bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of the quadripolar meiotic cytoskeleton in spore mother cells of the moss Funaria hygrometrica
Authors: Busby C. H. , Gunning B. E. S.
Published: April 2021

Evidence presented in the accompanying paper that plastids function as microtubule (MT)-organizing centres for development of the quadripolar cytoskeleton of pre-meiotic spore mother cells (SMCs) in the moss Funaria hygrometrica is complemented here by observations on the MT system in these cells.

Early in meiotic prophase numerous MTs align progressively along the two plastids as they elongate. Concomitant with (and perhaps causal for) plastid rotation, new MT arrays grow from each tip of each plastid to both tips of the other plastid. The ‘along-plastid’ and ‘between-plastid’ arrays ultimately form the edges of a tetrahedron, enclosing the prophase nucleus. MT breakdown at the centre of each edge leaves four cones of MTs, one emanating from each vertex, located at the plastid tips. These partially fuse in between-plastid pairs to give a twisted spindle with broad knifeedge poles oriented at right angles to one another, i.e. a condensed form of the quadripolar precursor.

The twist causes the metaphase plate and the subsequent phragmoplast and organelle band to be saddle-shaped, and the daughter nuclei to be elongated perpendicular to one another along the two knife edges. The tetrahedral array returns during interkinesis and again breaks down into four cones of MTs centred on the plastid tips; these, however, now become individual half spindles for the two perpendicularly arranged second division spindles. When meiosis is completed the four haploid nuclei thus come to lie at the vertices of a tetrahedron that was established by MT-mediated plastid positioning during meiotic prophase.

The tetrahedral cage of MTs precedes meiosis yet predicts the planes of division, and in these two respects it is the meiotic counterpart of the preprophase band of MTs, which develops before mitosis in most higher plant cells.
Cytoskeletal elements, cell shaping and movement in the angiosperm pollen tube
Authors: Heslop-Harrison J. , Heslop-Harrison Y. , Cresti M. , Tiezzi A. , Moscatelli A.
Published: April 2021

The ellipsoidal generative cell of the pollen grain of Endymion nonscriptus usually elongates further following germination and entry into the tube, producing attenuated extensions the forward one of which may reach into the vicinity of the vegetative nucleus. This shape change is accompanied by the stretching of the microtubule cytoskeleton of the cell, identified in the present work by immunofluorescence using monoclonal antibodies to tubulin. Complementary observations of living generative cells of Iris pseudacorus showed that they undergo slow undulatory movements accompanied by variation in shape and length during passage through the tube. Such changes must presumably be accompanied by modifications of the microtubule cytoskeleton.

Colchicine at 1 mM eliminated microtubules from tubes and most generative cells of E. nonscriptus,but did not radically affect pollen-tube shape or extension growth, nor arrest the movements of the vegetative nucleus and generative cell into and through the tube. Generative cells in colchicine-treated pollen of Galanthus nivalis rounded up and failed to undergo the usual changes in shape during passage through the tube. Secondary consequences were changes in precedence in movement through the tube, and a greater dispersal along its length. On the assumption that no other cytoskeletal elements remain to be discovered, it seems likely that microfilaments rather than microtubules provide the motive force for movement in the tube, although the latter are involved in shaping the generative cell and adapting it to its passage.
Cytoplasmic calcium measurements in intact higher plant cells: Results from fluorescence ratio imaging of fura-2
Authors: Clarkson David T. , Brownlee Colin , Ayling Sarah M.
Published: April 2021

Roots of seedling tomato (Lycopersicon esculenturn) and oilseed rape (Brassica napus) plants were grown on agarose slopes formed on microscope slides. Root hairs were used for microelectrode insertions, for observations of the rate of cytoplasmic streaming and for measurements of fluorescence of the calcium indicator fura-2

The resting potential across the plasma membrane of root hair cells was between −140 and − 160 mV in both species. Insertion of microelectrodes caused immediate cessation of cytoplasmic streaming; 1–4 min later streaming restarted and quickly recovered its normal velocity of 1–3 μm s−1. Once the electrode had been inserted, current pulses had no obvious effect on streaming. Thus, fura-2 injected by iontophoresis was quickly distributed throughout the length of the hair and the epidermal cell body. Injections were made into the cap of cytoplasm at the tip of the hair. Movement of fura-2 from the cytoplasm into the vacuole was observed; this was marked by a great increase in its fluorescence after excitation at 350 nm and occurred after about 15–30 min in tomato, but more rapidly (5–10 min) in oilseed rape hairs. No fluorescence was associated with the cell walls; this was demonstrated by plasmolysing the cells.

