bluuebunny/crossref_metadata_2025 huggingface dataset:
Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils
Authors: Klein Nigel J. , Shennan Graham I. , Heyderman Robert S. , Levin Michael
Published: April 2021

There is increasing evidence that the glycosaminoglycan (GAG) component of the vascular endothelium is important in regulating vascular permeability, throm-boresistance and cellular interactions. We have investigated the GAG metabolism of cultured human umbilical vein endothelial cells (HUVEC) in response to a range of inflammatory stimuli. Using both chemical measurement of cellular and supernatant GAGS and 35S labelling to identify newly synthesised GAGS, interleukin 1 (ILI), tumour necrosis factor (TNF) and interferon gamma (IFN γ) were shown to influence sulphated GAG metabolism significantly. ILI and TNF caused a marked increase in culture supernatant GAGS and a concomitant reduction in cell-associated GAGS.

This was shown histochemically to be associated with a marked reduction and redistribution of endothelial surface anionic sites. The addition of neutrophils to HUVEC pretreated with Escherichia coli endotoxin, ILI or TNF resulted in a further reduction in both cellular GAGS and surface anionic sites. These results suggest that changes in endothelial cell GAG metabolism during inflammation may contribute to the disturbance of vascular endothelial homeostasis associated with infectious and inflammatory states.
Anti-fibronectin antibodies that modify heparin binding and cell adhesion: evidence for a new cell binding site in the heparin binding region
Authors: Underwood P. Anne , Dalton B. A. , Steele J. G. , Bennett F. A. , Strike P.
Published: April 2021

A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/l and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAh concentrations as low as 1 μg/ml. This mAh was not so effective at inhibiting the adhesion of Bib mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep H (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep H and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 3050%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep H region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep H. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep H, other than peptide I. Recent independent evidence for an additional cell binding site in Hep H supports this view. Melanoma cellular receptor(s) for the Hep H region may be cell surface proteoglycans but do not appear to bind to areas of Hep H with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the ceUular receptor. These results are discussed in context with other reports in the literature.
Molecular characterisation of the gene for the 180 kDa subunit of the DNA polymerase-pri mase of Drosophila melanogaster
Authors: Melov Simon , Vaughan Helen , Cotterill Sue
Published: April 2021

We have cloned and sequenced the gene for the 180 kDa subunit of the a polymerase from Drosophila melanogaster. The protein shows high similarity to the 180 kDa subunits from other species. Comparative expression analysis for the transcript, protein and enzymic activity suggests that control occurs mainly at the level of transcription. In situ analyses of the RNA suggest that high levels of the transcript are synthesised in the ovaries and deposited uniformly in the egg. Immunolocalisation of the 180 kDa polypeptide in whole embryos shows that its location is mainly nuclear; however, dispersal of the protein can be seen to occur during mitotic phases of the cell cycle.
M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages
Authors: Racoosin Esther L. , Swanson Joel A.
Published: April 2021

