bluuebunny/crossref_metadata_2025 huggingface dataset:
A small molecular mass inhibitor of growth of 3T3 cells and porcine aortic smooth muscle cells released from the macrophage cell line P388D1
Authors: McMurray H. F. , Proudfoot D. , Davis J. B. , Parrott D. P. , Bowyer D. E.
Published: April 2021

Murine peritoneal macrophages and the macrophage-like cell line, P388D1, were found to release both mito-genic and inhibitory modulators of growth of cells in culture. These growth factors were effective against both murine Swiss 3T3 fibroblasts and porcine aortic smooth muscle cells as assessed by [3H]thymidine incorporation into DNA and by measurement of cell number.

Partial characterisation of the inhibitory activity demonstrated it to be lost on dialysis using a membrane with a 10 kDa cut-off, trypsin sensitive, heat stable, and slightly sensitive to freeze-thawing. The inhibitory activity not only affected cell growth but was found to change the morphology of porcine aortic smooth muscle cells. Gel permeation studies showed an estimated molecular mass in the range 2.5 to 6.5 kDa. The inhibitory activity could be partially purified using ion-exchange chromatography. Experiments with a neutralising anti-body against transforming growth factor beta (TGF-) showed that TGF-is not responsible for the activity observed. Indomethacin had no effect on the production of inhibitor suggesting that it is not an inhibitory prostanoid. The inhibitory activity was not due to a non-specific toxic mechanism as confirmed by a [3H]adenine release assay.

Incubation of P388D1 cells with cycloheximide pre-vented the release of inhibitory activity.
Regulation of pathways within cultured epithelial cells for the transcytosis of a basal membrane-bound peroxidase-polylysine conjugate
Authors: Taub Mitchell E. , Shen Wei-Chiang
Published: April 2021

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used as a non-specific adsorptive probe to study transcytosis in MDCK strain I and Caco-2 epithelial cells. As we have shown previously, HRP-PLL transcytosis proceeds via an intracellular, non-lyso-somal proteolytic compartment in MDCK cells; yet, this compartment is utilized for transcytosis only in the basal-to-apical direction (Taub, M. E. and Shen, W.-C. J. Cell. Physiol., 150, 283-290, 1992). Using size exclu-sion chromatography, we demonstrate that the PLL moiety of the conjugate is effectively cleaved during transcytosis, thus releasing free HRP from the apical surface of the cells. Pulse-chase studies indicate that ∼6% of basolateral surface-associated HRP-PLL conju-gate in Transwell-grown cell monolayers enters the basal-to-apical transcytotic pathway. Brief (1 hour) treatment with 160 nM phorbol ester (PMA), a protein kinase C stimulator, elicits a 2-fold increase in the rate and amount of HRP-PLL transcytosis following a 1 hour lag time. Treatment with 1.6 g/ml brefeldin A (BFA) inhibits HRP-PLL transcytosis by ∼30%; additionally, BFA is able to abolish completely the PMA stimulatory effect. Removal of BFA causes a re-establishment of the normal rate of transcytosis within 2 hours, demonstrat-ing the reversibility of BFA inhibition with respect to HRP-PLL transcytosis. Thus, PMA most likely elicits an increase in the amount of basally internalized conjugate delivered to BFA-sensitive transcytotic compartments. Leupeptin (50 g/ml), a protease inhibitor, which we have previously shown inhibits HRP-PLL transcytosis by ∼50%, fails to abolish the PMA effect, indicating that the protease involved in the PMA-enhanced pathway is either insensitive or inaccessible to basolaterally inter-nalized leupeptin. These results suggest that: (a) there exists more than one intracellular pathway for process-ing of endocytosed HRP-PLL conjugate; and (b) a strik-ingly similar mechanism for basal-to-apical transcytosis is utilized within two dissimilar species of polarized epithelium.
Orientation of macromolecules in the walls of elongating carrot cells
Authors: McCann M. C. , Stacey N. J. , Wilson R. , Roberts K.
Published: April 2021

When round cells from a carrot cell suspension culture are diluted into fresh medium without auxin, the cells elongate to almost 50 times their original diameter within three days. This process of elongation is accom-panied by changes in both the composition and the orientation of cell wall polymers. We have obtained infor-mation on the orientation of wall polymers in elongating cells by two complementary techniques, one using microscopy and one spectroscopy. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica tech-nique show that walls of round carrot cells have no net orientation of cellulose microfibrils, and that many thin fibres can be seen cross-linking microfibrils. Walls of elongated carrot cells, in contrast, show a marked net orientation of microfibrils at right angles to the axis of elongation.

