bluuebunny/crossref_metadata_2025 huggingface dataset:
Freeze-fracture and electrophysiological studies of newly developed acetylcholine receptors in Xenopus embryonic muscle cells.
Authors: Bridgman P C , Nakajima S , Greenberg A S , Nakajima Y
Published: May 2004

The development of acetylcholine receptors on Xenopus embryonic muscle cells both in culture and in situ was studied using electrophysiology and freeze-fracture electron microscopy. Acetylcholine sensitivity first appeared at developmental stage 20 and gradually increased up to about stage 31. Freeze-fracture of muscle cells that were nonsensitive to acetylcholine revealed diffusely distributed small P-face intramembraneous particles. When cells acquired sensitivity to acetylcholine, a different group of diffusely distributed large P-face particles began to appear. This group of particles was analyzed by subtracting the size distribution found on nonsensitive cells from that found on sensitive cells. We call this group of particles difference particles. The sizes of difference particles were large (peak diameter 11 nm). The density of difference particles gradually increased with development. The density of small particles (less than 9 nm) did not change with development. At later stages (32-36) aggregates of large particles appeared, which probably represent acetylcholine receptor clusters. The size distribution of difference particles was close to that of the aggregated particles, suggesting that at least part of difference particles represent diffusely distributed acetylcholine receptors. Difference particles exist mostly in solitary form (occasionally double), indicating that an acetylcholine receptor can be functional in solitary form. This result also shows that diffuse acetylcholine receptors that have previously been observed with 125I-alpha-bungarotoxin autoradiography do indeed exist in solitary forms not as microaggregates.
Importance of geometry of the extracellular matrix in endochondral bone differentiation.
Authors: Sampath T K , Reddi A H
Published: May 2004

Subcutaneous implantation of coarse powders (74-420 micron) of demineralized diaphyseal bone matrix resulted in the local differentiation of endochondral bone. However, implantation of matrix with particle size of 44-74 micron (Fine matrix) did not induce bone. We have recently reported that the dissociative extraction of coarse matrix with 4 M guanidine HCl resulted in a complete loss of the ability of matrix to induce endochondral bone; the total loss of biological activity could be restored by reconstitution of extracted soluble components with inactive residue. To determine the possible biochemical potential of fine matrix to induce bone, the matrix was extracted in 4 M guanidine HCl and the extract was reconstituted with biologically inactive 4 M guanidine HCl-treated coarse bone matrix residue. There was a complete restoration of the biological activity by the extract of fine matrix upon reconstitution with extracted coarse matrix. Polyacrylamide gel electrophoresis of the extract of fine matrix revealed similar protein profiles as seen for the extract of coarse matrix. Gel filtration of the 4 M guanidine HCl extract of fine powder on Sepharose CL-6B and the subsequent reconstitution of various column fractions with inactive coarse residue showed that fractions with proteins of 20,000-50,000 mol wt induced new bone formation. These observations demonstrate that although fine bone matrix contains, osteoinductive proteins, matrix geometry (size) is a critical factor in triggering the biochemical cascade of endochondral bone differentiation. Mixing of coarse matrix with Fine results in partial response and it was confined to areas in contact with coarse particles. The results imply a role for geometry of extracellular bone matrix in anchorage-dependent proliferation and differentiation of cells.
Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate.
Authors: Porrello K , Burnside B
Published: May 2004

We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.
Stimulated release of histamine by a rat mast cell line is inhibited during mitosis.
Authors: Hesketh T R , Beaven M A , Rogers J , Burke B , Warren G B
Published: May 2004

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine. Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca2+ that preceded secretion of histamine and a similar Ca2+ influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca2+ to the fusion of secretory granules with the plasma membrane.
Phosphorylation of microtubule-associated proteins by a Ca2+/calmodulin-dependent protein kinase.
Authors: Schulman H
Published: May 2004

