity is limited by the amount of available membrane, rather than by the availability of Fc-receptors. The capacity was also reduced by depolymerization of cytoplasmic microtubules with nocodazole. Nocodazole did not affect the area of maximal cell spreading during frustrated phagocytosis, but did alter the shape of the spread cells. Thus, microtubules may coordinate cytoplasm for engulfment of the largest particles.