Fourier Transform Infrared (FTIR) spectra obtained from defined areas of single cell walls show that walls of round carrot cells contain more protein, esters and phenolics in a given area (10 m 10 m) than walls of elongated carrot cells, that contain proportionally more carbohydrate. The orientation of particular func-tional groups, with respect to the direction of elongation of the cell, can be determined by inserting a polariser into the path of the infrared beam, before it passes through a cell wall sample mounted on the stage of the microscope accessory. In the walls of elongated cells, ester bands, amide bands characteristic of proteins, and stretching frequencies in the carbohydrate region of the spectrum all show a net orientation transverse to the long axis of the cells. In the walls of round carrot cells, however, there is no such net orientation of polymers. Spectra obtained from 25 m-thick fresh sections of the etiolated stem of a carrot seedling show that different wall components are polarised in different tissue types. These techniques have therefore enabled us to define dif-ferences in both the composition and the architecture of walls of elongating cells at the level of a single cell, and to suggest that polymers not previously thought to be ordered, such as pectin and protein, are strictly oriented in some wall types.
Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line caco-2: Evidence for glucose-dependent negative regulation
Authors: Chantret Isabelle , Rodolosse Annie , Barbat Alain , Dussaulx Elisabeth , Brot-Laroche Edith , Zweibaum Alain , Rousset Monique
Published: April 2021

The expression of the brush border-associated hydrolase sucrase-isomaltase was shown to increase from early to late passages of Caco-2 cells, concomitant with a decrease in the rates of glucose consumption. Twenty-six clones were isolated from early (P29) and late (P198) passages of the cell line. These clones show considerable and inverse differences in the levels of sucrase activities and rates of glucose consumption, without marked changes in other features of enterocytic differentiation of the cells (presence of an apical brush border, levels of expression of other brush border-associated hydrolases). Clones with low sucrase-isomaltase expression show a mosaic expression of the enzyme and a 38-fold higher rate of glucose consumption than clones with high sucrase-isomaltase expression. The clones with high expression show an homogeneous apical distribution of the enzyme and 70-fold and 35-fold higher levels of sucrase activities and sucrase-isomaltase mRNA, respectively. In contrast no differences were found from one clone to another in the enrichment of sucrase activity in brush border-enriched fractions as compared to cell homogenates. Switch to low glucose-containing medium (1 mM versus 25 mM in standard culture conditions) of cells with low sucrase-isomaltase results in an increased and more homogeneous expression of the enzyme and a tenfold augmentation of the levels of sucrase-isomaltase mRNA and sucrase activity. These results show that glucose interferes with the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level.
The processing pathway of prelamin a
Authors: Sinensky M. , Fantle K. , Trujillo M. , McLain T. , Kupfer A. , Dalton M.
Published: April 2021

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known preny-lated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subse-quent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine.

To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dex-amethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and car-boxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus.
CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment
Authors: Füllekrug Joachim , Sönnichsen Birte , Wünsch Ulrike , Arseven Kamran , Van Phuc Nguyen , Söling Hans-Dieter , Mieskes Gottfried
Published: April 2021

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H. D. and Hauri, H. P. (1993) Eur. J. Cell Biol. 60, 366-370). Our con-clusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and α-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In additional experiments, Vero cells were infected with VSV tsO45. At 15°C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.
Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase
Authors: Rimmington Georgina , Dalby Brian , Glover David M.
Published: April 2021