In an earlier study I demonstrated that rat brain cytosol contains a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates microtubule-associated protein 2 (MAP-2) but not MAP-1. Comparison of sites of phosphate incorporated in MAP-2 catalyzed by the Ca2+/calmodulin-dependent kinase activity and the cyclic AMP-dependent protein kinase activity in cytosolic extracts revealed distinct sites of phosphorylation (Schulman, H., 1984, Mol. Cell. Biol., 4:1175-1178; abstract by me and J.A. Kuret and K.H. Spitzer [1983, Fed. Proc., 42:2250]. I have now used MAP-2 as a substrate to purify the Ca2+/calmodulin-dependent protein kinase responsible for MAP-2 phosphorylation. The brain appears to contain a single predominant Ca2+/calmodulin-dependent protein kinase that phosphorylates MAP-2. The enzyme was purified to apparent homogeneity by column chromatography using DEAE-cellulose, phosphocellulose, hydroxylapatite, Sepharose 6B, and a calmodulin-Sepharose affinity column. The 580,000-dalton holoenzyme consists of 51,000- and 60,000-dalton subunits. The purified enzyme phosphorylates MAP-2 at the same "sites" that are phosphorylated in cytosolic extracts and thus has the same specificity as the activity present in cytosol. Analysis of phosphorylated MAP-2.1 and MAP-2.2, the two components of MAP-2, suggests considerable homology in their phosphorylated domains.
Role of microtubules in the organization and localization of the Golgi apparatus.
Authors: Sandoval I V , Bonifacino J S , Klausner R D , Henkart M , Wehland J
Published: May 2004

Normal interphase PtK2 and A549 cells display long microtubules radiating from the microtubule-organizing center (MTOC) to the plasma membrane. Both MTOC and Golgi apparatus are contained in the same perinuclear area. Treatment of cells with 1 microM colcemid for 2 h results in microtubule depolymerization and fragmentation of the Golgi apparatus into elements scattered throughout the cytoplasm. Both normal microtubules and the Golgi apparatus assemble again following removal of colcemid. Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'(alpha, beta-methylene)diphosphate [pp(CH2)pG], into interphase cells growing in normal medium results in the formation of microtubule bundles resistant to colcemid and prevents the fragmentation of the Golgi apparatus. Injection of pp(CH2)pG into cells incubated with colcemid results in substitution of tubulin ribbons for microtubules and has no effect on the Golgi-derived elements scattered throughout the cytoplasm. Removal of colcemid 1 h after the injection of pp(CH2)pG results in polymerization of large numbers of short, single randomly oriented microtubules, whereas the Golgi apparatus remains fragmented. Treatment of cells with 10 microM taxol for 3 h results both in polymerization of microtubule bundles without relation to the MTOC in the cell periphery and fragmentation of the Golgi apparatus. The Golgi-derived fragments are present exclusively in regions of the peripheral cytoplasm enriched in microtubules. The codistribution of microtubules and Golgi elements can be reversed in taxol-treated cells by injection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin. Cells incubated with colcemid after treatment with taxol have large numbers of Golgi-derived elements in close association with colcemid-resistant microtubule bundles. Incubation of cells with 50 microM vinblastine for 90 min results in microtubule dissembly, formation of tubulin paracrystals, and fragmentation of the Golgi apparatus into elements without relation to the tubulin paracrystals.
Taxol-induced rose microtubule polymerization in vitro and its inhibition by colchicine.
Authors: Morejohn L C , Fosket D E
Published: May 2004

Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephadex A50 chromatography, and the taxol-induced polymerization of microtubules in vitro was characterized at 24 degrees C by turbidity development, sedimentation analysis, and electron microscopy. Numerous, short microtubules were formed in the presence of taxol, and maximum levels of turbidity and polymer yield were obtained at approximately 2:1 molar ratios of taxol to tubulin. The critical concentration of rose tubulin for polymerization in saturating taxol was estimated to be 0.21 mg/ml. Colchicine inhibited the taxol-induced polymerization of tubulin as shown by sedimentation assays; however, much higher concentrations of colchicine were required for the inhibition of taxol-induced rose tubulin assembly than for inhibition of taxol-induced mammalian brain tubulin assembly. On the basis of the relative sensitivity of rose tubulin assembly to taxol and its insensitivity to colchicine, we propose that the taxol-binding site(s) on plant and animal tubulins have been more conserved over evolution than the colchicine-binding site(s).
Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells.
Authors: Connolly J A
Published: May 2004