The distribution and concentration of Ca2+ was estimated from the ratio of fura-2 fluorescence excited at 350 and 385 nm and from digital image analysis. Resting levels of calcium in the cytoplasm near the tip were in the range 30·90 nM, while much higher levels, which eventually saturated the fura-2 response (>5 μM), were seen in vacuoles once the indicator had crossed the tonoplast. This distribution was rapidly perturbed by the addition of the calcium channel blocker, verapamil, to the outer solution. There was a return to the original distribution as the verapamil was diluted. Separate experiments showed that verapamil inhibited cytoplasmic streaming over a time-course similar to these perturbations in cytoplasmic Ca2+ and caused a small depolarization (by approx. 30 mV) of the membrane potential. The application of 3×10−5 or 3 ×10−4 M-N-ethyl maleimide to the hairs strongly and irreversibly depolarized the plasma membrane electric potential, caused cytoplasmic streaming to stop or slow down and resulted in a very rapid rise in cytoplasmic Ca2+, which became uniformly distributed with saturating 350:385 nm ratios.
‘blebbing’ of the nuclear envelope of mouse zygotes, early embryos and hybrid cells
Authors: Szöllösi Maria S. , Szöllösi Daniel
Published: April 2021

In the mouse zygote and in two-cell stage embryos the inner leaflet of the nuclear envelope of pronuclei and that of blastomere and polar body II nuclei evaginate, forming multiple blebs within the perinuclear space, which contains a granular material. Blebbing exists only in oocytes activated by sperm in vivo or in vitro, or parthenogenetically by treatment with ethanol or puromycin. The germinal vesicle and an interphase nucleus formed after treatment of the oocyte at metaphase I by puromycin do not form blebs. Formation of blebs is specifically located in the cell cycle. The burst of the blebbing activity occurs during the first half of the cell cycle in one-cell embryos and in the earliest interphase period in the second cell cycle. Blebbing ceases from the beginning of the third cell cycle. The occurrence in the cytoplasm of ‘doublelayered’ vesicles containing granular material resembling bleb contents and the disappearance of blebs from the nuclear envelope by the end of the cell cycle provide evidence that blebs represent a step in the transport of some material from the nucleus to the cytoplasm. Ethanolic phosphotungstic acid does not stain blebs, suggesting the absence of basic protein in their contents.

Blebbing can be induced in somatic (thymocyte) and embryonic (blastomere of 8-cell stage embryo) nuclei following cell hybridization with activated oocytes. Their response to the oocyte cytoplasm by initiating blebbing depends on: (1) the position of the host cell in its cell cycle at the moment of hybridization, and (2) the time spent by the foreign nuclei in the host cytoplasm following cell fusion. If donor nuclei are introduced close to the time of activation, they start to produce blebs at the time corresponding to the initiation of blebbing by the female pronucleus in the first cell cycle. If foreign nuclei are introduced a few hours after activation they must be incubated in the host cytoplasm for some time before initiation of bleb formation, provided that the host pronucleus has initiated blebbing by that time.

The existence of blebbing in nuclei formed only after oocyte activation, and the timing and the general occurrence of this event during the earliest cleavage stages of almost every mammalian embryo, suggest that this special nucleocytoplasmic transport plays a specific role at the beginning of development.
The organization of the cytoskeleton during meiosis in eggplant (Solarium melongena (L)): microtubules and F–actin are both necessary for coordinated meiotic division
Authors: Traas J. A. , Burgain S. , Vaulx R. Dumas De
Published: April 2021

Because two division planes form at right angles, male meiosis in higher plants provides striking examples of both division control and spatial programming.

To investigate these processes we have stained microtubules and actin filaments during male meiosis in the eggplant. Our results indicate the following.

(1) That microtubules and their nucleation sites are involved in the establishment of polarity; this is supported by our observation that the drug CIPC affects spindle polarity.

(2) That actin microfilaments are involved in spindle formation and integrity, but not in the establishment of polarity: cytochalasin B and D affect the organization of the spindle microtubules, but not their polarized distribution.

(3) That microtubules radiating from the daughter nuclei at the cell poles during interkinesis probably establish the future division plane by concentrating actin in that plane (cf. the proposed role of asters in positioning the contractile ring in animal cells).