Although coated vesicles can mediate both solute and receptor-mediated endocytosis, there are other kinds of endocytic vesicles that contribute to these processes. The relative contributions of these other organelles, particularly regarding solute influx, remains unsettled. Here we describe a physiological uncoupling of solute and receptor-mediated endocytosis that occurs during growth factor-stimulated macropinocytosis. We examined how recombinant human macrophage colonystimulating factor (rM-CSF), which rapidly stimulates solute endocytosis in murine bone marrow-derived macrophages, affected ligand internalization via receptor-mediated endocytosis. Although rM-CSF stimulated internalization and accumulation of Lucifer Yellow (LY), a probe for solute endocytosis, it had no effect on accumulation of fluorescent acetylated low-density lipoprotein (acLDL), a ligand for the macrophage scavenger receptor, or on the endocytosis of 125I-labelled diferric transferrin. Video microscopy revealed that rM-CSF immediately induced active cell ruffling and the formation of phase-bright macropinosomes. Nocodazole pretreatment of macrophages inhibited both ruffling and macropinocytosis. Macropinosomes were fluorescently labelled by incubating macrophages briefly with probes for both solute endocytosis (fluorescent dextrans) and ligand endocytosis (fluorescein-labelled transferrin or dil-labelled acLDL). Macrophages incubated for one or two minutes formed macropinosomes that were labelled predominantly with the fluorescent solute probes but with little or none of the ligand probes; the latter were localized within smaller pinosomes. When cells pulsed with the fluorescent probes were washed and chased for an additional two minutes, solute and ligand probes occasionally co-localized in macropinosomes. Nocodazole inhibited macropinocytosis with little apparent effect on endocytosis via smaller vesicles. These experiments show that macropinosome formation is dependent on microtubules and also that the macropinosomes induced by rM-CSF are solute-rich and receptor-poor. Macropinosomes differ from coated vesicles in these respects, and therefore provide a physiologically regulated mechanism for uncoupling solute and receptor-mediated endocytosis.
Characterization of a 58 kDa Cis-Golgi protein in pancreatic exocrine cells
Authors: Lahtinen Ulla , Dahllöf Björn , Saraste Jaakko
Published: April 2021

We have studied the biochemical characteristics and localization of a 58 kDa cis-Golgi marker protein (p58) in rat pancreatic exocrine cells. The protein remained associated with membranes after extraction at alkaline pH and was largely resistant to proteases, added to intact microsomes. By electrophoresis p58 could be resolved into two bands which in two-dimensional gels separated into several charge variants around pI 5.5. This size and charge heterogeneity of p58 did not appear to be due to acylation, glycosylation or phosphorylation. In non-reduced gels p58 migrated as two kinetically related, high relative molecular mass forms, apparently corresponding to disulfide-linked homo-dimers and -hexamers. Immuno-electron microscopy localized p58 to both the fenestrated cis-Golgi cisternae and small Golgi vesicles or buds as well as large, pleiomorphic structures, scattered throughout the cells and associated with distinct smooth ER (endoplasmic reticulum) clusters. These findings correlated with cell fractionation results showing the concentration of p58 in two microsomal subfractions, banding at intermediate densities between the rough ER and trans-Golgi in sucrose gradients. Our results indicate that p58 is a major component of pre- and cis-Golgi elements and could be part of the transport machinery that operates in these membranes. Together with results obtained with other cell types, these observations suggest that the peripheral smooth ER clusters are involved in the early stages of the secretory pathway in the pancreatic acinar cells.
Plasticity of early endosomes
Authors: Parton Robert G. , Schrotz Petra , Bucci Cecilia , Gruenberg Jean
Published: April 2021

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometres and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.
Proteolytic processing of secretory proteins in Paramecium: Immunological and biochemical characterization of the precursors of trichocyst matrix proteins
Authors: Shih Shian-Jiun , Nelson David L.
Published: April 2021

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing.

Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.
The rhythm of protein synthesis does not depend on oscillations of ATP level
Authors: Brodsky Vsevolod Y. , Boikov Petry. , Nechaeva Natalia V. , Yurovitsky Yuri G. , Novikova Tatyana E. , Fateeva Valentina I. , Shevchenko Natalia A.
Published: April 2021

A rhythm of the [3H]Ieucine incorporation rate with a period of about one hour (circahoralian rhythm) has been found in rat hepatocytes grown in vitro as a monolayer and in liver organ culture. The periodicity of the incorporation rate remained after correction for changes in Ieucine pool size. A similar periodicity of the leucine incorporation rate was detected in a cell-free system prepared from rat hepatocytes. We have also found circahoralian oscillations of the ATP level and similar oscillations of the leucine tRNA aminoacylation rate in a hepatocyte monolayer. The addition of 1 mM ADP to the culture resulted in a considerable increase in the ATP level in the cells, but the rhythm of protein synthesis was retained under these conditions. The conclusion that there is a flexible association between changes in the ATP and GTP levels on the one hand, and oscillations of the protein synthesis rate on the other, is also supported by experiments with a cell-free system, in which the rhythm of protein synthesis rate was observed in the presence of excess ATP and GTP. We propose an hypothesis to explain the fractal pattern of circahoralian metabolic rhythms.
Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium
Authors: Vincent C. , Cheek T.R. , Johnson M.H.
Published: April 2021

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions.