We have examined the effects of microtubule- and microfilament-disrupting drugs on the stability, formation, and removal of acetylcholine (ACh) receptors and ACh receptor clusters on the surface of aneurally cultured chick embryonic myotubes. (a) In muscle cell cultures, cytochalasin D (0.2 microgram/ml) or B (2.0 micrograms/ml) causes the dispersal of 50-60% of the existing clusters over a 24-h period (visualized with rhodamine-conjugated alpha-bungarotoxin); Colcemid (0.5 micrograms/ml) has no affect on these clusters. The total number of cell surface ACh receptors does not decline during this period (measured by [125I]alpha-bungarotoxin binding) in the presence of either drug. (b) When cells are treated with biotinylated alpha-bungarotoxin and fluorescent avidin, ACh receptors are cross-linked and rapidly internalized (Axelrod, D., 1980, Proc. Natl. Acad. Sci. USA., 77: 4823-4827). Within 6 h, I have found that 0-15% of the existing large clusters remain. Cytochalasin D or B had no effect on this removal of clusters; however, Colcemid completely prevented the removal of clusters from the cell surface. (c) Addition of chick brain extract to chick myotubes causes an increase in the synthesis and clustering of ACh receptors (Jessell et al., 1979, Proc. Natl. Acad. Sci. USA. 76: 5397-5401). Cytochalasin D caused a slight increase in the number of receptors synthesized in the presence of brain extract whereas Colcemid had no effect on the synthesis and insertion of new receptors into the plasma membrane induced by the brain extract. However, both drugs prevented the increase in the number of receptor clusters. These results are consistent with the hypothesis that receptor clusters are stabilized by actin-containing filaments, but that the movement of receptors in the plane of the membrane requires Colcemid-sensitive microtubules.
Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.
Authors: Schedl T , Burland T G , Gull K , Dove W F
Published: May 2004

The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.
Receptor-mediated transport of IgG.
Authors: Rodewald R , Kraehenbuhl J P
Published: May 2004

The intestinal epithelium of the neonatal rat is a model system for the study of receptor-mediated endocytosis in which large amounts of IgG are transferred intact across polarized cells. This review summarizes the ultrastructural pathway followed by IgG during cellular transit and several important properties of the membrane receptor that recognizes the IgG.
Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus.
Authors: Leys E J , Crouse G F , Kellems R E
Published: May 2004

Methotrexate-resistant cells, which contain a 500-fold amplification of dihydrofolate reductase (DHFR) genes, were used as a model system for studying the regulation of DHFR gene expression during growth stimulation. We have shown that a threefold increase in DHFR mRNA levels following growth stimulation results from a corresponding increase in DHFR mRNA production (i.e., delivery to the cytoplasm) and is not the result of a change in DHFR mRNA half-life. We previously showed that the increase in DHFR mRNA production during growth stimulation is not accompanied by an increase in the relative rate of transcription of the DHFR gene. This suggested that changes in DHFR mRNA production during growth stimulation are due to changes in the stability of DHFR transcripts in the nucleus. Using continuous labeling experiments in vivo comparing the stability of DHFR RNA with specific reference sequences, we show that in growing cells most DHFR transcripts were converted to mRNA, whereas in resting cells the majority of DHFR transcripts were rapidly degraded in the nucleus. There was no significant difference in the rate of processing and transport of stable DHFR transcripts. Therefore, changes in the stability of DHFR RNA in the nucleus control the amount of mRNA available for translation in the cytoplasm.
Water near intracellular surfaces.
Authors: Parsegian V A , Rau D C
Published: May 2004

In this paper we make the following points: Water is perturbed within several angstroms of the surfaces of soluble molecules. Removal of this water can require significant amounts of work, seen as an exponentially varying "hydration force" with respect to molecular separation. The favorable and specific attractions that occur in molecular assembly or in ligand binding imply that the specific association between the molecular surfaces is stronger than the association of those surfaces with water. The specificity of biochemical association is not simply a matter of protein-protein interaction but also of competing protein-water interactions. Small structural changes in molecular surfaces can evoke large changes in the contact energy of hydrated surfaces; surface hydration and the energetics of water displacement are a likely mechanism for the contact specificity of intracellular associations integrating the cell matrix.
Synthesis of a low molecular weight collagen by chondrocytes from the presumptive calcification region of the embryonic chick sterna: the influence of culture with collagen gels.
Authors: Gibson G J , Beaumont B W , Flint M H
Published: May 2004

The mature chick sternum is divisible almost equally into cephalic calcified and caudal cartilagenous regions. Isolation and culture of cells derived from embryonic precursors of these regions has revealed two discrete populations of cells with distinct morphological features and synthetic capabilities. Both cell populations grew well in culture within or upon collagen gels or upon plastic and maintained morphologies similar to those observed in the parent tissue. Polyacrylamide gel electrophoresis of radiolabeled proteins synthesized by the cells in culture demonstrated large differences in the types of collagens synthesized. Both chondrocyte populations synthesized type II and minor cartilage collagens but only chondrocytes isolated from the presumptive calcification region synthesized the previously identified, low molecular weight collagen, termed G collagen. Synthesis of G collagen was stimulated by culture within or upon collagen gels such that it represented an average of 65% of the total collagen synthesized by presumptive calcification region chondrocytes after 7 d of culture within collagen gels. Light and scanning electron microscopy demonstrated that the two chondrocyte types exhibited distinct morphological features and accumulated different extracellular matrices in culture.
Assembly associated with the cytomatrix.
Authors: Fulton A B
Published: May 2004

Assembly in vivo has been studied both for endogenous cytoskeletal proteins and for several classes of viruses. Autoradiography of cytoskeletal proteins has shown that many associate with the cytoskeletal framework close to the time and place of synthesis. The cytoskeletal proteins rearrange after association with the cytoskeletal framework. Rearrangement in symmetrical giant cells occurs in a centrifugal and coherent pattern. Many of the cytoskeletal proteins associate cotranslationally, as shown by their puromycin resistance in a cell-free translation system. The assembly of several groups of viruses has been shown to be associated with various components of the cytoskeleton; whether such assembly is cotranslational has not yet been addressed directly.
Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.
Authors: Yamada K M , Kennedy D W
Published: May 2004

Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.
Two enzymes involved in the synthesis of O-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells.
Authors: Elhammer A , Kornfeld S
Published: May 2004

Microsomal membranes from mouse lymphoma BW5147 cells were fractionated on a continuous sucrose gradient and assayed for two enzymes involved in the synthesis of O-linked oligosaccharides. Both enzymes were recovered in membranes that were less dense than the membranes containing the endoplasmic reticulum marker enzymes, glucosidase I and II. UDP-Gal:N-acetylgalactosamine-beta 1, 3-galactosyltransferase had a distribution that coincided with that of the galactosyltransferase that acts on asparagine-linked oligosaccharides. This latter enzyme has been immunolocalized to the trans Golgi elements. The UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase was recovered in a membrane fraction of intermediate density, between the endoplasmic reticulum and trans Golgi markers. These findings are consistent with the assembly of O-linked oligosaccharides occurring in at least two different Golgi compartments.
Early cytoplasmic specialization at the presumptive acetylcholine receptor cluster: a meshwork of thin filaments.
Authors: Peng H B , Phelan K A
Published: May 2004

