Nucleosome packing in interphase chromatin.

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.

Membrane flow during pinocytosis. A stereologic analysis.

HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.

Nuclear pore complexes. Elimination and reconstruction during mitosis.

Nuclear structures similar to those of the nuclear pore complex were found on chromosomes. This finding indicates that part of the pore complex is retained by the chromosomes through mitosis in the absence of the nuclear membrane. The formation of approximately the same number of pore complexes in the presence and absence of protein synthesis during the first 4 h after mitosis proves the reassembly rather than new synthesis of the pore complex. The structure of pore complexes reconstructed in the absence of protein synthesis cannot be distinguished from the structure of those of control cells.

Pinocytosis in mouse L-fibroblasts: ultrastructural evidence for a direct membrane shuttle between the plasma membrane and the lysosomal compartment.

Mouse L-fibroblasts internalized large amounts of cationized ferritin (CF) by pinocytosis. Initially (60-90 s after addition of CF to cell monolayers at 37 degrees C), CF was found in vesicles measuring 100-400 nm (sectioned diameter) and as small clusters adhering to the inner aspect of the limiting membrane of a few large (greater than 600 nm) vacuoles. After 5-30 min, CF labeling of large vacuoles was pronounced and continuous. Moreover, 70-80% of all labeled structures were tiny (less than 100 nm) vesicles. However, the absolute frequency of tiny vesicles increased more than twofold from 5 min to 30 min. When the cells were incubated with CF for 30 min, then washed and further incubated for 3 h without CF, almost all CF was present in dense bodies (100-500 nm). When L-cells were first incubated with horseradish peroxidase (HRP), then washed and incubated with CF, double-labeled vacuoles were observed. Tiny vesicles also contained HRP-CF, and small HRP-CF patches were localized on the cell surface. Distinct labeling of stacked Golgi cisterns was not observed in any experiment. These observations suggest that the numerous tiny vesicles are not endocytic but rather pinch off from the large vacuoles and move towards the cell surface to fuse with the plasma membrane. Thus, ultrastructural evidence is provided in favor of a direct membrane shuttle between the plasma membrane and the lysosomal compartment.

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.

In situ localization of globin messenger RNA formation. I. During mouse fetal liver development.

Globin mRNA levels in 11-15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80-85% by days 13-15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.

Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines.

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.

Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.

Tau proteins are a class of low molecular mass microtubule-associated proteins that are specifically expressed in the nervous system. A cDNA clone of adult rat tau was isolated and sequenced. To analyze functions of tau proteins in vivo, we carried out transfection experiments. A fibroblast cell line, which was transfected with the cDNA, expressed three bands of tau, while six bands were expressed in rat brain. After dephosphorylation, one of the three bands disappeared, demonstrating directly that phosphorylation was involved in the multiplicity of tau. Morphologically, we observed a thick bundle formation of microtubules in the transiently and stably tau-gene-transfected cells. In addition, we found that the production of tubulin was prominently enhanced in the stably transfected cells. Thus, we suppose that tau proteins promote polymerization of tubulin, form bundles of microtubules in vivo, and play important roles in growing and maintaining nerve cell processes.

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.

Pinocytosis in fibroblasts. Quantitative studies in vitro.

Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032-0.0035% of the administered load per 10(6) cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6-8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2-38 degrees C. The Q(10) was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10-20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown.

Production of globules in mouse L cells penetrated with hamster sperms.

Globule formation has been observed in long-term cultures of mouse L cells penetrated with hamster spermatozoa. These numerous, uniform, and spherical structures are approximately 10 micrometers in diameter when formed, and are positive to stains specific for proteins and nucleic acids. They may be produced either by sperms within their target cells or by the cells in response to penetration by the spermatozoa.

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.

Characteristics of cell membranes from somatic cell hybrids between rat hepatoma and mouse L-cells.

Cell membranes from mouse L-cells (L-B82), rat hepatoma (HTC-H1), and three clones of their somatic cell hybrids (07, V4a, and V5) showing different degrees of density-dependent inhibition of growth were analyzed by polyacrylamide gel electrophoresis. The membrane polypeptides of the hybrid clones were all similar and all showed higher proportions of polypeptides with molecular weights of 56,000 and 45,000 than their parents of their normal counterparts. The major glycoprotein form cell hybrids appeared to be identical with that of rat liver or rat hepatoma cells and different from that of L-cells. One hybrid showed density-dependent inhibition growth; the other two, like both parents, did not. All produced tumors in nude mice, although tumor production by the hybrids was delayed. A large external protein (M.W. 240,000) iodinated by lactoperoxidase-catalyzed reaction was virtually missing in the parents but was present at high levels in all their hybrid clones. Thus, there was a lack of correlation between the presence of this protein, growth control in vitro, and tumorigenicity. Furthermore, no correlation was seen between agglutination of these cells by concanavalin A and tumorigenicity. The factors controlling these membrane properties thus are independent of density-dependent inhibition of growth and of those controlling the expression of cancer.