(4) That this concentration of F-actin in the division plane may be involved in preparing the cytoplasm for cytokinesis and in memorizing the division plane (much as the preprophase band observed in polarized tissues does).

(5) That phragmoplast formation is a two-step process. No phragmoplast forms after metaphase I, but a four-way phragmoplast forms after metaphase U, indicating that mitosis and cytokinesis are not obligatorily coupled.

These studies demonstrate that actin and microtubules are jointly involved in the spatial coordination of the division process.
Lectin binding of F9 embryonal carcinoma cells: evidence for population heterogeneity and developmentally regulated high–Mr cell surface proteins
Authors: Tienari Jukka , Virtanen Ismo , Lehtonen Eero
Published: April 2021

Undifferentiated F9 embryonal carcinoma (EC) cells bound fluorochrome-coupled Helix pomatia agglutinins (HPA) and peanut agglutinins (PNA) homogeneously, but were distinctly heterogeneous in their binding of Dolicbos biflorus agglutinin (DBA) conjugates. Upon chemically induced differentiation the proportion of cells binding the DBA conjugates increased, but a distinct heterogeneity in the intensity of binding remained among the parietal endoderm (PE)-like F9 derivatives. These cells were heterogeneous in their binding of HPA conjugates as well, and many of them failed to bind PNA conjugates, apparently due to sialylation of the PNA-binding sites.

Electrophoretic analysis of lectin-binding glycol-proteins in the detergent-soluble fraction of the cells revealed the appearance of a doublet of polypeptides of Mr 300 000-400 000 upon ddferentiation induced by retinoic acid (RA). In addition, an Mr 220 000 polypeptide appeared upon ddferentiation induced by RA and dibutyryl cyclic AMP (dbcAMP). These polypeptides were obtained from both metabolically labelled and surface-labelled cells. A major secreted glycoprotein, which comigrated with laminin, bound to DBA. This suggests that laminin secreted by the ddferentiated F9 derivatives contains O-glycosidic saccharides.

The results show that even though differentiation of F9 cells leads to changes in their binding of fluorochrome-coupled lectins, these lectin conjugates reveal distinct population heterogeneity among unddferentiated and differentiated F9 cells and are hence likely to be of limited value in the characterization of individual cells. At the whole cell population level, on the other hand, affinity binding to lectins reveals the appearance of high-Mr. cell surface proteins in differentiating F9 cells.
Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells
Authors: Díaz-nido Javier , Avila Jesús
Published: April 2021

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 22OK (K = 10?Mτ), which are immunologically related to brain MAP-IB. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-IB and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in viuo, which suggests that they may participate in microtubule assembly in vivo, the 325K protein could perform such a role during the entire cell cycle, while the 22OK protein could be implicated in the formation of the mitotic spindle.
Cingulin: Characterization and localization
Authors: Citi Sandra , Sabanay Helena , Kendrick-Jones John , Geiger Benjamin
Published: April 2021

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108000 (cingulin-108), with a Stokes’ radius of ∼15nm, and a molecular length of 130 nm ± 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. Ln permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vincu-lin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (fzonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.
Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei
Authors: Bülow Roland , Griffiths Gareth , Webster Paul , Stierhof York-Dieter , Opperdoes Fred R. , Overath Peter
Published: April 2021

Glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC) is a membrane-bound enzyme of bloodstream forms of Trypanosoma brucei, which cleaves the GPI-membrane anchor of the variant surface glycoprotein forming diacylglycerol and 1,2-cyclic phosphate on the inositol ring. The cellular localization of the enzyme was studied by fractionation of sub-cellular organelles and immunofluorescence microscopy and was found to be primarily cytoplasmic. This was confirmed by _mmune-electron microscopy using cryo-sections, which showed that the labelling was predominantly on the cytoplasmic side of intracellular membranes but was absent from the plasma membrane includeing the region lining the flagellar pocket. The significance of these results for the possible function of the phospholipase is discussed.
Conformation and movement of the vegetative nucleus of the angiosperm pollen tube: association with the actin cytoskeleton
Authors: Heslop-Harrison J. , Heslop-Harrison Y.
Published: April 2021

