bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of ensconsin, A novel microtubule stabilizing protein
Authors: Bulinski Jeannette Chloe , Bossler Aaron 
Published: April 2021

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACTEffective 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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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 (&gt;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

ABSTRACT
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&lt;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&lt;0.001) and to 55.4±17.5% (P&lt;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&lt;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

ABSTRACT
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.