LM fibroblast plasma membrane subfractionation by affinity chromatography on con A-sepharose.

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-sided-out orientation of the vesicles. Similarly and asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5'-nucleotidase activity, and (Na+ + K+)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.

Histone inhibition of DNA synthesis in eukaryotic cells permeable to macromolecules.

Mouse L Cells, grown in suspension culture can be rendered permeable to exogenous deoxynucleoside triphosphates by a cold shock in a near isotonic buffer system. These cells use the deoxynucleotides to synthesize DNA in a semiconservative fashion. The addition of 0.05% Triton X-100 to this system increases the permeability of the cells so that exogenously supplied macromolecules gain access to the DNA. When DNAase and phosphodiesterase are added to the detergent-permeabilized cells, the cell DNA is rapidly degraded, demonstrating that the enzymes reach the DNA within the first 2 min of the incubation period. Addition of whole calf thymus histone or histone fractions to the detergent-permeabilized cells inhibits DNA synthesis. The lysine-rich histone, F is a more effective inhibitor than the arginine-rich histone, F3. The other histone fractions including the slightly lysine-rich fractions, F2a and F2b, are intermediate between F1 and F3 as inhibitors of DNA- synthesis. Kinetic analysis demonstrates that the added histones increase apparent Km and reduce V of DNA synthesis in the permeabilized cells. These studies suggest the possibility that histones alter the association of the DNA replication complex and the DNA template in a manner that reduces the rate of DNA synthesis.

DNA synthesis in permeabilized mouse L cells.

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells were used to conform rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na+, ATP, all 4 deoxynucleotides, and Mg2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxy-urea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.

Evidence for an intracellular calcium store releasable by surface stimuli ifibroblasts (L cells).

A spontaneously occurring or electrically elicited hyperpolarizing activation (HA) in L cells was previously shown to be due to a specific increase in the membrane K+ permeability (Nelson et at. 1972. J. Gen. Physiol. 60:58--71). Intracellular injection of Ca++ elicits an identical hyperpolarizing response which suggests that the increased K+ permeability associated with the HA is mediated by an increase in cytoplasmic Ca++. In zero-Ca, EGTA-containing saline the proportion of cells in which HA's can be evoked decreases, but the amplitude of those HA's that are produced is comparable to that of HA's in normal Ca saline. Co++ does block the HA but only after a period of 2 h or longer; D-600 does not affect the HA. The observations, with others, suggest that the primary source of the Ca mediating the HA response is intracellular. In L cells the endoplasmic reticulum forms morphologically specialized appositions with the surface membrane which resemble structures at the triads of muscle that are thought to mediate coupling between surface membrane electrical activity and contraction via Ca release from the sarcoplasmic reticulum. The similar structures in L cells may mediate coupling between surface membrane electrical, mechanical, or chemical stimuli and the HA response via release of Ca from the endoplasmic reticulum. Surface-coupled release of Ca from intracellular stores might also regulate a number of other intracellular functions in nonmuscle cells.

Evidence for the nucleus-centriole association in living cells obtained by ultracentrifugation.

The association of centrioles with the interphase nuclei of L- and PE-cells has been studied using ultracentrifugation of a cell monolayer in a culture medium at 20 and 37 degrees C. Ultracentrifugation at 10 000 to 40 000 gav for 15 to 60 min did not cause any changes in the cell length or in the size of the nucleus, but entailed delocalization of nuclei. The distance between it and the centrioles hardly ever changes. Any nucleus-delocalizing centrifugation of non-treated cells also resulted in the centrioles being shifted towards the centripetal nucleus pole. After 30 to 60 min, at 40 000 gav, the cells remained viable and capable of mitosis. More intensive centrifugation (15 min at 70 000 gav) proved to be fatal to the cells. The distance between the centrioles and the nucleus became greater in the cells which were centrifuged after incubation with cytochalasin B. The results are interpreted as lending support to the previously demonstrated [28] association between the centrioles and the nucleus in the interphase cells.