Actin is present in the cytoplasm of the vegetative cell of angiosperm pollens in numerous fusiform, spiculate or toroidal bodies, and also as a sheath enveloping the vegetative nucleus. During activation following hydration, the compact cytoplasmic bodies are translated into skeins of extended fibrils, and circulatory movements begin in the cytoplasm. Throughout this period the vegetative nucleus, with fibrillar actin now associated with the surface, undergoes a continuous change of shape. In the extending tube following germination the actin cytoskeleton consists of numerous mainly longitudinally oriented fibrils. After entry into the tube the vegetative nucleus remains associated with the fibrils, usually extending greatly in length and developing attenuated, often pointed extensions. The observed conformations, which change continuously, suggest that varying local tensions are applied to the vegetative nucleus during passage through the tube. Cytochalasin D breaks up the actin fibril system and brings about a rapid contraction of the nucleus, at the same time eliminating the elastic extensions of the nuclear envelope. Nuclei isolated physically from unfixed tubes also contract in length as the fibrillar components of the cytoskeleton are detached. These findings indicate that the movement of the vegetative nucleus depends on local associations of the nuclear envelope with the actin cytoskeleton of the vegetative cell.
Effects of leukotrienes on phenotypic properties and growth of arterial smooth muscle cells in primary culture
Authors: Palmberg Lena , Claesson Hans-Erik , Thyberg Johan
Published: April 2021

During the first few days in primary culture arterial smooth muscle cells (SMCs) go through a transition from a contractile to a synthetic phenotype. Morphologically, this process includes loss of myofilaments and formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to the cells losing their contractility, beginning to secrete extracellular matrix components, and dividing in response to growth factor stimulation. Similar changes in the structure and function of the SMCs occur in the initial stages of atherogenesis. The object of the present investigation was to study the effects of leukotrienes on the differentiated properties and growth of rat aortic SMCs in primary culture. Enzymically isolated cells were seeded directly on a plastic surface in serum-containing medium or on a substratum of plasma fibronectin in serum-free medium. The change in cell morphology was followed by transmission electron microscopy, and the activation of cell growth by thymidine autoradiography and cell counting. The results demonstrate that 10pM-LTB4, -LTC4, -LTD4 and -LTE4 all speeded up the shift of the SMCs into a synthetic phenotype, whereas 5S,12S-DHETE (an isomer of LTB4) lacked effect. Further, LTB4, LTC4 and LTD4 stimulated the SMCs to enter the cell cycle earlier than in the controls, enhanced the proliferative response to serum mitogens, and under serum-free conditions induced DNA synthesis by themselves. Indomethacin did not interfere with the effect of LTB4 on the structural transformation of the cells but blocked its effect on DNA replication, suggesting that only the latter involved endogenous production of a cyclo-oxygenase product. When seeded on a substratum of laminin, a component of the basement membrane that normally surrounds smooth muscle, the cells were initially retained in a contractile phenotype and no clear effect of the leukotrienes was noted. Taken together, the findings indicate that leukotrienes are able to stimulate the phenotypic modulation and growth of arterial SMCs in primary culture.
Serotonin regulates cytosolic Ca2+ activity and membrane potential in a neuronal and in a glial cell line via 5-HT3 and 5-HT2 receptors by different mechanisms
Authors: Reiser Georg , Donié Frédéric , Binmöller Franz-Josef
Published: April 2021

The mechanisms of action of two different serotonin receptors, found in a neuronal cell line (neuroblastoma x glioma hybrid cells) and in a non-excitable glioma cell line, were explored. In both cell lines, serotonin induced a dose-dependent, transient rise of cytosolic Ca2+ activity (measured by fura-2 or indo-1 fluorescence). Ca2+ channel blockers (Ni2+ and La3+, not nifedipine) suppressed the Ca2+ response to serotonin in the hybrid cells but not in the glioma cells. After application of Ca2+ ionophores (ionomycin and A23187) in order to short-circuit internal Ca2+ stores, serotonin was still able to induce a Ca2+ response in the hybrid cells but not in the glioma cells. Serotonin dose-dependently stimulated the rate of 45Ca2+ uptake several-fold in the hybrid cells, but hardly at all in the glioma cells. Thus, in the neuronal cell line cytosolic Ca2+ activity is raised through enhancement of Ca2+ entry into the cells from the extracellular environment via 5-HT3 receptors (blocked by ICS 205-930, MDL 72222 and GR 38032 F). The depolarization response caused by serotonin in the hybrid cells is due to activation of cation conductance), obviously allowing entry of extracellular Ca2+. In contrast to the neuronal cell line, in the glial cell line the rise of Ca2+ activity is mediated by ketanserin-susceptible 5-HT2 receptors (not affected by treatment with pertussis toxin) mainly liberating Ca2+ from internal stores. In the glioma cells the release of Ca2+ from internal stores leads to opening of Ca2+-dependent K+ channels, responsible for the hyperpolarizing response. Thus, the neuronal and the glial cell lines might provide suitable systems in which to study the diverse cellular functions triggered by the rise of cytosolic Ca2+ activity, which is caused by different serotonin receptors.
The shape of mitochondria and the number of mitochondrial nucleoids during the cell cycle of Euglena gracilis
Authors: Hayashi Yasuko , Ueda Katsumi
Published: April 2021