When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.
Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5
Authors: Mardon Helen J. , Sebastio Gianfranco
Published: April 2021

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin 4 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5 ; CS1,CS5+ or CS1,CS5. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, premRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.
Capillary endothelial cells secrete a heparin-binding mitogen for pericytes
Authors: Swinscoe John C. , Carlson Edward C.
Published: April 2021

The cells of the retinal microvasculature consist predominantly of mesodermally derived pericytes and endothelial cells, and the regulatory factors which govern their co-ordinated growth and define their phenotypic characteristics in vivo may be regarded as key elements of the angiogenic process. An investigation of these cells in co-culture experiments has led to the identification of a potent mitogen for pericytes in medium conditioned by retinal endothelial cells (EC-FBS). EC-FBS activity was shown to be non-dialyzable, and stable to both heat and acid treatment. EC-FBS was inactivated by passage over a heparin-Agarose column. The column-bound activity could be eluted as a single peak at ∼1.0 M NaCl. Stimulation of pericyte growth was also achieved with platelet-derived growth factor (PDGF), acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) and could be blocked by using the appropriate antiserum (anti-PDGF or anti-aFGF). Neither antisera, however, blocked the activity of EC-FBS. The EC-FBS mitogen markedly altered the phenotypic behavior of pericytes compared with PDGF and the FGFs; yet, unlike them, it failed to stimulate the growth of smooth muscle cells (SMC) and Balb/c 3T3 cells.
Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture
Authors: Fukamachi Hiroshi
Published: April 2021

It has been a subject of controversy whether fibroblastic cells are necessary for the proliferation of intestinal epithelial cells in primary culture. To answer this question, we have developed a serum-free primary culture system which allows reproducible and quantitative assays of proliferation and differentiation of fetal rat intestinal epithelial cells in the absence of fibroblastic cells. Pure intestinal epithelial tissues were obtained from 16.5-day fetal rats without contamination of mesenchymal cells, and were successfully cultured on a collagen gel in a medium consisting of Ham’s F12, bovine serum albumin, epidermal growth factor (EGF), insulin, cholera toxin, transferrin and hydrocortisone. The epithelial nature of the cultured cells was confirmed by the presence of cytokeratin in the cells. Under optimal culture conditions, intestinal epithelial cells readily attached to the substratum in a day, and proliferated rapidly in vitro, increasing their number about 10 times in the first 5 days. EGF, insulin, cholera toxin, transferrin and hydrocortisone synergistically induced the epithelial proliferation, and lack of any one of them resulted in a significant reduction of the proliferation. In contrast, fetal bovine or horse serums, which have been widely used to supplement culture media, severely inhibited the epithelial proliferation.

Histological examination showed that the epithelial cells formed simple cuboidal epithelia with basallylocated nuclei when cultured on collagen gels. The intestinal epithelial nature of the cells was affirmed by the presence of villin on their luminal surface. Ultrastructurally, cells were connected by tight junctions and desmosomes at the subluminal region, and microvilli were projecting on the luminal surface, indicating that the cells in primary culture retained some characteristics of absorptive epithelial cells. Biochemical analysis showed that the cells expressed weak aminopeptidase activity while alkaline phosphatase or -glucosidase activities could not be detected, suggesting that they could not differentiate fully under these culture conditions. We concluded that the fetal intestinal epithelial cells can proliferate in the absence of heterologous cells, but that mesenchymal cells may be necessary for their full functional differentiation.
Growth requirements and expression of LDL receptor and HMG-CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium
Authors: Gherardi E. , Thomas K. , Cras T. D. Le , Fitzsimmons C. , Moorby C. D. , Bowyer D. E.
Published: April 2021

A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2.

Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 -ethinylestradiol but not by epidermal growth factor (EGF).

On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells.

Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37°C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro.

The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.
Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads
Authors: Peng H. Benjamin , Chen Qiming
Published: April 2021

The distribution of dystrophin in Xenopus myotomal muscle cells was examined in conventional and confocal immunofluorescence microscopy. By labeling dissoci-ated single muscle fibers with a monoclonal or a poly-clonal antibody against dystrophin, we found that dys-trophin is ten times more concentrated at the myotendinous junction (MTJ) than at the extrajunc-tional sarcolemma. At the MTJ, dystrophin lines the membrane invaginations where myofibrils attach to the membrane. It is colocalized with talin, but is not related to the distribution of acetylcholine receptors (AChRs) which are clustered at the postsynaptic membrane in the vicinity of the MTJ in these fibers. We found that the localization of dystrophin can be induced in cultured Xenopus myotomal muscle cells by treating them with polystyrene latex beads. Dystrophin is discretely local-ized at the bead-muscle contacts. With electron microscopy, a sarcolemma specialization with all the salient features of the MTJ, including basal lamina-lined membrane invaginations along which myofibrils make attachment. Although these beads also induce cluster-ing of AChRs, the patterns of dystrophin and AChR localization are distinct. The appearance of dystrophin at the bead-contacted sarcolemma is coincident with the development of the membrane invaginations. This, together with its concentration along membrane invagi-nations at the MTJ in vivo, suggests a role for dys-trophin in the formation of this junctional specializa-tion. Since the signal for MTJ development can be presented to cultured muscle cells in a temporally and spatially controlled manner by beads, this system offers a simple model for analyzing the mechanism of this sar-colemma specialization.
Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: Studies with Tetrahymena thermophila
Authors: Leick Vagn
Published: April 2021

Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD=4×10 −9 M to 1×10 −8 M). Moreover, Scatchard transformations of the binding data show that there are about 5×105 binding sites per cell on the cell surface.

Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdansLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl--endorphin, is internalized preferentially into small vesicles. The difference may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein.

Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).
Cell division in Aspergillus
Authors: Doonan John H.
Published: April 2021

Amenable to sophisticated genetic and molecular analysis, the simple filamentous fungus Aspergillus nidulans has provided some novel insights into the mechanisms and regulation of cell division. Mutational analysis has identified over fifty genes necessary for nuclear division, nuclear movement and cytokinesis. Molecular and cellular analysis of these mutants has led to the discovery of novel components of the cytoskeleton as well as to clarifying the role of established cytoskeletal proteins.

Mutations leading to defects in the kinases (i.e. p34cdc2) and phosphatases (i.e. cdc25 and PP1), which are known to regulate mitosis in other eukaryotes, have been identified in Aspergillus. Additional, as yet novel, mitotic regulatory molecules, encoded by the nimA and bimE genes, have also been discovered in Aspergillus.
Developmental stage- and tissue-specific expression of shibire, a Drosophila gene involved in endocytosis
Authors: Chen M. S. , Burgess C. C. , Vallee R. B. , Wadsworth S. C.
Published: April 2021

Dynamin, a microtubule-activated GTPase, has recently been identified as the product of the shibire gene in Drosophila. shits mutants are defective in synaptic vesicle recycling, which leads to rapid and reversible temperature-sensitive paralysis. In the present study, results from RNase protection assays and analysis of cDNA clones define a complex pattern of developmental- and tissue-specific alternative splicing at two sites within the coding region. This analysis is also supported by western blot analysis with two polyclonal antibodies. In situ hybridization data revealed a high concentration of shi transcripts in the central and peripheral nervous system throughout neuronal development. Other than the nervous system, shi transcripts are also expressed at a high level in early embryos, larval imaginal discs, and male and female gonads. These data provide a basis for interpreting the wide range of phenotypic effects of shi mutations in terms of the putative membrane-sorting properties of dynamin and for further functional study of different dynamin isoforms.
Localization of actin in the Tetrahymena basal body-cage complex
Authors: Hoey John G. , Gavin R. H.
Published: April 2021