We have introduced an N-terminally truncated form of cyclin B into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (<0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle; they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase.
Role of integrins in melanocyte attachment and dendricity
Authors: Hara Masahiro , Yaar Mina , Tang Aimin , Eller Mark S. , Reenstra Wende , Gilchrest Barbara A.
Published: April 2021

Integrins are a family of proteins known to mediate attach-ment of cells to extracellular matrix materials. The sub-stratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed β1, α2, α3, α5, α6 and αv integrin subunits as well as the αvβ3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but α6 and β1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while α2, α5 and αVβ3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-β1 and anti-α6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-α2, anti-α5 and anti-αvβ3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known char-acteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by α6β1 integrin in a Ca2+-independent, Mg2+- and/or Mn2+-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by αvβ3 as well as α2 and α5 integrins.
Purification and characterization of ensconsin, A novel microtubule stabilizing protein
Authors: Bulinski Jeannette Chloe , Bossler Aaron
Published: April 2021

In previous studies (Bulinski and Borisy (1979). Proc. Nat. Acad. Sci. 76, 293-297; Weatherbee et al. (1980). Biochemistry 19, 4116-4123) a microtubule-associated protein (MAP) of Mr ∼125,000 was identified as a prominent MAP in HeLa cells. We set out to perform a biochemical characterization of this protein, and to determine its in vitro functions and in vivo distribution. We determined that, like the assembly-promoting MAPs, tau, MAP2 and MAP4, the 125 kDa MAP was both proteolytically sensitive and thermostable. An additional property of this MAP; namely, its unusually tight association with a calcium-insensitive population of MTs in the presence of taxol, was exploited in devising an efficient purification strategy. Because of the MAP’s tenacious association with a stable population of MTs, and because it appeared to contribute to the stability of this population of MTs in vitro, we have named this protein ensconsin. We examined the binding of purified ensconsin to MTs; ensconsin exhibited binding that saturated its MT binding sites at an approximate molar ratio of 1:6 (ensconsin:tubulin). Unlike other MAPs char-acterized to date, ensconsin’s binding to MTs was insensitive to moderate salt concentrations (:S0.6 M). We further characterized ensconsin in immunoblotting experiments using mouse polyclonal anti-ensconsin antibodies and anti-bodies reactive with previously described MAPs, such as high molecular mass tau isoforms, dynamin, STOP, CLIP-170 and kinesin. These experiments demonstrated that ensconsin is distinct from other proteins of similar Mr that may be present in association with MTs. Immunofluorescence with anti-ensconsin antibodies demonstrated that ensconsin was detectable in association with most or all of the MTs of several lines of human epithelial, fibroblastic and muscle cells; its in vivo properties and distribution, especially in response to drug or other treatments of cells, were found to be different from those of MAP4, the pre-dominant MAP found in these cell types. We conclude that ensconsin, a MAP found in a variety of human cells, is bio-chemically - and perhaps functionally-distinct from other MAPs present in non-neuronal cells.
Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain
Authors: Lee Randall J. , Egelhoff Thomas T. , Spudich James A.
Published: April 2021

Conventional myosin (‘myosin II’) is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dic-tyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.
Supramolecular assemblies of the Ascaris suum major sperm protein (MSP) associated with amoeboid cell motility
Authors: King Karen L. , Stewart Murray , Roberts Thomas M.
Published: April 2021

Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of loco-motion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.
Exploitation of microfilament proteins byListeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets
Authors: Temm-Grove Constance J. , Jockusch Brigitte M. , Rohde Manfred , Niebuhr Kirsten , Chakraborty Trinad , Wehland Jürgen
Published: April 2021

Effective cell-to-cell spreading of the facultative intracellu-lar pathogen Listeria monocytogenes requires the interac-tion between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a ‘comet tail’ localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria- recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunore-activity as the brush border-like microvilli, when analysed by a specific actin antibody.