The shape of mitochondria and the number of mitochondrial nucleoids in Euglena cells were examined throughout the cell cycle by fluorescence microscopy. Both photoheterotrophic and heterotrophic cells contained a network of mitochondria that did not divide into fragments at any stage of the cell cycle. Mitochondrial nucleoids could be clearly detected in the mitochondria by staining with ethidium bromide and with DAPI. Half of the mitochondrial nucleoids entered each daughter cell during cytokinesis. Nucleoids in the newly produced daughter cells increased in number as the cells increased in size. The number of nucleoids reached double the initial value in cells at the stage just prior to mitosis. The total length of the mitochondrial net was proportional to the cell volume.
Inhibitors of topoisomerases I and II arrest DNA replication, but do not prevent nucleosome assembly in vivo
Authors: Annunziato Anthony T.
Published: April 2021

Specific inhibitors of eukaryotic DNA topoisomerases I and II (camptothecin and VM-26, respectively) were used to examine the involvement of topoisomerases in DNA replication and chromatin assembly in vivo. When used singly, either camptothecin or VM-26 inhibited DNA synthesis in HeLa cells by more than 80 %; when used simultaneously, the inhibitors effectively stopped replication, demonstrating that at least one class of topoisomerase must be active for fork propagation in vivo. To study nucleosome assembly during topoisomerase inhibition, three experimental strategies were employed: (1) pulse-chase experiments; (2) analyses of chromatin synthesized during residual replication in the presence of either camptothecin or VM-26; and (3) the assembly of previously replicated, unassembled DNA, generated in the presence of protein synthesis inhibitors. Using sensitivity to micrococcal nuclease and the maturation of non-nucleosomal replication intermediates as criteria, neither camptothecin nor VM-26, alone or in concert, inhibited nucleosome assembly under any experimental protocol tested. These data provide evidence that, although topoisomerase activity is essential for DNA replication, neither continuous fork propagation nor topoisomerase activity is required for chromatin assembly on new DNA.
Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs
Authors: Hutchison Christopher , Kill Ian
Published: April 2021

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.
Identification of proteins cross-reactive to phosphotyrosine antibodies and of a tyrosine kinase activity in boar spermatozoa
Authors: Berruti G. , Martegani E.
Published: April 2021

We used two different anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins in a germ cell, the boar spermatozoon. Ejaculated spermatozoa presented three major polypeptides, of Mr 43000, 40000 and 36000, respectively, that were immunorecognized on Western blots. These proteins were selectively enriched in the Triton X-100-soluble fraction and were released neither after an A23187-induced acrosome reaction nor after sperm homogenization. These findings suggest the presence of the three proteins in plasma membrane regions not involved in the acrosomal vésiculation. When epididymal boar spermatozoa were investigated, Western blot analysis of the detergent-soluble fractions from caput sperm did not reveal any detectable 43000, 40 000 and 36 000 Mr proteins cross-reactive with phosphotyrosine antibodies, whereas the detergent-soluble fractions from cauda sperm yielded very strong immunoreactive signals.