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify antichicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahy - mena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.
Differential regulation of βiii and other tubulin genes during peripheral and central neuron development
Authors: Jiang Yuan Qing , Oblinger Monica M.
Published: April 2021

Mammalian peripheral and central neurons differ considerably in the composition and properties of their axonal cytoskeletons. Recent reports of the selective expression of a high molecular weight (HMW) tau protein in neurons with peripherally projecting axons have furthered the idea that the microtubules in central and peripheral neurons are disparate. In the present study, we examined the possibility that the various tubulin genes are differentially expressed in central versus peripheral neurons. To examine this, we compared the expression of four of the β-tubulin mRNAs (classes βI, βII, βIII, βIV) and the α1-tubulin mRNA in rat dorsal root ganglion (DRG) neurons with their expression in cerebral cortex during postnatal development (P5-90), using northern blots and in situ hybridization. We document both similarities and differences in tubulin gene expression in these two regions of the neuraxis during postnatal development. In both DRG and cortex, the expression of the class β1- and βII-tubulin mRNAs and the α1-tubulin mRNA was higher at earlier stages of postnatal development than in the adult. However, class βIV-tubulin mRNA levels increased during cortical development but decreased during DRG postnatal development. The opposite pattern was found for the neuron-specific class βIII-tubulin gene, the mRNA levels of which were high in cortex, at birth and then decreased with increasing postnatal development. in DRG, the pm-tubulin mRNA levels generally increased during postnatal development. βIII-tubulin protein levels were examined qualitatively at different developmental stages (5–90 days) by immunoblotting and immunocytochemistry. These studies showed a robust reduction in Pm-tubulin levels from P5 to adult stages in cortex, but a more complex pattern of change in the DRG. The results suggest that differential utilization of the tubulin gene products exists in central and peripheral nervous tissue and that this may play a role in specifying differences in the functional properties of neuronal cytoskeletons in these regions.
Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein
Authors: Kagami Osamu , Kamiya Ritsu
Published: April 2021

Dynein was extracted from outer arm-less axonemes of the mutant odal and fractionated by high-pressure liquid chromatography on a MonoQ column into seven distinct subspecies (named a–g). Each subspecies contained one or two heavy chains and several mediumsized and light chains; by vanadate/UV-induced photocleavage and SDS-polyacrylamide gel electrophoresis, eight distinct heavy chains were identified. Analysis of the mutant axonemes indicated that the subspecies f (containing two heavy chains) is missing in the inner-arm mutant idal and the subspecies a, c and d are missing in the mutant ida4. Six subspecies (all but f supported microtubule translocation with the maximal rate ranging from 2 to 12 μm s−1 and the apparent Km for ATP ranging from about 10 to 100 μM. All the subspecies translocated microtubules with the plus end leading, indicating that all the inner-arm dyneins are minus end-directed motors. Five subspecies (all but b and f) displayed microtubule rotation during translocation at rates of up to about 10 Hz. Unexpectedly, the Km values for ATP for translocation and rotation did not always agree; because of this, the pitch of the movement was variable with some subspecies. These observations indicate that axonemes are equipped with several inner-arm subspecies and that torque generation is a feature common to many of them.
A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton
Authors: Woods Angela , Baines Anthony J. , Gull Keith
Published: April 2021

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210 000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.
Continuous growth of vimentin filaments in mouse fibroblasts
Authors: Coleman Thomas R. , Lazarides Elias
Published: April 2021

We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives - roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (>20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton.
Amphibian intestinal villin: isolation and expression during embryonic and larval development
Authors: Heusser S. , Colin S. , Figiel A. , Huet C. , Keller J.M. , Pornet P. , Robine S. , Vandamme J. , Vandekerckhove J. , DAUçA M.
Published: April 2021