Postsynaptic differentiation can be experimentally induced in cultured Xenopus myotomal muscle cells by polyornithine-coated latex beads (Peng, H. B., and P.-C. Cheng, 1982, J. Neurosci., 2:1760-1774). In this study, we examined the time course of this process. Small, punctate acetylcholine receptor (AChR) clusters were detectable as early as 1.5 h after the addition of the beads. Subsequently, both the size and the number of the clusters increased with time until a saturation level was reached between 8-24 h. Because the onset and the site of the AChR clustering could be precisely marked, we were able to examine the early structural specializations associated with presumptive AChR clusters. At 1 h, when less than 20% bead-muscle contacts displayed AChR clusters, 70% of the contacts already exhibited a meshwork of 5-6-nm filaments, which were of the same size as the thin filaments within the myofibrils and thus may contain actin. A system of cisternae similar to the smooth endoplasmic reticulum was suspended within this meshwork, but other organelles were excluded from it. This meshwork, being the earliest cytoplasmic specialization at the presumptive AChR clusters and appearing before the clusters, may be a mechanism for the clustering process.
Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins.
Authors: Wilson L , Miller H P , Pfeffer T A , Sullivan K F , Detrich H W
Published: May 2004

The colchicine-binding activity of tubulin has been utilized to distinguish the tubulins from two distinct microtubule systems of the same species, the sea urchin Strongylocentrotus purpuratus. We have analyzed the colchicine-binding affinities of highly purified tubulins from the unfertilized eggs and from the flagellar outer doublet microtubules by van't Hoff analysis, and have found significant differences in the free energy, enthalpy, and entropy changes characterizing the binding of colchicine to the two tubulins. The data indicate that significant chemical differences in the tubulins from the two functionally distinct microtubule systems exist, and that the differences are expressed in the native forms of the tubulins. Our findings are discussed in terms of the possibility that the colchicine-binding site may be an important regulatory site on the tubulin molecule.
Respective roles of centrosomes and chromatin in the conversion of microtubule arrays from interphase to metaphase.
Authors: Karsenti E , Newport J , Kirschner M
Published: May 2004

We report the results of studies in which partially purified centrosomes, nuclei, and DNA were injected into frog's eggs, which are naturally arrested in metaphase or interphase. These results have led to an independent assessment of the contributions of the centrosome and the chromatin to the formation of the mitotic spindle and suggest a simple explanation for the transition from interphase to metaphase microtubule arrays.
A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads.
Authors: Luna E J , Goodloe-Holland C M , Ingalls H M
Published: May 2004

In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluorescein-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads). Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at approximately 0.6 microgram of membrane protein per microgram of bead-bound F-actin, (c) rapid with a t1/2 of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads. This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E.J., V. M. Fowler, J. Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88:396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes. Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions. Using F-actin beads as an affinity column for actin-binding proteins, we show that at least 12 integral polypeptides in D. discoideum plasma membranes bind to F-actin directly or indirectly. At least four of these polypeptides appear to span the membrane and are thus candidates for direct transmembrane links between the cytoskeleton and the cell surface.
Specific inhibition of the differentiation of Trypanosoma cruzi.
Authors: Williams G T
Published: May 2004

The morphological transformation of Trypanosoma cruzi amastigotes was studied in both mammalian cells and an extracellular differentiation system. Inhibitors of ADP-ribosyl transferase were found to block differentiation in both cases, without affecting proliferation. The inhibitory effect was reversible and was not observed with chemical analogues that do not inhibit ADP-ribosyl transferase. As inhibitors of ADP-ribosyl transferase have recently been shown to block the differentiation of several cell types from vertebrates (Farzaneh, F., R. Zalin, D. Brill, and S. Shall, 1982, Nature (Lond.), 300:362-366; Johnstone, A. P., and G. T. Williams, 1982, Nature (Lond.), 300:368-370), our results suggest that the enzyme is of general importance in eucaryotic differentiation both in multicellular and unicellular organisms. In addition, since the compounds can block T. cruzi differentiation inside mammalian cells, these results suggest that it may be possible to exploit such inhibition in a new and potentially powerful approach to the chemotherapy of several important parasitic diseases.
Microfilament-organizing centers in areas of cell contact: cytoskeletal interactions during cell attachment and locomotion.
Authors: Geiger B , Avnur Z , Rinnerthaler G , Hinssen H , Small V J
Published: May 2004

In this article we discuss three aspects of cell contact formation: (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane- and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion. We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts: some proteins are specifically associated with focal contacts (vinculin and talin); alpha-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and alpha-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction. Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions.
Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.
Authors: Zucker-Franklin D , Petursson S
Published: May 2004