The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extra-cellular milieu. This is in contrast to nonpolarized epithe-lial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.
Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions
Authors: Had L. , Faivre-Sarrailh C. , Legrand Ch. , Méry J. , Brugidou J. , Rabié A.
Published: April 2021

Antipeptide antisera specific for TM-4 and TMBr-3, the two tropomyosin isoforms in neurons, were used to investigate the concentrations and distributions of these F-actin-binding proteins in neurons in vitro and in vivo. TM-4 and TMBr-3 tropomyosins had different developmental profiles. TM-4 was found mainly in immature stages, while the concentration of TMBr-3 increased with maturation. The two isoforms also had different subcellular distributions. TM-4 was concentrated in the growth cones of cultured neurons and, in vivo, in areas where neurites were growing. Later, when development was complete, TM-4 was restricted to postsynaptic sites in the cerebellar cortex, whereas TMBr-3 was found in the presynaptic terminals. These data suggest that the tropomyosin isoforms have different functions, through their interaction with the actin cytoskeleton. TM-4 may be involved in the motile events of neurite growth and synaptic plasticity, while TMBr-3 could play a role in stabilizing neuronal networks and synaptic functioning.
Nuclear import of serum response factor (SRF) requires a short aminoterminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site
Authors: Rech Jocelyne , Barlat Isabelle , Veyrune Jean Luc , Vie Annick , Blanchard Jean Marie
Published: April 2021

Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133-235 and 168-235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein.
A role for Ca2+-conducting ion channels in mechanically-induced signal transduction of airway epithelial cells
Authors: Boitano Scott , Sanderson Michael J. , Dirksen Ellen R.
Published: April 2021

Mechanical stimulation of a single cell in a cultured monolayer of airway epithelial cells initiates an intercellularly communicated increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates radially through adjacent cells via gap junctions, forming an intercellular Ca2+ wave. Mechanically-induced intercellular Ca2+ waves also occur in the absence of extracellular Ca2+. However, in Ca2+-free medium an increase in [Ca2+]i of the stimulated cell does not occur. Thus, mechanically-induced [Ca2+]i changes in the stimulated cell are influenced by the extracellular Ca2+ concentration. To investigate if a channel-mediated Ca2+ flux across the plasma membrane contributes to the elevation of [Ca2+]i in the stimulated cell we used digital image microscopy to measure mechanically-induced [Ca2+]i changes in the presence of Ca2+ channel blockers. In Ca2+-free medium containing Gd3+ (20 µM) mechanical stimulation resulted in an [Ca2+]i increase in the stimulated cell. The delay time between mechanical stimulation and increase in [Ca2+]i of the stimulated cell was dependent on extracellular [Gd3+], with a half-maximal effective concentration of approximately 40 µM. Mechanical stimulation in Ca2+-free medium containing La3+ (10 µM) or Ni2+ (100 µM) gave similar results. Mechanical stimulation in Ca2+-free medium containing the dihydropyridine Ca2+ channel blockers nifedipine (10 µM) and nimodipine (10 µM) also resulted in an increase of [Ca2+]i of the stimulated cell. Mechanical stimulation of cells treated with thapsigargin to deplete intracellular Ca2+ stores, in the presence of 1.3 mM extracellular Ca2+, results in an increase in [Ca2+]i of the stimulated cell without the propagation of an intercellular Ca2+ wave. Mechanical stimulation of thapsigargintreated cells in an extracellular medium buffered to 50 µM free Ca2+ still results in an increase in [Ca2+]i in the stimulated cell. However, the mechanically-induced Ca2+ increase in the presence of 50 µM extracellular [Ca2+] can be reversibly blocked by 100 µM Ni2+. From these results we conclude that a flux of Ca2+ across the plasma membrane and through Ca2+-conducting channels contributes to the mechanically-induced [Ca2+]i changes in the stimulated cell.
Fusion between large phagocytic vesicles: Targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in chinese hamster ovary cells
Authors: Veras Patricia S. T. , Chastellier Chantal de , Moreau Marie-Françoise , Villiers Veronique , Thibon Monique , Mattei Denise , Rabinovitch Michel
Published: April 2021

This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetii or the Protozoan flagellate Leishmania amazonensis. Infection by these organisms is compatible with cell survival and multiplication. Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles. Donor vesicles contained heat-killed yeast, zymosan, β-glucan or latex beads taken up by the host cells. Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp. Cell Res. 104, 325-334, 1977). Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy.

Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection. Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours). Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined. The fraction of uncoated or Con-canavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads. In addition, only rarely did latex beads enter these vacuoles. The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals.
Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling
Authors: Sardini A. , Mintenig G. M. , Valverde M. A. , Sepúlveda F. V. , Gill D. R. , Hyde S. C. , Higgins C. F. , McNaughton P. A.
Published: April 2021

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drugtransporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.
Plasmodesmata of maize root tips: structure and composition
Authors: Turner Adrian , Wells Brian , Roberts Keith
Published: April 2021

A procedure is described for obtaining clean maize cell wall preparations that contain embedded plasmodesmata. Negative staining and rotary shadowing have been used with transmission electron microscopy to visualise the plasmodesmata in these isolated walls, and to assess the effects of simple biochemical treatments on plasmodesmal components. Light protease treatment removes material from the exposed ends of plasmodesmata but does not extract the plasmodesmal core, which lies within the cell wall. However, heavy proteolysis occasionally removes the complete plasmodesma, including its enclosing collar structure, from the wall. Extraction with urea has a similar effect. The collar itself appears not to be proteinaceous in composition, although protein may bind it into the wall.

Callose is localised in the wall around plasmodesmata, but does not appear to be a constituent of the collar. The membrane components of the plasmodesma (plasma membrane and desmotubule) can be extracted with membrane-solubilising detergents. This treatment releases from the wall a small number of proteins that are regarded as being potentially of plasmodesmal origin. These results show that plasmodesmata from maize can be dissected biochemically and suggest a strategy for the characterisation of individual molecular components.
Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line
Authors: Tore Gjertsen Bjørn , Cressey Lill Irene , Ruchaud Sandrine , Houge Gunnar , Lanotte Michel , Døskeland Stein Ove
Published: April 2021

The protein phosphatase inhibitors okadaic acid and calyculin A at moderate concentrations induced three types of apoptotic promyelocytic leukemia cell death, distinct with respect to ultrastructure and polynucleotide fragmentation. Calyculin A at higher concentrations (>50 nM) induced a non-apoptotic death type with high ATP and pronounced micromitochondriosis. This suggests that protein phosphorylation pathways are involved in the triggering of several death pathways. Activation of the cAMP kinase induced yet another apoptotic death type, preferentially affecting cells in S-phase. In fact, cAMP acted in two ways to stop IPC promyelocyte proliferation: (1) block in late G1 (preventing new cells from entering DNA replication); and (2) induction of apoptosis in S-phase. cAMP and phosphatase inhibitors acted via distinct pathways. The inhibitors suppressed cAMP-induced death, but only at concentrations high enough to commit the cells to alternative, less conspicuous death types. The tumor-promoting activity of okadaic acid and calyculin A may therefore not be by protection against apoptosis. DNA fragmentation correlated with the novel feature of limited 28 S rRNA cleavage, suggesting co-ordinated polynucleotide cleavage, possibly directed against illegitimate polynucleotides, in some apoptotic death types.
Selective increase in the binding of the α1β1 integrin for collagen type IV during neurite outgrowth of human neuroblastoma TR 14 cells
Authors: Carmeliet Geert , Himpens Bernard , Cassiman Jean-Jacques
Published: April 2021

Regulation of β1 integrins in neurite outgrowth following N6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate (dBcAMP) treatment was investigated using the human neuroblastoma cell line TR 14. Three β1 integrins were identified: the α1β1 receptor bound collagen type I, collagen type IV and probably laminin; the α2β1 integrin bound collagen type I; and the αvβ1 receptor bound fibronectin.

Neurite extension was detectable as early as 30 minutes following dBcAMP treatment, was maximal after 24 hours and remained constant during treatment for 4 days. Adhesion-perturbing β1 subunit-specific antibodies, added together with dBcAMP, prevented the outgrowth of new neurites.