Labelling of freshly ejaculated spermatozoa with [32P] orthophosphate yielded a wide range of labelled phosphoproteins, but we failed to identify specific tyrosine phosphorylation under the experimental conditions employed. Tyrosine phosphorylation occurred when specific synthetic polymers of tyrosine, commonly used for studying tyrosine protein kinases, were assayed as substrates against both the Triton-soluble and Triton-insoluble sperm fractions. This is the first immunological and biochemical report on the presence of phosphotyrosine-containing proteins and protein kinase activities that phosphorylate tyrosine residues in a mammalian mature spermatozoon.
Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field
Authors: Gaertig Jacek , Iftode Francine
Published: April 2021

This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells. Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density. The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs. The course of further changes in cell organization depends on the polarity of fused cells. Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements. In contrast, cortical integration of heteropolar fused cells is limited. Cytoskeletal integration is particularly promoted if the cells are incubated in nongrowing conditions. Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.
Embryonic chicken fibroblast collagen binding proteins: distribution, role in substratum adhesion, and relationship to integrins
Authors: Ogle Roy C. , potts A. Jeannette , Yacoe Marshall , Little Charles D.
Published: April 2021

Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dosedependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin β subunits; and that antibodies to avian integrins recognize a 100 000 MrCBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family.
Biochemical characterisation of polycation-induced cytotoxicity to human vascular endothelial cells
Authors: Morgan David M. L. , Larvin Victoria L. , Pearson Jeremy D.
Published: April 2021

Pro-inflammatory effects of cationic proteins secreted by human granulocytes include induction of increased vascular permeability and oedema, which are likely to be mediated by damage to vascular endothelium. We have shown previously that a series of synthetic polycationic amino acids produce a dose-, time- and Mr-dependent inhibition of [3H] leucine or [3H] thymidine incorporation into macromolecules by human umbilical vein endothelial cells, and that the extent of inhibition was correlated with changes in cell morphology, with release of cytoplasmic constituents and was irreversible.

The experiments reported here characterise further the requirements for the induction of cytotoxicity by polycations. We have found that the extent of inhibition is related to both the identity of the monomer, for polymers of Mr 40 000 the order is ornithine>lysine>arginine, and to its configuration; poly-D-lysines are more potent inhibitors than poly-L-lysines of similar Mr. Only brief exposure to the agonist is required, 90% inhibition occurred after 10 min of exposure to poly-L-lysine (Mr 90000). Treatment of endothelial cells with neuraminidase, heparinase, hyaluronidase, chondroitinase or trypsin did not reduce their susceptibility to poly lysine. Inhibition of microtubule or microfilament formation also had no effect on polylysine cytotoxicity, indicating that internalisation of the polymer was not a prerequisite for the effect. Inhibition of protein synthesis or pretreatment with simple sugars likewise failed to block the effects of polylysine treatment. Natural cationic proteins exerted similar effects on endothelial cells, the extent of the effect apparently being related to the pI of the protein. We conclude that the extent of cytotoxicity induced by cationic polymers on proteins is related to the number of attachment sites to which an individual molecule binds, which in turn depends upon its size and conformation, and on the distribution of anionic domains on the cell surface.
Expression of anionic sites on tumour cells at different stages of tissue invasion in vivo: A comparative study by X-ray microanalysis
Authors: Evans Peter M. , Suker Dhamia K. , Gwynn Iolo ap
Published: April 2021

Quantification of colloidal iron hydroxide (CIH) labelling by X-ray microanalysis was used to investigate anionic sites at the surface of Ehrlich carcinoma cells from different locations in the mouse host. Individual tumour cells from peritoneal ascites suspensions (pre-invasion stage) varied up to threefold in their ability to bind CIH and a similar degree of intra-tumour heterogeneity was observed in different experimental animals. Pre-treatment of the cells with neuraminidase confirmed that binding was at least partly due to surface sialic acid. Invasive cells isolated from mesenteric tumour nodules were also heterogeneous with regard to the availability of surface anionic sites, as were tumour cells adhering to the surface of the mesentery; however, in both these populations CIH binding was significantly greater on average than for free ascites tumour cells. The results suggest that surface anionic sites are determinants of the invasiveness of malignant cells in vivo, and that both the number and topography of these sites may be important in modulating tumour cell behaviour.
Dissociation of senescence-associated changes in differentiated gene expression and replicative senescence in cultured adrenocortical cells
Authors: Yang Lianqing , Hornsby Peter J.
Published: April 2021

