bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton
Authors: Ursitti Jeanine A. , Wade James B. 
Published: February 2005

AbstractIsolated skeletons from human erythrocyte ghosts were studied using immunogold labeling; negative staining; and quick‐freeze, deep‐etch, rotary replication with Pt/C (QFDERR). Isolated skeletons visualized by QFDERR were similar to the negatively stained skeletons in that the proteins spectrin, actin, and ankyrin could be easily distinguished. However, the quick‐frozen skeletons had two fewer filaments (4.2 ± 0.7) at an actin junction. Immunogold labeling of skeletons with site‐specific spectrin antibodies not only confirmed the designation of these filaments as spectrin molecules, but indicated that about 30% of spectrin filaments form non‐actin junctions consistent with the hexameric organization of these filaments. Many of the filaments displayed a striking banding pattern indicative of underlying substructure. Isolated skeletons prepared by QFDERR also showed evidence of laterally associated spectrin filaments. These associations, as well as many hexamer junctions, are lost during negative staining. Negative staining also apparently caused ∼21% of the spectrin filaments to separate into their monomeric subunits. These results indicate that the surface tension imposed during negative staining of isolated skeletons can cause a loss of interactions normally present in the intact membrane skeleton. © 1993 Wiley‐Liss, Inc.
DiOC6 staining reveals organelle structure and dynamics in living yeast cells
Authors: Koning Ann J. , Lum Pek Yee , Williams Jennifer M. , Wright Robin 
Published: February 2005

AbstractWhen present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357–435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076–1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory‐deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild‐type cells could no longer grow on non‐fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time‐lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time‐lapse studies of living DiOC6‐stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or rotations of the nucleus/vacuole, DiOC6 staining also revealed more subtle dynamics, including the forces of the spindle on the nuclear envelope during mitosis. This technique should have broad application in analyses of yeast cell structure and function. © 1993 Wiley‐Liss, Inc.
Characterization of spontaneous and action potential‐induced calcium transients in developing myotubes in vitro
Authors: Flucher Bernhard E. , Andrews S. Brain 
Published: February 2005

AbstractWe have investigated the onset and maturation of action potential‐ and calcium‐induced calcium release from the sarcoplasmic reticulum during the differentiation of excitation‐contraction coupling in skeletal muscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo‐3 revealed the dynamics, time course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contractions in well‐differntiated myotubes are accompanied by brief (200–500 ms) increases in the concentration of free cytoplasmic calcium. These transients are modulated by sub‐threshold concentrations of caffeine, resulting in a plateau of elevated calcium. Two novel types of calcium transients were observed in non‐contracting myotubes. (1) Fast localized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. (2) Upon addition of caffeine, propagating calcium waves are generated (35–70 μm/s velocity), which are accompanied by contractures. Aside from caffeine sensitivity, calcium waves and contractionrelated sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, these transients may represent calcium‐induced calcium release in quiescent and active myotubes, respectively. Following one calcium‐induced calcium release event, myotubes become refractory to new calcium‐induced transients; however, action potential‐induced transients and FLTs are not blocked. This suggests that these transients occur by distinct release mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation. © 1993 Wiley‐Liss, Inc.
Tipping of flagellar agglutinins by gametes of Chlamydomonas reinhardtii
Authors: Goodenough Ursula W. 
Published: February 2005

AbstractThe migration of cross‐linked agglutinins to the gametic flagellar tips (tipping) is a hallnmark of the Chlamydomonas mating reaction. In this study, an assay was developed to analyze the kinetics and biological requirements for the tipping response: isolated flagella from mt− gametes of C. reinhardtii were allowed to agglutinate to the immotile flagella of pf‐18 mt+ gametes, and their migration to the tips was monitored by phase microscopy. The tipping process is shown to require both adhesion and elevated levels of cAMP. The cAMP may activate tipping motors directly. In addition, cAMP stimulates the recruitment of agglutinins to flagellar surface to replace those inactivated by adhesion. These results are compared with previous studies on the tipping of flagellar surface proteins cross‐linked by soluble ligands, and an integrated model is presented. © 1993 Wiley‐Liss, Inc.
Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages
Authors: Messier Jeanne M. , Shaw Leslie M. , Chafel Mark , Matsudaira Paul , Mercurio Arthur M. 
Published: February 2005

AbstractThe actin‐bundling protein fimbrin is homologous to l‐plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069–1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton‐X‐100 (soluble fraction), Tween 40‐deoxy‐cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone‐extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC‐extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins. © 1993 Wiley‐Liss, Inc.
Human fibroblast tropomyosin isoforms: Characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells
Authors: Novy Robert E. , Lin Jenny Li‐Chun , Lin Ching‐Shwun , Lin Jim Jung‐Ching 
Published: February 2005