During the first 24 hours of neurite outgrowth, no change was observed in the amount of β1 integrins nor in their topographic distribution. However, dBcAMP treatment increased the binding of α1β1 receptors to collagen type IV-Sepharose by a factor 2.3±0.6 (P<0.02), while no alteration in the binding to collagen type I was detected. Moreover, neurites and growth cones were immunoreactive for collagen type IV but not for collagen type I. Consistently, dBcAMP-induced neurite outgrowth was inhibited by adhesion-perturbing α1 subunit-specific antibodies.

Following maximal neurite outgrowth, the amount of β1 integrins determined by immunoprecipitation and by confocal microscopy decreased to 58.3±11.2% (P<0.001) and to 55.4±17.5% (P<0.001) of untreated levels, respectively, without any change in the level of β1 mRNA or de novo synthesized β1 precursor. However, pulse-chase experiments showed an increased turnover of the β1 subunit: the amount of β1 precursor that was degraded after 1 hour chase was 50.5±8.4% in cells treated for 4 days and 34.2±3.9% in untreated cells (P<0.02); the amount of mature β1 after 24 hours chase was smaller in cells treated for 4 days compared to untreated cells.

In conclusion, during neurite outgrowth, α1β1 integrins are required and acquire an enhanced binding activity for collagen type IV; but following maximal neurite outgrowth, expression of β1 integrins is reduced.
Identification of tau protein regions required for process formation in PC12 cells
Authors: Léger J. G. , Brandt R. , Lee G.
Published: April 2021

Tau is a neuronal microtubule-associated protein that is required for the development and maintenance of neuronal cell polarity. It promotes microtubule assembly in vitro and we have recently reported that a specific tau region, which spans amino acid residues 154-172 of human fetal tau, is not required for growth of existing microtubules, but is required for nucleation of new microtubules. These residues also confer stronger microtubule binding activity in 3T3 cells. The aim of this study was to investigate the functional organization of tau in relation to its role in promoting process formation in a neuronal model system. We transfected undifferentiated PC12 cells with vectors expressing tau fragments and treated the expressing cells with cytochalasin B to allow process extension. We found that deletion of amino acid residues 154-172 greatly reduced the percentage of transfected cells bearing processes compared to that of cells transfected with fulllength tau or with an amino-terminally deleted tau fragment containing residues 154-172. These differences do not appear to result from a quantitative difference in protein expression, as shown by immunoblot analysis of transfected cells. We also observed that while the presence of tau fragments increases acetylation of microtubules, the pattern of acetylation in cells transfected with the fragment missing residues 154-172 is less extensive, suggesting that it does not result in the same level of stabilization as the longer tau fragments. Taxol promoted process outgrowth in cells treated with cytochalasin and restored process outgrowth to cells transfected with the tau fragment lacking this activity. Therefore, process formation involves primarily the stabilization and nucleation of microtubules. We conclude that the residues necessary for confering microtubule nucleation activity of tau in vitro are important for process formation in vivo. It is likely that these residues influence the binding affinity and therefore the stabilization activity of tau.
Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression
Authors: Lütcke Anne , Parton Robert G. , Murphy Carol , Olkkonen Vesa M. , Dupree Paul , Valencia Alfonso , Simons Kai , Zerial Marino
Published: April 2021

Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell typedependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.
Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles
Authors: Cowles Christopher R. , Emr Scott D. , Horazdovsky Bruce F.
Published: April 2021

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete vacuolar hydrolases. A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble vacuolar proteins, yet maintain a near-normal vacuole structure. Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a protein family that includes the yeast proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms. These proteins are thought to participate in vesicle-mediated protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes. Significantly, this fraction lacks ER, vacuole and plasma membranes. Overexpression of Vps45p saturates the sites with which Vps45p associates. A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters. This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicles in the vacuolar protein sorting pathway.
Initiation of growth inhibition by TGFβ1 is unlikely to occur in G1
Authors: Kramer IJsbrand M. , Patel Raj , Spargo Donald , Riley Patrick
Published: April 2021

