bluuebunny/crossref_metadata_2025 huggingface dataset:
Autocrine signals enable chondrocytes to survive in culture.
Authors: Ishizaki Y , Burne J F , Raff M C
Published: May 2004

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage--which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.
Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts.
Authors: Bassell G J , Powers C M , Taneja K L , Singer R H
Published: May 2004

Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize "individual" poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (< 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a "microdomain" where mRNA is attached and functionally expressed.
In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.
Authors: Huang S , Deerinck T J , Ellisman M H , Spector D L
Published: May 2004

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.
Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.
Authors: Deerinck T J , Martone M E , Lev-Ram V , Green D P , Tsien R Y , Spector D L , Huang S , Ellisman M H
Published: May 2004

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.
Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex.
Authors: Feldheim D , Schekman R
Published: May 2004

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.
N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion.
Authors: Whiteheart S W , Rossnagel K , Buhrow S A , Brunner M , Jaenicke R , Rothman J E
Published: May 2004

The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 microliters assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42% the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.
Dictyostelium discoideum mutants with conditional defects in phagocytosis.
Authors: Cohen C J , Bacon R , Clarke M , Joiner K , Mellman I
Published: May 2004

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s).
Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells.
Authors: Matter K , Yamamoto E M , Mellman I
Published: May 2004

In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.
The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase.
Authors: Nilsson I , Whitley P , von Heijne G
Published: May 2004

Signal peptides (SPs) target proteins to the secretory pathway and are cleaved from the nascent chain once the translocase in the ER has been engaged. Signal-anchor (SA) sequences also interact transiently with the ER translocase, but are not cleaved and move laterally out of the translocase to become permanent membrane anchors. One obvious difference between SP and SA sequences is the considerably longer hydrophobic regions (h regions) of the latter. To study the interaction between SP/SA sequences and the ER translocase, we have constructed signal sequences with poly-Leu h regions ranging in length from 8 to 29 residues and have characterized their locations within the translocase using both a new assay that measures the minimum number of amino acids needed to span the distance between the COOH-terminal end of the h region and the active site of the oligosaccharyl transferase enzyme and an assay where the efficiency of signal peptidase catalyzed cleavage is measured. Our results suggest that SP and SA sequences are positioned differently in the ER translocase.
Characteristics of endoplasmic reticulum-derived transport vesicles.
Authors: Rexach M F , Latterich M , Schekman R W
Published: May 2004

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.
pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.
Authors: Orci L , Halban P , Perrelet A , Amherdt M , Ravazzola M , Anderson R G
Published: May 2004

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.
The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system.
Authors: Bosshart H , Humphrey J , Deignan E , Davidson J , Drazba J , Yuan L , Oorschot V , Peters P J , Bonifacino J S
Published: May 2004

To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule.
Mitochondrial Ca2+ homeostasis in intact cells.
Authors: Rizzuto R , Bastianutto C , Brini M , Murgia M , Pozzan T
Published: May 2004

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.
Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro.
Authors: Eilertsen K J , Kazmierski S T , Keller T C
Published: May 2004

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.
Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell.
Authors: Hill M A , Schedlich L , Gunning P
Published: May 2004

Cell motility is dependent upon the reorganization of the cellular cytoskeleton. Actin filaments form the major component of the cytoskeleton and respond rapidly to serum growth factors. We have previously shown that myoblasts sort the two cytoskeletal beta- and gamma-actin isoform mRNAs to different intracellular regions and that only beta-actin mRNA was associated with peripheral regions of cell motility (Hill, M.A. and P. Gunning. 1993. J. Cell Biol. 122: 825-832). We now show by in situ hybridization that 3T3 fibroblasts similarly sort actin isoform mRNAs and that peripheral beta-actin mRNA is regulated by serum. In the absence of serum, we could not detect beta-actin mRNA at the periphery. Addition of serum rapidly redistributed beta-actin mRNA to the periphery. gamma-actin mRNA distribution was not altered by serum addition at any time. Both proteins, as identified by immunochemistry with isoform-specific antibodies, were found in similar cellular structures. Serum-stimulated cell motility is mediated through the GTPase signal transduction pathway. We find that an RNA-binding protein, p62, that is part of this pathway, displays a localization pattern similar to beta-actin mRNA. Our results suggest a new biological mechanism which integrates signal transduction with the supply of an architectural component required for membrane remodeling. We propose that active transport of beta-actin mRNA to regions of cell motility is one possible objective of these signal transduction pathways.
Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin.
Authors: Menkel A R , Kroemker M , Bubeck P , Ronsiek M , Nikolai G , Jockusch B M
Published: May 2004

Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.
Elements of error correction in mitosis: microtubule capture, release, and tension.
Authors: Nicklas R B , Ward S C
Published: May 2004

The correction of certain errors in mitosis requires capture and release: new kinetochore microtubules must be captured and old, misdirected ones must be released. We studied capture and release in living grasshopper spermatocytes. Capture is remarkably efficient over a broad range in the angle at which a microtubule encounters a kinetochore. However, capture is inefficient when kinetochores point directly away from the source of properly directed microtubules. Capture in that situation is required for correction of the most common error; microtubule-kinetochore encounters are improbable and capture occurs only once every 8 min, on average. Release from the improper attachment caused by misdirected microtubules allows kinetochore movement and the completion of error correction. We tugged on kinetochores with a micromanipulation needle and found they are free to move less than one time in two. Thus error correction depends on two improbable events, capture and release, and they must happen by chance to coincide. In spermatocytes this will occur only once every 18 min, on average, but a leisurely cell cycle provides ample time. Capture and release generate only change, not perfection. Tension from mitotic forces brings change to a halt by stabilizing the one correct attachment of chromosomes to the spindle. We show that tension directly affects stability, rather than merely constraining kinetochore position. This implies that chromosomes are attached to the spindle by tension-sensitive linkers whose stability is necessary for proper chromosome distribution but whose loss is necessary for the correction of errors.
Stimulation of a glycosyl-phosphatidylinositol-specific phospholipase by insulin and the sulfonylurea, glimepiride, in rat adipocytes depends on increased glucose transport.
Authors: Müller G , Dearey E A , Korndörfer A , Bandlow W
Published: May 2004

Lipoprotein lipase (LPL) and glycolipid-anchored cAMP-binding ectoprotein (Gce1) are modified by glycosyl-phosphatidylinositol (GPI) in rat adipocytes, however, the linkage is potentially unstable. Incubation of the cells with either insulin (0.1-30 nM) or the sulfonylurea, glimepiride (0.5-20 microM), in the presence of glucose led to conversion of up to 35 and 20%, respectively, of the total amphiphilic LPL and Gce1 to their hydrophilic versions. Inositol-phosphate was retained in the residual protein-linked anchor structure. This suggests cleavage of the GPI anchors by an endogenous GPI-specific insulin- and glimepiride-inducible phospholipase (GPI-PL). Despite cleavage, hydrophilic LPL and Gce1 remained membrane associated and were released only if a competitor, e.g., inositol-(cyclic)monophosphate, had been added. Other constituents of the GPI anchor (glucosamine and mannose) were less efficient. This suggests peripheral interaction of lipolytically cleaved LPL and Gce1 with the adipocyte cell surface involving the terminal inositol-(cyclic)monophosphate epitope and presumably a receptor of the adipocyte plasma membrane. In rat adipocytes which were resistant toward glucose transport stimulation by insulin, the sensitivity and responsiveness of GPI-PL to stimulation by insulin was drastically reduced. In contrast, activation of both GPI-PL and glucose transport by the sulfonylurea, glimepiride, was not affected significantly. Inhibition of glucose transport or incubation of rat adipocytes in glucose-free medium completely abolished stimulation of GPI-PL by either insulin or glimepiride. The activation was partially restored by the addition of glucose or nonmetabolizable 2-deoxyglucose. These data suggest that increased glucose transport stimulates a GPI-PL in rat adipocytes.
Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase.
Authors: Arroyo A G , Campanero M R , Sánchez-Mateos P , Zapata J M , Ursa M A , del Pozo M A , Sánchez-Madrid F
Published: May 2004

Intercellular adhesion molecule (ICAM)-3, a recently described counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1-mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.
Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.
Authors: LaFlamme S E , Thomas L A , Yamada S S , Yamada K M
Published: May 2004

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.
Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium.
Authors: Ballock R T , Reddi A H
Published: May 2004

Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defined cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis.
An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II.
Authors: Meller V H , McConnell M , Fisher P A
Published: May 2004

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids.
Authors: Masaki R , Yamamoto A , Tashiro Y
Published: May 2004

Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536-19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli beta-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting.
Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium.
Authors: Hitt A L , Hartwig J H , Luna E J
Published: May 2004

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.
Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family.
Authors: Turunen O , Wahlström T , Vaheri A
Published: May 2004

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.
Microtubule dynamics in fish melanophores.
Authors: Rodionov V I , Lim S S , Gelfand V I , Borisy G G
Published: May 2004

We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X-rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport.
Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.
Authors: Li M , McGrail M , Serr M , Hays T S
Published: May 2004

The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte.