Expression of normal and mutant avian integrin subunits in rodent cells [published erratum appears in J Cell Biol 1989 Oct;109(4 Pt 1):1187]

We describe the expression of the beta 1 subunit of avian integrin in rodent cells with the purpose of examining the structure-function relationships of various domains within this subunit. The exogenous subunit is efficiently and stably expressed in 3T3 cells, and it forms hybrid heterodimers with endogenous murine alpha subunits, including alpha 3 and alpha 5. These heterodimers are exported to the cell surface and localize in focal contacts where both extracellular matrix and cytoskeleton associate with the plasma membrane. Hybrid heterodimers consisting of exogenous beta 1 and endogenous alpha subunits bind effectively and specifically to columns of cell-binding fragments of fibronectin. The exogenous avian beta 1 subunit appears to function as well as its endogenous murine equivalent, consistent with the high degree of conservation noted previously for integrins. In contrast, expression of a mutant form of avian integrin beta 1 subunit lacking the cytoplasmic domain produces hybrid heterodimers which, while efficiently exported to the cell surface and still capable of binding fibronectin, do not localize efficiently in focal contacts. This further implicates the cytoplasmic domain of the beta 1 subunit in interactions required for cytoskeletal organization.     

Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 1):1679]

The current model of cytokinesis proposes that spindle poles and associated microtubules determine the cleavage plane, and, once the signal has been delivered to the cortex, the entire mitotic apparatus can be removed without affecting cell division. While supported by compelling data from Echinoderm embryos, recent observations suggest that the model may not be universally applicable. In this study, we have examined the relationship(s) among microtubules, chromosomes, and cleavage activity in living normal rat kidney (NRK) cells with multipolar mitotic figures. We found that cleavage activity correlated with the distribution of midzone microtubule bundles and Telophase Disc 60 protein (TD60) rather than the position of spindle poles. In addition, reduction of midzone microtubules near the cortex, by either nocodazole treatment or spontaneous reorganization in tripolar cells, caused inhibition or regression of furrowing. These results demonstrate that continuous interaction between midzone microtubule bundles and the cortex is required for successful cleavage in tissue culture cells.     

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.     

A 13-A map of the actin-scruin filament from the limulus acrosomal process [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1621]

We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)- ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross- links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Primary structure of the brain alpha-spectrin [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Regulation of fibronectin receptor distribution [published erratum appears in J Cell Biol 1992 Jul;118(2):491]

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1623]

The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect cells infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B- rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for alpha 6 and beta 1 integrin chains. Since intercalation (a) restored a conformationally dependent alpha 6 beta 1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits.     

Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.     

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10(-9) cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10(-10)-10(-11) cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodamine-phosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltage-dependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.     

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 2):1925]

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small- budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal- Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

Alcohol inhibits cell-cell adhesion mediated by human L1 [published erratum appears in J Cell Biol 1996 Jun;133(5):1139-40]

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector- transfected controls. Ethanol potently and completely inhibited L1- mediated adhesion both in transfected L cells and NIH/3T3 cells. Half- maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n- butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1- mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N- CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.     

Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment.     

A mutation affecting basal body duplication and cell shape in Paramecium [published erratum appears in J Cell Biol 1987 Jun;104(6):1755]

The thermosensitive mutant sm19 of Paramecium tetraurelia undergoes a progressive reduction in cell length and basal body number over successive divisions at the nonpermissive temperature of 35 degrees C. In spite of these defects, sm19 cells retain the same generation time as wild-type cells at 35 degrees C. Cytological observations at both electron and light microscopy levels reveal no other perturbation than the rarefaction of basal bodies and the rare (3%) absence of one or two microtubules in basal bodies or ciliary axonemes. The temperature-sensitive period, during the last 30 min of the cell cycle, corresponds to the phase of basal body duplication. Upon transfer back to the permissive temperature, all basal bodies are normally duplicated. The mutational defect is transiently restored by microinjection of wild-type cytoplasm or of a soluble proteic fraction from wild-type cell homogenates. Altogether, the cytological and physiological data support the conclusion that the sm19+ gene codes for a diffusible product required for the initiation of basal body duplication and would thus be the first identified gene involved in this process. Our data also indicate that in Paramecium basal body number is not coupled with control of the cell cycle, but helps determine the shape of the cell via the organization of the cytoskeleton.