AbstractA tropomyosin‐specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen γgt10 and γgt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT‐14 and WI‐38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low Mr isoforms (hTM5a and hTM5b), and a high Mr isoform (hTMsmα). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsmα represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue‐specific expression suggests that different isoforms may perform distinct functions in different tissues. © 1993 Wiley‐Liss, Inc.
Expression of four myosin heavy chain isoforms with development in mouse uterus
Authors: Eddinger Thomas J. , Wolf Jane A. 
Published: February 2005

AbstractIn smooth muscle tissue, two or three isoforms of myosin heavy chain (MHC) have been reported (SM1, SM2, and/or NM). In mouse uterus tissue, four bands in the region of the MHC's can be resolved on high resolution SDS polyacrylamide gels. Western blots using smooth muscle (SM) MHC‐specific and nonmuscle (NM) MHC‐specific polyclonal antibodies show the upper two bands in the MHC region are SM isoforms, whereas the lower two bands are NM isoforms. One‐dimensional peptide maps of these four bands show each to have a unique pattern of polypeptide fragments following α‐chymotrypsin digestion. Developmental expression of myosin heavy chains (MHC) in mouse uterus, aorta, bladder, and stomach (6 ages, 10–150 days) was determined using tissue homogenates. In the uterus, both SM MHC's show an increase in relative content with increasing age, whereas the NM MHC's show a decrease. The mouse aorta shows a significant increase in the SM MHC's and a significant decrease in the NM MHC from day 10 to day 30, which is similar to data reported for the rat aorta. Whereas both the bladder and stomach contain relatively small amounts of NM MHC's (∼ 10% or less), these quantities do show decreases with development. The SM1:SM2 ratio for the uterus remains high (3.4 at 150 days) through development; the aorta, bladder, and stomach also start out high, but tend toward 1.0 in the 150‐day animals. The presence of four MHC isoforms in the uterus with unique developmental regulation of expression is consistent with hypotheses of unique functional roles for these isoforms. © 1993 Wiley‐Liss, Inc.
Biochemical and immunochemical identification of a microtubule‐binding protein from bovine pancreas
Authors: Michalik Liliane , Neuville Pascal , Vanier Marie‐Thérèse , Launay Jean‐François 
Published: February 2005

AbstractWe have identified a 67 kDa heat‐stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat‐stable microtubule‐associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau. © 1993 Wiley‐Liss, Inc.
PKC mediates 12(S)‐HETE‐induced cytoskeletal rearrangement in B16A melanoma cells
Authors: Timar J. , Tang D. , Bazaz R. , Haddad M. M. , Kimler V. A. , Taylor J. D. , Honn K. V. 
Published: February 2005

AbstractThe fatty acid 12(S)‐HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)‐HETE stimulates cytoskeleton‐dependent cellular responses such as adhesion and spreading. Analysis of 12(S)‐HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin‐binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)‐HETE. The 12(S)‐HETE‐induced cytoskeletal alterations were accompanied by centrifugal organelle‐translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)‐HETE effect indicated a PKC‐mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal‐associated protein. Optimal effects were obtained after 5 min treatment with 12(S)‐HETE at 0.1 μM concentration. 12(S)‐HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)‐HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton‐dependent cell activity such as spreading. © 1993 Wiley‐Liss, Inc.
Inhibition of microtubule sliding by Ni2+ and Cd2+: Evidence for a differential response of certain microtubule pairs within the bovine sperm axoneme
Authors: Kanous Kathleen S. , Casey Christina , Lindemann Charles B. 
Published: February 2005

AbstractBovine sperm, extracted with 0.1% Triton X‐100, frozen at −20°C for 48–120 hours, and thawed, disintegrated by microtubule sliding when 1 mM MgATP was added. Microtubules and outer dense fibers (ODFs) were usually extruded in groups or “bundles.” A total of 44.5% of the cells extruded two distinct bundles, one from each side of the connecting piece, exhibiting opposite curvatures. Only one bundle was observed in 46.2% of the cells, and 9.2% showed no signs of sliding. Transmission electron microscopy (T.E.M.) showed one group consisting of the 4,5‐6,7 elements, with the 9,1,2 elements on the other side of the axoneme making up the other bundle. T.E.M. revealed that when only one side of the axoneme had extruded elements, they were always from the 4,5‐6,7 group. The remainder of the axoneme (8,9,1,2,3 and the central pair) was left relatively intact, suggesting a difference in the sliding response of the nine pairs of axonemal microtubules. These results indicate a predisposition for sliding between elements 7 and 8 over that between doublets 2 and 3, perhaps due to a disparity in activation thresholds. Also, both Ni2+ and Cd2+ appear to selectively block activation of 2–3 interdoublet sliding.Incubation with 0.25 mM Ni2+ prior to adding MgATP modified the percentages of sliding patterns: 8.6% demonstrated two‐sided extrusion, 58.2% showed one‐sided, and 33.2% had no extruded bundles. Again, when half the axoneme was missing, it was always the 4,5‐6,7 group. Ten micromolar Cd2+ altered the sliding pattern similarly to Ni2+, with 28% two‐sided extrusion, 55.9% one‐sided extrusion and 16.1% with no extruded bundles.Either pretreatment regimen impeded extrusion of the 9,1,2 group in a high percentage of cells, compared to untreated cells. This specific inhibition of the 9,1,2 side by Ni2+ or Cd2+ is especially significant since Ni2+ also inhibits spontaneous wave initiation in bull sperm (Lindemann et al.: Journal of Cell Biology 87:420–426, 1980), and both Ni2+ and Cd2+ reportedly block the flagellar Ca2+‐response in rat sperm (Lindemann and Goltz: Cell Motility and the Cytoskeleton 10:420–431, 1988; Lindemann et al.: Cell Motility and the Cytoskeleton 20:316–324, 1991). © 1993 Wiley‐Liss, Inc.
Method of cell handling affects leakiness of cell surface labeling and detection of intracellular keratins
Authors: Riopel Carrie L. , Butt Isha , Omary M. Bishr 
Published: February 2005

AbstractKeratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well‐accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase‐catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water‐soluble sulfo–NHS–biotin. Partitioning of the keratins to a soluble and an insoluble pool after “cell surface” 125I‐labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti‐keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques. © 1993 Wiley‐Liss, Inc.
Neurofilaments move apart freely when released from the circumferential constraint of the axonal plasma membrane
Authors: Brown Anthony , Lasek Raymond J. 
Published: February 2005

AbstractSquid giant axons were used to obtain axonal cytoskeletons that had been separated from the confines of their plasma membranes. To remove the plasma membrane, axoplasm was extruded from the giant axon directly into an artificial axoplasm solution (AAS). This procedure produces a smooth axoplasmic cylinder in which neurofilaments (NFs) are the most prevalent cytological elements. The NFs scatter light strongly and thus dark‐field light microscopy can be used to quantify the volume occupied by these polymers. Measurements of the widths of the dark‐field images of the axoplasmic cylinders showed that the cross‐sectional area of the NF population increased by 60–110% (n = 8) between 1–100 min after plasma membrane removal, and then continued to increase more slowly for many hours. After 1,000 min, the cross‐sectional area was 75–160% (n = 8) larger than at 1 min. These light microscopic measurements of axoplasm suggest that the NF population disperses to occupy a continuously increasing volume after removal of the plasma membrane and immersion in AAS. This inference was confirmed by quantitative ultrastructural studies of NFs in axoplasmic cross‐sections, which demonstrated that the spacing between the NFs increased between 1–1,000 min after plasma membrane removal. Comparison of the NF density distribution after 1,000 min with a theoretical distribution calculated using the Poisson theorem indicated that the NFs dispersed randomly. These studies on NFs in isolated axoplasm suggest that ordinary thermal forces of Brownian motion are sufficient to move axonal NFs apart independently and thereby to disperse them. We propose that, in the intact axon, the dispersive movements of the NFs spread the NF cytoskeleton radially and expansively to fill out the cylindrical space contained by the axonal plasma membrane and its surrounding connective tissue elements. © 1993 Wiley‐Liss, Inc.
Role of phosphorylation in keratin and vimentin filament integrity in cultured thyroid epithelial cells
Authors: Deery William J. 
Published: February 2005

AbstractCytokeratin and vimentin intermediate filaments (IFs) possess relatively stable polymeric properties which can be affected by phosphorylation. The present study, using cultures of thyroid epithelial cells, shows by indirect immunofluorescence that these cells contain both keratin tonofilament and vimentin IF complexes. Immunoblots of Triton X‐100 insoluble cytoskeletal fractions show vimentin, and ∼52 kDa type II and 40/38 kDa type I keratins. Under “basal” conditions, following prelabeling of cells with [32PO4], vimentin is not significantly phosphorylated, while both type II and I keratins are phosphorylated. Treatment of cells for 20 min with 1 mM dbcAMP or 0.4 μM 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), to stimulate protein kinase A and C, respectively, has no effect on either the phosphorylation state or cytoplasmic filament integrity of vimentin. However, while dbcAMP also does not affect keratin filaments, TPA increases both type II and I phosphorylation ∼3‐fold, and concomitantly disrupts tonofilament complexes associated with the nucleus, cytoplasm, and desmosomes. TPA‐treated cells also show dramatic shape changes and protrusive activity. Tryptic peptide mappings show phosphorylations of at least 6 and ∼2 additional sites for type II and I keratins, respectively, vs. [32P]‐peptides from control cells. Treatment of [32PO4]‐labeled cells with 0.4 μM calyculin A to inhibit types 1 and 2A phosphatase activity causes hyperphosphorylation of both vimentin and keratin, disruption of IF complexes, and actomyosin/cell contraction within 20 min. Quantitatively, ∼50% of the type II/I keratin hyperphosphorylations are at some sites apparently also phosphorylated after TPA treatment. Thus, in these cells, IFs are specifically and differentially affected and regulated by the activity of several kinases. © 1993 Wiley‐Liss, Inc.