An actin-binding protein of Mr 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian Mr 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal Mr 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal Mr 105,000 protein is immuno - logically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.
Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells
Authors: Brunkener Mariann , Georgatos Spyros D.
Published: April 2021

Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a urea-extractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least twofold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2×10−7 and 4×10−7 M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis. Finally, heat-treatment of the trypsinized membranes, or digestion of urea-stripped membranes with a-chymotrypsin and V8 protease, affect filensin binding to a variable degree. Based on these data, we conclude that filensin and its COOH-terminal proteolytic product have the potential to directly associate with intrinsic elements of the lens cell membrane.
Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants
Authors: Mackie Eleanor J. , Tucker Richard P.
Published: April 2021

The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.
Adhesion of golgi cisternae by proteinaceous interactions: intercisternal bridges as putative adhesive structures
Authors: Cluett Edward B. , Brown William J.
Published: April 2021

We have investigated the nature of the component(s) responsible for holding the cisternal membranes of the Golgi complex into a stacked unit. Isolated Golgi complexes were treated with a variety of agents to induce the separation of intact Golgi stacks into single cisternal elements, i.e. “unstacking”, and the effects were analyzed and quantitated by electron microscopy. In control experiments, isolated, intact Golgi stacks were stable at 4°C and 20°C for ≥1 h; however, some unstacking occurred at 32°C. Treatment of intact Golgi stacks with a variety of proteolytic enzymes resulted in a time- and dose-dependent unstacking of the cisternae, although stacks were resistant to various other proteases. Following liberation from the stack, single cisternae remained flattened with dilated rims. The integrity of intact Golgi stacks was unaffected by treatment with various concentrations and combinations of monovalent and divalent cations, or chelators of divalent cations. Electron microscopic observations of tannic acid- or negatively stained Golgi complexes, revealed the presence of highly structured, intercisternal “bridges”. When seen within intact Golgi complexes, these bridges were only consistently found between closely apposed cisternae and were not observed on dilated rims or secretory vesicles. These bridges, on both intact stacks and physically disrupted cisternae, were rectangular, being ∼8.5 nm in width, ∼11 nm in height. Treatment with proteases under conditions that resulted in the unstacking of intact complexes also removed these bridge structures. These data show that proteinaceous components are responsible for holding Golgi cisternae together into a cohesive, stacked unit, and identify a candidate bridge structure that could serve this purpose.
Cell shape, chromosome orientation and the position of the plane of division in Vicia faba root cortex cells
Authors: Oud J. L. , Nanninga N.
Published: April 2021

Three-dimensional chromosome orientation was studied in thick sections of Vicia faba root meristem, using confocal microscopy and digital image analysis techniques. In the proliferative part of the root meristem, where the cells are organized in longitudinal files, it was expected to find dividing cells with a spindle axis parallel to the file axis and, occasionally, perpendicular to the file axis (resulting in a local file bifurcation). However, we observed a large number of oblique spindle axes. From metaphase to telophase there was a progressive increase in the rotation of the spindle axis. A 90° turn of the metaphase equator plane was never observed. Threedimensional measurements of both the space occupied by the ana- and telophase chromosome configurations, and the size of the corresponding cortex cells, showed that most cells were too flat for an orientation of the spindle parallel to the file axis. Apparently, cell size limitations forced the spindle to rotate during mitosis. Consequently, the nuclei in the daughter cells were positioned diagonally in opposite directions, instead of on top of each other. In the majority of these cells, a transverse plane of division would intersect the nuclei. Therefore, the new cell wall was sigmoid shaped or oblique. Most daughter cells remained within the original cell file but, occasionally, in extremely flat cells the position of the daughter nuclei forced the cell to set a plane of division parallel to the file axis. This resulted in file bifurcation. It has been concluded that cell shape, the extent of spindle rotation and the position of the division plane are related.