Type β transforming growth factors represent a family of polypeptides that modulate growth and differentiation. They exert their effect on target cells through interaction with multiple cell surface receptors. Transforming growth factor-β1 has a strong inhibitory action on cell division in mink lung CCl64 cells, a process that is initiated by immediate induction of junB and phosphorylation of nuclear protein followed by a reduced expression of cdk4. However, its signal transduction pathways are still unresolved. In this study we report a detailed analysis of cell kinetic events following addition of transforming growth factor-β1 to mink lung CCL64 cells. We show that transforming growth factor-β1 reduces [3H]thymidine incorporation after a delay of 8 hours, which reaches its nadir at 16 hours. The reduced growth rate is maintained for at least 48 hours as shown by flow cytometric analysis of DNA content. Using time-lapse video microscopy it was shown that control cells double on average every 14.4 hours, whereas the transforming growth factor-β1-treated cells have a doubling time of on average 20.3 hours. The difference in intermitotic time is a consequence of a prolonged G1 phase (a shift from 7.5 to 13.5 hours on average). However, changes in intermitotic times occur only after cells have undergone division in the presence of transforming growth factor-β1 and treated cells finish the ongoing cell cycle exactly like control cells. From these findings we conclude that transforming growth factor-β1 may change cell cycle parameters by interfering with cellular events prior to G1. Immunoprecipitation studies with antibodies against cyclin B demonstrate that transforming growth factor-β1 induces the association of a 20 kDa protein with the immune complex. This observation suggests that interference with the cell cycle may commence in late S or G2.
Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis
Authors: Hall* Peter A. , Coates Philip J. , Ansari Bijan , Hopwood David
Published: April 2021

The regulation of cell number in adult tissues is determined by the balance of cell production and cell loss. In the gastrointestinal tract, where there are well defined zones of proliferation and migration of both epithelial cells and associated fibroblasts, it is widely held that cell loss occurs by shedding into the gut lumen. Since the evidence for this is not compelling, we investigated the distribution and amount of apoptosis in the normal mammalian gut. In the stomach, small intestine and colon of rodents and man, there is a small number of apoptotic bodies in the epithelium and in the immediate sub-epithelial connective tissue. Engulfment by adjacent epithelial cells and sub-epithelial macrophages accounts for the removal of apoptotic bodies. Apoptotic bodies are not randomly distributed but are found towards the distal end of the known cellular migration routes of both epithelial and mesenchymal cells. Furthermore, consideration of the absolute numbers of apoptotic bodies, their rapid clearance and the dimensions of the small intestinal villi and colonic crypts indicates that the cell loss in the normal murine intestine can largely be explained on the basis of the observed apoptosis. Despite being inconspicuous in histological material, apoptosis probably accounts for the bulk of cell loss in the gut and is a central feature of the regulation of cell number in adult tissues.
Transcriptional regulation of a ras nucleotide-exchange factor gene by extracellular signals in fission yeast
Authors: Hughes David A. , Yabana Naoyuki , Yamamoto Masayuki
Published: April 2021

The ste6 gene of Schizosaccharomyces pombe encodes a putative GDP-GTP exchange factor for the ras1 gene product. Genetic analysis of the ste6 and ras1 genes has shown that they are required for mating and for the response to mating pheromones. In this study we show that expression of the ste6-encoded mRNA is induced by nitrogen starvation, the physiological signal that triggers mating and sexual differentiation. Exposure to mating pheromones enhances the induction of ste6 expression upon nitrogen starvation. Pheromone-induced expression requires not only the function of components of the pheromone-signalling pathway, but also ras1 function. Furthermore, mutants in which the Ras1 protein is activated have higher basal and induced levels of ste6 gene expression than wild-type cells. These observations indicate the existence of a positive-feedback loop through which Ras1 stimulates the expression of its own activator. Since Ste6 is likely to promote the exchange of guanine nucleotides on Ras1 protein, our results suggest an important role for GDP-GTP exchange in the regulation of Ras1 activity during the mating process in S. pombe.