In bovine adrenocortical cells senescing in culture, the decline in expression of cyclic AMP-inducible 17 α-hydroxylase in mass cultures and clones of bovine adrenocortical cells results from a decrease in the number of cells in the population capable of expressing this gene. As the population of cells decreases in proliferative potential, the fraction of the population that expresses 17 α-hydroxylase after induction with cyclic AMP decreases, until the population comprises entirely nonexpressing cells. We used in situ hybridization to study the relationship between the loss of the ability to replicate and the loss of expression of this differentiated function gene. Colonies of cells from early passage cultures comprised complex mosaics of expressing and nonexpressing cells, indicating a high-frequency switching event. At an individual cell level, there was a complete independence of replicative senescence and loss of 17 α-hydroxylase expression. Groups of cells with a morphology characteristic of replicative senescence, occurring frequently in early-passage cultures, comprised both expressing and nonexpressing cells, as did cells that appeared to be in rapid growth. This was confirmed by prior incubation of cells with bromodeoxyuridine to label cycling cells; both cycling and noncycling populations comprised mixtures of expressing and nonexpressing cells. In contrast, replication was required for the loss of 17 α-hydroxylase expression, since cells rendered incapable of division by treatment with mitomycin C maintained 17 α-hydroxylase expression over long time periods. Thus, the process that results stochastically in replicative senescence and the process that results in loss of 17α-hyroxylase expression are independent, but they have similar time-courses and both require replication for phenotypic switching to take place.
Steps in the morphogenesis of a polarized epithelium : I. uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts
Authors: Wang Allan Z. , Ojakian George K. , Nelson W. James
Published: April 2021

The development of cell polarity in Madin-Darby canine kidney (MDCK) cells has been analyzed under conditions in which cells are induced to form multicellular epithelial cysts in stages that mimic the ontogeny of epithelial tissues and organs in vivo. The morphogenesis of MDCK cysts in suspension culture or in a collagen gel proceeds in distinct stages involving the initial aggregation of cells followed by development of a closed monolayer of polarized epithelial cells that surrounds a central lumen. The polarity of cells was determined at each stage by analyzing the distributions of marker proteins of the apical (gpl35) and basal-lateral (Na+,K+-ATPase) domains of the plasma membrane, the tight junction (ZO-1) and proteins involved in cell-cell (uvomorulin) and cell-substratum contact (type IV collagen). We show that cells have a distinctive and opposite polarity in cysts formed in suspension culture compared to those formed in collagen gels. In suspension culture, the basal-lateral membrane faces the central lumen and the apical membrane faces the outside, whereas in collagen gel, the basal-lateral membrane faces the outside collagen and the apical membrane faces the central lumen. Detailed analysis of the distributions of marker proteins during the morphogenesis of these three-dimensional structures indicates that: (1) cell-cell contact is sufficient to trigger the segregation of marker proteins of the apical and basal-lateral membrane domains to distinct regions of the membrane; (2) cell-cell contact induces association of the tight junction protein ZO-1 with the contact zone between cells; (3) localization of the tight junction protein ZO-1 to the apex of the lateral membrane and the establishment of the epithelial axis, however, requires the formation of a basal lamina and cell-substratum contact; (4) in suspension culture, MDCK cysts secrete and establish a basal lamina in the central lumen. These results show that cell-cell and cell-substratum contact have distinct roles in the morphogenesis of polarized epithelia. We suggest that the mechanisms involved in triggering cell polarity may be common to different polarized epithelia in vivo.
Control of fluid transport in human rectal adenocarcinoma cells (hra-19) in monolayer and collagen gel cultures
Authors: Kirkland Susan C.
Published: April 2021

HRA-19a1.1. cells, derived from a primary human rectal adenocarcinoma, form polarised monolayers when grown on tissue-culture plastic. HRA-19 monolayers have a heterogeneous morphology even after 150 passages in vitro or single cell cloning. The morphological changes observed in HRA-19 monolayers were postulated to be the result of vectorial fluid transport leading to accumulation of fluid between cells. To test this hypothesis, a variety of agents that control ion transport in colorectal epithelium were tested for their effect on HRA-19 morphology.

Forskolin, cholera toxin and prostaglandin E2 all markedly changed HRA-19 monolayer morphology, with the rapid disappearance of intercellular spaces. These agents all stimulate Cl− secretion in colorectal epithelium, i.e. transport from basolateral to apical surface, and therefore would be expected to reduce fluid accumulation at the basolateral side of the cell. Conversely, vasopressin, which stimulates absorption of Na+ and water across colorectal epithelium, leads to a small increase in intercellular spaces in the monolayer.

In collagen gel cultures, addition of cholera toxin, forskolin or prostaglandin E2 resulted in a large increase in colony size. In such treated cultures, thecolonies were ‘bubble-like’, often composed of a single rim of flattened cells, which appeared to encompass a fluid-filled space. Similar morphological changes were observed when HRA-19 cells were co-cultured with 3T3 cells. This effect was probably due, at least in part, to prostaglandin production by the 3T3 cells, as the effect could be markedly reduced by the addition of indomethacin to these cultures.

These results strongly suggest that the morphological changes observed in HRA-19 monolayers are generated as cells change their fluid transport characteristics. Therefore, it would appear that vectorial fluid transport can lead to marked morphological changes in monolayer cultures, which differ from the more usually reported dome formation. In addition, these studies show that HRA-19 cells, derived from a primary human rectal adenocarcinoma, retain the ability to respond to many of the agents active in controlling transport in normal colorectal epithelium.
Phosphorylation-related accumulation of the 125k nuclear matrix protein mitotin in human mitotic cells
Authors: Zhelev N. Z. , Todorov I. T. , Philipova R. N. , Hadjiolov A. A.
Published: April 2021

The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI 6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named ‘mitotin’. Here, we show by short-term [35S] methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.
Lysosomal sorting mutants of coronavirus E1 protein, a Golgi membrane protein
Authors: Armstrong John , Patel Shaila , Riddle Peter
Published: April 2021

As a model for the intracellular sorting of Golgi membrane proteins, we are studying the El protein of the coronavirus Mouse Hepatitis Virus A59. The wild-type protein, when expressed from synthetic RNA, is localised in the Golgi complex. When the second and third of the three predicted membranespanning sequences were deleted from the protein, the resulting mutant was retained in the endoplasmic reticulum. In contrast, removal of the first and second membrane-spanning sequences allowed the protein to pass through the Golgi complex and reach the lysosomes. Likewise, when 40 amino acids were deleted from the C-terminal cytoplasmic part of El, the truncated protein was transported to the lysosomes. We discuss the implications of these results for the structure of the El protein and the mechanism by which it is localised in the cell.
Microtubule-associated protein 3 (MAPЗ) expression in non-neuronal tissues
Authors: Huber Gerda , Matus Andrew
Published: April 2021

Microtubule-associated protein 3 (MAPЗ, Mr 180 000), which in previous studies has been shown to be associated with glial processes and neurofilament-rich axons in rat brain, was examined in various non-neuronal rat tissues, immunoblots of adult rat tissues (brain, liver, heart, spleen, adrenal medulla and kidney) showed that MAPЗ is present in all organs tested. In addition we demonstrated that MAPЗ is a heat-stable protein. Using immunohistochemistry, we established the localIsation of MAPЗ in various cell types. MAPЗ-containing cells appeared to have in common an asymmetric morphology with long processes that need structural support. In kidney MAPЗ is limited to epithelial podocytes and in liver to Kupffer cells. In the adrenal gland, the cells of the cortex are devoid of MAPЗ compared to the cells of the medulla. High concentrations of MAPЗ are also found in cardiac muscle along the Z-disc and in the smooth muscle cells of the digestive tract. In spleen MAPЗ is found in cells of the white pulp surrounding central blood vessels. A co-distribution of MAPЗ with microtubules and intermediate filaments but not with microfilaments was found in each cell type examined. The widespread distribution pattern of MAPЗ together with its molecular size and heat-stability indicate that MAPЗ might be a member of the recently postulated family of homologous 200 000Mr mammalian tissue MAPs. Potential functions for MAPЗ in specific cell types are discussed.
The differentiation process of intestinal epithelial cells is associated with the appearance of statin, a non-proliferation-specific nuclear protein
Authors: Bissonnette Robert , Lee Menq-Jer , Wang Eugenia
Published: April 2021

The expression of statin, a 57 000 Mr nuclear protein specifically present in non-proliferating cultured fibroblasts, was studied in vivo in the differentiating epithelial cells of the rat intestine. Using immunofluorescence microscopy we found that undifferentiated, proliferating crypt enterocytes are statin negative, whereas the differentiated nonproliferating villus enterocytes are statin positive. The epithelial cells of the intestine were isolated according to different stages of differentiation and the expression of statin was studied biochemically by immunoblotting assays. The prominent band (57 000 Mr) was present in abundance in villus cell fractions but undetectable in crypt cell fractions. These findings were also confirmed by immunofluorescence microscopy on individual intestinal epIthelial cells of the different isolated fractions. The results presented here, which are similar to observations made in cell cultures, suggest that statin is a unique protein associated with the non-proliferative state of differentiated cells in tissue.