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.
Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.
Authors: Gilbert S P , Sloboda R D
Published: May 2004

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.
Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.
Authors: Green C R , Severs N J
Published: May 2004

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved. Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons. In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays. By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling. These experiments demonstrate that, contrary to the conclusions of some earlier studies (Baldwin, K. M., 1979, J. Cell Biol., 82:66-75; Peracchia, C., and L. L. Peracchia, 1980, J. Cell Biol., 87:708-718), the arrangement of gap junction connexons, in cardiac tissue at least, cannot be used as a reliable guide to the functional state of the junctions.
A study of chromosomal changes associated with amplified dihydrofolate reductase genes in rat hepatoma cells and their dedifferentiated variants.
Authors: Fougere-Deschatrette C , Schimke R T , Weil D , Weiss M C
Published: May 2004

We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R-banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection.
Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles.
Authors: Pepper D A , Kim H Y , Berns M W
Published: May 2004

We have devised a procedure for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. We prepared monoclonal antibody-producing hybridomas by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, one hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was found to be Colcemid sensitive. However, the 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear, and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide molecular weight of 240,000 is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.
Luminal material in microtubules of frog olfactory axons: structure and distribution.
Authors: Burton P R
Published: May 2004

The substructure and distribution of luminal material in microtubules of olfactory axons were studied in the bullfrog, Rana catesbeiana. By using numerous fixation methods, with and without osmium tetroxide, the luminal component was shown not to be an artifact of fixation. The material consists of globular elements 4-5 nm in diameter loosely arranged within the lumen in a discontinuous column. Counts of microtubules showing luminal material were obtained for axons in the proximal and distal ends of the olfactory nerve, and it was found that 16-18% more of the microtubules in the distal regions showed the luminal component. This raises the possibility that the material might be translocated within the microtubule lumen and tends to accumulate as it moves distally toward the axon terminal. In contrast to those of the olfactory axons, microtubules assembled in vitro from frog brain tubulin did not show luminal material. When microtubules in olfactory axons were depolymerized in situ by cold and calcium treatment and then induced to reassemble, most of those that were formed de novo showed empty lumina. Such evidence suggests that the luminal material is not an integral component of the microtubule. The hypothesis is discussed that material may be translocated within the lumina of microtubules. Furthermore, in the case of neuronal microtubules, the possibility is raised that they may serve as conduits for their own wall subunits.
Phalloidin enhances actin assembly by preventing monomer dissociation.
Authors: Coluccio L M , Tilney L G
Published: May 2004

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation with actin in the presence of equimolar phalloidin in 100 mM KCl, 1 mM MgCl2 and 0.5 mM ATP at pH 7.5 resulted in a slightly smaller association rate constant at the preferred end than in the absence of the drug (3.36 +/- 0.14 X 10(6) M-1 s-1 vs. 2.63 +/- 0.22 X 10(6) M-1 s-1, control vs. experimental). In the presence of phalloidin, the dissociation rate constant at the preferred end was reduced from 0.317 +/- 0.097 s-1 to essentially zero. Consequently, the critical concentration at the preferred end dropped from 0.10 microM to zero in the presence of the drug. There was no detectable change in the rate constant of association at the nonpreferred end in the presence of phalloidin (0.256 +/- 0.015 X 10(6) M-1 s-1 vs. 0.256 +/- 0.043 X 10(6) M-1 s-1, control vs. experimental); however, the dissociation rate constant was reduced from 0.269 +/- 0.043 s-1 to essentially zero. Thus, the critical concentration at the nonpreferred end changed from 1.02 microM to zero in the presence of phalloidin. Dilution-induced depolymerization at both the preferred and nonpreferred ends was prevented in the presence of phalloidin. Thus, phalloidin enhances actin assembly by lowering the critical concentration at both ends of actin filaments, a consequence of reducing the dissociation rate constants at each end.
Identification of calmodulin-binding proteins in chicken embryo fibroblasts.
Authors: Burgess W H , Watterson D M , Van Eldik L J
Published: May 2004

We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.